Discovery of phosphonamidate IDO1 inhibitors for the treatment of non-small cell lung cancer

Article abstract of DOI:10.1016/j.ejmech.2019.111629

Targeting indoleamine 2,3-dioxygenase 1 (IDO1) has been identified as an attractive approach for the development of cancer immunotherapy. In this study, a series of phosphonamidate ester containing compounds were designed, synthesized and evaluated for their inhibitory activities against IDO1. Among them, compounds 16, 17, and 26 with good IDO1 inhibitory (HeLa IDO1 IC₅₀ = 10–21 nM, hIDO1 IC₅₀ = 78–121 nM) activities were selected for further investigation and showed good physicochemical properties. Furthermore, based on comparable PK profile and excellent IDO2/TDO inhibitory potency, representative compound 16 was selected for further bio-evaluation and characterized with good efficacy in suppressing lung metastasis (77% inhibition rate) of Lewis cells in vivo. Thus, compound 16 could be a potential and efficacious agent for further evaluation.

Full text of DOI:10.1016/j.ejmech.2019.111629

Journal Pre-proof
Discovery of phosphonamidate Ido1 inhibitors for the treatment of non-small cell lung
cancer
Qianming Du, Xi Feng, Yinuo Wang, Xi Xu, Yan Zhang, Xinliang Qu, Zhiyu Li, Jinlei
Bian
PII:
S0223-5234(19)30763-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111629
EJMECH 111629
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 May 2019
Revised Date: 3 August 2019
Accepted Date: 14 August 2019
Please cite this article as: Q. Du, X. Feng, Y. Wang, X. Xu, Y. Zhang, X. Qu, Z. Li, J. Bian, Discovery of
phosphonamidate Ido1 inhibitors for the treatment of non-small cell lung cancer, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111629.
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2019 Published by Elsevier Masson SAS.

Discovery of Phosphonamidate IDO1 inhibitors for the
Treatment of Non-Small Cell Lung Cancer
a,b,c,1
a,1
d
a
e
f
Qianming Du,
Xi Feng, Yinuo Wang, Xi Xu, Yan Zhang, Xinliang Qu, Zhiyu
*,a
*,a
Li and Jinlei Bian
a
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing 210009, China
b
General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006,
P.R.China
c
Department of Clinical Pharmacy, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical
University, Nanjing 210009, China
d
Department of Biology, University of California San Diego, La Jolla, California 92093, United States
e
Department of Gastroenterology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210029,
P.R.China
f
Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210029, P.R.China
1
These two authors contributed equally to this work.
KEYWORDS: IDO1 inhibitors, phosphonamidate ester, epacadostat, IDO2, TDO
ABSTRACT:
Targeting indoleamine 2,3-dioxygenase 1 (IDO1) has been identified as an attractive approach for the
development of cancer immunotherapy. In this study, a series of phosphonamidate ester containing
compounds were designed, synthesized and evaluated for their inhibitory activities against IDO1.
Among them, compounds 16, 17, and 26 with good IDO1 inhibitory (HeLa IDO1 IC = 10–21 nM,
5
0
hIDO1 IC50 = 78–121 nM) activities were selected for further investigation and showed good
physicochemical properties. Furthermore, based on comparable PK profile and excellent IDO2/TDO
inhibitory potency, representative compound 16 was selected for further bio-evaluation and
characterized with good efficacy in suppressing lung metastasis (77% inhibition rate) of Lewis cells in
vivo. Thus, compound 16 could be a potential and efficacious agent for further evaluation.

1

1
. Introduction
Indoleamine 2,3-dioxygenase 1 (IDO1) is an extrahepatic cytosolic heme-containing enzyme that
catalyzes the initial and rate-limiting step of the L-tryptophan (L-Trp) catabolism through kynurenine
pathway (KP), which is expressed by cancer cells to induce immunosuppression in tumor
microenvironment [1–3]. It negatively modifies cellular immune response by a molecular mechanism of
depleting local tryptophan storages and producing immunologically active KP metabolites [4–7]. IDO1
catalyzes the oxidative cleavage of the 2,3-double bond of tryptophan pyrrole ring to produce N-
formylkynurenine (NFK) with participation of molecular oxygen [8,9]. Subsequently, the generated
oxidative product NFK is hydrolyzed to form kynurenine and other bioactive metabolites in KP, which
can further activate aryl hydrocarbon receptor (AhR) [10]. IDO1 is also involved in suppression of
+
+
CD8 T effector cells and natural killer (NK) cells and increases activity of CD4 regulatory T cells
(Treg) and myeloid-derived suppressor cells (MDSCs) [11,12]. Actually, IDO1 could be overexpressed
by majority cancers in a constitutive manner [2] and high IDO1 expression is usually associated with
poor prognosis in a variety of cancer types [13,14]. Furthermore, evidences revealed that IDO1 could
contribute to the arising of resistance in immune checkpoint blockade therapies [15,16], and
correspondingly, the combination of checkpoint inhibitors with IDO1 inhibition offers an advanced
strategy in cancer immunotherapy and showed efficiency in clinical trials [17–19]. Thus, targeting the
IDO1 pathway is an attractive approach for cancer immunotherapy [20].
Indeed, IDO1 has been developed as a potent target for anticancer research as several candidates have
entered clinical trials (Fig. 1) [1,21]. Epacadostat (INCB24360, 1), a N-hydroxyamidine derivative
developed by Incyte in 2012 through HTS strategy, is the first candidate compound entering phase III as
selective IDO1 inhibitor and now being tested alone and in combination with PD-1 inhibitors treating
melanoma, lung cancer, urothelial cancer, renal cell cancer [21,22]. The Trp analog indoximod (D-1MT,
2
) is the first reported IDO1 inhibitor and entered phase III in 2017 for the treatment of melanoma [23].
However, growing studies suggest that the primary mechanism of action of 2 possibly is regulating the
downstream signaling of IDO1 pathway [24]. The quinoline-containing compound BMS-986205
2

(
Flexus Biosciences, Inc./Bristol-Myers Squibb, 3) is the only “suicide-inhibitor” (irreversible inhibitor)
that exhibits the best cell-based potency and been evaluated in phase II/III clinical studies [19,25]. PF-
684003 (EOS-200271, 4) from Pfizer, which shares a similar indole scaffold chemotype to that of 3, is
0
in phase I clinical trials for the treatment of grade IV glioblastoma or grade III anaplastic glioma [1,26].
Recently, the negative outcomes of an ECHO-301 phase III trial with IDO1 inhibitor 1 revealed that
selectively inhibiting IDO1 enzyme in body may be not enough for sufficiently modulating KP.
However, the failure may relate to inapplicable pharmacodynamical index or mismatched drug
combination strategy [27]. There are still 35 clinical trials in the active state for several different IDO1
inhibitors. Furthermore, a meta-analysis of 2,706 patients showed the high expression of IDO1 was
significantly associated with tumor differentiation and adverse clinical results, indicating that IDO1
related therapy will continue to be an exciting area [28]. In addition, the failure of ECHO-301 phase III
trial may due to the selectivity toward IDO1 than TDO (tryptophan 2,3-dioxygenase) and IDO2
(indoleamine 2,3-dioxygenase 2), which also exert root functions in cancer inflammatory programming
[27].
Our group has focused on the discovery of potent IDO1 inhibitors for several years. In this work, we
would report a series of IDO1 inhibitors containing a phosphonamidate ester subunit. It is obvious that
the phosphorus-containing moieties (e.g. phosphonamidates, phosphonates, and phosphinates, Fig. 1)
resembles to sulfonamides and sulfonyl derivatives. The two types of functionalities are ideal structural
mimics of each other due to the similar shape and electron density [29,30]. Though the
phosphonamidate esters provide higher substituent variability than sulfonamides moieties, they are
rarely employed to replace sulfur-containing groups in medicinal chemistry. However,
phosphonamidate esters were induced in phosphonate prodrug analogs as the C–P bond in
phosphonamidate could increase the metabolic stability [31]. Furthermore, phosphonamidate esters were
also employed in lead molecules for structure optimization to improve the affinities and bioactivities
[29,32,33]. Herein, considering the isosteric relationship between sulfonamides and phosphonamidates,
a series of novel IDO1 inhibitors were designed and synthesized based on the molecule 1. The obtained
3

phosphonamidate ester derivatives were identified with potent IDO1 inhibitory activities as well as good
human serum stability. Furthermore, we designed to discover pan IDO/TDO inhibitors other than
selective IDO1 inhibitory activity by involving in the rarely applied functional groups and planned to
demonstrate the efficacy for the treatment of NSCLC. This study may also be a useful reference for the
application of IDO1 inhibitors in clinical.
Fig. 1. Structure of candidates targeting IDO1 in clinical trials currently, and the general structure of the phosphonamidate
moiety.
2
. Results and discussioin
2
.1. Chemistry
The synthetic pathways for phosphonamidates 16–48 are depicted in Scheme 1–3. The key
intermediate 10 was prepared by the oxidation reaction of intermediate 9, as the synthetic route of 9 was
an optimized consideration from literature starting with commercially available malononitrile (5,
Scheme 1) [34]. Initially, compound 5 underwent a three-stage transformation in a one-pot reaction
including nitrosation, rearrangement, and dehydration to afford intermediate 6 in 80–85% yield.
Intermediate 7 was obtained through diazo-reaction from intermediate 6 with sodium nitrite in water
with a yield of 50–55%. Heating 7 with sodium bicarbonate and substituted aniline in tetrahydrofuran at
reflux afforded 8a–k (84–98%). Then, the amidoxime of intermediate 8a–k was readily converted to the
4

oxadiazolone with the presence of N,N′-carbonyldiimidazole (CDI) in ethyl acetate at room temperature
to afford amino-furazan 9a–k (80–91%).
2
Scheme 1. Synthesis of amino-furazans 10a–k. Reagents and conditions: (a) (i) HCl, water, 0 °C, (ii) NH OH•HCl, water,
RT, (iii) NaOH, water, reflux; (b) NaNO , HCl, NaCl, AcOH, water, 0 °C; (c) substituted aniline a–h, NaHCO , THF, reflux;
3
3
(
d) N,N′-carbonyldiimidazole, EtOAc, RT; (e) H O (30%), TFA, 45–55 °C, 16 h.
2
2
The phosphonamidate subunit of these pursuing compounds was prepared via mixing
primary/secondary amino of intermediate 12a–h with appropriate phosphonochloridates 14a–h (Scheme
), which were prepared according to published methodologies involving two steps (Scheme 2) [29].
Phosphonate precursor 13a–h was obtained from either commercial source (13a–e) or Arbuzov reaction
13f–h) [35,36]. Subsequently, the obtained precursor was then treated with oxalyl chloride to form
4a–h as monochlorination product.
3
(
1
Scheme 2. Synthesis of phosphonochloridates 14a–h. Reagents and conditions: (a) Oxalyl chloride, DMF, CH Cl , 0 °C to
2
2
reflux, overnight; (b) DMF, 70 °C, 4 h; (c) n-Bu NI, neat, 125 °C, 24 h.
4
5

On the other hand, since the primary amino group adjacent to oxadiazole of 9 is deficient in
nucleophilicity, it is difficult to introduce further side-chains directly based on this group [34]. In
addition, reported synthetic routes consist of 4 extra steps utilizing rearrangement, diazotization, and
azidation which were considered time-consuming and low-yield [37]. Actually, in this regard, the amino
group can be readily converted to a more reactive nitro group with presence of specific oxidant. Such
transformation from amino-furazan to nitro-furazan is practically applied in the construction of
energetic compounds [38–40]. Subsequently, the obtained nitro-furazan 10 was treated with mono-Boc
protected diamine at room temperature to afford intermediate 11a–v (39–83%) which were followed
with deprotection to give 12a–v (74–91%). Next, the reaction of hydrochloride with phosphonchloridate
in anhydrous dichloromethane led to the phosphonamidate 15a–ag (corresponding to each target
compound 16–48, detailed in Experimental Section). Then, an additional deprotection was performed
immediately to give the desired compounds 16–48, Scheme 3.
Scheme 3. Synthesis of phosphonamidate derivatives 16–48. Reagents and conditions: (a) aqueous NaOH, THF, RT, 2 h; (b)
HCl, dioxane, RT, overnight; (c) TEA, CH Cl , 0 °C to RT, 1 h; (d) aqueous NaOH, THF, RT, overnight.
2
2
2
.2. IDO/TDO inhibitory activities
6

For a comparison of these series of phosphorus-based inhibitors with sulfonamide containing IDO1
selective inhibitor 1, an in vitro IDO1 inhibitory test was conducted in first intention. The TDO/IDO2
enzyme inhibitory profiles of the most potent derivatives were further determined, and then the in vivo
efficiency of the most representative compound was validated on non-small cell lung cancer model. In
addition, the preliminary drug-like and pharmacokinetic properties were also studied.
2
.2.1. IDO1 inhibitory activities.
Initial structure-activity relationship (SAR) studies were performed to investigate the exact effect of
the replacement of the sulfamide group to phosphonamidate group on the inhibitory activity of IDO1.
As shown in Table 1, initial 11 compounds 16–26 were designed and synthesized with substituted
phosphonamidate esters whereas 3-bromo-4-fluorophenyl moiety was kept. All of the synthesized
compounds were screened for their IDO1 inhibitory activities in IDO1 HeLa assay and IDO1 enzyme
assay in comparison with 1. It was glad to find that all compounds exhibited moderate to good
inhibitory activities against IDO1 and compound 16 (Fig. 2A) showed equal potency in HeLa assay with
IC50 of 10.1 nM and enzyme IC50 of 104 nM compared to the positive control 1 (HeLa IDO1 IC50 = 10.0
nM, hIDO1 IC = 72 nM). Substitution with ethyl in phosphonamidate group (17, HeLa IDO1 IC =
5
0
50
2
1
0.8 nM, hIDO1 IC = 78 nM) or replacement with a longer linking group (for 26, HeLa IDO1 IC =
50 50
8.9 nM, hIDO1 IC = 121 nM) on the structure of 16 also exhibited good inhibitory activities despite
5
0
slight loss of potency (Fig. 2B). In addition, a 4 to 14-fold decrease in inhibitory potency was observed
accompanied after the introduction of propyl or benzyl to phosphonamidate group respectively (18, 19
and 22). However, compounds containing piperidinyl linker (23–26) totally displayed reduced activities
(HeLa IDO1 IC50 > 100 nM).
7

Fig. 2. (A) Structure of phosphonamidate compounds 16, 17, and 26; (B) IDO1 inhibitory activities in enzyme assay and
HeLa assay by 16, 17, and 26.
Table 1
Screening of phosphonamidate 16–26 on IDO1 inhibitory activities.
1
2
a
a,b
Cpds
R
R
n
HeLa IDO1 IC50 (nM)
hIDO1 Inhibition (nM or %)
1
1
1
1
2
2
2
2
2
2
2
6
7
8
9
0
1
2
3
4
5
6
NHCH2
NHCH2
Me
Et
Me
Et
10.1 ± 0.5
20.8 ± 1.3
37.2 ± 2.7
119 ± 8
44 ± 4
104 ± 5
78 ± 4
58 %
79%
NHCH2
Pr
Et
NHCH2
Bn
Et
Et
NHCH2CH2
NHCH2CH2
4-piperidinyl
4-piperidinyl
4-piperidinyl
3-piperidinyl
NH(CH2)3
–
Et
77%
Pr
Et
143 ± 9
>200
74%
Me
Et
Me
Et
38%
>200
70%
Pr
Et
>200
42%
Me
Me
–
Me
Me
148 ± 8
18.9 ± 1.1
10.0 ± 0.5
23%
121 ± 7
72 ± 3
1
–
a
b
IC50 values are presented as mean ± SD. IDO1 enzyme IC50 or inhibition rates at 200 nM by compounds.
For further screening of phosphonamidate compounds as IDO1 inhibitor, a series of derivatives were
designed and synthesized with varying substituents and linkers (Table 2). To examine the effect of aryl
substituents on activity, phenyl and naphthalen-1-ylmethyl containing compounds were tested (27 and
2
8) and ordinary potency (HeLa IDO1 IC = 132 nM and 126 nM, respectively) was both observed
50
consistent with compound 19 (benzyl, HeLa IDO1 IC50 = 119 nM). Next, compounds with small
8

substituents including ethyl and
i-propyl on phosphomonoester displayed moderate to good activities
(
29 and 30, HeLa IDO1 IC = 25 and 19 nM, hIDO1 IC = 120 and 115 nM, respectively). On the
5
0
50
other hand, we evaluated the longer 1,5-pentyl and the smaller pyrrolyl (compound 31 and 32) as a
supplement of SAR. Compound 31 still exhibited good activity (HeLa IDO1 IC = 26 nM, hIDO1 IC
5
0
50
=
130 nM) and compound 32 had an improved activity (HeLa IDO1 IC50 = 104 nM) against the
compounds containing piperidine (22–25). Unexpectedly, there was a significant decrease in potency (>
00-fold) when piperazine or piperidine directly connected to the furazan ring (compound 33–35). This
1
could be explained that the formation of tertiary amine contributed to the transition of former
preferential conformation (cis-conformation of N-OH) as there is hydrogen bond between this furazan
NH and the imino nitrogen of the hydroxyamidine [34,41]. Hence, methyl or ethyl was picked for both
1
2
R and R in phosphonamidate group meanwhile ethylidene and propylidene were chosen as linker.
Table 2
Additional SAR of substituted phosphonamidates and likers on IDO1 inhibitory activities.
1
2
a
a,b
Cpds
R
R
n
HeLa IDO1 IC50 (nM)
hIDO1 Inhibition (nM or %)
2
2
2
3
3
3
3
3
3
7
8
9
0
1
2
3
4
5
Phenyl
Et
Et
1
1
1
1
4
-
132 ± 8
126 ± 7
25 ± 1
63%
50%
Naphthalen-1-methyl
Me
Me
Me
Me
Me
Me
Me
-
i-Propyl
Et
120 ± 8
120 ± 10
115 ± 9
130 ± 7
NA
19.1 ± 1.0
26 ± 2
Me
Me
104 ± 7
>1000
Me
-
Me
-
>1000
NA
Me
-
>1000
NA
1
-
-
10.0 ± 0.5
72 ± 3
a
b
IC50 values are presented as mean ± SD. IDO1 enzyme IC or inhibition rates at 200 nM by compounds.
50
9

With the optimized phosphonamidate group and linker in hand, an additional screening was
performed to evaluate the phenyl derivatives (Table 3). The meta substitution was vital for inhibitory
potency as more than 50-fold loss was demonstrated in cellular assay when
substitution was removed (42, HeLa IDO1 IC = 850 nM). Further optimization revealed that meta-
m-bromo or m-chloro
5
0
halogens (36–41, including Br and Cl, HeLa IDO1 IC ranging from 10.5 to 35 nM) and electron-
5
0
withdrawing groups (43–46, including trifluoromethyl and cyano, HeLa IDO1 IC ranging from 17 to
5
0
9
4 nM) provided preferable IDO1 inhibitory activities (among them, compound 40 displayed the best
potency of HeLa IDO1 IC50 = 10.5 nM, hIDO1 IC50 = 114 nM). Subsequently, when we introduced a
weak electron-donating group of methyl, a reduced potency was determined by compound 47 and 48
(
HeLa IDO1 IC = 23 nM and 94 nM, respectively). Interestingly, the para-substitution with fluorine
50
showed decreasing contribution to the inhibitory potency, which seemed not be consist with the
observation that F atom could interact with Cys129 of IDO1 [41]. In general, the scope of phenyl
substitution showed that electron-withdrawing groups in meta position were crucial for inhibition and
the priority was Br > Cl > CF > CN > CH > H.
3
3
Table 3
SAR of substituted phenyl on IDO1 inhibitory activities.
3
4
a
a,b
Cpds
R
R
n
HeLa IDO1 IC50 (nM)
hIDO1 Inhibition (nM or %)
3
3
3
3
6
7
8
9
F
F
Cl
Cl
Cl
Br
Br
Br
H
1
2
1
1
1
2
1
1
1
1
17.9 ± 1.4
35 ± 2
34%
57%
H
H
H
H
F
14.4 ± 1.0
10.5 ± 0.4
14.6 ± 0.8
24 ± 2
44%
114 ± 9
109 ± 7
99 ± 7
NA
c
4
0
4
4
4
4
4
1
2
3
4
5
850 ± 64
17.1 ± 1.2
21 ± 1
F
CF
CF
3
102 ± 7
121 ± 7
101 ± 5
H
F
3
CN
24 ± 2
1
0

4
4
4
6
7
8
H
F
H
-
CN
1
1
1
-
36 ± 3
23 ± 2
40%
48%
CH
CH
-
3
3
94 ± 5
29%
1
10.0 ± 0.5
72 ± 3
a
b
c
IC50 values are presented as mean ± SD. IDO1 enzyme IC or inhibition rates at 200 nM by compounds. Compound with
50
P2 group.
2
.2.2. Representative IDO1 inhibitors showed good activity toward IDO2 and TDO
Encoded by Ido2, Indoleamine 2,3-dioxygenase 2 (IDO2) is a peripheral metabolic enzyme which is
also associated with modulating immunity through Trp catabolism as an IDO1 paralogue [42,43]. It was
suggested that IDO2 shows a lower activity on Trp degradation than IDO1 and functions in a different
manner with restricted expression [1]. Although relatively little studied yet, IDO2 has been found to be
an important contributor to IDO1-mediated immune tolerance [44]. On the other hand, tryptophan 2,3-
dioxygenase (TDO) is a hepatic/tetrameric enzyme that catalyzes the rate-limiting step in Trp
catabolism [45]. Studies have shown that TDO is overexpressed in some tumors as a means of immune
escape [46]. Emerging evidence reveals that developing combo or pan IDO1/IDO2/TDO inhibitors may
broaden impact in cancer treatment and overcome the deficiency in clinical trials of IDO1 selective
inhibitors [46,47]. Considering this, representative compound 16, 17, and 26 with potencies of 20 nM or
less were evaluated on IDO2 and TDO inhibition activities (Table 4, Fig. S1). First, all compounds
tested displayed improved potency against IDO2 comparing to positive control 1 and compound 17
exhibited the best potency that ever reported (hIDO2 enzyme IC = 0.28
µ
M). On the other hand,
M and compound 17/26 exhibited
M respectively), despite the obvious
structural difference between IDO1 and TDO. It should be noted that the control 1 showed weak
inhibitory activity toward TDO with IC50 > 10 M. In general, selected compounds 16, 17 and 26
5
0
compound 16 displayed moderate TDO enzyme IC of 3.96
µ
5
0
promising inhibitory potency (hTDO IC50 = 2.54 and 3.32
µ
µ
showed pan inhibition of IDO1/IDO2/TDO comparing to the clinical candidate 1 which has been
confirmed as selective IDO1 inhibitors [1]. The characteristic of this series of compounds may plumb
the full potential of blunting Trp catabolism which may be beneficial for the therapy in clinical.
1
1

Table 4
IDO1, IDO2 and TDO inhibition activities of compound 16, 17, and 26.
a
a
a
Cpd
hIDO1 enzyme IC50 (µM)
hIDO2 enzyme IC50 (µM)
hTDO enzyme IC50 (µM)
1
1
2
6
7
6
0.104 ± 0.005
0.078 ± 0.004
0.121 ± 0.007
0.072 ± 0.003
0.49 ± 0.034
0.28 ± 0.074
0.59 ± 0.044
0.71 ± 0.064
3.96 ± 0.37
2.54 ± 0.40
3.32 ± 0.25
>10
1
a
IC50 value represents as mean ± SD from two trials.
2
.3. Molecular modeling
For further illustrating the structure-activity relationship, we performed a docking study to elucidate
the possible binding mode of IDO1 with the representative compound 16 (Fig. 3). A crystal structure of
IDO1 (PDB id: 5WN8) was selected as receptor. Considering the phosphorus atom in compound 16 is
one chiral center (Fig. 3A), then we utilized two possible enantiomers for the in silico docking
respectively. Initially, both (R)-16 and (S)-16 enantiomer oriented in a similar pose with the native
ligand 1 (Fig. 3B) [41]. As shown in Fig. 3C and 3D, the phosphonamidate moiety of both enantiomers
occupied the pocket B and hydrogen bonds formed between the oxygen atoms with the key residue
Arg231. On the other hand, the halogen-substituted phenyl stretched into pocket A and the hydroxy of
hydroxyamidine posed toward the ion-containing porphyrin. The preliminary docking results indicate
that the chiral phosphonamidate group may show limiting influence on the general conformation and
support the fact that compound 16 was an excellent inhibitor of IDO1.
1
2

Fig. 3. (A) The phosphorus atom in phosphonamidate moiety is one chiral center; (B) Co-crystal structure of IDO1 bound to
compound 1 (shown in purple, PDB id: 5WN8). The benzene group occupies the pocket A and is stabilized by Cys129. The
side arm extends out into pocket B and interacts with IDO1 between the sulfamide group and Arg231. The oxygen atom of
hydroxyamidine group coordinates to the heme iron. (C) and (D) The The docking mode of compound 16 (shown in yellow)
against IDO1. The (R)-16 (C) and (S)-16 (D) both docked well in IDO1 and showed similar conformation and binding mode.
The interaction mode was obtained through molecular docking (PDB id: 5WN8) and depicted using MOE 2018.01. The H-
bonds were shown as black dot lines.
2
.4. Physiochemical assay
Subsequently, the representative compounds 16, 17 and 26 were evaluated for the physiochemical
properties (Table 5). Firstly, modification with phosphonamidate group obviously reduced the
molecular polarity of all tested compounds which could be demonstrated by the lower polar surface area
2
(
PSA, < 140 Å ) and higher cLogP. Following this, moderated permeabilities were confirmed as
−
6
expected in Caco-2 assay and compound 16 displayed the best property (Papp A→B = 2.9 × 10 cm/s)
1
3

with no significant efflux (efflux ratio = 1.07). As reported in Table 5, the synthesized compounds were
characterized by improved water solubility values and compound 17 exhibited an advanced solubility of
2
-fold against compound 1 (0.25 mg/mL for 17 and 0.13 mg/mL for 1). In addition, this modification
resulted in agreeing with Lipinski’s rule-of-five (HBD < 5, HBA < 10, MW < 500) and the optimized
molecules proved to be more “drug-like” on the base of the result. Besides, ligand efficiency (LE) and
lipophilic ligand efficiency (LLE) were calculated based on cell IC s which were instructive in drug
5
0
candidate development [48] and result revealed that all compounds displayed ideal values (LE > 0.3,
LLE > 5) except that compound 17 had a lower LLE (LLE = 4.4). Considering the chemical/metabolic
stability of phosphonamidate esters, a human serum stability assay of lead compound 16 was then
performed. Interestingly, result showed that compound 16 was well tolerated in human serum and barely
reduced throughout the experimental period (t1/2 > 24 h, Fig. S2). Till now, present result demonstrates
that the introduction of phosphonamidate is an appropriate strategy.
Table 5
Selected SAR and calculated property of phosphonamidates
b
a
S
HeLa IDO1
IC (nM)
hIDO1 IC50 Caco-2
c
d
d
e
e
Cpds
PSA
cLogP HBD
HBA
MW LE
LLE
(nM)
(cm/s)
50
(mg/mL)
1
1
2
6
7
6
10.1
20.8
18.9
10.0
104
78
2.9 (1.07)
1.9 (1.12)
2.1 (1.16)
3.1 (1.09)
0.21
129
129
129
2.2
3.3
2.2
1.5
4
4
4
6
10
10
10
11
451
479
479
438
0.42
0.38
0.38
0.44
5.8
4.4
5.5
6.5
0.25
121
0.16
1
72
0.13
163
a
−6
b
Caco-2 values: Papp A→B × (10) cm/s with efflux ratio in bracket. Aqueous solubility and each value was represented as a
c
2
d
e
mean from two trials. PSA in Å (calculated using ChemBioDraw Ultra 13.0). Hydrogen bond donor and acceptor. Ligand
efficiency (LE) and lipophilic ligand efficiency (LLE) calculated using HeLa pIC50
.
2
.5. Pharmacokinetic studies
1
4

∗
Based on the biochemical and cellular activities and the drug-like profile, compound 16 was selected
for further PK evaluation (through iv and po routes, Table 6). In general, compound 16 showed a
moderate PK profile comparing to 1 [22]. In the iv route (2.5 mg/kg), compound 16 showed a half-life
of 0.66 h and an AUC of 902 ng/mL × h. While through the po route (10.0 mg/kg), 16 showed moderate
exposure and oral bioavailability (%F = 11%) with a volume of distribution (Vss) of 2.09 L/kg. On the
basis of these results, compound 16 was projected into further evaluation of the in vivo antitumor
activity.
Table 6.
a,b
In vivo pharmacokinetic profile of 16
Cpd
Cl (L/h/kg)
V (L/kg)
ss
iv t1/2 (h)
AUC (h*ng/mL)
%F
po t1/2 (h)
1
6
2.81
2.09
0.66
902
11
0.91
a
b
Each value represents as mean from five trials. Intravenous administration of compound with dosing of 2.5 mg/kg and oral
dosing at 10 mg/kg.
2
.6. Compound 16 dose-dependently suppressed lung metastasis of Lewis cells in vivo
With promising in vitro properties and moderate PK profiles, compound 16 was evaluated in a lung
metastasis of Lewis cells assay for its in vivo antitumor activity (Fig. 4). Initially, grouped
immunocompetent C57BL6 mice were challenged with Lewis cells through lateral tail vein and treated
with 16 (15, 30, 60 mg/kg) and epacadostat (1, 60 mg/kg) by gavage. As shown in Fig. 4A,
administration of 16 (30 and 60 mg/kg) and eapcadostat remarkably increased the body of mice against
the control group. Importantly, both 16- and epacadostat-treated mice displayed a significant increase in
survival relative to vehicle-treated mice at the day 30 (Fig. 4B). Subsquently, pneumonectomy on mice
was performed and the lung metastatic nodules of each lungs were counted. As shown in Fig. 4C and
4
D, compound 16 suppressed lung metastasis of Lewis tumor cells in a dose-dependent manner.
∗
Unfortunately, we did not find an applicable dosage form of compound 17 for further in vivo evaluation
despite its promising properties in vitro
.
1
5

Furthermore, H.E. staining suggested that the area of metastases was much smaller in the lungs from 16-
treated mice comparing to the control group. Significantly, compound 16 exhibited an equal efficacy in
inhibiting lung metastasis of Lewis cells comparing to epacadostat at the dose of 60 mg/kg, as the
average lung metastatic nodules of each compound per mice is 21 (referring to an inhibition rate of
7
7%).
Fig. 4. Compound 16 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 = 8 for 16-15 mg/kg, n = 9 for 16-30 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. Scare bar = 50 µm. (D) The corresponding
statistical plots were presented (n = 7 for Ctrl group, n = 8 for 16-15 mg/kg, n = 9 for 16-30 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).
3
. Conclusions
Several phosphonamidate derivatives of epacadostat (1) were designed and synthesized in this work.
SARs were performed for IDO1 inhibitory activities and the synthesized compounds were proved to be
potent IDO1 inhibitors. Then the selected compounds 16, 17, and 26 were characterized with improved
properties in physiochemistry and proved to be pan IDO/TDO inhibitors targeting Trp/Kyn pathway.
Further bio-evaluation of model compound 16 was performed and favorable outcomes in PK and PD
1
6

validated its efficacy in vivo. Featured compounds (e. g. compound 17) and derivatives of this series
deserve further investigation based on the finding described in this paper. The unexpected pan
IDO/TDO inhibitory activities and the potential for further optimization of these phosphonamidate
derivatives could be an important solution in this field. The separation of 16 enantiomers, further studies
toward improving IDO/TDO inhibitory activities and physiochemical properties are under way in our
laboratory.
1
7

4
. Experimental
4
.1. Chemistry
Unless otherwise noted, all reagents were obtained from commercial suppliers and used without
further purification. Reactions were monitored by thin layer chromatography (TLC) visualized under
1
UV light. NMR spectra were measured on a 300 MHz Bruker unit (300 MHz for H NMR, 75 MHz for
1
3
31
C NMR, 121 MHz for P NMR) using CDCl or DMSO-d6 as the solvent at room temperature.
3
Chemical shifts (δ) are reported in parts per million (ppm) from tetramethylsilane (TMS) using the
residual solvent resonance and constants (
J
) are given in hertz. 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. Column chromatography was performed with 100–200 mesh silica gel and yields
refer to chromatographically and spectroscopically pure compounds.
4
.1.1. General protocol for the synthesis of phosphonchloride 14a–14h
The phosphonchlorides (14a–14h) were prepared from substituted phosphonates [49]. The
corresponding materials 13a–13e were obtained from commercial source whereas 13f–13h were
prepared via Arbuzov reaction [33]. 13f and 13g were synthesized according to the following procedure.
A 10 mL Schlenk tube with a magnetic stir bar was charged with triisopropyl phosphite or triethyl
phosphite (20 mmol), methyl iodide (30 mmol, 1.5 equiv) and N,N-dimethylformamide (10 mL). The
reaction mixture was sealed under N atmosphere and stirred at 80 °C for 4 h. The reaction was
2
monitored by TLC. Upon completion, the mixture was diluted with 2 N sodium hydroxide (15 mL) and
extracted with dichloromethane (2 × 20 mL). The organic phases were combined and washed with
2 4
water, brine and dried over anhydrous Na SO . After being filtered and concentrated, the residue was
purified by column chromatography on silica gel to give the desired phosphonates (2.80 g, 78% for 13f
and 2.64g, 87% for 13g) as colorless oil. 13h were prepared according to the method described by Ma et
al [36] as a colorless oil (1.47 g, 53%). 14a–14h were prepared according to following procedure.
Phosphonate (20 mmol) was dissolved in dry dichloromethane. The mixture was cooled to 0 °C, and
1
8

oxalyl chloride (30 mmol, 1.5 equiv) was added slowly. The mixture was stirred at ambient temperature
for 30 min and then allowed to warm to room temperature. Several drops of N,N-dimethylformamide
were added as catalyst and the reaction mixture was then refluxed for 8 h. The solvent was evaporated
under reduced pressure and additional dichloromethane (15 mL) was evaporated three more times to
obtain the desired product as a garnet oil. The product was used for the next step without further
purification.
4
.1.2. Protocol for the Synthesis of 4-amino-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide (6).
A 500 mL three-necked flask with a magnetic bar was charged with sodium nitrite (20.7 g, 300 mmol)
and water (40 mL). To the resulting solution was added malononitrile (5, 9.9 g, 150 mmol) predissolved
in 150 mL of 2 N hydrochloric acid by dropwise, and the mixture was stirred overnight at room
temperature. Then the reaction mixture was cooled in an ice bath to 0 °C and aqueous hydroxylamine
hydrochloride (23 g, 340 mmol in 40 mL water) was added and stirred for 30 min. The reaction mixture
was cooled in an ice bath and 10 N sodium hydroxide was added slowly to pH 10. When the alkalization
finished, the ice bath was removed and stirring was continued for 2 h at 35 °C and it was brought to
reflux for 2 h. Then the reaction mixture was allowed to cool and the precipitate was collected by
filtration, washed well with water and dried completely to give the desired product (14.9 g, 68%) as a
1
white solid. H NMR (300 MHz, DMSO-
d
6
): δ = 10.46 (s, 1H), 6.24 (s, 2H), 6.02 (s, 2H) ppm. HRMS
+
+
(
ESI ): calcd for C
3
H
5
N O
5 2
(M + H) 143.0516; found, 143.0518.
4
.1.3. Protocol for the synthesis of 4-amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (7)
A 250 mL three-necked flask with a magnetic stirred bar was charged with compound 6 (4.2 g, 29.5
mmol), 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). The resulting suspension was stirred and heated at 45 °C until the
complete solution was achieved. The reaction was cooled in an ice-salt-bath to –8 °C and a solution of
sodium nitride (2.0 g, 29 mmol) in water (7 mL) was added dropwise while maintained the temperature
under 0 °C for 3 h. Upon completion, the reaction mixture was allowed to warm to room temperature.
1
9

The precipitate was collected by filtration, washed well with water and dried. The crude product was
mixed with toluene (25 mL) and the obtained slurry was stirred at 80 °C for 1 h. After filtration, the
1
desired product was obtained (2.5 g, 53%) as an off-white solid. H NMR (300 MHz, DMSO-d6): δ =
+
+
1
3.39 (s, 1H), 6.29 (s, 2H); HRMS (ESI ): calcd for C H ClN O (M + H) 161.9872; found, 161.9872.
3 3 4 2
4
3
.1.4. Protocol for the synthesis of 4-amino-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-
-carboximidamide (8a)
A 150 mL four-necked flask with a magnetic stir bar was charged with compound 7 (3.24 g, 20
mmol), sodium bicarbonate (3.36 g, 40 mmol, 2 equiv) and tetrahydrofuran (70 mL). At 45 °C, a
solution of 3-bromo-4-fluoroaniline (4.18 g, 22 mol, 1.1 equiv) in tetrahydrofuran (15 mL) was added to
the reaction mixture in 10 min. Then the reaction mixture was heat to reflux for another 3 h. On
completing of the reaction monitored by TLC, the sodium bicarbonate was filtered and the filtrate was
evaporated in vacuo. The residue was dissolved with ethyl acetate (70 mL), washed with water (40 mL),
brine, dried over anhydrous Na SO and concentrated in vacuo. The crude product was slurried in
2
4
petroleum ether (50 mL) for 1 h and the precipitate was collected by filtration and dried to give the
1
desired product (5.75 g, 91%) as a white solid. H 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. HRMS (ESI ): calcd for C
9
8 5 2
H BrFN O (M + H) 315.9840; found, 315.9842.
4
.1.5. Protocol for the synthesis of 3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one (9a)
A 150 mL round-bottomed flask with a magnetic stir bar was charged with compound 8a (5.0 g, 15.8
mmol), ethyl acetate (100 mL) and
N,N′-carbonyldiimidazole (5.12 g, 31.6 mmol, 2 equiv). The
resulting mixture was stirred at ambient temperature for 2 h. On completing of the reaction monitored
by TLC, the mixture was washed with 1 N hydrochloric acid (60 mL), water (70 mL), brine and
evaporated in vacuo. The crude product was recrystallized in ethanol to give the desired product (4.80 g,
1
8
9%) as a white needle-like crystal. H NMR (300 MHz, DMSO-d6): δ = 8.10 (dd, J1 = 6.2 Hz, J2 = 2.4
2
0

1
3
Hz, 1H), 7.74–7.69 (m,1H), 7.61 (t,
J
= 8.6 Hz, 1H), 6.60 (s, 2H) ppm. C NMR (75 MHz, DMSO-
d
6
):
δ = 160.6, 157.3, 155.1, 148.8, 134.2, 133.3, 130.0 (
J
= 8.4 Hz), 128.6 (
J
= 3.4 Hz), 117.5 (J
= 23.9
+
+
Hz), 108.3 (
J
= 22.8 Hz) ppm. HRMS (ESI ): calcd for C H BrFN O (M + H) 341.9633; found,
1
0
6
5
2
3
41.9638.
4
.1.6. Protocol for the synthesis of 4-(3-bromo-4-fluorophenyl)-3-(4-nitro-1,2,5-oxadiazol-3-yl)-1,2,4-
oxadiazol-5(4H)-one (10a)
A 150 mL round-bottomed flask with a magnetic stir bar was charged with compound 9a (4.5 g, 13.1
mmol), 30% hydrogen peroxide (30 mL) and trifluoroacetic acid (90 mL). The resulting suspension was
stirred at 45 °C overnight and the substrate gradually dissolved over time. On completing of the reaction
2 3
monitored by TLC, the reaction mixture was then quenched with saturated aqueous Na SO and
precipitation appeared. The precipitate was collected by filtration and well washed with water. The
crude product was then purified by column chromatography on silica gel (petroleum ether/ethyl acetate
1
=
8/1) to give the desired product (2.19 g, 45%) as a yellow solid. H NMR (300 MHz, DMSO-d6): δ =
1
3
8
.06 (dd, J1 = 6.0 Hz, J2 = 2.3 Hz, 1H), 7.69–7.64 (m, 1H), 7.60 (t,
MHz, DMSO- 6): 160.6, 157.3, 156.4, 145.7, 137.3, 133.0, 129.6 (
Hz), 108.6 ( = 22.7 Hz) ppm.
J
= 8.6 Hz, 1H) ppm. C NMR (75
d
J
= 8.1 Hz), 127.7, 117.6 ( = 23.9
J
J
4
1
.1.7. Protocol for the synthesis of tert-butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-
,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]carbamate (11a)
A 100 mL round-bottomed flask with a magnetic stir bar was charged with compound 10a (1.86 g, 5
mmol), tetrahydrofuran (50 mL) and
N-Boc-1,2-ethylenediamine (1.60 g, 10 mmol, 2equiv). To the
mixture was added 2 N sodium hydroxide (5 mL, 2 equiv). The reaction mixture was stirred at room
temperature for 1 h. On completing of reaction monitored by TLC, the mixture was acidified with 1 N
hydrochloric acid to pH 4.0 and evaporated in vacuo. The residue was then diluted with water (30 mL)
and extracted with ethyl acetate (2 × 25 mL). The organic phases were combined and washed with water
(
30 mL), brine, dried over anhydrous Na SO and concentrated in vacuo. The residue was then purified
2 4
2
1

by column chromatography on silica gel (petroleum ether/ethyl acetate = 4/1) to give the desired
1
product (1.89 g, 78%) as a pale-yellow solid. H NMR (300 MHz, DMSO-d6):
δ = 8.09–8.07 (m, 1H),
7
9
.72–7.70 (m, 1H), 7.60 (t,
J
= 8.4 Hz, 1H), 6.93 (s, 1H), 6.51 (s, 1H), 3.29 (s, 2H), 3.17 (s, 2H), 1.37 (s,
–
–
H) ppm. HRMS (ESI ): calcd for C H BrFN O (M – H) 485.0413; found, 485.0422.
1
7
17
6
5
4
.1.8. Protocol for the synthesis of 3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-
fluorophenyl)-1,2,4-oxadiazol-5(4H)-one hydrochloride (12a)
A 50 mL round-bottomed flask with a magnetic stir bar was charged with compound 11a (1.45 g, 3
mmol), hydrochloric acid solution (10 mL, 4.0 M in ethyl acetate). The mixture was stirred at room
temperature overnight. On completing of the reaction monitored by TLC, the resulting slurry was
filtered and well washed with ethyl acetate. The isolated solid was dried completely to afford the desired
1
product (1.12 g, 89%) as an off-white solid. H NMR (300 MHz, DMSO-
d
6
): δ = 8.16–8.14 (m, 4H),
7
.80–7.76 (m, 1H), 7.60 (t,
J
= 8.6 Hz, 1H), 6.79 (t,
J
= 5.8 Hz, 1H), 3.37 (sbr, 2H), 3.06–3.00 (m, 2H)
+
+
ppm. HRMS (ESI ): calcd for C H BrFN O (M + H) 385.0055; found, 385.0051.
1
2
11
6
3
4
.1.8.1.
3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12b). It was prepared by deprotection and salification of 11b (1.2
1
g, 2.40 mmol) according to the method of 12a, giving 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. HRMS
+
+
(
ESI ): calcd for C H BrFN O (M + H) 399.0211; found, 399.0209.
1
3
13
6
3
4
.1.8.2.
3-{4-[(4-Aminobutyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12c). It was prepared by deprotection and salification of 11c (1.0 g,
+
1
.94 mmol) according to the method of 12a, giving an off-white solid (0.71 g, 81%). HRMS (ESI ):
+
calcd for C14
H
15BrFN
6
O
3
(M + H) 413.0368; found, 413.0367.
2
2

4
.1.8.3.
3-{4-[(5-Aminopentyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12d). It was prepared by deprotection and salification of 11d (0.70
+
g, 1.32 mmol) according to the method of 8, giving an off-white solid (0.47 g, 77%). HRMS (ESI ):
+
calcd for C H BrFN O (M + H) 427.0524; found, 427.0522.
1
5
17
6
3
4
5
.1.8.4. 4-(3-Bromo-4-fluorophenyl)-3-[4-(piperidin-4-ylamino)-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-
(4H)-one (12e). 11e (1.50 g, 2.85 mmol) was treated with a solution of trifluoracetic acid (6 mL) in
dichloromethane (30 mL) and the result mixture was stirred at room temperature for 3 h. The reaction
was monitored by TLC and then the mixture was quenched with saturated aqueous sodium bicarbonate,
extracted with ethyl acetate (3 × 15 mL), washed with water, brine and dried over anhydrous Na SO
2
4.
1
After filtered and concentrated in vacuo, 12e was obtained as a pale-yellow solid (1.08 g, 83%). H
NMR (300 MHz, DMSO-
d
6
): δ = 8.08 (dd,
J
1
= 6.0 Hz,
J
2
= 2.1 Hz, 1H), 7.68–7.56 (m, 2H), 6.22 (d,
J
=
7.32 Hz, 1H), 3.35–3.33 (m, 2H), 2.93 (d,
J
= 12.2 Hz, 3H), 2.44 (s, 1H), 1.83 (d, J = 10.2 Hz, 2H),
+
+
1
.40–1.30 (m, 2H) ppm. HRMS (ESI ): calcd for C H BrFN O (M + H) 425.0368; found, 425.0361.
15
15
6
3
4
5
.1.8.5. 4-(3-Bromo-4-fluorophenyl)-3-[4-(piperidin-3-ylamino)-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-
(4H)-one (12f). It was prepared by deprotection of 11f (0.77 g, 1.47 mmol) by trifluoracetic acid
+
according to the procedure of 12f, giving a pale-yellow solid (0.48 g, 77%). HRMS (ESI ): calcd for
+
C H BrFN O (M + H) 425.0368; found, 425.0368.
15
15
6
3
4
5
.1.8.6. 4-(3-Bromo-4-fluorophenyl)-3-[4-(pyrrolidin-3-ylamino)-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-
(4H)-one hydrochloride (12g). It was prepared by deprotection and salification of 11g (1.0 g, 1.95
1
mmol) according to the method of 12a, giving a tan solid (0.71 g, 81%). H NMR (300 MHz, DMSO-
d6): δ = 9.47 (sbr, 3H), 8.14 (d, J = 6.0 Hz, 1H), 7.75–7.72 (m, 1H), 7.63 (t, J = 8.6 Hz, 1H), 7.03 (d, J =
6
.8 Hz, 1H), 4.24 (d,
J
= 5.3 Hz, 1H), 3.33–3.12 (m, 4H), 2.28–2.16 (m, 1H), 2.04–2.00 (m, 1H) ppm.
+
+
HRMS (ESI ): calcd for C14
H
13BrFN
6
O
3
(M + H) 411.0211; found, 411.0209.
2
3

4
.1.8.7. 4-(3-Bromo-4-fluorophenyl)-3-[4-(piperazin-1-yl)-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-
one (12h). It was prepared by deprotection of 11h (1.1 g, 2.15 mmol) according to the method of 12f,
+
+
giving an off-white solid (0.68 g, 77%). HRMS (ESI ): calcd for C H BrFN O (M + H) 411.0211;
1
4
13
6
3
found, 411.0220.
4
.1.8.8.
3-[4-(4-Aminopiperidin-1-yl)-1,2,5-oxadiazol-3-yl]-4-(3-bromo-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12i). It was prepared by deprotection and salification of 11i (0.80
1
g, 1.52 mmol) according to the method of 12a, giving a white solid (0.58 g, 84%). H NMR (300 MHz,
DMSO-d6): δ = 8.99 (s, 3H), 8.05 (s, 1H), 7.76–7.57 (m, 2H), 6.76 (d,
J
= 6.1 Hz, 1H), 3.79 (d, J = 12.8
+
Hz, 1H), 3.28–2.96 (m, 4H), 1.98 (s, 2H), 1.82–1.61 (m, 2H) ppm. HRMS (ESI ): calcd for
+
C
15
H15BrFN
6
O
3
(M + H) 425.0368; found, 425.0361.
4
.1.8.9.
3-[4-(3-Aminopiperidin-1-yl)-1,2,5-oxadiazol-3-yl]-4-(3-bromo-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12j). It was prepared by deprotection and salification of 11j (0.69
+
g, 1.31 mmol) according to the method of 12a, giving an off-white solid (0.53 g, 88%). HRMS (ESI ):
+
calcd for C15
H
15BrFN
6
O
3
(M + H) 425.0368; found, 425.0366.
4
.1.8.10. 3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromophenyl)-1,2,4-oxadiazol-5(4H)-
one hydrochloride (12k). It was prepared by deprotection and salification of 11k (0.73 g, 1.5 mmol)
1
according to the method of 12a, giving an off-white solid (0.52 g, 85%). H NMR (300 MHz, DMSO-
d6): δ = 8.09 (sbr, 3H), 7.96 (s, 1H), 7.79 (d,
J
= 7.5 Hz, 1H), 7.68 (d,
J
= 7.4 Hz, 1H), 7.52 (t, J = 7.9
+
Hz, 1H), 6.80 (s, 1H), 3.54 (d,
J
= 5.2 Hz, 2H), 3.03 (s, 2H) ppm. HRMS (ESI ): calcd for
+
C H BrN O (M + H) 367.0149; found, 367.0146.
12
12
6
3
4
5
.1.8.11.
3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromophenyl)-1,2,4-oxadiazol-
(4H)-one hydrochloride (12l). It was prepared by deprotection and salification of 11l (0.96 g, 2 mmol)
1
according to the method of 12a, giving a white solid (0.68 g, 82%). H NMR (300 MHz, DMSO-d6):
δ
=
8.03 (sbr, 3H), 7.92 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 8.0 Hz,
2
4

+
1
H), 6.75 (s, 1H), 3.33 (s, 2H), 2.81 (s, 2H), 1.88 (t, J = 6.5 Hz, 2H) ppm. HRMS (ESI ): calcd for
+
C H BrN O (M + H) 381.0305; found, 381.0305.
13
14
6
3
4
.1.8.12. 3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chlorophenyl)-1,2,4-oxadiazol-5(4H)-
one hydrochloride (12m). It was prepared by deprotection and salification of 11m (0.9 g, 2.12 mmol)
1
according to the method of 12a, giving a white solid (0.69 g, 91%). H NMR (300 MHz, DMSO-d6):
δ
=
8.02 (sbr, 3H), 7.82 (s, 1H), 7.67–7.58 (m, 3H), 6.78 (s, 1H), 3.55–3.49 (m, 2H), 3.03 (t, J = 6.1 Hz,
+
+
2
H) ppm. HRMS (ESI ): calcd for C H ClN O (M + H) 323.0654; found, 323.0650.
12 12 6 3
4
.1.8.13.
3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12n). It was prepared by deprotection and salification of 11n (1.0
+
g, 2.26 mmol) according to the method of 12a, giving a white solid (0.63 g, 74%). HRMS (ESI ): calcd
+
for C H ClFN O (M + H) 341.0560; found, 341.0552.
1
2
11
6
3
4
.1.8.14.
3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12o). It was prepared by deprotection and salification of 11o (1.44
1
g, 3.16 mmol) according to the method of 12a, giving a white solid (1.08 g, 88%). H NMR (300 MHz,
6
DMSO-d ): δ = 8.02 (sbr, 4H), 7.70–7.62 (m, 2H), 6.75 (s, 1H), 3.34 (s, 2H), 2.81 (s, 2H), 1.89 (t, J =
+
+
6
6 3
.4 Hz, 2H) ppm. HRMS (ESI ): calcd for C13H13ClFN O (M + H) 355.0716; found, 355.0712.
4
.1.8.15. 3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-
one hydrochloride (12p). It was prepared by deprotection and salification of 11p (1.0 g, 2.46 mmol)
1
according to the method of 12a, giving a white solid (0.72 g, 88%). H NMR (300 MHz, DMSO-
d
6
):
δ
=
8.13 (sbr, 3H), 7.73–7.69 (m, 2H), 7.43–7.37 (t,
J
= 7.8 Hz, 2H), 6.81 (s, 1H), 3.53 (d,
J
= 5.5 Hz,
+
+
2
H), 3.02 (s, 2H) ppm. HRMS (ESI ): calcd for C H FN O (M + H) 307.0949; found, 307.0944.
1
2
12
6
3
4
1
.1.8.16.
3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(4-fluoro-3-(trifluoromethyl)phenyl)-
,2,4-oxadiazol-5(4H)-one hydrochloride (12q). It was prepared by deprotection and salification of 11q
2
5

1
(
1.20 g, 2.5 mmol) according to the method of 12a, giving a white solid (0.91 g, 88%). H NMR (300
MHz, DMSO- 6
.40 (s, 2H), 3.05 (d,
found, 375.0822.
d
): δ = 8.31 (d,
J
= 5.9 Hz, 1H), 8.15 (sbr, 4H), 7.81 (t,
J
= 9.5 Hz, 1H), 6.81 (s, 1H),
+
+
3
J
= 5.0 Hz, 2H) ppm. HRMS (ESI ): calcd for C H F N O (M + H) 375.0823;
1
3
11
4
6
3
4
.1.8.17.
3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12r). It was prepared by deprotection and salification of 11r (1.64
1
g, 3.59 mmol) according to the method of 12a, giving a white solid (1.19 g, 85%). H NMR (300 MHz,
DMSO-d6): δ = 8.17 (sbr, 4H), 8.01 (t,
J
= 8.6 Hz, 2H), 7.84 (t,
J
= 7.8 Hz, 1H), 6.85 (t,
J
= 5.3 Hz,
+
+
1
3
H), 3.55 (d,
J = 5.8 Hz, 2H), 3.03 (s, 2H) ppm. HRMS (ESI ): calcd for C H F N O (M + H)
13 12 3 6 3
57.0917; found, 357.0911.
4
.1.8.18.
5-(3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl)-2-
fluorobenzonitrile hydrochloride (12s). It was prepared by deprotection and salification of 11s (1.98 g,
1
4
.6 mmol) according to the method of 12a, giving a pale-yellow solid (1.52 g, 90%). H NMR (300
MHz, DMSO-
d
6
): δ = 8.38 (dd,
J
1
= 5.4 Hz,
J
2
= 2.4 Hz, 1H), 8.15 (sbr, 4H), 7.82 (t, J = 9.0 Hz, 1H),
+
6
.80 (s, 1H), 3.55 (d,
J
= 5.8 Hz, 2H), 3.30 (s, 2H) ppm. HRMS (ESI ): calcd for C H FN O (M +
1
3
11
7
3
+
H) 332.0902; found, 332.0899.
4
.1.8.19.
3-(3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-
yl)benzonitrile hydrochloride (12t). It was prepared by deprotection and salification of 11t (2.30 g, 5.5
1
mmol) according to the method of 12a, giving a white solid (1.50 g, 77%). H NMR (300 MHz, DMSO-
d6): δ = 8.24 (s, 1H), 8.16 (sbr, 3H), 8.08–8.03 (m, 2H), 7.82 (t,
J
= 7.9 Hz, 1H), 6.83 (t,
J
= 5.5 Hz,
+
+
1
3
H), 3.55 (d,
J
= 5.8 Hz, 2H), 3.04 (s, 2H) ppm. HRMS (ESI ): calcd for C H N O (M + H)
1
3
12
7
3
14.0996; found, 314.1002.
4
.1.8.20.
3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(4-fluoro-3-methylphenyl)-1,2,4-
oxadiazol-5(4H)-one hydrochloride (12u). It was prepared by deprotection and salification of 11u (2.3
2
6

1
g, 5.5 mmol) according to the method of 12a, giving a white solid (1.50 g, 77%). H NMR (300 MHz,
DMSO-d6): δ = 8.19 (sbr, 4H), 7.61–7.52 (m, 2H), 7.33 (t,
J
= 9.1 Hz, 1H), 6.82 (s, 1H), 3.54 (s, 2H),
+
+
3
3
.20 (s, 2H), 2.26 (s, 3H) ppm. HRMS (ESI ): calcd for C H FN O (M + H) 321.1106; found,
1
3
14
6
3
21.1102.
4
.1.8.21.
3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(m-tolyl)-1,2,4-oxadiazol-5(4H)-one
hydrochloride (12v). It was prepared by deprotection and salification of 11v (2.3 g, 5.8 mmol) according
1
to the method of 12a, giving a white solid (1.57 g, 79%). H NMR (300 MHz, DMSO-d6): δ = 8.12 (sbr,
3
H), 7.42–7.37 (m, 4H), 6.84 (t,
J
= 5.3 Hz, 1H), 3.52 (d, J = 5.9 Hz, 2H), 3.31 (s, 2H), 2.34 (s, 3H)
+
+
ppm
.
HRMS (ESI ): calcd for C H N O (M + H) 303.1200; found, 303.1196.
1
3
15
6
3
4
1
.1.9. Protocol for the synthesis of methyl N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-
,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]-P-methylphosphonamidate (15a)
A 50 mL round-bottomed flask with a magnetic stir bar was charged with hydrochloride 12a (0.42 g,
1
2
mmol), dry dichloromethane (30 mL) under N atmosphere. The resulting suspension was cooled in an
ice bath and stirred. At 0 °C, a solution of phosphonochloridate 14a (0.38 g, 3 mmol, 3 equiv) in
dichloromethane (5 mL) was syringed to the reaction mixture and stirring continued for 30 min. Next a
solution of triethylamine (0.30g, 3 mmol, 3 equiv) in dichloromethane (5 mL) was then syringed to the
mixture while maintained the temperature below 3 °C. After stirring for 30 min, the reaction mixture
was allowed to warm to room temperature and the hydrochloride gradually dissolved over time. On
completing of the reaction monitored by TLC, the solvent was removed under reduced pressure and the
residue was diluted with 1 N hydrochloric acid (10 mL) and extracted with ethyl acetate (2 × 20 mL).
2
The organic phases were combined and washed with water, brine and dried over anhydrous Na SO4.
After being filtered and concentrated in vacuo, the desired product was obtained was a pale-yellow solid
without further purification and used for next step directly.
2
7