Synthesis and evaluation of oxindoles as promising inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1

Article abstract of DOI:10.1039/c7md00226b

Indoleamine 2,3-dioxygenase 1 (IDO1) is considered as an important therapeutic target for the treatment of cancer, chronic infections and other diseases that are associated with immune suppression. Recent developments in understanding the catalytic mechanism of the IDO1 enzyme revealed that conversion of l-tryptophan (l-Trp) to N-formylkynurenine proceeded through an epoxide intermediate state. Accordingly, we synthesized a series of 3-substituted oxindoles from l-Trp, tryptamine and isatin. Compounds with C3-substituted oxindole moieties showed moderate inhibitory activity against the purified human IDO1 enzyme. Their optimization led to the identification of potent compounds, 6, 22, 23 and 25 (IC₅₀ = 0.19 to 0.62 μM), which are competitive inhibitors of IDO1 with respect to l-Trp. These potent compounds also showed IDO1 inhibition potencies in the low-micromolar range (IC₅₀ = 0.33-0.49 μM) in MDA-MB-231 cells. The cytotoxicity of these potent compounds was trivial in different model cancer (MDA-MB-231, A549 and HeLa) cells and macrophage (J774A.1) cells. Stronger selectivity for the IDO1 enzyme (124 to 210-fold) over the tryptophan 2,3-dioxygenase (TDO) enzyme was also observed for these compounds. These results suggest that the oxindole moiety of the compounds could mimic the epoxide intermediate state of l-Trp. Therefore, the structural simplicity and low-micromolar inhibition potencies of these 3-substituted oxindoles make them quite attractive for further investigation of IDO1 function and immunotherapeutic applications.

Full text of DOI:10.1039/c7md00226b

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ARTICLE
Synthesis and evaluation of oxindoles as promising inhibitors to
the immunosuppressive enzyme indoleamine 2,3-Dioxygenase 1
Saurav Paul,†a Ashalata Roy,†a Suman Jyoti Deka,b Subhankar Panda,a Gopal Narayan Srivastava,a
Vishal Trivedib and Debasis Manna*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Indoleamine 2,3-dioxygenase 1 (IDO1) is considered as an important therapeutic target for the treatment of cancer,
chronic infections and other diseases that are associated with immune suppression. Recent developments in
understanding the catalytic mechanism of IDO1 enzyme revealed that conversion of L-tryptophan (L-Trp) to N-
formylkynurenine proceed through an epoxide intermediate state. Accordingly, we synthesized a series of 3-substituted
oxindoles from L-Trp, tryptamine and isatin. Compounds with C3-substituted oxindole moiety showed moderate inhibitory
activity against purified human IDO1 enzyme. The optimizations directed to the identification of potent compounds, 6, 22,
23 and 25 (IC50 = 0.19 to 0.62 μM), which are competitive inhibitors of IDO1 with respect to L-Trp. These potent
compounds also showed IDO1 inhibition potencies in the low-micromolar range (IC50 = 0.33-0.49 μM) in MDA-MB-231
cells. The cytotoxicity of these potent compounds were trivial in different model cancer (MDA-MB-231, A549 and HeLa)
cells and macrophase (J774A.1) cells. Stronger selectivity for IDO1 enzyme (124 to 210-fold) over tryptophan 2,3-
dioxygenase (TDO) enzyme was also observed for these compounds. These results suggest that oxindole moiety of the
compounds could mimic the epoxide intermediate state of L-Trp. Therefore, structural simplicity and low-micromolar
inhibition potencies of these 3-substituted oxindoles make them quite attractive for further investigation of IDO1 function
www.rsc.org/
and
immunotherapeutic
applications.
7,
8
encephalitis and others.4,
Depletion of the local
Introduction
concentration of L-Trp and generation of kynurenine and other
metabolites including, excitotoxin quinolinic acid, N-methyl-D-
aspartate receptor antagonist kynurenic acid, and
nicotinamide adenine dinucleotide (NAD) help IDO1 to restrain
local immune response by perturbing the proliferation of T-
lymphocytes.4, 9
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-
dioxygenase (TDO) enzymes are the two important heme-
containing redox enzymes that catalyse the initial and rate-
determining step for the transformation of
L-Trp to N-
formylkynurenine through kynurenine pathway.1-3 IDO1 is
expressed in several tissues all throughout the body, but over-
expressed in a variety of non-hepatic tissues, including lung,
epididymis, gut and placenta. Cytokines like interferon-γ are
mostly accountable for this over-expression of IDO1 enzyme in
non-hepatic tissues.1 TDO is expressed predominantly in
Cancer immunotherapy by targeting IDO1 enzyme is
recognized as an exciting approach for drug development.
Unlike TDO the active site of IDO1 enzyme is amenable to
small molecules. Recent studies in different animal models of
cancer described that the perturbation of IDO1 activity by
using small molecules successfully restrain the abnormal
growth of tumours. This IDO1 inhibition approach also showed
hepatic tissues and its activity regulates L-Trp balance in
response to dietary intake. The increased level of IDO1
expression is interrelated with reduced prognosis in different
cancers, including pancreatic, ovarian and others.3, 4 However,
it is recently reported that endothelial IDO1 expression in
augmented effect with chemotherapeutic and radio-
11
Currently,
therapeutic treatment of malignant tumours.10,
IDO1 inhibitors INCB024360 and NLG919 in combination with
humanized antibodies are under clinical trials for the
treatment of cancer and other diseases.12, 13 Tryptophan-based
compound, D-1-MT is also under clinical trial as kynurenine
pathway inhibitor, but its mechanism of action is uncertain. It
is described that high concentrations of D-1-MT restrain the
kynurenine generation, but it fails to successfully reinstate
IDO1-promoted obstruction of T-cell proliferation and other
6
kidney tumours is associated with a better prognosis.5,
Stimulated expression of IDO1 is also related with the
neurodegenerative disorder, age related cataract, HIV
14
biological activities.4, IDO1 is also a promising therapeutic
target for the treatment of chronic viral infections and others
that are related with the pathological immune suppression.13,
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15 Therefore, there is a clear and unmet need to develop small Fe(III)-superoxide intermediate (Fig. 1).23 Consequently,
molecule-based IDO1 inhibitors that will adequately address oxindole derivatives could hinder the IDO1 enzyme-dependent
this cancer immunotherapeutic approach.
oxidative cleavage reaction of the pyrrole ring of L-Trp.
There are several known L-Trp or indole-based IDO1 Therefore, we hypothesized that oxindole-based derivatives
inhibitors. 1-Methyl-L-tryptophan (L-1-MT) is one of the could be used as potent inhibitor of IDO1 enzyme (Fig. 1).
commonly used IDO1 inhibitors but with moderate activity.
Brassinin, tryptamine, carboline, keto-indoles, indol-2-yl
ethanones, 1-methyl-tryptophan-tirapazamine, isatin and
other indole derivatives also showed poor to moderate IDO1
inhibitory activities.16-22 Comprehensive mechanistic studies of
IDO1 induced L-Trp catabolism revealed that the addition of
ferrous heme-iron coordinated molecular oxygen to the C2-C3
double bond of the pyrrole ring is the prerequisite for the IDO1
supported oxidation of
formation of epoxide intermediate state during the
transformation of -Trp to N-formylkynurenine by IDO1
L-Trp. These studies also proposed the
L
enzyme.23-25 Therefore, development of Trp or indole-based
IDO1 inhibitors that could block this enzyme-dependent
oxidative cleavage reaction of the pyrrole ring could construct
mechanism-based effective IDO1 inhibitors (Fig. 1).
In an attempt to find effective IDO1 inhibitors, we
synthesized oxindole and explored their enzyme inhibition
potentials. Several of our tested oxindole-based compounds
showed low-micromolar inhibitory activities against the
purified IDO1 enzyme. Selected compounds also showed low-
micromolar IDO1 enzyme inhibitory activity in MDA-MB-231
cells and almost no/negligible amount of cytoxicity. Additional
studies showed that these potent compounds were more
selective toward the IDO1 enzyme in comparison with the TDO
enzyme, making oxindoles of compelling value for further
development as therapeutic agents targeting IDO1.
Fig. 1 Proposed epoxide intermediate for the IDo1 induced
metabolism of L-Trp (A, B) and its mimic (C).
Initially, we synthesized oxindole derivatives
1 and 2 of L-Trp
and tryptamine, respectively by treating with the oxidizing
mixture of DMSO/HCl in AcOH solution.32 To explore the role
of hydrophobic substitution of the amine groups, oxindole
derivatives of L-Trp and tryptamine 3, 4, 5, 6, and 7 were
synthesized from N-Cbz, N-Boc, N-benzoyl and N-acetyl
protected L-Trp and triptamine, respectively under the similar
experimental conditions. To understand the importance of
Result and discussion
Synthesis of oxindole derivatives
free acid group compounds,
8 and 9 were synthesized from
Earlier, L-Trp derivative 5-((1H-indol-3-yl)methyl)-3-methyl-2-
only C-methyl and both C-benzyl and N-CbZ protected L-Trp
(Scheme 1).32 For the same purpose, 2-amino-3-(2-oxoindolin-
thioxoimidazolidin-4-one,
1-methyltryptophantirapazamine
and tryptamine derivatives were reported as moderate
inhibitor of IDO1 enzyme.4 However, no L-Trp analog showed
low-micromolar activities, probably because of moderate
affinity of L-Trp for IDO1 enzyme (Kd ~ 300 μM).4 We
hypothesize that L-Trp derivative that could mimic the
transition/intermediate state of the enzymatic reaction, offer a
better approach in developing potent IDO1 inhibitors. In this
regard, we developed oxindole derivatives of L-Trp and
tryptamine. Recently, several oxindole derivatives have been
reported as potent inhibitors of TAK1 kinase and activators of
AMP kinase.26, 27 Oxindole derivatives also show antiglycation,
antifungal, and other biological activities.28, 29 Oxindole -based
3-yl)propanamide (10) was also synthesized from compound
according to the reported procedures.33, 34 To explore the role
3
Scheme 1 Synthesis of oxindole derivatives from L-Trp and tryptamine.
alkaloids from Uncaria tomentosa have been described to
suppress L-Trp degradation.30,
31
Therefore, oxindole moiety
could be considered as an useful tool in drug discovery.
thorough understanding of the IDO1 assisted
A
transformation of L-Trp to N-formayl-kynurenine recommend
that the presence of carbonyl group at the C2-position of the
pyrrole ring of L-Trp could perturb further radical addition of
2 | J. Name., 2012, 00, 1-3
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of C3-substitution of the oxindole moiety at inhibiting the IDO1 and/or α-carboxyl groups of
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DOI: 10.1039/C7MD00226B
ARTICLE
L
-Trp and tryptamine structure
enzyme activity, we also synthesized hydrazone (compound and (2) C3-substitution of the isatin.
11 16), phenylimino (17 19) and alkene-oxindole (20 and 21
-
-
)
derivatives of isatin. C3-Substituted 3-hydroxy-3-alkyl
derivatives of isatin and 5-chloroisatin were synthesized
according to the reported procedure to explore the role
substitution in the oxindole ring on IDO1 enzyme activity.35
Condensation of substituted hydrazine or aniline with isatin in
refluxing ethanol (in the presence of catalytic amount of
AcOH) directly yielded hydrazones and phenylimino derivatives
Scheme 3 Synthesis of alkene and 3-hydroxy-3-alkyl derivatives of 5-chloroisatin.
(Scheme 2).35 The alkene-oxindole derivatives (20 and 21
)
were synthesized from isatin in the presence of either
piperidineacetate in water or piperidine in EtOH (Scheme 2).36
The 3-hydroxy-3-alkyl compounds
(
24 and 25
)
were
synthesized from isatin according to the similar procedure
(Scheme 2).36 Condensation of 5-chloroisatin with acetone first
yielded 3-hydroxy-3-alkyl compound 26 in the presence of
K2CO3. Dehydration of compound 26 in the presence of
concentrated HCl and catalytic amount of AcOH in ethanol
yielded compound 22 (Scheme 3). Condensation of 5-
chloroisatin with acetylacetone in the presence of K2CO3
yielded both alkene and 3-hydroxy-3-alkyl derivatives 23 and
27, respectively (Scheme 3).37 Hence, a range of oxindole
Modification of the α-amino and/or α-carboxyl groups of L-
Trp and tryptamine structure ― We explored the role of α-
amino and/or α-carboxyl groups of oxindolealanine and 3-(2-
aminoethyl)indolin-2-one on IDO1 enzyme activity (Table 1).
The IC50 values of the compound
at the free amino-group and the presence of free carboxyl-
group ( ) plays an important role in their IDO1 inhibitory
activity. The IC50 values of compounds 10 further support
these findings. The stronger IDO1 inhibitory activity of
compound, (IC50 = 0.62 μM) could be due the presence of
1-10 suggest that substitution
derivatives was synthesized from
5-chloroisatin.
L-Trp, tryptamine, isatin and
3-6
7-
Scheme 2 Synthesis of hydrazone, phenylimino and alkene derivatives of isatin.
6
free α-carboxyl group and substituted α-amino group with
lesser bulkiness. The bulkiness of substituents at the α-amino
Table 1 Inhibitory activity of the L-Trp and tryptamine-based 2-indolinone derivatives
against purified human IDO1 enzyme.
Compound
IDO1 inhibition
IC50 (μM)a
Compound
IDO1 inhibition
IC50 (μM)a
3.39 ± 0.29
3.97 ± 0.39
1.28 ± 0.28
2.31 ± 0.21
13.59 ± 0.19
2.25 ± 0.23
Inhibitory activities of the oxindole derivatives against purified
IDO1 enzyme
1.95 ± 0.32
1.58 ± 0.27
0.62 ± 0.11
8.33 ± 0.21
The inhibitory activity of the synthesized oxindole derivatives
was first examined using standard spectrophotometric method
monitoring the differences in absorbance values of the
product generated from kynurenine and Ehrlich's reagent (at
480 nm) in the acidic medium.3, 12, 38 Absorption spectra of the
pure compounds (100 nM to 1 mM) showed no or little
interference with this enzyme activity assay. The calculated Km
COOH
NHCOPh
385.41 ± 35.31
O
N
5
H
COOH
NHCOCH3
O
N
6
H
and kcat values of the enzyme with L-Trp were 47.8 ± 2.5 μM
aIC50 values are the mean of five independent assays.
and 3.7 ± 0.1 Sec-1, respectively.12 To improve the efficacy of
the oxindole lead, we investigated two general modifications
of the oxindole structure: (1) modification of the α-amino
group could also play a crucial role in proper fitting of the
compounds within the active site of the IDO1 enzyme. Overall,
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oxindole moiety and its C3-substitutions play an important role procedures.12 The amount of
Journal Name
L
-Trp catabolism by IDO1 enzyme
in their IDO1 inhibitory activity. Under the experimental in the absence or presence of the potent inhibitors was
conditions, the IC50 value of L-1-MT was 385 μM, which is in monitored by HPLC analyses after quenching the reaction by
accordance with the reported values.39,
40
Interestingly, acidification and hydrolysis of N-formylkynurenine to
oxindolealanine (
products of
-Trp.8 Whether feedback inhibition of IDO1 generated using pure kynurenine and then amount of
enzyme by compound is physiologically relevant or it plays kynurenine formation from -Trp in the absence or presence of
-Trp metabolism remains to be compounds under enzymatic reaction conditions was
1) has been shown to be one of the oxidized kynurenine (Table S1 in the ESI‡). A standard curve was
L
1
L
any important role in
investigated.
L
measured to investigate their inhibitory efficacies. The
calculated concentrations of the tested compounds required
C3-substitution of the isatin ― Our oxindolealanine and 3-(2- to inhibit the kynurenine generation from L-Trp under similar
aminoethyl)indolin-2-one derivatives showed that oxindole experimental conditions were in the low-micromolar range
moiety is very important for their IDO1 inhibition activity. and in accordance with the inhibition, activities calculated
However, we presume that rotation at the C3-position could using PDMAB-method.12 The differences in IC50 values of the
be one the reasons for its moderate inhibitory activity. compounds between the spectroscopic and HPLC-based
Therefore, for further optimization of its inhibition efficacy, we methods could be due to the methylene blue-ascorbate
used a series of oxindole derivatives with restricted rotation at regeneration system, which preserves IDO1 in its active state
the
C3-position.
However,
tested
hydrazones, (Fe2+). The IC50 values from HPLC-based assay also revealed
phenylindolinone and alkene derivatives of isatin showed that the presence of carbonyl group at the C2-position of the
moderate IDO1 inhibitory activities. In terms of structure- 5-member pyrrole ring is beneficial for the inhibitory activity of
activity relationships with the hydrazones, it appears that the the tested compounds, whereas appropriate substitution at
presence of 4-chloro substitution (12) is favourable for the the C3-position of the oxindole ring assists the compounds to
inhibitory activity with IC50 values of 0.63 μM (Table 2). interact strongly with the IDO1 enzyme.
Whereas, 2-Br substituted phenylimino derivative of isatin 19
Table 2 Inhibitory activity of the isatin-based oxindole derivatives against purified
showed stronger IDO1 inhibitory activity (IC50 = 1.24 μM)
human IDO1 enzyme.
among the tested phenylimino compounds (Table 2). These
results suggest that halogen substitution on the aryl ring of the
hydrazone and phenylimino derivatives of oxindole also plays
an important role on their IDO1 inhibition activity. The effect
of halogen substitutions on the oxindole ring was also explored
for probable interactions with the hydrophobic residues
present within the pocket-‘A’ (comprises of Y126, F163 and
S167 residues) of IDO1 enzyme. Several research groups had
successfully taken advantage of these hydrophobic
interactions in the optimization of the IDO1 inhibition
efficacies.4, 12, 13 The alkene oxindole derivatives of isatin and 5-
chloroisatin showed moderate to strong inhibitory activity (IC50
values 0.19 to 1.69 μM). Compound 23 showed the
Compound
IDO1
Compound
IDO1 inhibition
IC50 (μM)a
inhibition
IC50 (μM)a
1.95 ± 0.29
0.63 ± 0.15
0.93 ± 0.06
1.69 ± 0.13
1.51 ± 0.15
3.74 ± 0.32
1.58 ± 0.12
0.36 ± 0.08
0.19 ± 0.07
4.73 ± 0.61
considerably higher IDO1 enzyme inhibition potency (IC50
=
0.19 μM) among the tested compounds. We tested 3-hydroy-
3-alkyled derivatives of isatin and 5-chloroisatin for further
improvement of the efficacy of the oxindoles. Our activity
assay showed that 3-hydroxy-3-alkyl- oxindole derivatives of
isatin and 5-chloroisatin had moderate IDO1 inhibition
activities (IC50 values 0.45 to 4.73 μM) among the tested
oxindoles. Therefore, both 5-chloro and suitable substitutions
at the C3-position of the oxindole ring had considerable effect
on IDO1 enzyme inhibition activity. However, 3-hydroxy-3-alkyl
derivatives do not substantial effect of IDO1 inhibition efficacy.
Recently 3-hydroxy-3-alkyl isatin derivatives were developed
as comparable inhibitors for both IDO1 and TDO enzymes.21
This also endorses the importance of C3-substituted oxindole
moiety in designing inhibitors for these enzymes.
1.78 ± 0.39
2.85 ± 0.42
0.45 ± 0.09
1.97 ± 0.41
2.56 ± 0.22
1.24 ± 0.38
0.89 ± 0.07
The inhibition efficacies of these oxindole derivatives were
performed by spectrophotometric method. Therefore,
additional IDO1 activity assay for selected compounds was
performed by HPLC analysis according to the reported
aIC50 values are the mean of five independent assays.
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Spectroscopic studies for binding analysis of 2-indolinones enzyme. The calculated EC50 values of the compounds are
with IDO1 The UV-vis absorption properties of the within 0.33-1.26 μM range in MDA-MB-231 cells (Table 3).
―
porphyrin-ring are highly sensitive and useful in understanding Control compound, L-1-MT showed EC50 values of 120 μM
the ligand/substrate binding ability to IDO1 enzyme.41-44 The under the similar experimental conditions.47 The differences in
UV-vis absorption spectra of ferric-IDO1 and deoxy-ferrous- EC50 values of the compounds between the enzymatic assay
IDO1 were recorded in the absence and presence of against purified IDO1 and cellular assays could be due to the
compounds, 3, 6, 23, and 25 (Fig. 2A and B). The absorption difficulty in controlling IDO1 redox activity, which maintains
spectrum of only ferric-IDO1 showed a Soret peak at 404 nm, IDO1 in active state (Fe2+) and/or environmental effect.
which is in accordance with the reported results.13, 42-45 In the Overall, a good correlation between these assays corroborates
presence of compounds, this Soret peak showed a slight blue the IDO1 inhibition potencies of these oxindoles. MTT assay of
shift with increase in intensity, under the similar experimental the compounds in MDA-MB-231 (breast cancer), A549 (lung
condition. Compound 25 showed a maximum blue shift of 9 cancer), HeLa (cervical cancer) and J774A.1 (macrophage) cells
nm, indicating its strong binding to the ferric-IDO1 enzyme, (concentrations of IC50 and 2 × IC50 values from the enzymatic
possibly through the carbonyl-oxygen of the oxindole-ring (Fig. assay) also revealed no/negligible level of toxicity of the
2A). The blue-shift of the Soret peak could be associated with compounds under the tested conditions (Fig. S1 –S5 in the
the presence of an electron-withdrawing group close to the ESI‡).
porphyrin-ring, which is also in accordance with their mode of
Table 3 IDO1 enzyme inhibitory activity of the selected compounds in MDA-MB-231
interaction. Fig. 2B showed that in the absence of any inhibitor
cells.
the deoxy-ferrous-IDO1 enzyme exhibit Soret and Q-band at
421 and 558 nm.13, 42, 43 However, in the presence of inhibitors
the Soret band shifted to 419-425 nm and appearance of new
Q bands around 527/558 nm (Fig. 2B). This indicates their
probable binding with the Fe2+-IDO1 enzyme possibly via
carbonyl-oxygen of the oxindole-ring. The absorption spectra
of only compounds did not show any peak in this region
(spectra not shown here). Although further studies are
required to prove the coordination of the oxindole derivatives
to heme-group of IDO1, but the results strongly support the
Compound
IDO1 inhibition in MDA-MB-231 cella
EC50 (μM)b
3
6
1.26 ± 0.17
0.48 ± 0.12
12
0.42 ± 0.14
22
0.46 ± 0.11
23
0.33 ± 0.09
25
L-1-MTc
0.49 ± 0.19
119.66 ± 11.31
aIDO1 protein expression in MDA-MB-231 cells was induced by human IFN-γ (20 ng/
mL). bEC50 values are the mean of three independent assays. cReported compound.
proposed mimic of the epoxide intermediate state for the
Trp oxidation by these oxindoles.
L-
Mode of IDO1 enzyme inhibition by the potent oxindoles
―
To understand the mode of IDO1 inhibition by the compounds,
we performed enzyme kinetics in the presence of eight potent
compounds
against inhibitor concentrations ([I]) showed that 12 and 27
followed uncompetitive inhibition and 19 22 23 and 25
3, 6, 12, 19, 22, 23, 25, and 27. The plots of [S]/V
3,
6,
,
,
followed competitive inhibition modes (Fig. 3 and S6). [S] and
V represent the substrate concentration and initial rate of the
reaction, respectively. Detailed mechanistic studies for the
IDO1 mediated transformation of L-Trp to N-formylkynurenine
showed that formation of ferric superoxide intermediate due
to binding of O2 to the Fe(III) of the heme-group is the
prerequisite for this oxidation reaction. Therefore, although
these tested compounds follow competitive/uncompetitive
Fig. 2 Absorption spectra of ferric-IDO1 (A) and deoxy-ferrous-IDO1 enzyme in the
absence and presence of the 50 μM compounds in 100 mM potassium phosphate
buffer at pH 6.5. Y-axis is not same for both the spectra. IDO1 enzyme concentration =
5 μM. Ferrous-deoxy reaction environment was generated by adding Na2S2O4 under N2
atmosphere.
mode of inhibition with respect to L-Trp but may not be
competitive/uncompetitive with respect to O2. For this reason,
additional kinetics measurement with respect to O2 is required
to understand the comprehensive mode of IDO1 inhibition by
Cellular IDO1 inhibitory activities of oxindoles ― To explore these compounds.
the therapeutic potential of these oxindoles, six of the most
potent compounds were tested for the IDO1 cellular activity in Probable mode of interaction of the potent oxindoles with
MDA-MB-231 breast cancer cells. It is well documented that IDO1 enzyme ― With confirmation that potent oxindoles
interferon (IFN)-γ appreciably induce the expression of native inhibit IDO1 enzyme activity, we performed molecular docking
human IDO1 enzyme from its mRNA in MDA-MB-231 cells.12, 46 analyses of the three potent compounds to explore their
The cellular inhibition activities of the compounds are in probable mode of interaction with the IDO1 enzyme (PDB
accordance with that of in vitro data against purified IDO1 code: 2D0T).48 Molecular docking analysis revealed that the
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Journal Name
potent compounds
6
,
23 and 25 presumably interact with the in interaction with Ser263 and Ala264 residues. Recently, 3-
-Trp (Fig. 4).47 The hydroxy-3-alkyl derivatives of isatins were developed as
23 and 25 is -153.18 favourable inhibitors of TDO enzyme. This study also showed
IDO1 enzyme in a similar pattern like
interaction energy of compound
L
6,
kJ/mol, -142.33 kJ/mol, -146.22 kJ/mol respectively. The that 3-hydroxy group plays a crucial role in proper binding of
oxindole-moiety of the compounds is presumably placed in the inhibitors within the active site of the TDO enzyme.21
pocket-‘A’ and interacts with Ser167 residue through H- Therefore, H-bonding, hydrophobic and other interactions play
bonding. Hydrophobic amino acids like Phe163, Phe164, important roles in stronger binding of the oxindole derivatives
Tyr126 and others present in pocket-‘A’ could be involved pi- of
stacking or halogen bonding and hydrophobic interactions with energies of the oxindoles
the oxindole moiety. Recently, it was reported that agreement with the measured IC50 values of these compounds.
hydrophobic residues Phe163 and Phe164 are important for The differences in the IC50 values among compounds 23
the activity of the IDO1 enzyme.49 Whereas, polar residues and 25 could be due to their efficiency in proper fitting into
Ser167 and Tyr126 are crucial for the high -Trp affinity of the binding pocket. During docking analysis the binding pocket
IDO1 enzyme.50 Therefore, the mode of interactions clearly
L
-Trp to the IDO1 active site. Molecule docking interaction
6
,
23 and 25 are not in complete
6,
L
was considered as ‘rigid-body’. In solution the IDO1 binding
pocket may have different conformation(s), which may
possibly explain the reason for their different IC50 values.
Therefore, the mode of interaction of these compounds with
IDO1 could be different or volume/interaction pattern of C3-
substituents could be crucial for their binding under the
experimental conditions. Overall, molecular docking analyses
support that suitable C3-substituted oxindoles may act as a
600
550
500
450
400
350
300
250
200
1400
(A)
(B)
Compound 6
Compound
0 nM
3
1200
1000
800
0 nM
200 nM
500 nM
1000 nM
1500 nM
200 nM
500 nM
1000 nM
1500 nM
600
mimic of the epoxide intermediate for the transformation of
Trp to -formylkynurenine by IDO1 enzyme.
L-
400
N
200
-0.015 -0.01 -0.005
0
0.005 0.01 0.015 0.02 0.025
-0.02
-0.01
0
0.01
0.02
0.03
1/ [S]
1/ [S]
1400
600
(C)
1200
(D)
550
Compound 22
Compound 23
0 nM
0 nM
200 nM
500 nM
1000 nM
1500 nM
200 nM
500 nM
1000 nM
1500 nM
500
450
400
350
300
250
1000
800
600
400
200
Fig. 4. Probable mode of interaction of the compounds, 6 (A), 23 (B) and 25 (C) with the
active site of the IDO1 enzyme (2D0T). The model structures were generated using
MoleGro Virtual Docker, version 6.0. The oxygen and nitrogen atoms are shown in red
and blue, respectively. Residues involved in interactions through hydrogen bond
formation are shown using dashed lines (yellow). Images were generated using PyMol.
-0.015 -0.01 -0.005
0
0.005 0.01 0.015 0.02 0.025
-0.015 -0.01 -0.005
0
0.005 0.01 0.015 0.02 0.025
1/ [S]
1/ [S]
Fig. 3 Determination of mode of inhibition of the potent compounds. Plot of 1/V
against 1/[S] for compounds 3 (A), 6 (B), 22 (C) and 23 (D). Concentration of L-Trp was
varied from 50 to 150 μM. The concentrations of compounds were varied from 200 to
1500 nM. All the absorption measurements were performed in 50 mM phosphate
buffer pH 6.5 at room temperature.
Inhibitory activities of oxindoles against purified TDO enzyme
― TDO is the other enzyme, which also catalyses the initial,
rate-limiting step of the kynurenine pathway. TDO also
suggests that oxindole-moiety is mimicking the intermediate
state for the L-Trp oxidation. The model structures also predict
catabolizes more than 90% of the L-Trp in the liver to regulate
that the carbonyl group of the oxindole ring is orientated
towards the heme-group, which is in accordance with our
predicted mode of the interaction of these mechanism-based
inhibitors. The substituents at the C3-position may interact
with IDO1 through different modes. The free α-carboxyl
its level. To investigate the efficacy of these oxindoles in
selectively inhibiting IDO1 enzyme, TDO enzyme inhibition
studies were also performed (Table 4). Screening against
purified TDO enzyme revealed that these oxindoles were
considerably inactive (IC50 ≥ 20 μM) against TDO enzyme.
Substitution at the 3-position of the oxindole ring also plays a
significant role in inhibiting TDO enzyme activity. Selected
groups of compound
6 could be involved in H-bonding with
Ser263 and Ala264 residues, whereas its acyl-protected α-
amino group could be involved in interaction with heme-
group. The carbonyl (from acetylacetone) of the alkene
oxindole derivative 23 could be involved in H-bonding with the
Ser263 and Ala264 residues.5-Chloro substation in the
oxindole ring could assist compound 23 to interact with the
residues in pocket-‘A’ through halogen bonding, pi-stacking
and hydrophobic interactions. For compound 25 the carbonyl
(from acetylacetone) and 3-hydroxyl group could be involved
oxindole derivatives of L-Trp and tryptamine showed 18 to
135-fold stronger IDO1 inhibition in comparison with TDO
enzyme under similar experimental conditions. Oxindole
derivatives of isatins showed 20 to 200-fold stronger IDO1
inhibition in comparison with TDO enzyme. The most effective
oxindole-based compound, 23 (based on the enzymatic and
cellular assay) exhibited over 200-fold increase in inhibitory
6 | J. Name., 2012, 00, 1-3
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ARTICLE
activity toward IDO1 enzyme when compared against TDO improve the potency as well as the selectivity of IDO1
enzyme. Recently a series of 3-hydroxy substituted isatin inhibitors.
derivatives were developed as inhibitors for TDO enzyme.
However, our studies suggest that suitable C3-subtittuents is
crucial in proper binding with the IDO1 over TDO enzyme.21
In this study, oxindole derivatives were designed as
mechanism-based inhibitors of IDO1 enzyme. Subsequent
modification at the C3-position of the oxindole ring directed to
the identification of potent inhibitors with low-micromolar
IDO1 enzyme inhibitory activities under in vitro conditions.
Low-micromolar IC50 values of the compounds and smaller
differences in their inhibitory activities, suggest that the
presence of oxindole-moiety could be the driving force for
their moderate potencies. Overall, activity studies showed that
Conclusion
In summary, we synthesized oxindole derivatives with
moderate inhibitory activity against purified human IDO1
enzyme. Activity measurements showed that the oxindole ring
plays an important role in their IDO1 inhibitory potencies.
Halogen substitution and restricted rotation around C3-
position of the oxindole ring were effective in improving their
efficacies. Spectroscopic studies supported the interaction of
potent compounds with the heme-group of IDO1 enzyme,
indicating its preliminary role in mimicking the epoxide-
N-acyl protected oxindolealanine
(
6)
could considerably
intermediate for the IDO1 catalysed transformation of L-Trp to
enhance the inhibition potency of the
L-Trp derivatives.
N-formylkynurenine. IDO1 activity in the interferon-γ-induced
MDA-MB-231 cells showed that the tested compounds have
minimal cytotoxicity and low-micromolar potencies. These
oxindole derivatives also showed selectivity for IDO1 enzyme
over TDO enzyme. Overall, these observations suggest that
oxindole derivatives are potential inhibitor of IDO1 enzyme
and could be of interest as drug target in cancer and other
human diseases.
Table 4 Inhibitory activity of the selected compounds against purified human IDO1 and
TDO enzymes.
Compound
Mode of
IDO1
IDO1
TDO
Selectivity
ratiob
inhibition
inhibition
inhibition
NM
(IC50 (μM))
3.39 ± 0.29
1.28 ± 0.28
0.62 ± 0.11
2.31 ± 0.21
0.63 ± 0.15
1.51 ± 0.15
1.58 ± 0.12
1.78 ± 0.39
1.24 ± 0.38
0.93 ± 0.06
0.36 ± 0.08
0.19 ± 0.07
0.45 ± 0.09
(IC50 (μM))a
60.22 ± 2.02
27.20 ± 3.08
83.47 ± 3.27
58.33 ± 3.58
53.38 ± 0.51
46.81 ± 4.21
38.31 ± 4.56
52.90 ± 3.31
46.24 ± 6.06
71.01 ± 5.09
54.69 ± 2.59
39.92 ± 1.03
56.12 ± 6.14
1
18
21
3
competitive
competitive
NM
6
135
25
7
Experimentals
12
13
15
16
19
20
22
23
25
uncompetitive
NM
85
31
General information
NM
24
All reagents were purchased from different commercial
sources and used directly without further purification. Column
chromatography was performed using 60―120 mesh silica gel.
Reactions were monitored by thin-layer chromatography (TLC)
on silica gel 60 F254 (0.25 mm). 1H NMR and 13C NMR were
recorded at 400 and 100 MHz respectively with Varian AS400
spectrometer at 600 and 151 MHz respectively with Brucker
spectrometer, using TMS as an internal standard with CDCl3
and DMSO-d6. The coupling constant (J values) and chemical
shifts (δppm) were reported in Hertz (Hz) and parts per million
(ppm) respectively. Multiplicities are reported as follows: s
NM
30
competitive
NM
37
76
competitive
competitive
competitive
152
210
124
aIC50 values are the mean of three independent assays against purified enzymes.
bSelectivity ratio is calculated as (IC50 value of TDO)/(IC50 value of IDO1). NM = not
measured.
Suitable substitution and restricted rotation at the C3-position
of the isatin/5-chloroisatin could also augment their inhibition
potencies. UV-vis absorption properties of the heme-
containing porphyrin-ring in the absence and presence of the
compounds, suggest the interaction of carbonyl-oxygen of the
oxindole-ring with the heme-group of IDO1 enzyme.
Molecular model structures also proposed that additional
hydrogen bond interaction of the oxindole ring with Ser167
residue along with the interaction with the residues present in
“pocket-A” of the IDO1 enzyme could be the primary
contributing factor for the stronger inhibitory properties. The
potent compounds also exhibited a 15 to 210-fold stronger
inhibition in IDO1 enzyme inhibition compared to TDO
enzyme. Until now limited indole/tryptophan, based
derivatives have been reported with such selective and
stronger IDO1 enzyme inhibitors.16-22 These results clearly
suggest that designing compounds, which could perturb the
electrophilic addition of O2 of the oxygenated-heme group to
(singlet),
d (doublet), t (triplet), m (multiplet) and br
(broadened). High-resolution mass spectra (HRMS) were
recorded at Agilent Q-TOF mass spectrometer with Z-spray
source using built-in software for analysis of the recorded
data.
General procedure for the synthesis of CbZ-protected L-
tryptophan and tryptamine
L-Tryptophan or tryptamine (29.4 mmol) was first dissolved in
1M NaOH (60 mL) and stirred at 0 °C for 30 min. Then
benzylchloroformate (32.3 mmol) and 1M NaOH (30 mL)
where simultaneously added to the solution in drop-wise
fashion.32 The mixture was stirred for 12 hours at room
temperature. The solution was acidified with 6 (M) HCl to pH
1-2 and extracted with EtOAc (3 × 200 mL). The combined
organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The product was used
directly in the following reactions.
the pyrrole ring of L-Trp, could be an efficient approach to
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Journal Name
Synthesis of 2-amino-3-(1H-indol-3-yl)propanamide (10)
General procedure for the synthesis of benzyl-protected L- To a stirring solution of compound
tryptophan
3
(1.5 mmol) in DMF (10
mL) was added 1-hydroxybenzotriazole (1.5 mmol) and EDCI
To a stirring solution of L-tryptophan (2.0 mmol) in toluene (50 (1.65 mmol). The whole mixture was then stirred for 2 hours at
mL) PTSA (4.8 mmol) was added. Then benzylalcohol (23.08 room temperature under N2 atmosphere.34 Then aqueous
mmol) was added in dropwise fashion under stirring condition ammonia solution (0.5 mL, 7.0 mmol) was added drop wise
and the reaction mixture was refluxed for 24 hours using dean under continuous stirring condition and the resulting solution
stark apparatus. Excess toluene was removed under reduced was allowed to stir for another 18 hours at room temperature.
pressure and the reaction mixture was diluted with water (50 Then water (20 mL) was added and washed with EtOAc (3 × 10
mL). The compound was then extracted with EtOAc (3 × 100 mL). The organic phase was again washed with 5% NaHCO3
mL). The combined organic layer was dried over anhydrous solution, dried over anhydrous Na2SO4, and concentrated
Na2SO4 and concentrated under reduced pressure. Column under reduced pressure. The crude mixture was further
chromatography with silica gel and a gradient solvent system hydrogenated (1 atmosphere pressure) in MeOH using Pd/C as
of MeOH to DCM (5-10%) yielded the target products (75%).
catalytic amount (10 mol%) to obtain the crude product 10.
The pure compound was isolated using column
chromatography with silica gel and a gradient solvent system
Synthesis of 2-acetylamino-3-(1H-indol-3-yl)propanoic acid
A solution of L-tryptophan (1.5 mmol) and acetic anhydride (3 of methanol to dichloromethane (10-35%).
mmol) in methanol (20 ml) was refluxed for overnight and
then concentrated under reduced pressure.51 The crude General procedure for the synthesis of hydrazones and
mixture was then diluted with water and extracted with ethyl phenylimino derivatives of isatin
acetate (3×30 mL). The combined organic layer was dried over To a stirring solution of isatin (1 mmol) in ethanol was added
anhydrous Na2SO4 followed by concentration under reduced appropriate amount of the aromatic hydrazine or primary
pressure to obtain the N-acetylated product, which was amine (1 mmol) and a catalytic amount of acetic acid.35 The
directly used for next step without further purification.
whole solution was refluxed for 0.5-4 hours under constant
stirring. After the completion of the reaction, the solvent was
evaporated under reduced pressure followed by washing the
Synthesis of 2-benzoylamino-3-(1H-indol-3-yl)propanoic acid
To a stirred solution of L-tryptophan (1.5 mmol) in 2 ml water crude product with water (3 × 10 mL) and dried under reduced
was added 2 ml of 1M NaOH aqueous solution followed by the pressure. Column chromatography with silica gel and
a
addition of benzoyl chloride (1.8 mmol) dissolved in 0.5 (M) gradient solvent system of ethyl acetate to hexane (5-20%)
NaOH (8 ml) and chloroform (8 mL) drop wise at 0 °C under yielded the target products. Recrystallization from ethanol also
continuous stirring. Then the whole solution was allowed to yielded desired pure product.
stir at room temperature for 12 hours.52 After completion of
the reaction (monitored by TLC), the residual solvent was General procedure for the synthesis of 2-indolinone
removed under reduced pressure and the reaction mixture derivatives, 21 and 25
was further acidified with 0.5 (M) HCl to pH 3-4. The aqueous To an ice cooled mixture of isatin (2.5 mmol) and acetone or
layer was then extracted with ethyl acetate (3 × 30 mL) and acetyl acetone (2.5 mmol) was added 0.5 mL of piperidine
the combined organic layer was dried over anhydrous Na2SO4 under continuous stirring.36 The whole mixture was stirred at 0
and concentrated under reduced pressure to obtain the °C for 4 hours and then cold ethanol was added. The resulted
desired solid product, which was directly used for the next solid was filtered and dried under reduced pressure. Column
step.
chromatography with silica gel and a gradient solvent system
of ethyl acetate to hexane (5-20%) yielded the target products.
General procedure for the synthesis of 2-indolinone
derivatives of L-tryptophan and tryptamine
General procedure for the synthesis of 2-indolinone
The desired amount of substituted L-tryptophan or tryptamine derivatives, 20 and 24
(1.5 mmol) was first dissolved in acetic acid (1.5 mL) and then A solution of isatin (1 mmol) and diethyl malonate (1 mmol) or
a mixture of DMSO and concentrated HCl (1:3 approx.) was dimedone (1 mmol) in water was refluxed with a catalytic
slowly added under continuous stirring conditions.32 The amount of piperidinium acetate for 3-4 hours at 100 ° C.36 The
resulting solution was then stirred at room temperature for solid thus obtained was filtered, washed properly with water
another 1-2 hours. After completion of reaction (monitored by and dried in oven. Further purification was performed with
TLC), the reaction mixture was diluted with water (8 mL) and silica gel column chromatography using ethyl acetate: hexane
further extracted with ethyl acetate (3 × 30 mL). The combined gradient solvent system (5-20%) or recrystallized from ethanol
organic layer was dried over anhydrous Na2SO4 and to obtain the desired pure product.
concentrated
under
reduced
pressure.
Column
chromatography with silica gel and a gradient solvent system General procedure for the synthesis of 5-chloroisatin
of methanol to dichloromethane (5-20%) yielded the target derivatives
products.
The compounds were synthesized according to reported
procedure with slight modification.37 The desired amount of 5-
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ARTICLE
chloro isatin (1.1 mmol) was dissolved in acetone (10 mL) and 2-((Tert-butoxycarbonyl)amino)-3-(2-oxoindolin-3-yl)propanoic
1
then appropriate amount of K2CO3 (1.3 mmol) was added acid (4) ― As brown semisolid (68% yield; rotamers); H NMR
under stirring condition. The whole solution was then refluxed (400 MHz, CDCl3 + CD3OD) δppm7.61-7.59 (m, 1H), 7.13-7.09 (m,
for 14 hours at 50 °C. After completion of reaction (monitored 1H), 6.51-6.45 (m, 2H), 4.28 (s, 1H), 3.61-3.43 (m, 1H), 2.92-
by TLC), the reaction mixture of 3-hydroxy 5-chloroisatin was 2.91 (m, 2H), 1.27 (s, 9H); 13C NMR (151 MHz,CDCl3 + CD3OD)
concentrated under reduced pressure and the residue δppm 202.0, (201.9), 156.3, (156.3), 150.7, 134.8, 131.5, (131.4),
obtained was triturated with diethyl ether resulting the alcohol 117.7, (117.5), 115.9, (115.8), 79.6, 52.2, 42.2, 29.7, 28.3;
intermediate as a white solid, which was directly used for the HRMS [ESI] calcd. for C16H20N2O5 [M +H]+ 321.1445, found
next step.
To a stirring solution of the 3-hydroxy 5-chloroisatin in
ethanol (5 mL), was added concentrated HCl (approx. 1 ml) 2-Benzamido-3-(2-oxoindolin-3-yl)propanoic acid (isomeric
drop wise and a catalytic amount of acetic acid. Then the mixture of ) ― As yellow semi-solid (60% yield; rotamers, mp:
321.1506.
5
1
mixture was refluxed for 12 hours at 60 °C under continuous 162-165 °C); H NMR (400MHz, CDCl3 + DMSO-d6) δppm 10.15
stirring.37 After the reaction was complete (monitored by TLC), (s, 1H), 10.07 (br s, 1H), 8.81-8.80 (m, 1H), 8.43 (br s, 1H), 7.91-
the mixture was concentrated and washed with saturated 7.84 (m, 2H), 7.47-7.33 (m, 3H), 7.20-7.09 (m, 1H), 6.97-6.91
sodium bicarbonate solution. The aqueous layer was then (m, 1H), 6.84-6.80 (m, 1H), 4.96-4.90 (m, 1H), 4.82-4.78 (m,
extracted with ethyl acetate (3×30 mL) and the combined 1H), 2.61-2.51 (m, 1H), 2.40-2.24 (m, 1H); 13C NMR (151
organic layer was further dried over anhydrous Na2SO4 and MHz,CDCl3 + DMSO-d6) δppm 180.4, (179.8), 173.3, (173.2),
concentrated
under
reduced
pressure.
Column 167.3, (166.8), 141.9, 133.6, (133.5), 131.3, (131.2), 128.0,
chromatography with silica gel and a gradient solvent system 127.9, (127.7), 127.2, 124.2, (123.5), 122.0, (121.9), 109.8,
of ethyl acetate to hexane (5-25%) yielded the target products. (109.7), 51.2, 43.3, (43.1), 32.3, (31.3); HRMS [ESI] calcd. for
C18H16N2O4 [M +H]+ 325.1183, found 325.1155.
Characterization of the synthesized compounds
2-Amino-3-(2-oxoindolin-3-yl) propanoic acid (1) ― As pink 2-Acetamido-3-(2-oxoindolin-3-yl)propanoic acid (isomeric
solid (45% yield; rotamers, hygroscopic); 1H NMR (600MHz, mixture of
1
) ― As brown semi-solid (70% yield; rotamers); H
6
CDCl3 + DMSO-d6) δppm 10.46 (s, 1H), 8.82 (br, s, 1H), 7.47-7.44 NMR (400 MHz, CDCl3 + CD3OD) δppm 9.28 (br, s, 1H), 6.59-6.55
(m, 1H), 7.18 (d, 1H, J = 12 Hz), 7.13 (d, 1H, J = 6), 7.09-7.08 (m, (m, 1H), 6.49-6.40 (m, 1H), 6.26-6.22 (m, 1H), 6.16-6.13 (m,
1H), 6.92-6.88 (m, 1H), 6.83-6.80 (m, 1H), 3.75-3.73 (m, 1H), 1H), 4.07-4.02 (m, 1H), 3.89-3.86 (m, 1H), 1.89 (s, 3H), 1.69-
3.54-3.52 (m, 1H), 1.93-1.89 (m, 2H); 13C NMR (100MHz, CDCl3 1.61 (m, 1H), 1.54-1.48 (m, 1H); 13C NMR (151 MHz, CDCl3 +
+ DMSO-d6) δppm 179.8, (179.5), 169.8, 141.5, (141.4), 127.8, CD3OD) δppm 180.3, (180.2), 172.2, (171.9), 141.7, (141.6),
(127.7), 123.3, (123.2), 121.7, 109.8, (109.7), 51.3, (50.6), 43.5, 128.3, (128.1), 124.2, 123.7, 110.1, (110.0), 50.3, (50.1), 39.8,
(42.0), 30.2, (29.8); HRMS [ESI] calcd. for C11H12N2O3 [M + H]+ 31.9, (31.1), 22.0, (21.9); HRMS (ESI) calcd. for C13H14N2O4 [M
221.0921, found 221.0920.
+H]+ 263.1026, found 263.1012.
3-(2-Aminoethyl)indolin-2-one (
2
) ― As light brown solid (70% Benzyl [2-(2-oxoindolin-3-yl)ethyl]carbamate (
7
) ― As light
yield; rotamers, mp: 245-247 °C); H NMR (400 MHz, CDCl3 + brown solid (70% yield; rotamers, mp: 161-163 °C); 1H NMR
DMSO-d6) δppm 10.41 (br s, 1H), 8.38 (br s, 2H), 7.24-7.16 (m, (400 MHz, CDCl3 + DMSO-d6) δppm 9.65 (br s, 1H), 7.39-7.25 (m,
2H), 7.00-6.96 (m, 1H), 6.90 (d, 1H, J = 8 Hz), 3.56-3.53 (m, 1H), 5H), 7.20-7.15 (m, 1H), 7.01-6.95 (m, 1H), 6.87-6.86 (m, 2H),
3.11-3.06 (m, 2H), 2.38-2.29 (m, 1H), 2.24-2.14 (m, 1H); 13C 5.95 (br s, 1H), 5.07 (s, 2H), 3.43-3.38 (m, 1H), 2.74-2.65 (m,
NMR (151 MHz, CDCl3 + DMSO-d6) δppm 178.3, (178.2), 141.6, 2H), 2.12-2.09 (m, 2H); 13C NMR (151 MHz, CDCl3 + DMSO-d6)
(141.5), 127.5, (127.4), 127.2, 122.9, (121.1), 121.0, 109.2, δppm 179.2, (179.1), 155.9, 141.6, 136.2, 128.7, 127.9, (127.4),
(109.1), 43.0, (42.9), 36.4, 27.1, (27.0); HRMS [ESI] calcd. for 127.3, 123.5, (123.4), 121.4, 109.2, 65.8, 43.3, 37.8, 30.0;
1
C10H12N2O [M +H]+ 177.1022, found 177.1026.
HRMS [ESI] calcd. for C18H18N2O3 [M +H]+ 311.1390, found
311.1398.
2-[((Benzyloxy)carbonyl)amino]-3-(2-oxoindolin-3-yl)propanoic
1
acid (
3
) ― As light brown semi-solid (70% yield; rotamers); H Benzyl 2-amino-3-(2-oxoindolin-3-yl)propanoate
(
8)
―
As
NMR (600 MHz, CDCl3 + DMSO-d6) δppm 9.48 (br s, 1H), 7.33- yellow semi-solid (55% yield; rotamers); 1H NMR (400MHz,
7.32 (m, 2H), 7.25-7.10 (m, 5H), 6.97-6.92 (m, 1H), 6.80 (d, 2H, CDCl3 + CD3OD) δppm 7.57 (d, 2H, J = 8 Hz), 7.37 (d, 1H, J = 8 Hz),
J = 6 Hz), 5.04 (s, 2H), 4.59 (t, 1H), 3.48-3.34 (m, 1H), 2.36-2.31 7.27-6.92 (m, 6H), 5.05 (s, 2H), 4.03 (t, 1H), 3.23-3.18 (m, 1H),
(m, 2H); 13C NMR (151 MHz, CDCl3 + DMSO-d6) δppm 180.8, 2.19 (s, 2H); 13C NMR (151 MHz, CDCl3 + CD3OD) δppm 174.6,
(180.6), 174.1, (174.0), 156.9, (156.5), 141.6, (141.5), 136.5, (174.6), 170.6, 140.4, (141.3), 136.5, 128.7, 128.5, 128.3,
(136.3), 129.1, 128.5, (128.4), 128.3, 128.1, (128.0), 127.9, 125.5, 124.4, 121.7, 119.1, 117.7, 111.5, (111.4), 67.7, 64.1,
124.7, 124.1, 122.8, (122.7), 110.2, 67.1, (66.9), 52.1, 40.3, 53.5, 39.7; HRMS [ESI] calcd. for C18H18N2O3 [M +H]+ 311.1390,
33.2, (32.0); HRMS [ESI] calcd. for C19H18N2O5 [M +H]+ found 311.1395.
355.128.8, found 355.1289.
Benzyl
2-(((benzyloxy)carbonyl)amino)-3-(2-oxoindolin-3-
yl)propanoate (
9) ― As light brown oil (55% yield; rotamers);
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1H NMR (600 MHz, CDCl3 + DMSO-d6) δppm 9.18 (br s, 1H), 7.33- 110.6, 108.1; HRMS [ESI] calcd. for C14H10N4O3 [M +H]+
7.10 (m, 10H), 6.99-6.94 (m, 2H), 6.84 (d, 1H, J = 12 Hz), 6.40 283.0826, found 283.0833.
(d, 1H, J = 12 Hz), 6.24 (d, 1H, J = 12 Hz), 5.06 (s, 4H), 4.66-4.64
(m, 1H), 3.53-3.51 (m, 1H), 2.38-2.31 (m, 1H); 13C NMR (151 3-[2-(2-(Trifluoromethyl)phenyl)-hydrazono]indolin-2-one
(15)
1
MHz, CDCl3 + DMSO-d6) δppm 175.8, 174.8, (174.7), 156.7, ― As light yellow solid (65% yield; mp: 252-254 °C); H NMR
(156.5), 141.3, 136.5, (136.3), 128.7, (128.6), 128.5, (128.4), (600 MHz, CDCl3 + DMSO-d6) δppm 8.04-8.02 (m,1H), 7.66-7.58
128.3, (128.2), 124.6, (124.3), 123.1, 110.6, (110.5), 67.3, (m, 2H), 7.44-7.39 (m, 2H), 7.27-7.26 (m, 1H), 7.11-7.10 (m,
(67.1), 53.2, (51.9), 43.5, (43.0), 40.3, (40.2), 32.8, (31.9); 1H), 6.94-6.92 (m, 1H); 13C NMR (151 MHz, CDCl3 + DMSO-d6)
HRMS [ESI] calcd. for C26H24N2O5 [M +H]+ 445.1758, found δppm 163.1, 140.1, 140.0, 132.7, 130.4, 128.6, 125.5, 121.5,
445.1762.
120.5, 118.8, 114.4, 113.9, 110.2; HRMS [ESI] calcd. for
C15H10F3N3O [M +H]+ 306.0849, found 306.0850.
2-Amino-3-(2-oxoindolin-3-yl)propanamide (10) ― As yellow
semi-solid (45% yield; rotamers); 1H NMR (400MHz, CDCl3
+
4-Methyl-N'-(2-oxoindolin-3-ylidene)benzenesulfonohydrazide
DMSO-d6) δppm 7.91-7.86 (m, 1H), 7.81-7.73 (m, 1H), 7.66-7.60
(16) ― As yellow solid (65% yield; mp: 196-198 °C (lit. mp 188
(m, 1H), 7.49-7.36 (m, 1H), 3.67-3.63 (m, 1H), 3.01-2.97 (m, °C)36); 1H NMR (600 MHz, CDCl3 + DMSO-d6) δppm 8.10-8.09 (m,
1H), 2.27-2.23 (m, 1H), 2.13-2.09 (m, 1H); 13C NMR (151 MHz, 2H), 8.01-7.99 (m, 2H), 7.58 (br s, 1H), 7.50-7.42 (m, 2H), 7.20-
CD3OD + DMSO-d6) δppm 181.2, 180.1, 144.4, 133.0, (132.9), 7.17 (m, 1H), 7.03-7.01 (m, 1H), 2.57 (s, 3H); 13C NMR (151
128.2, 123.7, 118.7, (118.4), 111.8, (111.3), 52.9, 43.1, 37.1; MHz, CDCl3 + DMSO-d6) δppm 148.1, 138.8, 135.0, 133.3, 133.1,
HRMS [ESI] calcd. for C11H13N3O2 [M + NH4]+ (237.1346), found 131.8, 131.2, 126.3, 125.8, 124.7, 123.2, 114.5, 24.9; HRMS
237.0859
[ESI] calcd. for C15H13N3O3S [M +H]+ 316.0750, found
316.0751.
3-(2-Phenyl-hydrazono)indolin-2-one (11) ― As yellow solid
(85% yield; mp: 208-210 °C (lit. mp 214-216 °C)32); 1H NMR 3-(Phenylimino)indolin-2-one (17) ― As yellow solid (75% yield;
(600 MHz, CDCl3 + DMSO-d6) δppm 12.69 (br s, 1H), 7.77 (br s, mp: 230-232 °C); 1H NMR (600 MHz, CDCl3 + DMSO-d6) δppm
1H), 7.64-7.63 (m, 1H), 7.38-7.34 (m, 4H), 7.23-7..20 (m, 1H), 10.26 (br s, 1H), 7.36-7.34 (m, 1H), 7.30-7.29 (m, 1H), 7.20-7.16
7.10-7.08 (m, 1H), 7.06-7.04 (m, 1H), 6.90-6.88 (m, 1H); 13C (m, 2H), 7.93-7.92 (m, 2H), 6.82-6.80 (m, 1H), 6.61-6.60 (m,
NMR (151 MHz, CDCl3 + DMSO-d6) δppm 163.6, 142.7, 138.2, 1H), 6.48-6.47 (m, 1H); 13C NMR (151 MHz, CDCl3 + DMSO-d6)
129.6, 128.3, 126.9, 123.6, 122.9, 122.3, 119.4, 114.6, 110.4; δppm 164.7, 155.2, 150.4, 146.8, 134.1, 129.3, 126.1, 125.1,
HRMS [ESI] calcd. for C14H11N3O [M +H]+ 238.0975, found 122.0, 117.7, 116.0, 111.6; HRMS [ESI] calcd. for C14H10N2O [M
238.0977.
+H]+ 223.0866, found 223.0870.
3-[2-(4-Chlorophenyl)hydrazono]indolin-2-one
(
12
)
―
As 3-[(4-Chlorophenyl)-imino]indolin-2-one (18) ― As yellow solid
yellow solid (88% yield; mp: mp 262-264 °C (lit. mp 266-267 (70% yield; mp: 237-239 °C); 1H NMR (600 MHz, CDCl3
+
1
°C)32); H NMR (600MHz, CDCl3 + DMSO-d6) δppm 10.59 (br s, DMSO-d6) δppm 10.32 (br s, 1H), 7.26-7.24 (m, 1H), 7.16-7.12
1H), 7.58-7.56 (m, 1H), 7.37-7.33 (m, 4H), 7.25-7.22 (m, 1H), (m, 1H), 6.83-6.81 (m, 2H), 6.76-6.75 (m, 1H), 6.59-6.57 (m,
7.07-7.05 (m, 1H), 6.94-6.93 (m, 1H); 13C NMR (151 MHz, CDCl3 1H), 6.50-6.49 (m, 2H);13C NMR (151 MHz, CDCl3 + DMSO-d6)
+ DMSO-d6) δppm 163.6, 140.9, 139.3, 128.8, 127.9, 126.9, δppm 164.4, 155.7, 148.7, 147.1, 134.4, 129.4, 126.0, 122.0,
121.5, 121.0, 118.5, 114.7, 110.2; HRMS [ESI] calcd. for 120.6, 119.4, 115.8, 111.8; HRMS [ESI] calcd. for C14H9ClN2O
C14H10ClN3O [M +H]+ 272.0585, found 272.0584.
[M +H]+ 257.0476, found 257.0476.
3-[2-(3-Chlorophenyl)-hydrazono]indolin-2-one
(
13
)
―
As 3-[(2-Bromophenyl)-imino]indolin-2-one (19) ― As yellow solid
yellow solid (80% yield; mp: 237-239 °C (lit. mp 233-234 °C)32); (45% yield; mp: 237-239 °C); 1H NMR (600 MHz, CDCl3
+
1H NMR (600 MHz, CDCl3 + DMSO-d6) δppm 10.14 (br s, 1H), DMSO-d6) δppm 10.51 (br s, 1H), 7.69-7.67 (m, 1H), 7.39-7.36
7.44-7.43 (m, 1H), 7.11-7.04 (m, 3H), 6.98-6.97 (m, 1H), 6.90- (m, 1H), 7.34-7.31 (m, 1H), 7.13-7.10 (m, 1H), 6.98-6.95 (m,
6.88 (m, 1H), 6.82-6.80 (m, 1H), 6.76-6.75 (m, 1H); 13C NMR 2H), 6.77-6.74 (m, 1H), 6.50-6.49 (m, 1H); 13C NMR (151 MHz,
(151 MHz, CDCl3 + DMSO-d6) δppm 163.9, 135.0, 130.3, 128.9, CDCl3 + DMSO-d6) δppm 165.3, 156.4, 148.9, 146.0, 135.2, 133.7,
128.9, 128.5, 122.3, 122.0, 121.3, 119.0, 113.7, 112.3, 110.6; 128.7, 126.6, 123.3, 118.8, 116.5, 112.3; HRMS [ESI] calcd. for
HRMS [ESI] calcd. for C14H10ClN3O [M +H]+ 272.0585, found C14H9BrN2O [M +H]+ 300.9971, found 300.9974.
272.0587.
Diethyl 2-(2-oxoindolin-3-ylidene)malonate (20) ― As red semi-
1
3-[2-(3-Nitrophenyl)-hydrazono]indolin-2-one (14) ― As yellow solid (60% yield); H NMR (600 MHz, CDCl3 + DMSO-d6) δppm
solid (70% yield; mp: 263-265 °C (lit. mp 267-269 °C)35); 1H 8.81 (br s, 1H), 8.33 (d, 1H, J = 12 Hz), 7.30 (t, 1H), 6.70 (t, 1H),
NMR (600 MHz, CDCl3 + DMSO-d6) δppm 10.53 (br s, 1H), 8.20 6.82 (d, 1H, J = 6 Hz), 4.44-4.35 (m, 4H), 1.38-1.24 (m, 6H); 13C
(s, 1H), 7.83-7.82 (m, 1H), 7.64-7.60 (m, 1H), 7.52-7.49 (m, 2H), NMR (151 MHz, CDCl3 + DMSO-d6) δppm 168.0, 165.6, 163.1,
7.24-7.23 (m, 1H), 7.08-7.06 (m, 1H), 6.94-6.93 (m, 1H); 13C 143.8, 135.0, 133.4, 129.7, 129.2, 123.0, 119.9, 110.5, 62.5,
NMR (151 MHz, CDCl3 + DMSO-d6) δppm 163.7, 149.0, 143.8, 14.1; HRMS [ESI] calcd. for C15H15NO5 [M +H]+ 290.1023, found
140.1, 138.2, 130.1, 128.9, 122.0, 120.8, 119.4, 119.2, 116.4, 290.1025.
10 | J. Name., 2012, 00, 1-3
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6.65-6.62 (m, 1H), 6.36 (br, s, 1H), 3.69 (s, 2H), 3.28 (s, 2H),
3-(2-Oxopropylidene)indole-2-one (21) ― As red solid (88% 1.69 (s, 3H); 13C NMR (151 MHz, CDCl3) δppm 193.5, 165.1,
yield; mp: 172-174 °C); 1H NMR (400 MHz, CDCl3) δppm 8.44 (d, 150.2, 136.2, 132.0, 120.8, 118.9, 115.1, 47.4, 42.4, 26.4, 24.6;
1H, J = 8 Hz), 7.27 (t, 1H), 7.20 (s, 1H), 6.97 (t, 1H), 6.79 (d, 1H, HRMS [ESI] calcd. for C13H12ClNO4 [M +H]+ 282.0528 found
J = 8 Hz), 2.42 (s, 3H); 13C NMR (100 MHz, CDCl3) δppm198.7, 282.0398.
170.0, 143.6, 135.7, 133.3, 128.8, 128.0, 123.2, 120.9 110.3,
32.6; HRMS [ESI] calcd. for C11H9NO2 [M +H]+ 188.0706, found Purification of the compounds by HPLC analysis
188.0708.
All synthesized compounds were further purified by analytical-
HPLC analyses (with a purity level ≥ 94-95%) before performing
5-Chloro-3-(2-oxopropylidene)indole-2-one (22) ― As red solid in vitro enzyme activity, cellular activity, cell viability assays,
(75% yield; mp: 183-185 °C); 1H NMR (400MHz, CDCl3) and others. Waters 600 HPLC system with Ascentis® Express
δppm8.59(d, 1H, J = 4 Hz), 7.40-7.38 (m, 1H), 6.89 (s, 1H), 6.87 C18 2.7 μM analytical column (Sigma) at a flow rate of 0.5
(s, 1H), 2.60 (s, 3H); 13C NMR (100 MHz, CDCl3 + CD3OD) δppm mL/min was used for the purification of the compounds. All
198.8, 169.8, 142.7, 135.3, 132.8, 128.9, 128.4, 121.9, 111.3, the compounds (~1mg) were dissolved in MeOH (1 mL) for
32.4; HRMS [ESI] calcd. for C11H8ClNO2 [M +H]+ 222.0316, HPLC analyses. All the compounds have a strong absorption
found 222.0322.
peak at 280 nm. Hence, HPLC analyses were performed using a
UV-detector at 280 nm. During each injection 20 µL of the
1-(5-Chloro-2-oxoindolin-3-ylidene)pentane-2,4-dione (23) ― compound solution was used and fractions were collected.
As brown solid (60% yield; mp: 128-130 °C); 1H NMR (400 MHz, This step was repeated for more than 10-times to get sufficient
CDCl3 + DMSO-d6) δppm 9.45 (br, s, 1H), 7.23 (s, 1H), 7.12 (d, 1H, amount of the pure compounds. A total run time was 10 min.
J = 8 Hz), 6.76-6.73 (m, 1H), 5.41 (s, 1H), 2.88 (s, 1H), 2.09 (s, All the collected fractions for each compound were dried
1H), 1.93 (s, 3H); 13C NMR (100 MHz, CDCl3 + DMSO-d6) under reduced pressure and verified by HRMS analyses. The
δppm191.8, 189.2, 178.6, 140.0, 132.1, 129.6, 127.8, 124.9, mobile phase for HPLC measurements was 60% MeOH and
111.6, 101.5, 74.9, 45.6, 24.1; HRMS [ESI] calcd. for 40% H2O (isocratic mode).
C13H10ClNO3 [M +H]+ 282.0528, found 282.0398.
Expression and purification of recombinant human IDO1 and
2-(3-Hydroxy-2-oxoindolin-3-yl)cyclohexane-1,3-dione (24) ― TDO enzymes
As light brown solid (55% yield; mp: 296-298 °C); 1H NMR (600 The cDNA of human IDO1 (in the vector pQE30) and TDO (in
MHz, CDCl3 + DMSO-d6) δppm 10.44 (br s, 1H), 9.01 (s, 1H), 7.16- the vector pET28a) were used for expression of recombinant
7.14 (m,1H), 6.93-6.88 (m, 2H), 6.85-6.84 (m, 1H), 3.48 (s, 1H), human IDO1 and TDO enzymes enzyme. The human cDNAs of
2.39-2.10 (m, 4H), 1.98-1.96 (m, 2H); 13C NMR (151 MHz, CDCl3 IDO1 and TDO enzymes were a generous gift from Professor
+ DMSO-d6) δppm 202.5, 170.2, 144.1, 121.7, 120.4, 112.8, Emma Raven (University of Leicester). Both the enzymes were
109.8, 100.2, 60.3, 46.7, 36.0, 20.1; HRMS [ESI] calcd. for purified according the reported procedures with minor
C14H13NO4 [M +H]+ 260.0917, found 260.1440.
modifications.12, 38, 45 Briefly, the protein expression isopropyl
β-D-1-thiogalactopyranoside (IPTG), hemin and
1-(3-Hydroxy-2-oxoindolin-3-yl)pentane-2,4-dione (25) ― As phenylmethylsulfonyl fluoride (PMSF) were added to the
brown semi-solid (55% yield; rotamers); 1H NMR (600 MHz, culture at the final concentration of 1 mM, 7 µM and 1 mM,
CDCl3 + DMSO-d6) δppm 7.44-7.42 (m, 1H), 7.30-7.26 (m, 1H), respectively. The cells were grown for 3 hours at 25 ºC and 120
6.67-6.62 (m, 2H), 6.36 (br, s, 1H), 3.67 (s, 2H), 3.28-3.26 (m, rpm. After this incubation period, ethylenediaminetetraacetic
2H), 1.69-1.65 (m, 3H); 13C NMR (151 MHz, CDCl3 + DMSO-d6) acid (EDTA) was added to the culture at a final concentration
δppm 194.5, 165.8, 151.7, 136.1, (136.0), 133.4, (133.2), 117.3, of 1 mM and cells were grown for another 12 hours. Cells were
(117.2), 116.5, (116.3), 114.4, 47.3, (47.2), 42.2, (42.0), 26.3, collected by centrifugation at 5000 rpm for 10 minutes at 4 ºC.
25.6, (24.5); HRMS [ESI] calcd. for C13H13NO4[M + Na]+ The cell pellet was re-suspended in 20 mL of ice-cold
271.0769 found 271.0906.
phosphate-buffered saline (PBS) containing 1 mM PMSF, 1 mM
EDTA and then centrifuged at 15000 rpm for 15 minutes at 4
(26) ― As ºC. The pellet was re-suspended in 20 mL of ice-cold PBS
5-Chloro-3-hydroxy-3-(2-oxopropyl)indolin-2-one
white solid (75% yield; mp: 188-190); 1H NMR (400MHz, CDCl3, buffer containing 1 mM PMSF and centrifuged at 15,000 rpm
DMSO-d6) δppm 9.58 (br, s, 1H), 7.32 (s, 1H), 7.23 (d, 1H, J = 8 for 10 minutes at 4 ºC for the removal of EDTA. The pellet was
Hz), 6.87-6.84 (m, 1H), 5.49 (s, 1H), 3.29-3.11 (m, 2H), 2.19 (s, stored at -80 ºC. The washed pellet, obtained as described
3H);13C NMR (100 MHz, CDCl3 + CD3OD) δppm 205.8, 178.9, above, was re-suspended in 20 mL of ice-cold 50 mM
140.4, 133.2, 129.7, 127.1, 124.0, 111.1, 73.0, 49.4, 29.1; potassium phosphate buffer (KPB) at pH 6.5 containing 300
HRMS [ESI] calcd. for C11H10ClNO3 [M +H]+ 240.0422, found mM potassium chloride (KCl), 10 mM imidazole, 10 mM
240.0420.
magnesium chloride (MgCl2), protease inhibitors (complete
EDTA free) and DNase (˂1mg). The cell lysate was centrifuged
at 20,000 rpm for 30 minutes at 4 ºC, followed by filtering the
1-(5-Chloro-3-hydroxy-2-oxoindolin-3-yl)pentane-2,4-dione
(
27) ― As yellow semi-solid (40% yield; mp: 142-144 °C); 1H supernatant through 0.22 µm filter. After that, 1 mL of nickel-
NMR (600 MHz, CDCl3) δppm 7.41 (s, 1H), 7.25-7.24 (m, 1H), nitrilotriacetic acid resin (Qiagen) was added to the clear
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Journal Name
supernatant and incubated on ice for 2 hours at 80 rpm. Then, concentrations. The amount of generated N-formylkynurenine
the mixture was poured into the column filled with 50 mM KPB was monitored at different time interval. The mode of
at pH 6.5 containing 300 mM KCl and 10 mM imidazole. For inhibition was determined from the plot of [S]/V against
IDO1 enzyme column was sequentially washed with 10 mL of inhibitor concentration [I]. Where, [S] and V represent L-
KPB at pH 6.5 containing 300 mM KCl and 20 and 30 mM tryptophan concentration and initial rate of enzyme catalysis,
imidazole, respectively to remove the non-specifically bound respectively.
protein. The IDO1 protein was eluted with 50 mM KPB at pH
6.5 containing 300 mM KCl and 190 mM imidazole. For TDO Molecular docking analysis
enzyme column was sequentially washed with 10 mL of KPB at Molecular docking analyses studies of the interaction of the
pH 8.0 containing 300 mM KCl and 20 and 30 mM imidazole compounds with IDO1 enzyme (PDB code: 2D0T) was
respectively, to remove the non-specifically bound protein. performed using MoleGro Virtual Docker version 6.0 (Molegro
The TDO protein was eluted with 50 mM KPB at pH 8.0 ApS, Aarhus, Denmark).12, 45, 48, 54-56 To generate apo-protein,
containing 300 mM KCl and 190 mM imidazole. Both IDO1 and the ligands were first removed from the co-crystal structures
TDO proteins were then buffer exchanged by 50 mM Tris- and then were processed by energy minimization. The energy
buffer at pH 7.4 and 50 mM Tris-buffer at pH 8.0, respectively, minimized three-dimensional structure of the ligands was
using Sephadex-G25 column. The purity of the enzyme was prepared by using the GlycoBioChem PRODRG2 server
confirmed by Coomassie-blue stained SDS-PAGE analysis and it (http://davapc1.bioch.dundee.ac.uk/prodrg/). The occupied
showed 85-90 % purity. The ratio of absorbance of the purified position of the ligand (in the crystal structure) was used as the
IDO1 and TDO enzyme at 404 nm to that at 280 nm was center of docking site (radius: 12 Å; and center: x = 61, y = 51, z
around 1.3 and 1.2, respectively.
= 19). Other parameters were set default during docking
analyses. In each docking run, two hundred docked structures
were generated for an individual ligand. Energetically favored
IDO1 and TDO inhibition assay by spectrometric method
Both, IDO1 and TDO inhibition assays were performed docked conformations were evaluated based on the moledock
according to the earlier reported procedures procedure.2, 12, 38, and re-rank scores (docking score-based on energy function
45
The solubility of the compounds in water was either such as a force field with repulsive and attractive Van-der
moderate or poor. Hence, stock solution of the compounds Waals terms and electrostatic term). The docking poses were
were prepared by first dissolving in DMSO and then diluted exported and examined with PyMOL software (The PyMol
with buffer. In the assay system the minimum and maximum Molecular Graphics System, Version 1.0r1, Schrödinger, LLC).
amount of DMSO were 0.02% and 2%, respectively. The
standard reaction mixture (500 μL) contained KPB (100 mM, IDO1 and TDO inhibition assay by HPLC analysis
pH 6.5 for IDO1 enzyme and 50 mM, pH 8 for TDO enzyme), The enzymatic reaction (100 μL) was performed in 100 mM
sodium ascorbate (20 mM), methylene blue (10 µM), catalase potassium phosphate buffer at pH 6.5 using sodium-ascorbate
(240 nM, from brovin liver), L-Trp (150 µM), purified enzyme (20 mM), catalase (240 nM), methylene blue (10 μM), purified
(40 nM for IDO1 and 25 nM for TDO), DMSO (0.05 %, v/v), recombinant IDO1 (41 nM), L-Trp (150 μM), DMSO (0.05%, v/v)
triton-X 100 (0.01 %, v/v) and inhibitors. The reaction was and triton-X 100 (0.01%, v/v). First, the assay was performed
quenched using 100 µL of 30 % (w/v) trichloroacetic acid. The using the inhibitors at different concentrations of 0.5 to 1.5
amount of kynurenine formation was quantified using 2 % μM of the compounds. The reaction was performed at 37 °C
(w/v) p-dimethylaminobenzaldehyde (pDMAB) in acetic acid. for 1 h and quenched by addition of 30% (w/v) trichloroacetic
The absorbance of the reaction mixture was recorded by at acid (20 μL). The reaction mixture was incubated at 50 °C for
480 nm. All these experiments were repeated for three times 30 minutes and then centrifuged at 10000 rpm for 10 minutes.
for each compound.
Then, 20 μL of supernatant from each reaction mixture was
used for HPLC analyses. The mobile phase for HPLC
measurements was 50% sodium citrate buffer (40 mM, pH
Binding analysis by spectroscopic measurement
The absorption spectra were recorded at room temperature 2.25) and 50% methanol with 400 μM SDS. The rate of flow
using a Perkin Elmer Lambda-25 UV-vis spectrophotometer. All through the Ascentis® Express C18, 2.7 μm HPLC column was
the measurements were performed in 100 mM Tris buffer pH 0.5 mL/min, and kynurenine was detected at a wavelength of
7.4 with IDo1 enzyme concentration of 5 μM and compound 321 nm. A similar HPLC analyses were performed using pure
concentration of 50 μM. The deoxy-reaction system was kynurenine and a standard curve was prepared. The IC50 values
prepared by injecting sodium dithionite (
the samples pre-purged with N2 gas.12, 13, 45
∼
10-fold excess) into of the compounds were calculated from this standard curve.
Similarly, TDO enzyme inhibition activity assay was performed
for these selected compounds.
Determination of modes of enzyme inhibition by the
compounds
Cellular activity assay
The IDO1 enzyme inhibition mode of the selected compounds Breast cancer cells, MDA-MB-231 were selected for the in vitro
was measured according to the reported method.12, 45, 53. The cellular assay.12, 45 50,000 cells were seeded in each well of a
IDO1 enzyme kinetics was performed using 50, 100 and 150 24-well plate in DMEM F12 complete media and were allowed
μM of L-Trp and 100, 500, 1000 and 1500 nM of inhibitor to adhere overnight. First, cells were treated with different
12 | J. Name., 2012, 00, 1-3
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ARTICLE
concentration of human IFN-γ (from 5-1000 ng/mL) in mg/mL in PBS) for 4h at 37 °C with 5% CO2. Then, MTT solution
complete media for a period of 48 h. Following this, 150 μM was removed and the formazan crystals were dissolved in
tryptophan was added and treated for additional period of 5 h. 100μL cell culture grade DMSO. The absorbance was
Post treatment phase, the cells were washed with sterile cell- determined using a spectrophotometer (SpectraMax M2) at
culture grade PBS and were trypsinized and centrifuged at 570 nm and 660 nm (to subtract scattering effects of crystals).
1000 rpm. The cell pellet was dissolved again in sterile PBS and MDA-MB-231 and J774A.1 cell lines were procured from
centrifuged at 1000 rpm as a period of washing. The pellet was national cell culture facility, Central Drug Research Institute,
hypotonically lysed in 10 mM HEPES buffer by passing through Lucknow, India. A549 and HeLa Cell lines were procured from
a sterile syringe 10 times. This lysate was used for standard National Center for Cell Science, Pune, India.
IDO1 assay as mentioned earlier. The results showed that 20
ng/mL of human IFN-γ is sufficient enough to show that
activity in the cellular conditions.12 Therefore, all cell works
were performed using this concentration of IFN-γ. After that,
the cells were treated with human IFN-γ (20 ng/mL) in
complete media for a period of 48 h. This treatment is
Acknowledgements
We are thankful to SERB, Govt. of India (EMR/2016/005008)
for financial support and CIF, IIT Guwahati for instrumental
support.
reported to allow over-expression of IDO1 enzyme in MDA-
MB-231 cells.12,
46
Next, the cells were treated with
Conflict of Interest
appropriate concentrations of the compounds (20 nM to 1
mM) for a period of 4 h. Following this, 150 μM tryptophan
was added and treated for additional period of 5 h. Cells
stimulated with IFN-γ alone served as negative control while
cells stimulated with IFN-γ and then with 150 μM tryptophan
served as positive control. Post treatment phase, the cells
were washed with sterile cell-culture grade PBS and were
trypsinized and centrifuged at 1000 rpm. The cell pellet was
dissolved again in sterile PBS and centrifuged at 1000 rpm as a
period of washing. The pellet was hypotonically lysed in 10
mM HEPES buffer by passing through a sterile syringe 10
times. This lysate was used for standard IDO1 assay as
mentioned earlier.12, 38, 43 IC50 values were determined for each
inhibitor accordingly.
The authors declare no competing interests
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TOC text:
Structurally simple C3-Substituted oxindoles showed inhibitory activity against immunosuppressive
indoleamine-2,3-dioxygenase-1 (IDO1) enzyme.

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