Fused Heterocyclic Compounds as Potent Indoleamine-2,3-dioxygenase 1 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00359

Uncontrolled metabolism of l-tryptophan (l-Trp) in the immune system has been recognized as a critical cellular process in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) enzyme plays an important role in the metabolism of a local l-Trp through the kynurenine pathway in the immune systems. In this regard, IDO1 has emerged as a therapeutic target for the treatment of diseases that are associated with immune suppression like chronic infections, cancer, and others. In this study, we synthesized a series of pyridopyrimidine, pyrazolopyranopyrimidine, and dipyrazolopyran derivatives. Further lead optimizations directed to the identification of potent compounds, 4j and 4l (IC₅₀ = 260 and 151 nM, respectively). These compounds also exhibited IDO1 inhibitory activities in the low nanomolar range in MDA-MB-231 cells with very low cytotoxicity. Stronger selectivity for the IDO1 enzyme (>300-fold) over tryptophan 2,3-dioxygenase (TDO) enzyme was also observed for these compounds. Hence, these fused heterocyclic compounds are attractive candidates for the advanced study of IDO1-dependent cellular function and immunotherapeutic applications.

Full text of DOI:10.1021/acsmedchemlett.6b00359

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Letter
Fused Heterocyclic Compounds as Potent
Indoleamine-2,3-dioxygenase 1 Inhibitors
Subhankar Panda, Ashalata Roy, Suman Jyoti Deka, Vishal Trivedi, and Debasis Manna
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00359 • Publication Date (Web): 15 Oct 2016
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Fused Heterocyclic Compounds as Potent Indoleamine-2,3-dioxy-
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†
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,a
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Subhankar Panda, Ashalata Roy, Suman Jyoti Deka,b Vishal Trivedib and Debasis Manna*,a
aDepartment of Chemistry, Indian Institute of Technology Guwahati, Assam 781039, India
bDepartment of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Assam 781039, India
KEYWORDS: Indoleamine 2,3-dioxygenase 1 inhibition, fused heterocyclic compounds, halogen substituents, low IC50 and EC50 values, low
cytotoxicity
ABSTRACT: Uncontrolled metabolism of L-tryptophan (L-Trp) in the immune system has been recognized as a critical cellular process in
immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) enzyme plays an important role in the metabolism of a local L-Trp through the
kynurenine pathway in the immune systems. In this regard, IDO1 has emerged as a therapeutic target for the treatment of diseases that are
associated with immune suppression like chronic infections, cancer and others. In this study, we synthesized a series of pyridopyrimidine, py-
razolopyranopyrimidine and dipyrazolopyran derivatives. Further lead optimizations directed to the identification of potent compounds, 4j
and 4l (IC50 = 260 and 151 nM, respectively). These compounds also exhibited IDO1 inhibitory activities in the low nanomolar range in MDA-
MB-231 cells with very low cytotoxicity. Stronger selectivity for the IDO1 enzyme (> 300-fold) over tryptophan 2,3-dioxygenase (TDO) en-
zyme was also observed for these compounds. Hence, these fused heterocyclic compounds are attractive candidates for the advanced study of
IDO1-dependent cellular function and immunotherapeutic applications.
Immunotherapy is currently considered as one of the most promis-
ing approaches in the battle against cancer.1-3 Recent accomplish-
ment with the immune checkpoint inhibitors against a wide range of
cancers has made cancer immunotherapy as one of the most exciting
developments. It has been also shown that cancer immunotherapy
and traditional chemotherapy or radiotherapy could also be bene-
fited from combinatorial strategies against tumor-induced immuno-
ety of preclinical studies with cancer models suggest that IDO1 as-
sists cancer progression and metastasis.9, 10 All these findings high-
light the effectiveness of IDO1 in cancer and other diseases. Recent
developments have shown that inhibition of IDO1 enzyme activity
improves the efficacy of chemotherapeutic and radio therapeutic
treatment of malignant tumors.6, 7, 9, 10
There are a number of reported small molecules-based IDO1 in-
hibitors with different structural classes, including tryptophan, imid-
azole, triazole, N-hydroxyamidine, iminoquinone and others. Natu-
ral products like norharman, β-carboline, benzomalvin and their de-
rivatives also showed IDO1 inhibitory potencies.8, 11-13 IDO1 inhibi-
tors INCB024360, NLG919 and 1-methyl-L-tryptophan (L-1MT)
are currently under clinical trials for the treatment of different types
of cancers.8, 14, 15 Successful use of ipilimumab and nivolumab and
current clinical development of the inhibitors of IDO1 enzyme in-
spire researchers to develop IDO1 inhibitors for cancer immuno-
therapy.14
2,
4
suppression.1,
Induced metabolism of L-tryptophan (L-Trp)
through kynurenine pathway and consequential production of
kynurenine, 3-hydroxy kynurenine, kynurenic acid, excitotoxin
quinolinic acid and other metabolites are primarily responsible for
local immunosuppression.4-6
In non-hepatic cells, indoleamine 2,3-dioxygenase 1 (IDO1) cat-
alyze the rate limiting step of the L-Trp catabolism through
kynurenine pathway. IDO1 activity is generally low in healthy hu-
mans and has insignificant physiological effects. However, within the
immune system IDO1 gets highly up-regulated in response to in-
flammatory signals under pathophysiological conditions (e.g. in tu-
mor cells). Up-regulation of IDO1 is interrelated with poor progno-
sis in different types of cancers, including pancreatic, ovarian, colo-
rectal, and others.5, 6 Recent reports also suggest that neurodegener-
ative disorder, HIV-1 encephalitis and other diseases are also associ-
ated with the up-regulated IDO1 activity.7, 8 IDO1 promotes a toler-
ogenic state in the tumor cells and its lymph nodes by suppressing
the T cells and enhancing the local regulatory T cells. IDO1 expres-
sion is strongly up-regulated by cytokines like interferon-γ. Cyto-
toxic T lymphocytes produce this interferon-γ, which perhaps in-
dulges the efficacy of other immune therapy against cancer.3 A vari-
To identify a new structural class of IDO1 inhibitors that could be
optimized to afford potent and selective inhibitors of IDO1, we syn-
thesized a series of fused di- and tri-heterocyclic compounds. Activ-
ity studies of these compounds revealed their strong and selective
IDO1 inhibitory potency under the in vitro conditions with no/neg-
ligible cytotoxicity.
The 4-phenyl-1,4-dihydropyridine (1), 4-phenyl-2-pyridone (2)
and 4-phenyl-4H-pyran (3 and 4) derivatives were synthesized ac-
cording to the reported procedures (Scheme 1).16 Condensation of
the substituted aromatic aldehyde with either malononitrile and 6-
amino uracil or ethyl cyanoacetate and 6-amino uracil or 5-methyl-
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2,4-dihydro-3H-pyrazol-3-one and 6-amino uracil or methyl-2,4-di- residues (Y126, C129 and F163) present in the active site of IDO1
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hydro-3H-pyrazol-3-one one or methyl-2,4-dihydro-3H-pyrazol-3-
one, hydrazine hydrate and ethyl cyanoacetate or hydrazine hydrate
and ethyl cyanoacetate yielded these heterocyclic compounds under
mild basic conditions. This single-step synthetic method is highly
beneficial for structural modifications and large-scale production.
Inhibitory activity of the compounds was first explored using
standard spectrophotometric method.8, 11, 17 Absorption spectra of
the compounds (10 nM to 50 μM) showed no or little interference
with this enzyme activity assay. The calculated Km and kcat values of
the enzyme with the L-Trp were 68.9 3.5 μM and 4.2 0.2 Sec-1,
respectively. To improve the efficacy of the fused heterocyclic com-
pounds, we investigated two general modifications of the structure
of the compounds: alteration of the fused heterocyclic ring and sub-
stitution of the phenyl ring.
enzyme.11, 17, 18 In our previous work, we successfully exploited these
interactions and showed that halogen substituted aryl ring increased
the inhibitory potencies of N′-hydroxyamidines.8 We showed that
the halogen substitution at the meta- and/or para-positions of the
aryl ring of N′-hydroxyamidines had greater impact on their IDO1
inhibitory activity. Several reports also described such benefits of
halogen substituted aryl ring on the IDO1 inhibitory efficacies.8, 14, 17,
19
For these fused heterocyclic compounds halogen substituents to
9
meta and/or para-positions of the aryl ring proved to be quite bene-
ficial (Table 1 and 2). Such stronger activity of the halogen substi-
tuted compounds could be due to the hydrophobic interactions, pi-
stacking interaction with aromatic amino acids or halogen bonding
with the Lewis bases present within the active site.
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Table 1. Inhibitory activity of the aryl substituted 4-phenyl-1,4-
dihydropyridine and 4-phenyl-2-pyridone derivatives against
purified human IDO1 enzyme.
Scheme 1. Synthetic scheme of 4-phenyl-1,4-dihydropyridine,
4-phenyl-2-pyridone and 4-phenyl-4H-pyran derivativesa.
Compound
IDO1
Compound
IDO1
IC50 (nM)a
IC50 (nM)a
1a; R1 = R2 = R3 2784 72
2a; R1 = R2 = R3 = 2092 54
= H
H
1b; R1 = R3 = H,
R2 = F
718 19
473 23
668 30
909 56
942 78
680 26
577 26
879 22
2b; R1 = R3 = H,
R2 = F
383 15
296 18
1c; R1 = R3 = H,
R2 = Cl
2c; R1 = R3 = H,
R2 = Cl
1d; R1 = R2 = H,
R3 = F
2d; R1 = R2 = H, 1149 41
R3 = F
1e; R1 = R2 = H,
R3 = Cl
2e; R1 = R2 = H, 1365 47
R3 = Cl
1f; R1 = R2 = H,
R3 = Br
2f; R1 = R2 = H,
R3 = Br
790 26
559 36
650 35
362 15
1g; R1 = H, R2 =
R3 = F
2g; R1 = H, R2 =
R3 = F
1h; R1 = H, R2 =
R3 = Cl
2h; R1 = H, R2 =
R3 = Cl
aReagents and conditions: (a) malononitrile, 6-amino uracil, EtOH,
reflux, 4h. (b) ethyl cyanoacetate, 6-amino uracil, EtOH, reflux, 4h. (c)
5-methyl-2,4-dihydro-3H-pyrazol-3-one, 6-amino uracil, EtOH, reflux,
4h. (d) 5-methyl-2,4-dihydro-3H-pyrazol-3-one, EtOH, reflux, 4h. (e)
ethyl cyanoacetate, hydrazine, 5-methyl-2,4-dihydro-3H-pyrazol-3-one,
6-amino uracil, EtOH, reflux, 4h. (f) ethyl cyanoacetate, hydrazine,
EtOH, reflux, 4h.
1i; R1 = H, R2 =
2i; R1 = H, R2 =
Cl, R3 = F
Cl, R3 = F
a IC50 values are the mean of three independent assays.
The halogen substituted pyridopyrimidines 1b-i and 2b-i showed
1.5 to 7-fold stronger IDO1 inhibitory activities than the lead com-
pounds 1a and 2a (Table 1). Among these tested compounds, 3-
chloro substituted pyridopyrimidines 1c (IC50 = 473 nM) and 2c
(IC50 = 296 nM) showed stronger IDO1 inhibitory activity than the
other tested pyridopyrimidines. 3-Chloro-4-fluoro substituted pyri-
dopyrimidine 2i showed moderate inhibitory activity with IC50 value
of 362 nM. However, no considerable preference for certain halogen
substitutions at the meta- and/or para-positions were observed pyr-
idopyrimidines. Halogen substituted compounds with pyrazolopy-
ranopyrimidine and dipyrazolopyran moieties showed 1.3 to 8-fold
stronger IDO1 inhibitory activities than the original lead com-
pounds 3a and 4a. For these compounds a substantial preference for
the fluoro-substitution at the meta- and/or para-positions of the aryl
We first investigated the role of fused di- and tri-heterocyclic ring
on IDO1 enzyme activity (Table 1 and 2). The IC50 values of the lead
compounds 1a, 2a, 3a and 4a suggest that not only the presence of
di- and tri-heterocyclic ring, but also substitution and electronic
properties of these fused heterocyclic rings plays an important role
in their inhibitory efficacy against purified IDO1 enzyme (Table 1
and 2).
To optimize the efficacies of these fused heterocyclic compounds,
we tested the substitution effect of the aryl ring on IDO1 activity. We
presume that the presence of a substituted phenyl ring in the com-
pound's core structural unit could also play an important role in their
inhibitory efficacies because of their interactions with the amino acid
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IDO1 inhibitory activities. Similar inhibitory activities were also ob-
ring were observed for these pyrazolopyranopyrimidines and dipyra-
1
2
3
4
5
6
7
8
zolopyrans (Table 2). Dipyrazolopyran 4j showed lowest IC50 value
of 260 nM among the tested compounds with 4-phenyl-1,4-dihydro-
pyridine, 4-phenyl-2-pyridone and 4-phenyl-4H-pyran moieties.
In addition, we also investigated how the electronic properties of
the dipyrazolopyran moiety affect IDO1 inhibitory activities. In this
regard, halogen substituted phenyl ring containing mono- and dia-
mino-dipyrazolopyrans (at 3 and/or 5 position) 4k-p were selected
(Table 2). The results showed that
served among the dipyrazolopyrans 4j, 4m, and 4p. Isovanillin de-
rivative of dipyrazolopyran, 4n also showed strong IDO1 inhibitory
activity. The aryl ring of compound 4n contains 3-hydroxy and 4-
methoxy group. However, its higher/similar IC50 value than the di-
halogen substituted dipyrazolopyrans, also suggest that halogen sub-
stitution of the aryl ring plays an important role at inhibiting IDO1
activity. The IC50 value of the reported potent compound 4-amino-
N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-car-
boxymidimade, 5l under the experimental conditions was 91 nM
(Table 2), which is in accordance with the reported values.13 IDO1
inhibitor L-1MT, which is under clinical trial for the treatment of
several types cancer showed IC50 value of 385 µM, which is in ac-
cordance with the reported values.15
IDO1 activity in the presence of selected compounds was also an-
alyzed by HPLC-based assay for further confirmation of their inhi-
bition potencies. In this assay the amount of kynurenine generated
from L-Trp was directly measured to calculate the inhibitory activi-
ties of the compounds.8 The results showed that the inhibitory activ-
ities of the compounds are within 78-561 nM range (Table S1) and
the differences in the IC50 values of the compounds are in agreement
with that of measured using the spectrophotometric method. We
presume that the accuracy of the methylene blue-ascorbate regener-
ation system to keep IDO1 in its active state (Fe2+) could be the pri-
mary reason for the differences in the inhibitory activity values be-
tween the pDMAB- and HPLC-based methods.8, 14 The HPLC-
based assay also showed that mono-amino substituted dipyrazolopy-
ran derivative 4l displayed strongest inhibitory activity (IC50 = 78
nM) among all the investigated compounds.
Overall, the inhibitory efficacy of the fused heterocyclic com-
pounds is sensitive to the size and position of the halogen substitu-
ent(s) on the aryl ring, possibly due to the restricted space in "pocket
A" of the IDO1 enzyme. Stronger inhibitory activities of the 3-chloro
substituted pyridopyrimidines 1c and 2c could be due to both elec-
tronic properties of the fused di-heterocyclic ring, hydrophobic in-
teraction between the halogen substituted aryl ring, and the hydro-
phobic residues present within the IDO1 binding pocket. Preference
for the fluoro-substitution at the meta- and/or para-positions of the
aryl ring of the dipyrazolopyran derivatives (4j and 4l) could be due
to the pi-stacking interaction with aromatic amino acids (Y126 and
F163). Electronic properties of the fused heterocyclic ring and the
interactions of the fused heterocyclic ring with the heme-group and
polar residues of the IDO1 enzyme plays important roles for the in-
hibitory potencies of the dipyrazolopyran derivatives. This could ex-
plain why dipyrazolopyran derivative 4j or 4l showed lower IC50 val-
ues than compounds 1c, 2c and 3g.
The optical properties of the heme-group are very much sensitive
to the local environment and crucial in understanding the ligand
binding aptitude to the IDO1 enzyme.14 Although, IDO1 enzyme in-
hibition studies demonstrate the inhibition capability of these fused
heterocyclic compounds but it fails to provide any direct evidence of
ligand binding to the active site of the enzyme. In this regard, we rec-
orded the absorption spectra of ferric-IDO1 and deoxy-ferrous-
IDO1 in the absence and presence of the selected compounds (Fig-
ure 1A and B).14 Figure 1A showed that in the presence of com-
pounds, the Soret peak (404 nm) showed no substantial shift, indi-
cating its trivial binding to the ferric-IDO1 enzyme. Figure 1B
showed that in the absence of compound the deoxy-ferrous-IDO1
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Table 2. Inhibitory activity of the aryl substituted 4-phenyl-4H-
pyran derivatives against purified human IDO1 enzyme.
Compound
IDO1
Compound
IDO1
IC50 (nM)a
IC50 (nM)a
3a; R1 = R2 = R3 3275 96
= H
4e; R1 = R2 = H,
R3 = F
479 20
889 32
652 40
441 22
505 24
260 11
546 18
3b; R1 = R3 = H, 1347 41
R2 = F
4f; R1 = R2 = H,
R3 = Cl
3c; R1 = R3 = H,
R2 = Cl
932 39
571 61
862 32
969 55
410 24
4g; R1 = R2 = H,
R3 = Br
3d; R1 = R2 = H,
R3 = F
4h; R1 = H, R2 =
R3 = F
3e; R1 = R2 = H,
R3 = Cl
4i; R1 = H, R2 =
R3 = Cl
3f; R1 = R2 = H,
R3 = Br
4j; R1 = H, R2 =
Cl, R3 = F
3g; R1 = H, R2 =
4k; R1 = R2 = H,
R3 = F, R4 = NH2,
R5 = Me
R3 = F
3h; R1 = H, R2 =
R3 = Cl
593 35
438 22
4l; R1 = H, R2 =
R3 = F, R4 = NH2,
R5 = Me
151 15
345 21
397 21
3i; R1 = H, R2 =
Cl, R3 = F
4m; R1= H, R2 =
Cl, R3 = F, R4 =
NH2, R5 = Me
4a; R1 = R2 = R3 1139 52
4n; R1 =H, R2 =
OH, R3 = OMe,
R4 = NH2, R5 =
Me
= H
4b; R1 = F, R2 = 693 19
R3 = H
4o; R1 = H, R2 =
R3 = F, R4 = R5 =
NH2
239 27
378 18
91 02
4c; R1 = R3 = H, 808 22
R2 = F
4p; R1 = H, R2 =
Cl, R3 = F, R4 =
R5 = NH2
4d; R1 = R3 = H, 910 16
5lb
R2 = Cl
a IC50 values are the mean of three independent assays.
b Reported compound.13
mono-amino substituted dipyrazolopyran derivative 4l exhibited
lowest IC50 values of 151 nM among all the investigated compounds.
A comparison of the IC50 values of 4h, 4l and 4o clearly showed that
the electronic properties of the dipyrazolopyran moiety also affect
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enzyme displayed Soret and Q-band at 421 and 556 nm.14 In the concentration, respectively.8, 21 However, this observed uncompeti-
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3
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7
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presence of compounds, 4j and 4l the Soret band (421 nm) got blue
shifted and new Q bands appeared around 520/550 nm (Figure 1B).
This indicates their probable binding to the Fe2+-IDO1 enzyme. The
UV-Vis spectra of only compounds did not show any peak in this re-
gion (Figure S1). Although additional studies are necessary to con-
firm the binding of these compounds to the IDO1 enzyme, but these
spectral analyses undoubtedly, support the binding of compounds to
the ferrous-IDO1 enzyme.
tive mode of IDO1 inhibition does not demonstrate their actual
mode of enzyme binding. There are several reports of uncompetitive
and noncompetitive inhibitors of IDO1 enzyme. 4-Phenylimidazole
is a noncompetitive inhibitor of IDO1, but its crystal structure and
enzyme activity studies clearly demonstrate its binding to IDO1 ac-
tive site.14, 22 Recently reported o-alkylhydroxylamines are also un-
competitive inhibitors of IDO1 with activities in the nanomolar
range.14 Mechanistic studies of the IDO1 induced catabolism of L-
Trp revealed that the formation of ferric-superoxide intermediate is
the primary requirement for this oxidation process. Hence, addi-
tional kinetic studies with respect to O2 are needed to understand
the definite mode of IDO1 enzyme inhibition by these fused heter-
ocyclic compounds.8, 11, 14
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Table 3. EC50 values of the selected compounds in MDA-MB-
231 cells.
Compound
MDA-MB-231 Compound
MDA-MB-231
cellsa
EC50
cellsa
EC50
(nM)b
(nM)b
Figure 1. Absorption spectra of ferric-IDO1 (A) and deoxy-ferrous-
IDO1 enzyme in the absence and presence of the compounds (270 μM)
in 50 mM Tris-Hcl buffer at pH 8.0. [IDO1] = 5 μM. Ferrous-deoxy re-
action environment was generated by adding Na2S2O4 to the solution
under N2 atmosphere.
1c
2c
3g
3i
75 09
52 07
76 11
83 10
135 12
71 10
52 12
4l
48 08
81 12
113 12
73 11
139 13
59 04
4m
4n
4o
4p
5lc
To determine the therapeutic potential of these potent com-
pounds, cellular IDO inhibitory activities were tested in MDA-MB-
231 breast tumor cells. It is reported that interferon (IFN)-γ sub-
stantially promote the expression of native IDO enzyme from its
mRNA in MDA-MB-231 cells.8, 20 Calculated inhibitory activities of
the compounds under these cellular environments follow similar
pattern as that of against purified IDO1 enzyme. The calculated cel-
lular EC50 values of the compounds are within the range of 48-139
nM (Table 3). Control compound, 5l and L-1MT displayed EC50
values of 59 nM and 120 µM, respectively under the similar experi-
mental conditions, which are in accordance with the reported val-
ues.13, 15 Subtle differences in the compound's inhibitory activity val-
ues between the enzymatic assay against purified IDO1 and under
cellular conditions could be because of the complications in regulat-
ing the IDO1 redox activity and/or environmental effect. In general,
a good correlation between these assays validates the IDO1 inhibi-
tion potencies of these fused heterocyclic compounds. We also pre-
sume that for the same reason the cell-based activity assay of the
compounds shows lower EC50 values than the IC50 values against pu-
rified enzyme. Cytotoxicity assay of the compounds in MDA-MB-
231 cells (concentrations of IC50 and 2 × IC50 values from the enzy-
matic assay) also displayed negligible level of toxicity of the com-
pounds under the experimental conditions (Figure S2 and S3).
The inhibition studies reveal that few of these heterocyclic com-
pounds strongly inhibit the L-Trp catabolic activity of the IDO1 en-
zyme. Therefore, to understand the mode of IDO1 inhibition we
performed enzyme kinetics in the absence and presence of these po-
tent compounds. The plots of [S]/V against inhibitor concentra-
tions ([I]) showed that tested compounds 1c, 2c, 3i, 4h, 4j, 4l, 4m,
4n and 4p followed uncompetitive inhibition, whereas 3g and 4o
followed competitive inhibition modes (Table S2 and Figure S4). V
and [S] represent the initial rate of the reaction and the substrate
4e
4h
4j
a IDO protein expression in MDA-MB-231 cells was induced by human
IFN-γ (20 ng/mL).
b EC50 values are the mean of three independent assays.
c Reported compound.13
Our enzyme inhibition studies clearly demonstrate that the po-
tent compounds strongly interact with the enzyme through its active
site. Spectroscopic studies also supported their binding capabilities
with the IDO1 enzyme. To determine their plausible mode of inter-
actions, we performed molecular docking analyses with the IDO1
enzyme (PDB code: 4PK5).23 The model structures propose that the
pyridopyrimidines 1c and 2c might have two equally possible bind-
ing modes; in "pocket A" near F163 and S167 residues or "pocket B"
near F226 and 7-propionate of the porphyrin ring. A similar mode of
interaction for the pyrazolopyranopyrimidines 3g and 3i was ob-
served. However, the molecular models predicted that dipyra-
zolopyrans 4j, 4l and 4m interact with IDO1 enzyme preferably
through its "pocket A". The halogen substituted aryl rings of 4j and
4l could be involved in interaction with the amino acids like F163,
F164, Y126 and others present in pocket-‘A’ through pi-stacking and
hydrophobic interactions (Figure 2). The fused tri-heterocyclic ring
of these dipyrazolopyran derivatives could be involved in interaction
with the heme group. This proposed interaction is in accordance
with the spectroscopic based binding studies. The pyrazole ring
could be also involved in interaction with S167 residue and 7-propi-
onate group of heme, through hydrogen bonding. An additional in-
teraction between the 3-amino pyrazole-ring of compound 4l with
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gests that the electronic properties of dipyrazolopyran ring and hal-
S167 residue was observed (Figure 2B). A similar mode of interac-
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tion was also observed for compound 4m. Model structures also pro-
pose that the presence of additional 5-amino group of 3,5-diamino
substituted dipyrazolopyran derivatives may not have any substan-
tial role in interaction with the IDO1 through its binding site, which
is in accordance with its effectiveness for IDO1 inhibitory activity
(4o). Electronic properties of the fused heterocyclic ring and halo-
gen substitution on the aryl ring also play crucial role in binding of
the compounds to the active site of IDO1 enzyme. Hence, hydrogen
bonding, pi-stacking and hydrophobic interactions play important
roles in stronger binding of the compounds. The differences in in-
hibitory activities among the compounds under the experimental
conditions suggest that their mode of interaction with the IDO1 en-
zyme active site could be different that the proposed one by molec-
ular docking analysis. Molecular volume and interaction pattern of
the compounds could be also critical for their binding under the ex-
perimental conditions.
TDO is the other enzyme that also catalyzes the rate-limiting step
of the kynurenine pathway in the liver. TDO enzyme directly regu-
late the cellular level of L-Trp. Therefore, to determine the selectiv-
ity of the compounds at inhibiting the IDO1 enzyme, we measured
the inhibitory activities of the selected compounds against purified
TDO enzyme. Activity studies showed that these fused heterocyclic
compounds have variable TDO inhibition properties. The IC50 val-
ues of the selected compounds against purified TDO enzyme vary
from 3 to 79 μM (Table S3). Compound 4j and 4l showed higher
inhibitory activity for IDO1 (> 300 fold) over TDO enzyme under
the similar experimental conditions. The results also showed that
substitution of the aryl ring and electronic properties of the fused
heterocyclic ring play crucial roles in their TDO inhibition. HPLC
analysis also showed similar TDO inhibitory activities of these po-
tent compounds (Table S1).
ogen substituted aryl ring assist these compounds to interact with
the IDO1 through hydrogen bonding, pi-stacking and hydrophobic
interactions. Additional calculation of inhibitory constant (Ki) val-
ues from the enzyme kinetics measurements revealed that the Ki val-
ues of these potent compounds are within 35-297 nM range. Com-
pound 4l showed the lowest Ki value of 35 nM. Reported compound,
5l showed Ki value of 22 nM under the similar experimental condi-
tions (Table S2).13 The strong potencies of compounds 4j and 4l
also yielded very good ligand efficiencies of 0.42 and 0.43, respec-
tively.14 Hence, dipyrazolopyran represent a promising class of
IDO1 inhibitors. The potent compounds also displayed > 300-fold
stronger inhibition for IDO1 enzyme inhibition in comparison with
the TDO enzyme. IDO1 activity in the MDA-MB-231 cells showed
that the tested compounds have minimal cytotoxicity and low-nano-
molar potencies. Low cytotoxicity and inactivity for TDO enzyme
support further development of dipyrazolopyran derivatives as in-
hibitor of IDO1 enzyme.
In summary, to find potent inhibitors of IDO1 enzyme we synthe-
sized a series of fused heterocyclic compounds. Several of these com-
pounds showed strong inhibitory activity against human IDO1 en-
zyme. The presence of fused di- or tri-heterocyclic moieties and its
electronic properties could be the key factor for their strong in vitro
inhibitory activities. Halogen substitutions in the aryl ring were ef-
fective in improving the potency of these compounds. These com-
pounds also could be considered as good molecular probes based on
their ligand efficiency values. Overall, these observations suggest
that these simple fused heterocyclic compounds are potential inhib-
itor of IDO1 enzyme and could be of interest as drug target in cancer
and other human diseases.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Pub-
lication website at DOI:
Experimental sections, IDO1 and TDO inhibition potencies,
13
characterization and 1H and C NMR spectra of the synthesized com-
pounds.
AUTHOR INFORMATION
Figure 2. Probable mode of interaction of the compounds, 4j (A), 4l
(B) and 4o (C) with the active site of the IDO1 enzyme (4PK5). The
model structures were generated using MoleGro Virtual Docker, ver-
sion 6.0. The oxygen and nitrogen atoms are shown in red and blue, re-
spectively. Residues involved in interactions through hydrogen bond
formation are shown using dashed lines (yellow). Images were gener-
ated using PyMol.
Corresponding Author
*(D.M.) E-mail: dmanna@iitg.ernet.in.
Author Contributions
†S.P. and A.R. contributed equally to this work.
Funding Sources
In this study, fused heterocyclic compounds were designed as
IDO1 inhibitor. Subsequent modification of the electronic proper-
ties of the fused heterocyclic ring and substitution of the aryl ring di-
rected to the identification of potent inhibitors with nanomolar level
IDO1 enzyme inhibitory activities under the in vitro conditions.
Overall, activity studies showed that the 3,5-dimethyl-dipyrazolopy-
ran or 3-amino-5-methyl-dipyrazolopyran moiety and dihalogen
substituted aryl ring could considerably enhance the inhibition po-
tency of these fused heterocyclic compounds. Spectroscopic studies
suggest that the dipyrazolopyran derivatives preferably interact with
the deoxy-ferrous-IDO1 enzyme. Molecular model structure sug-
The authors gratefully acknowledge DST, Govt. of India (SB/FT/CS-
131/2012) for financial support. We are thankful to Central Instrument
Facility and Department of Chemistry for instrumental support.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
IDO1, indoleamine 2,3-dioxygenase 1; TDO, tryptophan 2,3-dioxygen-
ase; L-Trp, L-tryptophan.
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Synthetic scheme of 4-phenyl-1,4-dihydropyridine, 4-phenyl-2-pyridone and 4-phenyl-4H-pyran
derivativesa.
Scheme 1.
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Figure 1. Absorption spectra of ferric-IDO1 (A) and deoxy-ferrous-IDO1 enzyme in the absence and
presence of the compounds (270 µM) in 50 mM Tris-Hcl buffer at pH 8.0. [IDO1] = 5 µM. Ferrous-deoxy
reaction environment was generated by adding Na2S2O4 to the solution under N2 atmosphere.
Figure 1
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Probable mode of interaction of the compounds, 4j (A), 4l (B) and 4o (C) with the active site of the IDO1
enzyme (4PK5). 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.
Figure 2.
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Graphical Abstract
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