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

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Synthesis and evaluation of oxindoles as promising inhibitors to

the immunosuppressive enzyme indoleamine 2,3-Dioxygenase 1

Saurav Paul,†^aAshalata Roy,†^aSuman Jyoti Deka,^bSubhankar Panda,^aGopal Narayan Srivastava,^a

Vishal Trivedi^band 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.^1-3IDO1 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.¹TDO 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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¹⁵Therefore, there is a clear and unmet need to develop small Fe(III)-superoxide intermediate (Fig. 1).²³Consequently,

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.^16-22Comprehensive 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.^23-25Therefore, 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.³²To 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).³²For the same purpose, 2-amino-3-(2-oxoindolin-

thioxoimidazolidin-4-one,

1-methyltryptophantirapazamine

and tryptamine derivatives were reported as moderate

inhibitor of IDO1 enzyme.⁴However, no L-Trp analog showed

low-micromolar activities, probably because of moderate

affinity of L-Trp for IDO1 enzyme (K_d~ 300 μM).⁴We

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

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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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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).³⁵The 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).³⁶Condensation 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).³⁷Hence, 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 IC₅₀values 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 IC₅₀values 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 K_m

COOH

NHCOPh

385.41 ± 35.31

O

N

5

H

COOH

NHCOCH₃

O

N

6

H

and k_catvalues of the enzyme with L-Trp were 47.8 ± 2.5 μM

^aIC₅₀values are the mean of five independent assays.

and 3.7 ± 0.1 Sec^-1, respectively.¹²To 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.¹²The 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 IC₅₀value 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.⁸Whether 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.¹²The differences in IC₅₀values 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 IC₅₀values 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 IC₅₀values 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 (IC₅₀values 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

^aIC₅₀values are the mean of five independent assays.

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Spectroscopic studies for binding analysis of 2-indolinones enzyme. The calculated EC₅₀values 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 EC₅₀values of 120 μM

the ligand/substrate binding ability to IDO1 enzyme.^41-44The under the similar experimental conditions.⁴⁷The differences in

UV-vis absorption spectra of ferric-IDO1 and deoxy-ferrous- EC₅₀values 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 IC₅₀and 2 × IC₅₀values 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 cell^a

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-MT^c

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). ^bEC₅₀values 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 O₂to 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 Na₂S₂O₄under 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 O₂is 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).⁴⁸Molecular docking analysis revealed that the

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potent compounds

6

,

23 and 25 presumably interact with the in interaction with Ser263 and Ala264 residues. Recently, 3-

-Trp (Fig. 4).⁴⁷The 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 IC₅₀values of these compounds.

hydrophobic residues Phe163 and Phe164 are important for The differences in the IC₅₀values among compounds 23

the activity of the IDO1 enzyme.⁴⁹Whereas, 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.⁵⁰Therefore, 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 IC₅₀values.

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]

1400

600

(C)

1200

(D)

550

Compound 22

Compound 23

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]

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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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 IC₅₀values 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

TDO

Selectivity

ratio^b

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

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 (δ_ppm) were reported in Hertz (Hz) and parts per million

(ppm) respectively. Multiplicities are reported as follows: s

NM

30

competitive

NM

37

76

competitive

152

210

124

^aIC₅₀values are the mean of three independent assays against purified enzymes.

^bSelectivity ratio is calculated as (IC₅₀value of TDO)/(IC₅₀value 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.^16-22These results clearly

suggest that designing compounds, which could perturb the

electrophilic addition of O₂of 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.³²The 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 Na₂SO₄and

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 N₂atmosphere.³⁴Then 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

Na₂SO₄and 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.⁵¹The 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 Na₂SO₄followed 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.³⁵The

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.⁵²After 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 Na₂SO₄under continuous stirring.³⁶The 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.³²The amount of piperidinium acetate for 3-4 hours at 100 ° C.³⁶The

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 Na₂SO₄and 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.³⁷The desired amount of 5-

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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) δ_ppm7.61-7.59 (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 δ_ppm202.0, (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) δ_ppm10.15

stirring.³⁷After 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 Na₂SO₄and MHz,CDCl₃+ DMSO-d₆) δ_ppm180.4, (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) δ_ppm10.46 (s, 1H), 8.82 (br, s, 1H), 7.47-7.44 NMR (400 MHz, CDCl₃+ CD₃OD) δ_ppm9.28 (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, CDCl₃1.61 (m, 1H), 1.54-1.48 (m, 1H); ¹³C NMR (151 MHz, CDCl₃+

+ DMSO-d₆) δ_ppm179.8, (179.5), 169.8, 141.5, (141.4), 127.8, CD₃OD) δ_ppm180.3, (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) δ_ppm10.41 (br s, 1H), 8.38 (br s, 2H), 7.24-7.16 (m, (400 MHz, CDCl₃+ DMSO-d₆) δ_ppm9.65 (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) δ_ppm178.3, (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, δ_ppm179.2, (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) δ_ppm9.48 (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) δ_ppm7.57 (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) δ_ppm180.8, 2.19 (s, 2H); ¹³C NMR (151 MHz, CDCl₃+ CD₃OD) δ_ppm174.6,

(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) δ_ppm9.18 (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) δ_ppm175.8, 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) δ_ppm8.04-8.02 (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 δ_ppm163.1, 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) δ_ppm7.91-7.86 (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) δ_ppm8.10-8.09 (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) δ_ppm181.2, 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) δ_ppm148.1, 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) δ_ppm12.69 (br s, 1H), 7.77 (br s, mp: 230-232 °C); ¹H NMR (600 MHz, CDCl₃+ DMSO-d₆) δ_ppm

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) δ_ppm163.6, 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; δ_ppm164.7, 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) δ_ppm10.59 (br s, DMSO-d₆) δ_ppm10.32 (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, CDCl₃1H), 6.50-6.49 (m, 2H);¹³C NMR (151 MHz, CDCl₃+ DMSO-d₆)

+ DMSO-d₆) δ_ppm163.6, 140.9, 139.3, 128.8, 127.9, 126.9, δ_ppm164.4, 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) δ_ppm10.14 (br s, 1H), DMSO-d₆) δ_ppm10.51 (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) δ_ppm163.9, 135.0, 130.3, 128.9, CDCl₃+ DMSO-d₆) δ_ppm165.3, 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) δ_ppm

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) δ_ppm10.53 (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) δ_ppm168.0, 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) δ_ppm163.7, 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) δ_ppm193.5, 165.1,

yield; mp: 172-174 °C); ¹H NMR (400 MHz, CDCl₃) δ_ppm8.44 (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) δ_ppm198.7, 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

δ_ppm8.59(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) δ_ppmmL/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) δ_ppm9.45 (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

δ_ppm191.8, 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) δ_ppm10.44 (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, CDCl₃IDO1 and TDO enzymes were a generous gift from Professor

+ DMSO-d₆) δ_ppm202.5, 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) δ_ppm7.44-7.42 (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

δ_ppm194.5, 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, CDCl_3,buffer containing 1 mM PMSF and centrifuged at 15,000 rpm

DMSO-d₆) δ_ppm9.58 (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) δ_ppm205.8, 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) δ_ppm7.41 (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 IC₅₀values

prepared by injecting sodium dithionite (

the samples pre-purged with N₂gas.^{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.¹²Therefore, 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}IC₅₀values 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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Synthesis and evaluation of oxindoles as promising inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1

DOI: 10.1039/c7md00226b

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