Synthesis and in vivo antitumor evaluation of an orally active potent phosphonamidate derivative targeting IDO1/IDO2/TDO

Accepted Manuscript

Synthesis and in vivo antitumor evaluation of an orally active potent phospho-

namidate derivative targeting IDO1/IDO2/TDO

Xi Feng, Pei Shen, Yinuo Wang, Zhiyu Li, Jinlei Bian

PII:

S0006-2952(19)30266-7

https://doi.org/10.1016/j.bcp.2019.07.011

BCP 13582

DOI:

Reference:

Biochemical Pharmacology

To appear in:

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Accepted Date:

18 May 2019

10 July 2019

Please cite this article as: X. Feng, P. Shen, Y. Wang, Z. Li, J. Bian, Synthesis and in vivo antitumor evaluation of

an orally active potent phosphonamidate derivative targeting IDO1/IDO2/TDO, Biochemical Pharmacology (2019),

doi: https://doi.org/10.1016/j.bcp.2019.07.011

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Synthesis and in vivo antitumor evaluation of an

orally active potent phosphonamidate derivative

targeting IDO1/IDO2/TDO

Xi Feng,^aPei Shen,^aYinuo Wang,^bZhiyu Li^*,aand Jinlei Bian^*,a

^aJiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, School of

Pharmacy, China Pharmaceutical University, Nanjing 210009, China

^bDepartment of Biology, University of California San Diego, La Jolla, California 92093, United States

KEYWORDS: IDO1 inhibitors, Trp-Kyn, Phosphonamidate, epacadostat, NSCLC

ABSTRACT:

Targeting Trp-Kyn pathways has been identified as an attractive approach for the cancer

immunotherapies. In this study, a novel phosphonamidate containing compound was designed,

synthesized and evaluated for its inhibitory activity against key dioxygenases in Trp-Kyn pathway,

including IDO1, IDO2 and TDO. This compound showed potent IDO1 inhibitory activity with an IC₅₀

value of 94 nM in an enzymatic assay and 12.6 nM in HeLa cells. In addition, this compound showed

promising IDO2 inhibition and TDO inhibition with IC₅₀values of 310 nM and 2.6 μM, respectively,

in enzyme assay. Based on the promising enzyme inhibitory activity toward IDO/TDO, compound

F04 was evaluated of its antitumor effects in two tumor models. Further evaluation of mechanism

demonstrated compound F04 with the remarkable capacity of reducing kynurenine level in

plasma/TME and restoring anti-tumor immune response. F04 could be further developed as a potential

immunotherapeutic agent combined with immune checkpoint inhibitors or chemotherapeutic drugs

for cancer treatment.

1. Introduction

L-tryptophan (L-Trp) is the least abundant of the essential amino acids but is essential for

maintaining several cellular functions. L-Trp could be metabolized through four mechanisms,

including decarboxylation to tryptamine, protein synthesis, the serotonin pathway and the kynurenine

pathway (KP) [1,2]. Among them, the KP is responsible for the metabolism of approximately 95% of

all mammalian dietary tryptophan [3].

The heme-containing enzymes, indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-

dioxygenase 2 (IDO2) and tryptophan 2,3-dioxygenase (TDO) are cytosolic heme-containing

enzymes that catalyze the initial and rate-limiting step of the L-tryptophan (L-Trp) catabolism through

KP [4,5]. These heme-containing enzymes negatively modify cellular immune response by a

molecular mechanism of depleting local tryptophan storages and producing immunological active KP

metabolites [6,7]. Among the three enzymes, IDO1 has been studied for over 25 years which has been

proved to be overexpressed in majority cancers [5]. High IDO1 expression is usually associated with

poor prognosis in a variety of cancer types [8,9]. Increasing evidence revealed that IDO1 contribute

to the arising of resistance against immune checkpoint blockade therapies [10,11], and

correspondingly, the combination of checkpoint inhibitors with IDO1 inhibition offers an advanced

strategy in cancer immunotherapy and showed efficiency in clinical trials [12–14]. Thus, targeting the

IDO1 pathway is an attractive approach for cancer immunotherapy [15]. Indoleamine 2,3-dioxygenase

2 (IDO2), comparing to IDO1, is known as the other isoform of IDO which shares 43% sequence

identity and functions with distinct biochemical features [16,17]. Although been less thoroughly

studied and restrictively expressed in both normal or tumor tissues, IDO2 has been found to be a

contributor to IDO1-mediated immune tolerance [18]. As for intrahepatic enzyme tryptophan 2,3-

dioxygenase (TDO), it is strongly expressed in hepatocarcinoma and catalyzes the same reaction as

IDO in rate-limiting manner and overexpressed in some tumors as a mean of immune escape [19].

1

Recently, great attention has been paid on IDO1 inhibitors development both in academia and

pharmaceutical industry. Especially for the recent negative outcome of an ECHO-301 phase 3 trial

with selective IDO1 inhibitor epacadostat (INCB24360), many researchers were frustrated with the

result. However, the failure may relate to inapplicable pharmacodynamical index or mismatched drug

combination strategy [20]. There are still 35 clinical trials in the active state for several IDO1

inhibitors, indicated that IDO1 related therapy will continue to be an exciting area [21]. In addition,

the failure of ECHO-301 phase 3 trial may due to the highly selective toward IDO1 than TDO and

IDO2 [20]. Thus, developing combo or pan IDO1/IDO2/TDO inhibitors may broaden the impact in

cancer treatment and overcome the deficiency in clinical trials of IDO1 selective inhibitors [22,23].

Our group has been focused on the discovery of potent IDO1 inhibitors for several years. In this

work, we would report a novel IDO1/IDO2/TDO inhibitor F04 containing a phosphonamidate subunit.

Our objective was to synthesize potent inhibitor targeting key dioxygenases in Trp-Kyn pathway.

Interestingly, the compound showed potent IDO1 inhibitory activity as well as moderate IDO2/TDO

inhibitory activity by involving in the rarely applied functional group. In addition, F04 showed an

improved drug-like property than clinical candidate epacadostat. The further in vivo investigation

demonstrated that F04 could remarkably suppress tumor progression both in immunocompetent

C57BL6 mice and lung metastasis of Lewis cells model, which was more potent than epacadostat.

Furthermore, the mechanism study showed that tumors from F04-treated mice at 60 mg/kg exhibited

a markedly reduced Kyn/Trp ratio comparing to epacadostat. Therefore, F04 was deserved further

optimization with the aim to obtain antitumor agents through targeting Trp-Kyn pathway.

2

Phosphonamidate

O

H

O

H

O

N

S

N

P

N

NH₂

O

H

N

Br

N

OH

F

Epacadostat, INCB24360

Incyte, 2012

F04

Phase III Clinical Trials

Fig. 1. Structure of candidate epacadostat and the structure of compound F04 contained in the rectangle with

broken line was modified from epacadostat with the phosphonamidate moiety.

2. Materials and methods

2.1. General experimental methods

Unless otherwise noted, all reagents were obtained from commercial suppliers and used without

further purification. NMR spectra were measured on a 300 MHz Bruker unit (300 MHz for ¹H NMR,

13

31

75 MHz for C NMR, 121 MHz for P NMR) using CDCl₃or DMSO-d₆as the solvent at room

temperature. MS spectra were recorded on a LC/MSD TOF HR-MS Spectrum. Melting points were

determined on a Mel-TEMP II melting point apparatus without correction.

2.1.1. Synthesis of phosphonchloride 10

Phosphonchloride 10 was prepared according to the method described by McAnoy et al [24].

Dimethyl methylphosphonate (20 mmol) was treated with oxalyl chloride (30 mmol, 1.5 equiv) and

additional drops of N,N-dimethylformamide were added as catalyst. The desired product was obtained

as garnet liquid (1.2 g, 49%) and used for the next step without further purification.

3

2.1.2. Synthesis of 4-amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (5)

Intermediate 5 was prepared strictly according to literature procedure described by Tao et al [25].

(a) Commercially available malononitrile (9.9 g, 150 mmol), sodium nitrite (20.7 g, 300 mmol),

hydroxylamine hydrochloride (23 g, 340 mmol) were employed and 4-amino-N′-hydroxy-1,2,5-

oxadiazole-3-carboximidamide 2 was generated in a one-pot reaction as while solid (14.9 g, 68%). ¹H

NMR (300 MHz, DMSO-d₆): δ = 10.46 (s, 1H), 6.24 (s, 2H), 6.02 (s, 2H) ppm. (b) Next, intermidiate

2 (4.2 g, 29.5 mmol) was mixed with sodium chloride (5.2 g, 88.5 mmol), water (59 mL), acetic acid

(29 mL) and 6 N hydrochloric acid solution (14.6 mL, 88.5 mmol) then sodium nitride (2.0 g, 29

mmol) was added to the resulting suspension by drops under 0 °C. The target compound 3 was

obtained as an off-white solid (2.5 g, 53%). ¹H NMR (300 MHz, DMSO-d₆): δ = 13.39 (s, 1H), 6.29

(s, 2H). (c) 4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride 3 (3.24 g, 20 mmol) was

mixed with water (70 mL) and 3-bromo-4-fluoroaniline (4.18 g, 22 mol, 1.1 equiv). At 60 °C, a

solution of sodium bicarbonate (3.36 g, 40 mmol, 2 equiv) in water (15 mL) was added to the mixture

by drops then resulting mixture was heated to reflux. After purification, compound 4 was obtained as

a white solid (5.75 g, 91%). ¹H NMR (300 MHz, DMSO-d₆): δ = 11.46 (s, 1H), 8.89 (s, 1H), 6.99 (t,

J = 8.8 Hz, 1H), 6.81 (dd, J₁= 6.0 Hz, J₂= 2.7 Hz, 1H), 6.56–6.51 (m, 1H), 6.28 (s, 2H) ppm. (d)

Last, 4 (5.0 g, 15.8 mmol) was treated with N,N′-carbonyldiimidazole (5.12 g, 31.6 mmol, 2 equiv) at

room temperature to give 3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-

oxadiazol-5(4H)-one 5 as white solid (4.80 g, 89%). ¹H NMR (300 MHz, DMSO-d₆): δ = 8.10 (dd,

J₁= 6.2 Hz, J₂= 2.4 Hz, 1H), 7.74–7.69 (m,1H), 7.61 (t, J = 8.6 Hz, 1H), 6.60 (s, 2H) ppm.

4

2.1.3. Synthesis of 4-(3-bromo-4-fluorophenyl)-3-(4-nitro-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-

5(4H)-one (6)

5 (4.5 g, 13.1 mmol) was mixed with 30% hydrogen peroxide (30 mL) and trifluoroacetic acid (90

mL) then the resulting mixture was stirred at 45 °C overnight. After transformation, 4-(3-bromo-4-

fluorophenyl)-3-(4-nitro-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one 6 was obtained as a yellow

1

solid (2.19 g, 45%). H NMR (300 MHz, DMSO-d₆): δ = 8.06 (dd, J₁= 6.0 Hz, J₂= 2.3 Hz, 1H),

7.69–7.64 (m, 1H), 7.60 (t, J = 8.6 Hz, 1H) ppm.

2.1.4. Synthesis of 3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-

1,2,4-oxadiazol-5(4H)-one hydrochloride (8).

6 (1.86 g, 5 mmol) and N-Boc-1,3-propanediamine (1.74 g, 10 mmol, 2equiv) was dissolved in

tetrahydrofuran (50 mL) followed by the addition of aqueous 2N NaOH (5 mL, 2 equiv) and the

reaction mixture was stirred at room temperature for 2 h. After purification by column

chromatography on silica gel, tert-butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-

1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)propyl]carbamate 7 was obtained as a pale-yellow

solid (1.37 g, 55%). ¹H NMR (300 MHz, DMSO-d₆): δ = 8.09–8.07 (m, 1H), 7.72–7.70 (m, 1H), 7.60

(t, J = 8.4 Hz, 1H), 6.93 (s, 1H), 6.51 (s, 1H), 3.31 (s, 2H), 3.08 (s, 2H), 1.85 (s, 2H), 1.37 (s, 9H)

ppm. Next, 7 (1.2 g, 2.40 mmol) was treated with hydrogen chloride (10 mL, 4.0 M in ethyl acetate)

at room temperature to afford 3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-

1

fluorophenyl)-1,2,4-oxadiazol-5(4H)-one hydrochloride 8 as an off-white solid (0.93 g, 89%). H

NMR (300 MHz, DMSO-d₆): δ = 8.12 (dd, J₁= 6.0 Hz, J₂= 2.2 Hz, 1H), 8.04 (sbr, 3H), 7.77–7.72

(m, 1H), 7.61 (t, J = 8.6 Hz, 1H), 6.75 (s, 1H), 3.35–3.33 (m, 2H), 2.83–2.81 (m, 2H), 1.91–1.87 (m,

2H) ppm.

5

2.1.5. Synthesis of methyl N-[3-({4-[N-(3-bromo-4-fluorophenyl)-N′-hydroxycarbamimidoyl]-1,2,5-

oxadiazol-3-yl}amino)propyl]-P-methylphosphonamidate (F04)

Hydrochloride 8 (0.44 g, 1 mmol) was treated with phosphonochloridate 10 (0.38 g, 3 mmol, 3

equiv) at 0 °C in dry dichloromethane (30 mL) then triethylamine (0.30g, 3 mmol, 3 equiv) was added.

The reaction mixture was allowed warm to ambient temperature and stirred for 1 h. 11 was then

obtained as a pale-yellow solid without further purification and used for the next step immediately.

Crude 11 was dissolved in tetrahydrofuran (20 mL) and treated with 2 N NaOH (4 mL). Deprotection

was performed at room temperature and target compound F04 was ultimately obtained after

purification by column chromatography on silica gel as a pale-yellow solid (0.17 g, 37% for two

1

steps). mp = 103–105 °C. ³¹P NMR (121 MHz, DMSO-d₆): δ = 34.6 ppm. H NMR (300 MHz,

DMSO-d₆): δ = 11.58 (s, 1H), 8.91 (s, 1H), 7.19 (t, J = 8.7 Hz, 1H), 7.11 (d, J = 3.3 Hz, 1H), 6.78–

6.77 (m, 1H), 6.39 (s, 1H), 4.78–4.73 (m, 1H), 3.35 (d, J = 11.1 Hz, 3H), 3.27–3.26 (d, J = 5.7 Hz,

13

2H), 2.86–2.83 (m, 2H), 1.71–1.69 (m, 2H), 1.35 (d, J = 16.3 Hz, 3H) ppm. C NMR (75 MHz,

DMSO-d₆): δ = 155.5, 152.1, 139.9, 139.1, 137.9, 124.7, 121.4 (J = 6.9 Hz), 116.0 (J = 23.2 Hz),

107.1 (J = 22.0 Hz), 49.7 (J = 6.3 Hz), 41.7, 37.7, 30.1 (J = 5.6 Hz), 12.0 (J = 129.2 Hz) ppm. HRMS

(ESI⁺): cacld for C₁₄H₁₉BrFN₆O₄P (M + H)⁺, 465.0446; found, 465.0444.

2.2. Materials

2.2.1. Cell culture and reagents.

Lewis lung carcinoma cells was purchased from Cell Bank of Shanghai Institute of Biochemistry

and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and grown in DMEM (Gibco,

Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Grand

6

Island, NY) and 1% penicillin/streptomycin (all from Invitrogen). Ki67 was obtained from R&D

System (MN, USA). Epacadostat was purchased from CSNpharm (CSN13892, USA).

2.2.2. Mice.

Eight-week-old male C57BL/6 mice, weighing 18–22 g, were supplied by Model Animal Genetics

Research Center of Nanjing University (Nanjing, China). Mice were maintained under standard

specific-pathogen-free (SPF) conditions (23 ± 2°C and 12-h light:dark cycle with a relative humidity

of 45 ± 10%) and all research involving animals strictly complied with protocols approved by the

Animal Welfare and Ethics Committee (AWEC, China Pharmaceutical University). In the tumor

transplanting experiments, the mice were inoculated subcutaneously with 6 × 10⁵Lewis tumor cells.

Mice bearing Lewis tumor were randomly divided into 5 groups. F04 at doses of 15 mg/kg, 30 mg/kg

and 60 mg/kg or epacadostat at 60 mg/kg, dissolved in 0.5% carboxymethylcellulose sodium (CMC-

Na), were administered i.g. daily until termination of the experiment, and in the control group, the

same volume of CMC-Na was gavaged as vehicle control. Tumor volume was measured using a

digital vernier caliper every 3 days post tumor inoculation using the formula V = π × length × width²/6.

Tumors were harvested on day 30 for further use. In the Tumor metastasis experiments, Lewis tumor

cells were prepared as a suspension of 10⁶cells in 150 µL saline and injected into the lateral tail vein

using a 29-gauge needle under sterile conditions. Mice were weighed every 3 days and lung metastasis

was determined by counting the total number of metastatic nodules in each lung.

2.3. Cell-based IDO1 activity assay.

Performed according to the literature protocol [26]. HeLa cells were seeded in 96-well culture plates

at a density of 5 × 10⁴per well. The following day, after growth media was aspirated, human hIFN-γ

(20 ng/well, Sigma-Aldrich SRP3058-100UG), L-Trp (15 μg/mL) and serial dilutions of test

7

compounds were added to the 96-well plates (200 μL final volume per well). After incubation at 37

°C for 48 h, 140 μL of the supernatant was transferred to a new 96-well plate and mixed with 10 μL

of 6.1 N trichloroacetic acid for 30 min at 50 °C to hydrolyze N-formlylkynurenine. Then the mixture

was cooled at 0 °C and centrifuged at 4000 rpm for 20 min. Subsequently, another 100 μL of the

supernatant was taken and added to a new 96-well plate and 100 μL of 2% (w/v) p-

dimethylaminobenzaldehyde (p-DMAB, Ehrlich's reagent) in acetic acid was added. The amount of

generated kynurenine was determined by the absorbance at 480 nm. Graphs of inhibition curves with

IC₅₀values were generated using Prism GraphPad. Epacadostat was purchased from CSNpharm (Cat.

CSN13892, USA).

2.4. hIDO1, hIDO2 and hTDO enzymatic inhibition assay.

Human IDO1 with an N-terminal His Tag was expressed in E. coli and purified by Ni-NTA Agarose

(Invitrogen R90101). The assay was performed by UV absorption using hIDO1 and L-Trp as

substrates. The UV absorption signal at 321 nm is correlated with the amount of generated N-

formylkynurenine and determined by EnSpire. The initial reaction rates were recorded by

continuously following the absorbance increase at 321 nm as a consequence of the formation of N-

formylkynurenine. The percent inhibition at individual concentrations was calculated by the slopes

and the IC₅₀values were generated using nonlinear regression (Prism GraphPad). hIDO2 and hTDO

were purchased from commercial source (BPS Bioscience, Cat. #71194-2 and #71195) and the

inhibition assay was detailed described in ref 27.

2.5. Caco-2 assay.

Permeability was studied with the Caco-2 monolayer cultured for 4-5 days (>500 cm²in each well)

and added the HBSS buffer containing 10 mM HEPES and test compounds. The test sample was taken

8

from both apical and basolateral chambers at 0 and 90 min after incubation at 37 °C and analyzed by

LC-MS/MS. The calculation formula of efflux ratio is P_app(B→A)/ P_app(A→B).

2.6. Histological analyses

For histology, lung tissues were fixed in 4% paraformaldehyde (PFA) and paraffin-embedded

sectioned were deparaffined and rehydrated, followed by hematoxylin and eosin (H&E) staining.

2.7. FACS analysis

Transplanted tumors were dissected into pieces and digested in 1 × HBSS buffer including 2% FBS,

1 mg/ml collagenase I (Sigma-Aldrich) and 0.5 mg/ml dispase (Invitrogen), followed by further

digestion in 10 μg/mL DNase (Invitrogen) for 45 min, and in 0.64% ammonium chloride

(STEMCELL Technologies) for 5 min at 37 ℃. Cells were filtered through a 70-μm cell strainer (BD

Biosciences), and resuspended in 1 × HBSS buffer containing 2% FBS, followed by a gradient

centrifugation in Ficoll-Pague (Sigma-Aldrich). Purified lymphocytes were stained using a fixation

and permeabilization kit (eBioscience) and analyzed by FACS analysis to detect expressions of CD45,

CD4, CD8, Foxp3 (Biolegend) and Ki67 (Cell Signaling Technology).

2.8. Tryptophan/kynurenine measurement

In IDO1 enzyme activity measurement, blood and Tumors from vehicle-, F04- and epacadostat-

treated mice were collected. Tumors were homogenized in 3 volumes of saline with 0.1% formic acid.

Following protein-precipitation extraction with methanol, plasmas and tumor homogenates were

collected and 20 μL of the supernatants were subjected to LC/MS/MS analysis. Aqueous standards

were prepared for adjustment of endogenous tryptophan and kynurenine levels.

9

2.9. Immunofluorescence

Immunofluorescence was performed on paraffin-embedded colonic tissue sections. The sections

were deparaffinized, rehydrated and washed in 1% PBS-Tween. Then they were treated with 3%

hydrogen peroxide, blocked with 10% goat serum and incubated with Ki67 and CD8 primary antibody

(Abcam) overnight at 4 °C, followed by incubation with goat anti-rat Alexa 488, goat anti-rabbit Alexa

594 secondary antibodies (all from Invitrogen) for 1 h at room temperature. The slides were stained

with DAPI. Images were acquired by confocal laser-scanning microscope (Olympus, Lake Success,

NY). Settings for image acquisition were identical for control and experimental tissues. Immunoblots

with the tissue of interest were used to determine whether the primary antibody can bind to a single

protein of the correct molecular weight.

2.10. Statistical analysis

Most results are presented as the mean ± SD. Data sets consisting of more than 2 groups were

analyzed by analysis of variance (ANOVA) with Tukey-Kramer HSD post test for multiple

comparisons if significance was determined. For data not conforming to a normal distribution,

Kruskal-Wallis test was used for multiple comparisons. P value that was less than 0.05 was considered

statistically significant for all data sets. All statistical analysis was performed using GraphPad Prism

software.

2.11. Water solubility assay

Compound (1 mg) was added to 1 mL of water and the result mixture was shaked at 25 °C for 30 h.

The suspensions were filtered and determined by HPLC in 254 nm. Quantification of each compound

was calibrated by comparison with linear standard curve established from standard solution in

methanol. Determination was performed in duplicate.

10

3. Results

3.1. Chemistry

The synthetic pathway for phosphonamidates F04 is depicted in Fig. 2. Intermediate 2 was

synthesized in a one-pot reaction from commercially available malononitrile (1, Fig. 2) in a yield of

68%. 2 was then treated with sodium nitrite in water to afford 3 in 53% yield. 4 was obtained through

a nucleophilic substitution reaction with substituted aniline and subsequently transformed to amino-

furazan 5 with the addition of N,N′-carbonyldiimidazole in adorable yields of 91% and 89%

respectively. Next, the amino group of 5 was oxidized by hydrogen peroxide in trifluoroacetic acid to

afford nitro-furazan 6 in a moderate yield of 45%. Subsequently, 7 was obtained in good yield by

treating 6 with N-Boc-1,3-propanediamine and then deprotected in hydrogen chloride to give 8 as a

hydrochloride salt. Amidation of 8 with phosphonchloride 10 generated intermediate 11 and an

additional deprotection was performed to give the target compound F04 in a yield of 37%.

3.2. F04 showed good activity toward IDO1, IDO2 and TDO

F04 was first evaluated on IDO1, IDO2 and TDO inhibition activity. The IDO1 inhibitory activity

was determined in IDO1 HeLa assay and IDO1 enzyme assay while IDO2/TDO inhibitory activity

were determined in enzyme assay. Clinical candidate epacadostat was selected as control. As shown

in Fig. 3, F04 was first evaluated for its IDO1 inhibitory activities in IDO1 HeLa assay and IDO1

enzyme assay. It was found that F04 showed equal potency in HeLa assay with IC₅₀of 12.6 nM and

enzyme IC₅₀of 94.5 nM comparing to the control epacadostat (HeLa IDO1 IC₅₀= 10.1 nM, hIDO1

IC₅₀= 72.2 nM). In addition, compound F04 displayed improved potency against IDO2 (IC₅₀= 310

nM) comparing to positive control epacadostat (IC₅₀= 0.71 μM) and moderate TDO enzyme IC₅₀of

2.6 μM, in spite of the evident diversity between two protein structures of IDO1 and TDO. It should

11

be noted that the control epacadostat showed low inhibitory activity toward TDO with IC₅₀> 10 μM.

In general, the new compound F04 showed pan inhibition of IDO1/IDO2/TDO comparing to the

clinical candidate epacadostat which confirmed as selective IDO1 inhibitors [4]. This characteristic

of compound F04 may plumb the full potential of blunting Trp catabolism which may be benefit for

the therapy in clinical.

H₂N

Br

F

(i) HCl, 0 ^o

C

OH

N

OH

N

OH

N

(ii) NH₂•HCl, rt

H

N

NaNO₃, NaCl, 0 ^o

53%

C

NaHCO₃, reflux

91%

Br

F

(iii) NaOH, reflux

CDI, rt

89%

NH₂

Cl

H₂N

NC

CN

68%

H₂N

N

O

N

O

1

2

3

4

O

H₂N

N

O

H

O

N

O

H₂O₂, 45 ^o

45%

C

NaOH, rt

55%

N

HCl, rt

89%

N

Br

F

H

N

H

N

O₂N

H₂N

Boc

F

N

O

N

Br

N

O

5

6

7

O

N

OH

O

H

N

, TEA, 0 ^oC

N

10

NaOH, rt

37%

N

HCl•

H₂N

H

N

H

N

H

N

P

F

P

N

F

O

N

O

N

O

Br

N

Br

8

11

F04

O

(COCl)₂, reflux

O

P

Cl

10

9

Fig. 2. Synthetic scheme for F04

12

Fig. 3. The inhibitory curves of compound F04 and epacadostat targeting IDO1-HeLa (A); hIDO1 (B); hIDO2 (C)

and TDO (D).

3.3. Physicochemical properties of compound F04

In an effort to identify potential drug-like compounds prior to the time-consuming and costly

development to optimize derivatives that may ultimately fail in in vivo experiments, then F04 was

employed to determine its physicochemical properties (Table 1). Introducing phosphonamidate group

comparing to sulfamide, F04 exhibited lower polarity with the support of reduced polar surface area

(PSA < 140 Å², F04 PSA = 129 Å²) [28] and favorable cLogP. Following this, moderated

permeabilities were confirmed as expected in Caco-2 assay (P_{app A-B}= 2.0 × 10⁻⁶cm/s) with no

significant efflux (efflux ratio = 1.10). In addition, F04 was characterized by improved water

solubility values (S = 0.18 mg/mL) than compound 1 (S = 0.13 mg/mL). Further, this compound F04

was in line with Lipinski’s rule-of-five (HBD < 5, HBA < 10, MW < 500) [29]. Subsequently, ligand

efficiency (LE) and lipophilic ligand efficiency (LLE) were calculated based on cell IC₅₀which were

13

instructive in drug candidate development[30] and result revealed that F04 displayed ideal values (LE

= 0.40, LLE = 5.3). Till now, findings aforementioned demonstrate that F04 displays good potential

as an oral drug.

Table 1

Calculated property of phonphonamidate derivative F04.

IDO1

IC₅₀

(nM)

S^b

HeLa IDO1

IC₅₀(nM)

Caco-2^a

(cm/s)

Cpds

PSA^c

cLogP

HBD^d

HBA^d

MW

LE^e

LLE^e

(mg/mL)

F04

12.6

10.0

94

72

2.0 (1.10) 0.18

3.1 (1.09) 0.13

129

163

2.5

1.5

4

6

10

11

465

438

0.40

0.44

5.3

6.5

epacadostat

^aCaco-2 values: P_app× (10)⁻⁶cm/s with efflux ratio in bracket. ^bAqueous solubility and each value was represented

d

as a mean from two trials. ^cPSA in Å²(calculated using ChemBioDraw Ultra 13.0). Hydrogen bond donor and

acceptor. ^eLigand efficiency (LE) and lipophilic ligand efficiency (LLE) calculated using HeLa pIC₅₀.

3.4. Molecular modeling.

Utilizing the crystal structure of IDO1 (PDB id: 5WN8), computational study was performed to

elucidate the possible binding mode of IDO1 with the representative compound F04 (Fig. 4A). The

compound oriented with the similar pose with the native ligand epacadostat (Fig. 4B) [31]. The

phosphonamidate group bound in pocket B and formed two strong hydrogen bonds with Arg231. In

addition, the NH at the oxadiazole could form a hydrogen bond with Gly262 (Fig. 4A). On the other

hand, we performed a in silico docking of F04 with a TDO protein structure (PDB id: 6A4I) which

was released recently (no IDO2 protein structure reported yet). As expected, as shown in Fig. 4C and

4D, compound F04 was well tolerated in TDO ligand-binding pocket comparing to epacadostat.

Additional hydrogen bonds were formed between phosphonamidate moiety and Arg144 with the help

of extended side chain which may be responsible for a higher affinity of F04 toward TDO than

14

epacadostat. The preliminary docking results may support the fact that the compound F04 was

excellent inhibitor of IDO1 and explain the improved inhibitory activity against TDO.

Fig. 4. The docking poses of compound F04 (A and C, shown in yellow) and epacadostat (B and D, shown in purple)

in IDO1 and TDO. The interaction mode was obtained through molecular docking (PDB id:5WN8 for IDO1 and

PDB id: 6A4I for TDO) and depicted using MOE 2018.01. The key residues are shown as cyan sticks and H-bonds

are shown as black dot lines.

3.5. F04 dose-dependently inhibited outgrowth of IDO1-expressing Lewis cells inoculated in

immunocompetent C57BL6 mice.

To investigate whether F04 could inhibit the outgrowth of transplanted syngeneic tumors in vivo,

immunocompetent C57BL6 mice were challenged with IDO1-expressing Lewis lung carcinoma cells

and treated with different doses (15/30/60 mg/kg) of F04 or epacadostat (60 mg/kg). F04 inhibited

15

tumor volume in a dose-dependent manner through reducing tumor volume by 32%, 50% and 73% at

15, 30 and 60 mg/kg, respectively (Fig. 5A and 5B). Interestingly, positive control epacadostat

treatment at 60 mg/kg reduced tumor volume by 68%, displaying a weaker antitumor efficacy as

comparing to F04 (60 mg/kg) (p > 0.05). Similarly, a significant decrease in tumor growth was

observed in F04- or epacadostat-treated mice comparing to vehicle-treated mice (Fig. 5C). Meanwhile,

body weight was markedly increased in mice that received both F04 (30 mg/kg and 60 mg/kg) and

epacadostat treatments comparing to mice received vehicle treatment (Fig. 5D). In the context of these

results, treatment with compound F04 resulted in a significant inhibitory effect on transplanted tumor

outgrowth.

Fig. 5. F04 dose-dependently inhibited outgrowth of IDO1-expressing Lewis cells inoculated in

immunocompetent C57BL6 mice. Mice were administered i.g. daily with the indicated compounds following s.c.

challenge with 6×10⁵Lewis tumor cells. (A) Representative photograph of excised Lewis tumors (n = 10 for each

treatment group). (B) Tumor mass in immunocompetent mice (n = 10 for each treatment group). (C) Tumor growth

in immunocompetent mice (n = 10 mice, each. The average tumor volume of Ctrl, F04 15/30/60 mg/kg, and

epacadostat 60 mg/kg are 1066, 729, 530, 312, and 359 mm³respectively). (D) Body weight of immunocompetent

mice for each treatment group. Statistical significance was evaluated by one-way ANOVA test (* P <0.05; ** P <

0.01; *** P < 0.001 vs Ctrl group; n.s., not significant).

16

3.6. F04 treatment reduced the Kyn/Trp ratio both in plasmas and tumors.

As the tryptophan degradation and kynurenine production are indicators of IDO1/TDO/IDO2

activity which can be reflected by the Kyn/Trp ratio, we sought to determine the Kyn/Trp ratio in mice

treated with compound F04. As shown in Fig. 6A and 6B, the kynurenine/tryptophan ratio in plasmas

and tumor tissues were reduced by F04 treatment in a dose-dependent manner, displaying that F04

effectively inhibited IDO1/TDO/IDO2 enzyme activity. Importantly, tumors from F04-treated mice

at 60 mg/kg exhibited a markedly reduced Kyn/Trp ratio comparing to epacadostat (p < 0.05).

3.7. F04 treatment increased accumulation and infiltration of T cells in transplanted syngeneic tumors.

Moreover, we observed a significantly increased number of tumor-infiltrating CD8 effector T cells

(CD8⁺CD45⁺) in mice treated with F04 as comparing to mice from Ctrl group (Fig. 6C). The

infiltration of proliferative CD8 effector T cells within the transplanted tumors was also increased by

F04 treatment, as measured by the expression of Ki67, a widely used cell proliferating marker (Fig.

6D). By contrast, tumors from F04-treated mice displayed a robustly reduced infiltration of regulatory

T cells (CD4⁺Foxp3⁺CD45⁺) comparing to vehicle-treated mice (Fig. 6E). A dramatic decrease in the

percentage of proliferative regulatory T cells was also detected within transplanted tumors in F04-

treated mice (Fig. 6F). Intratumoral ratios of CD4 effector T cells (CD4⁺Foxp3^–CD45⁺) to regulatory

T cells (CD4⁺Foxp3⁺CD45⁺) were significantly elevated in F04-treated mice as comparing to Ctrl

group (Fig. 6G). Additionally, a robustly elevated infiltration of proliferative CD8 effector T cells

(Ki67⁺CD8⁺) within transplanted tumors was confirmed with the immunofluorescence analysis in

F04-treated mice (Fig. 6H). Taken together, these results suggest that F04 treatment increased the

number of accumulation and infiltration of T cells in transplanted tumors and indicated that the pan

inhibitor of the Trp catabolizing enzymes may possess more advantage than the selective inhibitor.

17

18

Fig. 6. F04 treatment enhances infiltration and accumulation of T cells in transplanted syngeneic tumors. T

cells in transplanted tumors from the indicated groups were subjected to FACS analyses. (A) Ratio of tryptophan to

kynurenine concentration in plasmas from immunocompetent mice (n = 10). (B) Ratio of tryptophan to kynurenine

concentration in transplanted tumors from immunocompetent mice. (C) Percentage of CD8⁺effector T cells of total

CD45⁺cells (n = 10). (D) Percentage of CD8⁺effector T cells expressing Ki67 for the indicated mice (n = 10). (E)

Percentage of CD4⁺Foxp3⁺regulatory T cells of total CD45⁺cells for the indicated mice (n = 10). (F) Percentage

of CD4⁺Foxp3⁺regulatory T cells expressing Ki67 for the indicated mice (n = 10). (G) Ratio of CD4⁺Foxp3^-effector

T cells to CD4⁺Foxp3⁺regulatory T cells in tumors of indicated mice (n = 10). (H) Representative

immunofluorescent images (left panels) and percentage of CD8⁺T cells expressing Ki67 for tumors of indicated

mice (right panel; 1000 ~ 2000 cells were counted in 10 random fields of each slide). Statistical significance was

evaluated by One-way ANOVA test (* P< 0.05, ** P < 0.01 vs Ctrl; n.s., not significant).

3.8. F04 dose-dependently suppressed lung metastasis of Lewis cells

To further evaluate the in vivo antitumor activity of compound F04, lung metastasis of Lewis cells

model was employed. Immunocompetent C57BL6 mice were challenged with Lewis tumor cells via

the tail vein and treated with F04 or epacadostat. A marked increase in body weight was observed in

mice treated with F04 (30 mg/kg and 60 mg/kg) and epacadostat (Fig. 7A). Importantly, both F04-

and epacadostat-treated mice displayed significantly increased survival relative to vehicle-treated

mice (Fig. 7B). As shown in Fig. 7C and 7D, lung metastatic nodules of mice in Ctrl group receiving

pneumonectomy was detected, and F04 suppressed lung metastasis of Lewis tumor cells in a dose-

dependent manner. Similarly, H.E. staining suggested that the area of metastases was much smaller

in the lungs from F04-treated mice comparing to mice from Ctrl group. Interestingly, an average of

12 lung metastatic nodules per mice was observed in F04 (60 mg/kg) group and 21 was observed in

epacadostat group, implying that F04 exhibited a more potent inhibitory effect on lung metastasis of

Lewis cells than epacadostat at the same dose of 60 mg/kg (Fig. 7D).

19

Fig. 7. F04 dose-dependently suppressed lung metastasis of Lewis cells. Lewis tumor cells were injected into the

immunocompetent C57BL6 mice via the tail vein. (A) Body weight of immunocompetent mice for each treatment

group (on day 30, n = 7 for Ctrl group, n = 9 for F04-15 mg/kg, and n = 10 for other groups). (B) Kaplan-Meier

survival curves for metastasis tumor-bearing mice. (C) Representative images of lung metastatic nodules in the

indicated groups. Arrowheads denote the lung metastatic nodules stained by H&E. Scale bar = 50 μm. (D) The

corresponding statistical plots were presented (n = 7 for Ctrl group, n = 9 for F04-15 mg/kg, and n = 10 for other

groups). Statistical significance was evaluated by One-way ANOVA test (* P< 0.05, ** P < 0.01, *** P < 0.001 vs

Ctrl).

4. Discussion

In this work, a novel phosphonamidate derivative F04 of epacadostat was designed and synthesized.

This compound could be considered as bioisostere of sulfonamide-based IDO1 inhibitor. F04 has been

proved to be a pan IDO1/TDO/IDO2 inhibitor targeting Trp/Kyn pathway with potent IDO1 inhibitory

activity and moderate potency for IDO2/TDO. Although its inhibitory activity is slightly lower than

the best IDO1 inhibitors in the literature, its unexpected pan IDO1/TDO/IDO2 characteristics make

lower Kyn/Trp ratio in tumors which was worth for further studying. Indeed, the recent failure of the

IDO1-selective inhibitor epacadostat in the ECHO-301 Phase 3 trial in melanoma encouraged the

20

researchers to orient to discover pan inhibitors targeting Trp/Kyn which may be important to the

development of effective therapeutic inhibitors of tryptophan catabolism.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: bianjl@cpu.edu.cn (J. Bian).

*E-mail: zhiyuli@cpu.edu.cn (Z. Li).

Acknowledgements

We gratefully acknowledge financial support from the National Natural Science Foundation

of China (no. 81703347, 21672260, and 21372260), the National Natural Science Foundation

of Jiangsu Province of China (no. BK20170743 and BK20171393), and the “Double First-

class” University Project (CPU2018GY07).

ABBREVIATIONS USED

IDO1, indoleamine 2,3-dioxygenase 1; IDO2, indoleamine 2,3-dioxygenase 1; TDO, tryptophan

dioxygenase; -Trp, -tryptophan; KP, kynurenine pathway; NFK, N-formylkynurenine; AhR, aryl

L

hydrocarbon receptor; NK, natural killer; Treg, regulatory T cell; MDSC, myeloid-derived suppressor

cell; HTS, high-throughput screening; SAR, structure-activity relationship; CDI, N,N′-

carbonyldiimidazole; rt, room temperature; PSA, polar surface area; HBD, hydrogen bond donor;

HBA, hydrogen bond acceptor; LE, ligand efficiency; LLE, lipophilic ligand efficiency; SEM,

standard error of the mean; hIFN-γ, huaman interferon γ; p-DMAB, p-dimethylaminobenzaldehyde.

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Fig. 1. Structure of candidate epacadostat and the structure of compound F04 contained in the rectangle with

broken line was modified from epacadostat with the phosphonamidate moiety.

Fig. 2. Synthetic scheme for F04.