Discovery of IDO1 and DNA dual targeting antitumor agents

Article abstract of DOI:10.1039/c7ob02529g

The development of small molecules for cancer immunotherapy is highly challenging and indoleamine 2,3-dioxygenase 1 (IDO1) represents a promising target. Inspired by the synergistic effects between IDO1 inhibitors and traditional antitumor chemotherapeutics, the first orally active dual IDO1 and DNA targeting agents were designed by the pharmacophore fusion strategy. The bifunctional hybrids exhibited enhanced IDO1 enzyme inhibitory activity and in vitro cytotoxicity as compared to IDO1 inhibitor 1-methyl-tryptophan and DNA alkylating agent melphalan. In a murine LLC tumor model, the dual targeting agents demonstrated excellent antitumor efficacy, highlighting the advantages of this novel design strategy to improve the efficacy of small molecule cancer immunotherapy.

Full text of DOI:10.1039/c7ob02529g

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: K. Fang, S. Wu, G.
Dong, Y. Wu, S. Chen, J. Liu, W. Wang and C. Sheng, Org. Biomol. Chem., 2017, DOI:
10.1039/C7OB02529G.
This is an Accepted Manuscript, which has been through the
Volume 14 Number
1 7 January 2016 Pages 1–372
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 6
View Article Online
DOI: 10.1039/C7OB02529G
Journal Name
COMMUNICATION
Discovery of IDO1 and DNA Dual Targeting Antitumor Agents
Kun Fang†ab, Shanchao Wu†b, Guoqiang Dongb, Ying Wub, Shuqiang Chenb, Jianhe Liu*d, Wei
Wang*ac and Chunquan Sheng*b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The development of small molecules for cancer immunotherapy is immunotherapy has remarkable advantages such as oral
highly challenging and indoleamine 2,3-dioxygenase 1 (IDO1) administration and suitable pharmacokinetic profiles. IDO1 is a
represents a promising target. Inspired by the synergistic effects monomeric heme-containing enzyme which catalyzes the
between
IDO1
inhibitors
and
traditional
antitumor degradation of L-tryptophan (Trp) in the initial rate-limiting
chemotherapeutics, the first orally active dual IDO1 and DNA step of the kynurenine (Kyn) pathway.3, 4 The depletion of Trp
targeting agents were designed by the pharmacophore fusion can impede T lymphocytes proliferation and increase their
strategy. The bifunctional hybrids exhibited enhanced IDO1 susceptibility to apoptosis.5, 6 Subsequently, accumulation of
enzyme inhibitory activity and in vitro cytotoxicity as compared to toxicity metabolites, including Kyn, kynurenic acid, 3-hydroxy-
IDO1 inhibitor 1-methyl-tryptophan and DNA alkylating agent kynurenine, and quinolinic acid, can also increase the
melphalan. In a murine LLC tumor model, the dual targeting apoptosis of both T helper 1 (TH1) lymphocytes and natural
agents demonstrated excellent antitumor efficacy, highlighting killer (NK) cells.7, 8 Ultimately, the combined effects of the two
the advantages of this novel design strategy to improve the mechanisms result in cancer immune escape in tumor
efficacy of small molecule cancer immunotherapy.
microenvironment.
Numerous studies indicated that low IDO1 activity was
observed in most tissues.9 In contrast, increased IDO1
expression was found in various cancer types, such as
Immune checkpoint therapy has become a new weapon for
clinical treatment of cancer.1 The approach does not aim to
attack particular targets in the tumor cells, but rather to affect
T cells to enhance antitumor immune responses.1 Currently,
there are a number of immune checkpoint inhibitors under
clinical investigation, such as programmed death (PD-1),
cytotoxic T-lymphocyte antigen CTLA, T-cell immunoglobulin
and mucin 3 domain (TIM3), and indoleamine 2,3-dioxygenase
1 (IDO1). Three immune checkpoint antibodies (ipilimumab,
pembrolizumab and nivolumab) have been approved for the
treatment of melanoma and achieved great success in clinic.1, 2
As compared to antibodies, the small molecule
colorectal cancer, pancreatic cancer, non-small cell lung cancer
and glioblastoma.4,
10
Moreover, up-regulation of IDO1 in
tumor cells is correlated with poor prognosis and reduced
survival.11-13 Thus, IDO1 has attracted great interests as a
potential cancer immunotherapy target and a number of IDO1
inhibitors have been reported.14-17 Five of them, namely
indoximod (D-1-MT,
1), INCB024360 (2), PF-06840003 (3),
GDC-0919 (structure not disclosed) and F001287 (structure not
disclosed), are in the stage of clinical trials (Fig. 1). The racemic
1-methyl-tryptophan
(4, 1-MT) was first identified as a
competitive IDO1 inhibitor in 1991.18 Interestingly, the R-
enantiomer (D-1-MT) displayed less IDO1 inhibitory activity
but higher in vivo antitumor activity than the S-enantiomer (L-
1-MT).19 Although the detailed mechanisms of D-1-MT are still
poorly understood, it is now evaluated as a clinical candidate
in combination with traditional cancer chemotherapies.20
H
O
N
OH
COOH
NH2
H
O
N
O
O
N
H
S
F
N
H2N
N
H
N
F
N
N
N
O
Br
H
2
INCB024360
1 Indoximod
3 PF-06840003
Fig. 1 Chemical structures of IDO1 inhibitors in clinical trials.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 6
View Article Online
DOI: 10.1039/C7OB02529G
COMMUNICATION
Journal Name
From a mechanistic point of view, IDO1 inhibitors can kill
cancer cells by restoring immune recognition and enhancing T
cell immune responses.21 As a single agent, IDO1 inhibitors
generally exhibited modest antitumor activities in preclinical
studies.4,
22
Therefore, the combination of IDO1 inhibitor-
based immunomodulation with chemotherapy, radiotherapy
and/or immunotherapy, may be a promising strategy against
an array of human cancers.23,
24
Numerous studies have
suggested that the efficacy of traditional cancer
chemotherapies were improved by concomitant
administration of an IDO1 inhibitor.22,
In mouse tumor
25
Fig. 2 Design of dual IDO1- and DNA- targeting agents.
models, 1-MT displayed remarkable synergism with various
chemotherapeutic (e.g. platinum compounds, taxane
derivatives and cyclophosphamide) without increased
toxicity.22, 26
The synthetic route of hybrid compounds 17 and 18 is
outlined in Scheme 1. Starting from commercially available 1-
and 2-nitro-1H-indole 6a and 6b, they were transformed to 5-
and 6-nitrogramine 7a and 7b by the Mannich reaction, which
reacted with diethyl acetamidomalonate in the presence of
NaOH and toluene to give 8a and 8b. Intermediates 10a and
The
alkylating
antitumor
agents,
including
cyclophosphamide, chlorambucil and melphalan, are widely
used as first line treatment for various cancers in clinic.27-29 On
the basis of the synergistic antitumor effects between IDO1
inhibitors and nitrogen mustards, we envisioned that dual
IDO1 and DNA targeting agents might take advantages of
immune checkpoint therapy and chemotherapy, which could
enhance the anticancer efficacy and reduce systemic toxicity.
10b were obtained by monomethylation of 8a and 8b
,
affording 9a and 9b, which were subsequently saponifed and
decarboxylated under the alkaline condition. Then, after
hydrolysis under the acid condition, 5- and 6-nitro-1-methyl-
tryptophan 11a and 11b were obtained. Followed by
esterification, Boc protection and catalytic reduction,
intermediates 14a and 14b were afforded, which were reacted
with excessive ethylene oxide, and subsequently treated with
POCl3 in the presence of CHCl3 to give nitrogen mustards 16a
and 16b. Finally, deprotection and hydrolysis of 16a and 16b
with 2M HCl under the reflux condition gave the target
The anticancer ability and systemic toxicity of melphalan (5)
is ascribed to the extent of its interstrand cross-linking with
DNA. In consideration of low toxicity of aromatic nitrogen
mustard, we introduced the alkylating pharmacophore N,N-
bis(2-chloroethyl)-amine to the C1- or C2- position of 1-MT (4)
using the pharmacophore fusion strategy (Fig. 2). As a result,
two novel hybrids of 1-MT-bearing nitrogen mustards were
designed and synthesized, which showed potent in vivo
antitumor activity.
compounds 17 and 18
.
Scheme 1. Synthesis of compounds 17 and 18a
O
OH
COOC2H5
NHCOCH3
COOC2H5
COOC2H5
NHCOCH3
COOC2H5
R1
R1
R2
R1
R1
R2
C
R1
R2
N
C
O
d
a
b
c
HN
R2
R2
N
N
N
H
N
N
H
H
7a: R1 = NO2, R2 = H
10a: R1 = NO2, R2 = H
6a: R1 = NO2, R2 = H
8a: R1 = NO2, R2 = H
9a: R1 = NO2, R2 = H
7b
10b
: R1 = H, R2 = NO2
6b
: R1 = H, R2 = NO2
: R1 = H, R2 = NO2
8b
9b
: R1 = H, R2 = NO2
: R1 = H, R2 = NO2
O
O
i
O
O
O
O
R1
OH
f
R1
R2
O
R1
R1
h
g
e
HN
Boc
HN Boc
NH2
NH2
R2
R2
R2
N
N
N
N
13a: R1 = NO2, R2 = H
14a: R1 = NH2, R2 = H
14b
11a: R1 = NO2, R2 = H
12a: R1 = NO2, R2 = H
12b
: R1 = H, R2 = NO2
13b
: R1 = H, R2 = NO2
: R1 = H, R2 = NH2
11b
: R1 = H, R2 = NO2
O
O
O
O
OH
O
R1
R1
R1
j
k
NH2
HN Boc
HN Boc
R2
R2
R2
N
N
N
15a: R1 = N(CH2CH2OH)2, R2 = H
15b
16a: R1 = N(CH2CH2Cl)2, R2 = H
16b
17: R1 = N(CH2CH2Cl)2, R2 = H
18
: R1 = H, R2 = N(CH2CH2Cl)2
: R1 = H, R2 = N(CH2CH2OH)2
: R1 = H, R2 = N(CH2CH2Cl)2
aReagents and Conditions: (a) Dimethylamine, formaldehyde, AcOH, 30 min, 0 oC; 3 d, rt; 15% aq. NaOH solution, 0 oC, yield 73%;
(b) Diethyl acetamidomalonate, NaOH, PhMe, 110 C, 10 h, yield 64%; (c) MeI, NaH, DMF, rt, overnight, yield 77%; (d) 4% aq.
o
o
NaOH solution, EtOH, N2, 76 C, 6 h; (e) 6M aq. HCl solution, reflux, 10 h, two steps yield 76%; (f) SOCl2, EtOH, reflux, 6 h, yield
93%; (g) (Boc)2O, Et3N, DCM, rt, overnight, yield 95%; (h) Pd/C, H2, Ethyl acetate, rt, overnight; (i) Ethylene oxide, MeOH, rt,
overnight, two steps yield 95%; (j) POCl3, CHCl3, 60 oC, 3 h; (k) 2M aq. HCl solution, reflux, 2.5 h, two steps yield 73-80%.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Please do not adjust margins
Organic & Biomolecular Chemistry
Page 3 of 6
View Article Online
DOI: 10.1039/C7OB02529G
Journal Name
COMMUNICATION
Initially, we investigated their inhibitory efficacy against apoptotic cells were 38.3% (30 μM for 17), 55.6% (50 μM for
purified IDO1 enzyme (Table 1). Compound was a poorly 17), 39.0% (80 μM for 18) and 48.9% (100 μM for 18),
4
active IDO1 inhibitor with an IC50 value of 667 μM. respectively.
Interestingly, after the introduction of the nitrogen mustard
substitution on the indole C5- or C6- position, the IDO1
inhibitory activity was significantly improved. Compounds 17
(IC50 = 14 μM) and 18 (IC50 = 227 μM) was 47.6–fold and 2.7–
fold more active than compound 4, respectively.
Furthermore, the hybrids as well as melphalan and 1-MT
were screened for in vitro cytotoxicity against three cancer cell
lines (LLC lewis lung cancer, CT-26 mouse colon cancer, and
HCT-116 human colon cancer) with a constitutive IDO1
expression by the standard CCK8 assay. As shown in Table 1
,
compound 4 was nontoxic to the three cell lines even at a
concentration of 500 μM. For LLC and HCT-116 cell lines,
compounds 17 and 18 possessed stronger inhibitory efficacy
than compound 5. Compound 17 exhibited the most potent
antiproliferative activity against all the cell lines with the IC50
values ranging from 23.7 to 47.2 μM. Thus, the nitrogen
mustard substitution on 1-MT was beneficial for enhancing
both IDO1 inhibitory activity and in vitro cytotoxicity.
Fig. 3 Effects of compounds 17 and 18 on cell cycle (A) and cell
Table 1. IDO1 inhibitory activities and in vitro antitumor
activities of compounds 17 and 18 (IC50, μM)a
apoptosis (B).
Compds
rhIDO-1
667 ± 34
NT
LLC
>500
CT-26
>500
HCT116
>500
To investigate whether dual IDO1 and DNA targeting agents
were effective in vivo, immunocompetent mice bearing IDO1-
expressing LLC tumors were treated with 1-MT-coupled
nitrogen mustards. The model was widely used to evaluate the
antitumor efficacy of 1-MT.30 Female C57BL/C mice were
randomized into five groups (n = 6), and orally received the
vehicle (control), compound 17 (20 mg/kg, qd), compound 18
4
5
>100
21.7 ± 3.7
37.5 ± 5.1
88.5 ± 7.4
>100
17
14 ± 1.3
23.7 ± 3.6
47.2 ± 4.6
18
227 ± 23 63.9 ± 6.2
73.5 ± 8.3
aIC50 values are the mean of at least three independent assays,
presented as mean ± SD.
(20 mg/kg, qd), compound
4 (400 mg/kg, bid) or compound 5
(20 mg/kg, qd) treatments. As shown in Fig. 4A, both
compounds 17 and 18 demonstrated excellent antitumor
efficacy with tumor growth inhibition (TGI) values of 63.8%
and 73.5%, respectively, which was significantly more potent
DNA damage induced by nitrogen mustards is known to
arrest the cell cycle progression mainly at the G2/M phase. To
assess whether the suppression of LLC cells growth by
compounds 17 and 18 is caused by a cell-cycle effect, DNA cell
cycle analysis was performed (Fig. 3). After treating with
compounds 17 (10 μM) and 18 (30 μM) for 24 h, the
percentage of cells in G2 fraction increased from 7.24% to
75.37% and 79.02%, respectively, with a concomitant decrease
of cells in other phases of the cell cycle.
than compound 4 (TGI = 24.2%) and compound 5 (TGI =36.5%).
Moreover, compounds 17 and 18 exhibited potent antitumor
activity without significant effects on the body weight (Fig. 4B).
Also it should be noted that compound 18 showed better in
vivo antitumor potency than compound 17, although it was
less active in vitro, indicating that they might have different
pharmacokinetic properties.
The induction of apoptosis by the two compounds was
further evaluated using an annexin V-FITC/propidium iodide
(PI) binding assay. The percentages of apoptotic LLC cells were
determined by flow cytometry. As shown in Fig. 3, compounds
17 and 18 induced LLC cells apoptosis in a concentration-
dependent manner. After exposure to the various
concentrations of 17 and 18 for 24 h, the percentages of
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins