Nitrobenzofurazan derivatives of N′-hydroxyamidines as potent inhibitors of indoleamine-2,3-dioxygenase 1

Article abstract of DOI:10.1016/j.ejmech.2016.05.061

Tryptophan metabolism through the kynurenine pathway is considered as a crucial mechanism in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tryptophan catabolism in the immune system and it is also considered as an important therapeutic target for the treatment of cancer and other diseases that are linked with kynurenine pathway. In this study, a series of nitrobenzofurazan derivatives of N′-hydroxybenzimidamides (1) and N′-hydroxy-2-phenylacetimidamides (2) were synthesized and their inhibitory activities against human IDO1 enzyme were tested using in-vitro and cellular enzyme activity assay. The optimization leads to the identification of potent compounds, 1d, 2i and 2k (IC₅₀ = 39-80 nM), which are either competitive or uncompetitive inhibitors of IDO1 enzyme. These compounds also showed IDO1 inhibition potencies in the nanomolar range (IC₅₀ = 50-71 nM) in MDA-MB-231 cells with no/negligible amount of cytotoxicity. The stronger selectivity of the potent compounds for IDO1 enzyme over tryptophan 2,3-dioxygenase (TDO) enzyme (312-1593-fold) also makes them very attractive for further immunotherapeutic applications.

Full text of DOI:10.1016/j.ejmech.2016.05.061

Accepted Manuscript
Nitrobenzofurazan derivatives of N′-hydroxyoximidines as potent inhibitors of
indoleamine-2,3-dioxygenase 1
Saurav Paul, Ashalata Roy, Suman Jyoti Deka, Subhankar Panda, Vishal Trivedi,
Debasis Manna
PII:
S0223-5234(16)30459-7
10.1016/j.ejmech.2016.05.061
EJMECH 8653
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 March 2016
Revised Date: 25 May 2016
Accepted Date: 26 May 2016
Please cite this article as: S. Paul, A. Roy, S.J. Deka, S. Panda, V. Trivedi, D. Manna,
Nitrobenzofurazan derivatives of N′-hydroxyoximidines as potent inhibitors of indoleamine-2,3-
dioxygenase 1, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.061.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Nitrobenzofurazan Derivatives of N′-Hydroxyoximidines as potent
Inhibitors of Indoleamine-2,3-Dioxygenase 1
Saurav Paul,‡,a Ashalata Roy,‡, a Suman Jyoti Deka,b Subhankar Panda,a Vishal Trivedi
,a
,b and Debasis Manna
aDepartment of Chemistry, Indian Institute of Technology Guwahati, Assam 781039,
India
bDepartment of Bioscience and Bioengineering, Indian Institute of Technology Guwahati,
Assam 781039, India
Corresponding Author
Department of Chemistry, Indian Institute of Technology Guwahati, Assam-781039,
India. E-mail: dmanna@iitg.ernet.in; Fax: +91 03 61258 2350; Tel: +91 03 61258 2325.
ABBREVIATIONS USED
IDO1, indoleamine 2,3-dioxygenase 1; TDO, tryptophan 2,3-dioxygenase;
L
-Trp, -
L
tryptophan; HTS, High throughput screening; UV-Vis, ultraviolet-visible; HPLC, high-
performance liquid chromatography.
1

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Abstract
Tryptophan metabolism through the kynurenine pathway is considered as a crucial
mechanism in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role
in tryptophan catabolism in the immune system and it is also considered as an important
therapeutic target for the treatment of cancer and other diseases that are linked with
kynurenine pathway. In this study, a series of nitrobenzofurazan derivatives of N'-
hydroxybenzimidamides (1) and N'-hydroxy-2-phenylacetimidamides (2) were
synthesized and their inhibitory activities against human IDO1 enzyme were tested using
in-vitro and cellular enzyme activity assay. The optimization leads to the identification of
potent compounds, 1d, 2i and 2k (IC50 = 39-80 nM), which are either competitive or
uncompetitive inhibitors of IDO1 enzyme. These compounds also showed IDO1
inhibition potencies in the nanomolar range (IC50 = 50-71 nM) in MDA-MB-231 cells
with no/negligible amount of cytotoxicity. The stronger selectivity of the potent
compounds for IDO1 enzyme over tryptophan 2,3-dioxygenase (TDO) enzyme (312 to
1593-fold) also makes them very attractive for further immunotherapeutic applications.
KEYWORDS
N′-Hydroxyoximidines, Mechanism-based drug design, IDO1 inhibition, high selectivity,
low cytotoxicity.
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1. Introduction
Cancer immunotherapy by targeting indoleamine 2,3-dioxygenase 1 (IDO1) and
tryptophan 2,3-dioxygenase (TDO) enzymes is considered as an exciting approach for
drug development [1, 2]. Both IDO1 and TDO enzymes catalyze the initial and rate
limiting step in the catabolism of L-tryptophan (
oxidative cleavage of the pyrrole ring through kynurenine pathway [3, 4]. Uncontrolled
metabolism of -Trp abets tumor cells to escape from the immune responses [1, 4].
Reduction in local concentrations of -Trp and uninhibited formation of kynurenine and
L-Trp) to N-formylkynurenine by
L
L
other metabolites, including neurotransmitters serotonin and melatonin, excitotoxin
quinolinic acid, N-methyl-D-aspartate receptor antagonist kynurenic acid, and the
production of nicotinamide adenine dinucleotide (NAD) assists IDO1 enzyme to suppress
the local immune response by hindering the proliferation of T-lymphocyte in the G1-
phase of the cell cycle [1, 2, 5, 6]. Cytokines like interferon-γ are primarily responsible
for the over-expression of IDO1 enzyme in the macrophages, epithelial and dendritic
cells [7]. TDO enzyme is mainly expressed in the liver and catabolizes more than 90% of
the
L-Trp. This catabolism of
L-Trp in the liver by the TDO enzyme systematically
regulates
L-Trp balance in response to dietary intake. The over-expression of IDO1
enzyme is interconnected with poor prognosis in different cancers, including, ovarian and
pancreatic [1, 3, 8]. However, recently it is reported that endothelial IDO1 expression in
kidney tumors is associated with a better prognosis [9, 10]. Cellular IDO1 activities are
also related with neurodegenerative disorder HIV-1 encephalitis and age related cataract
[1, 11-14].
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Recent studies demonstrated that inhibition of IDO1 activity with small molecules
successfully restrain the abnormal growth of tumors and also showed complemented
effect with chemotherapeutic and radiotherapeutic treatment of malignant tumors [6, 15].
TDO enzyme is highly selective and preferably binds to -Trp. Whereas, the active site of
L
IDO1 enzyme is amenable to small molecules. Hence, IDO1 has emerged as an attractive
target in cancer immunotherapy. Presently, two IDO1 inhibitors, INCB024360 and
NLG919 are under clinical trials for the treatment of cancer and other diseases [16]. Even
though 1-D-MT is under clinical trial as kynurenine pathway inhibitor but its mechanism
of action is doubtful. Although, it is reported that D-1MT inhibit the kynurenine
production at high concentrations but 1-D-MT does not effectively restore IDO-induced
arrest of T-cell proliferation [1, 16-19]. Therefore, developments of small molecule-based
IDO1 inhibitors are essential to satisfactorily address this cancer immunotherapeutic
opportunity.
Incyte Corporation identified N-hydroxyamidines as reversible and potent
inhibitors of IDO1 enzyme. One of the most potent compounds 4-amino-N-(3-chloro-4-
fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboxymidimade (5l) showed IC50 values
of 67 and 19 nM for in-vitro and cellular enzyme activity assay, respectively [20]. This
potent compound lowered blood plasma level of kynurenine and restricted tumor growth
in a mouse model. N-hydroxyamidine moiety containing compound INCB024360 is
currently under clinical trial for the treatment of several types of cancer and other
diseases [1, 16]. Compound CBR703 with N-hydroxyamidine moiety notably alter the
activity of RNA polymerase from bacteria [21]. N-hydroxyamidines can be also used as
pro-drugs of amidines, which is an important tool in drug discovery [5]. Therefore,
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hydroxyamidine-based compounds show several biological activities and found to be a
useful drug scaffold. So far there are no other reports on the modifications of
hydroxyamidine scaffold for the improvement of IC50 values (for IDO1 enzyme
inhibitory activity) of the compounds like 4-amino-N-(3-chloro-4-fluorophenyl)-N′-
hydroxy-1,2,5-oxadiazole-3-carboxymidimade (5l) [20].
Here, we investigated IDO1 enzyme inhibitory activity by a series of N′-
hydroxyamidine moiety containing nitrobenzofurazan derivatives. Interestingly, the
resulting compounds proved to be potent inhibitors of IDO1 enzyme with inhibitory
potency in the nanomolar range under in vitro conditions. Selected compounds also
showed strong IDO1 enzyme inhibitory activity in MDA-MB-231 cells and almost
no/negligible amount of toxicity at the cellular level. Additional counter screening against
TDO enzyme showed their selectivity for IDO1 enzyme.
2. Results and Discussion
2.1. Chemistry
High throughput screening (HTS) by Incyte Corporation led to the discovery of
hydroxyamidine-based potent, competitive inhibitors of IDO1 enzyme [1, 20].
Hydroxyamidine containing compound INCB024360 in combination with
pembrolizumab is now in phase-I/II clinical trials for the treatment of several types of
cancers [1]. The other hydroxyamidine-based potent compound, 4-amino-N-(3-chloro-4-
fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboxymidimade (5l) showed enzymatic
and cellular IC50 values of 67 and 19 nM (in Hela cell), respectively and inhibit
melanoma growth in a mouse model [20]. A thorough structure–activity-relationship
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(SAR) studies revealed that the oxygen atom of these hydroxyamidines interact with the
heme-iron and play a decisive role in inhibiting IDO1 activity. Model structure also
proposed that the substitute phenyl ring of compound INCB024360 or 5l placed itself in
the hydrophobic “pocket-A”, whereas the furazan ring interacts with the 7-propionate of
the heme-group of IDO1 enzyme [1, 20]. Detailed mechanistic studies suggested that
addition of ferrous heme iron coordinated molecular oxygen across the C2-C3 double
bond is the prerequisite for the IDO1 promoted oxidative cleavage of
L-Trp in
extrahepatic tissues. These studies also proposed the formation of alkylperoxy
transition/intermediate state or dioxetane intermediate state during the transformation of
L-Trp to N-formylkynurenine by IDO1 enzyme [1, 16].
In our attempt to synthesize compounds containing hydroxyamidine scaffold with
better inhibitory potencies and to mimic the alkylperoxy transition/intermediate state, we
developed series of nitrobenzofurazan derivatives of substituted N'-
a
hydroxybenzimidamides (1) and N'-hydroxy-2-phenylacetimidamides (2), where one
oxygen atom of the peroxo-moiety is substituted with a nitrogen atom (Fig. 1). We
hypothesized
that
substituted
hydroxybenzimidamides
and
hydroxy-2-
phenylacetimidamides would be placed inside the hydrophobic “pocket A” and interact
with heme iron and can act as a mimic of the alkylperoxy transition/intermediate state for
the transformation of -Trp to N-formylkynurenine by IDO1 enzyme. Whereas, the
L
benzofurazan moiety may interact with the 7-propionate of the heme group. The
compounds 1 and 2 were synthesized in two steps under mild conditions. First,
condensation of hydroxylamine with corresponding benzonitrile and phenylacetonitrile
under basic condition produced substituted N'-hydroxybenzimidamide and N'-hydroxy-2-
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phenylacetimidamides with good yields [22, 23]. Further treatment of these
hydroxyamidines with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in the presence of
NaHCO3 under ethanolic solution produced the target compounds 1 and 2 with moderate
to good yields (Scheme 1) [24].
NO2
X
Y
+
+
+
NH3
COO-
NH3
COO-
NH3
COO-
3
HN
N
N
2
Z
HN
HN
N
HN
N
N
O
n
O
O
O
IDO1
O
n = 0, 1
O
O
HO
N
N
Fe2+
N
N
N
N
Fe2+
(A)
Fe2+
Fe2+
(C)
N
N
N
N
N
N
N
N
(B)
Fig. 1. Proposed alkylperoxy transition/intermediate state of IDO1 (A and B) and its
mimic (C).
2.2. Inhibitory activities of hydroxyamidines against purified IDO1 enzyme
The potencies of the synthesized nitrobenzofurazan derivatives in inhibiting the human
IDO1 enzyme activity were initially evaluated by measuring the changes in absorbance
values of the product produced from kynurenine and 4-dimethylaminobenzaldehyde (at
480 nm) in acidic medium [25-27]. Uv-Vis spectra of the pure compounds (50 nM to
1ꢀM) showed no or little interference with the activity assay. The measured Km and kcat
values of the purified hIDO1 enzyme with the -Trp as substrate were 47.8 ± 2.5 ꢀM and
L
3.7 ± 0.1 s-1, respectively. In an effort to further improve the efficacy of the
nitrobenzofurazan derivatives of N′-hydroxyamidines, we explored two general
modifications of the N′-hydroxyamidine structure: (1) modification of the carbon linker
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between the aryl ring and N′-hydroxyamidine moiety; (2) substitution of the aryl ring
(Fig.1).
Scheme 1: Synthesis of nitrobenzofurazan derivatives of N'-hydroxybenzimidamides and
N'-hydroxy-2-phenylacetimidamides.
Cl
NO2
X
Y
OH
X
N
X
Z
NH2
OH
N
N
CN
NH2OH.HCl,
O
Y
n
N
H
NO2
Y
n
Z
n
N
Et3N, MeOH,
60 °C, 12 h
NaHCO3,
EtOH-H2O
50 °C, 2 h
Z
N
N
O
[n = 0, 1]
2.2.1. Modification of the carbon linker between the aryl ring and N′-hydroxyamidine
moiety
We explored the role linker length and flexibility between the aryl ring and N′-
hydroxyamidine moiety on the potencies of the compounds (Table 1). The IC50 values of
compounds 1a and 2a against purified IDO1 enzyme clearly suggest that the
incorporation of an additional methylene group between the aryl ring and N′-
hydroxyamidine moiety resulted in a slender decrease in its inhibitory efficacy.
2.2.2. Substitution of the aryl ring
The effect of substitutions on the aryl ring was investigated for potential interactions with
the hydrophobic residues positioned within the “pocket-A”, by incorporating methyl or
halogen substitutions. Several research groups had successfully exploited these
hydrophobic interactions in the optimization of the IDO1 inhibition efficacies [1, 26, 28-
30].
Table 2 and 3 shows the IC50 values of nitrobenzofurazan derivatives of the
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substituted N'-hydroxybenzimidamides (1a-n) and N'-hydroxy-2-phenylacetimidamides
(2a-k), respectively. The IC50 value of the reported potent compound 5l under the
experimental conditions was 91 nM (Table 3), which is in accordance with the reported
values [20]. The N'-hydroxybenzimidamide compounds, 1b-n displayed strong hIDO1
Table 1: Inhibitory activity of the nitrobenzofurazan derivatives of N'-
hydroxybenzimidamide and N'-hydroxy-2-phenylacetimidamide against purified
human IDO1 enzyme.
Compound
IDO1 inhibition
IC50 (nM)a
Ligand efficiency
(LE) = 1.4 × pIC50/HACb
OH
NO2
N
268 ± 23
0.418
N
H
N
O
N
1a
NO2
OH
N
307 ± 22
0.396
N
N
O
H
N
2a
aIC50 values are the mean of five independent assays.
bHAC = heavy atom count.
inhibitory activities with IC50 values ranging from 63 to 204 nM (Table 2). The SAR
studies demonstrated that halogen substituted phenyl derivative 1b, 1d, 1f, 1j and 1n
have stronger IDO1 enzyme inhibition capabilities than the other synthesized N'-
hydroxybenzimidamides. Among these tested N'- hydroxybenzimidamides derivatives,
ortho-fluoro 1b showed stronger hIDO1 inhibitory activity (IC50 = 63 nM) than original
benzimidamide lead 1a. A substantial preference for the fluoro-substitution at the ortho-
(1b) meta- (1f) and para- (1j) positions of the phenyl ring was also observed from IDO1
enzyme inhibition studies. Various other substitutions showed moderate inhibition
potencies but with IC50 values still in the nanomolar range. Further optimizations
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revealed that di-halogen (3-Br, 4-F) substituted compound, 1n showed stronger IDO1
inhibition potency [20]. However, other disubstituted compound, 1m failed to show
stronger inhibition efficiency. Nitrobenzofurazan derivatives of N'-hydroxy-2-
phenylacetimidamides, 2b-k also exhibited strong hIDO1 inhibitory activities with IC50
values ranging from 39 to 307 nM (Table 3). The SAR studies demonstrated that
halogen-substituted benzyl derivatives 2b, 2f, 2i and 2k were more potent than the
original benzyl lead 2a. Among these tested N'-hydroxy-2-phenylacetimidamide
derivatives, 3,4-dichloro substituted compound, 2k showed (IC50
= 39 nM) stronger
hIDO1 inhibitory activity than the lead compound 2a (IC50 307 nM). 3,4-Difluoro
=
substituted compound 2i showed (IC50 = 59 nM) stronger hIDO1 inhibition potency than
unsubstituted parent compound, 2a. Preference for fluoro-substitution at meta-, ortho-
and para- positions were also demonstrated by the modest improvement in hIDO1
enzyme inhibitory activity. Interestingly 3,4-dichloro-substitution (2k) considerably
enhances the hIDO inhibitory potency than its respective mono substituted derivatives
(2d, 2f). Similarly, synergistic effect of 3,4-difluoro substitution was also observed for
compound (2j).
All of these IDO1 inhibition studies were performed by spectrophotometric
technique. For further confirmation of their efficacies, IDO1 activity assay was also
performed by HPLC analysis. The amount of kynurenine generated due to the catabolism
of
L-Trp, was directly quantified by HPLC analysis [28]. A standard curve generated from
pure kynurenine under similar experimental conditions was used to calculate the
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Table 2: Inhibitory activity of the nitrobenzofurazan derivatives of N'-
hydroxybenzimidamides against purified human IDO1 enzyme.
OH
NO2
R1
N
R2
R3
N
H
N
O
N
1b-n
Compound
IDO1 inhibition
IC50 (nM)a
Ligand efficiency
(LE) = 1.4 × pIC50/HACb
1b
R1 = F; R2 = R3 = H
1c
R1 = Cl; R2 = R3 = H
1d
R1 = Br; R2 = R3 = H
1e
R1 = H; R2 = CH3; R3 = H
1f
R1 = H; R2 = F; R3 = H
1g
R1 = H; R2 = Cl; R3 = H
1h
R1 = H; R2 = Br; R3 = H
1i
R1 = R2 = H; R3 = CH3
1j
R1 = R2 = H; R3 = F
1k
R1 = R2 = H; R3 = Cl
1l
R1 = R2 = H; R3 = Br
1m
R1 = H; R2 = Cl; R3 = F
1n
63 ± 3
135 ± 17
80 ± 6
0.438
0.418
0.431
0.413
0.433
0.410
0.425
0.407
0.426
0.423
0.422
0.397
0.411
163 ± 8
75 ± 5
184 ± 6
105 ± 3
204 ± 11
100 ± 9
113 ± 7
117 ± 1
158 ± 26
92 ± 1
R1 = H; R2 = Br; R3 = F
aIC50 values are the mean of five independent assays.
bHAC = heavy atom count.
inhibitory activity of the compounds. The calculated concentrations of the compounds
required to inhibit the kynurenine production from the -Trp were in the nanomolar range
L
(Table S1) and in accordance with the measured IC50 values calculated using the
pDMAB-method. The IC50 values of compounds 1b, 1d, 1j, 2i and 2k (as measured by
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HPLC method) are within 49-64 nM which is lower than the reported potent compound
5l, under the similar experimental conditions [20].
Therefore, most of the compounds displayed stronger hIDO1 inhibitory activities.
The common characteristic of these compounds is that, all of them possess
nitrobenzofurazan and N'-hydroxyamidine moieties, which emerge to be critical for their
stronger hIDO1 inhibitory activity. Separate inhibition activity studies with 4-chloro-7-
nitrobenzofurazan and 2-(3,4-difluorophenyl)-N'-hydroxyacetimidamide, a precursor for
compound 2i showed very weak or no inhibition of IDO1 enzyme activity under the
similar experimental conditions (data not shown here). This implies the importance of
both
nitrobenzofurazan
and
N'-hydroxybenzimidamides
or
N'-hydroxy-2-
phenylacetimidamide moiety for stronger inhibition of hIDO1 activity. The compounds
from 1a-n and 2a-k families are separated by one methylene group. Although the
structures of the compounds from both the series are highly related, but their IC50 values
indicate a substantial differences among their inhibition activities against purified IDO1
enzyme. However, the IC50 values of the compounds suggest that the strong interaction of
N'-hydroxy or nitrobenzofurazan moiety of the compounds with the IDO1 enzyme could
suppress the effect of substitution on the phenyl ring of N'-hydroxybenzimidamides or N'-
hydroxy-2-phenylacetimidamides. ESI-MS spectral analyses of the selected potent
compounds in water, 100 mM phosphate buffer at pH 6.5 and enzymatic assay solution
revealed that these compounds are also stable under the experimental conditions
(Supporting Information, Fig. S1-S5).
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Table 3: Inhibitory activity of the nitrobenzofurazan derivatives of N'-hydroxy-2-
phenylacetimidamides against purified human IDO1 enzyme
NO2
R3
R2
OH
N
N
H
N
N
R1
O
2b-k
Compound
IDO1 inhibition
IC50 (nM)a
Ligand efficiency
(LE) = 1.4 × pIC50/HACb
2b
R1 = F; R2 = R3 = H
102 ± 5
131 ± 4
199 ± 11
128 ± 3
127 ± 1
112 ± 7
98 ± 10
59 ± 5
0.408
0.401
0.391
0.386
0.402
0.405
0.409
0.405
0.380
2c
R1 = H; R2 = F; R3 = H
2d
R1 = H; R2 = Cl; R3 = H
2e
R1 = R2 = H; R3 = OCH3
2f
R1 = R2 = H; R3 = Cl
2g
R1 = R2 = H; R3 = Br
2h
R1 = R2 = H; R3 = F
2i
R1 = H; R2 = R3 = F
2j
R2 = H; R1 = R3 = F
160 ± 12
39 ± 1
2k
R1 = H; R2 = R3 = Cl
0.415
0.547
5lc
F
OH
91 ± 2
N
NH2
Cl
N
H
N
N
O
aIC50 values are the mean of five independent assays.
bHAC = heavy atom count.
cReported compound
2.3. Cellular IDO1 inhibitory activities of hydroxyamidines
To investigate the therapeutic potential of these hydroxyamidines, the ability of the
potent compounds in inhibiting the IDO1 activity under the in vitro cellular environment
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was measured using MDA-MB-231 breast cancer cell line. It is well demonstrated that
MDA-MB-231 cells express mRNA of native human IDO1 enzyme and interferon (IFN)-
γ considerably induce the IDO1 enzyme expression level in MDA-MB-231 cells [31]. In
this regard, MDA-MB-231 cells were first treated with human IFN-γ (20 ng/mL, Fig. S6)
for 48 h and then cells were grown in the presence of tested compounds (20, 50, 100 and
500 nM) and -Trp (150ꢀM). The IDO1 activity was determined by measuring the
L
formation of L-kynurenine using the pDMAB-method (Table 4) [27, 28, 31]. The
calculated cellular IC50 values of most of the tested compounds are in accordance with
our in vitro enzyme assay data. The calculated IC50 values of 1d, 2i and 2k are within 50-
71 nM range in MDA-MB-231 cells. Control compound 5l showed IC50 values of 59 nM
(Table 4) under the similar experimental conditions [20]. The deviation in IC50 values of
the compounds between the enzymatic assay against purified IDO1 and cellular assays
could be due to the methylene blue-ascorbate regeneration system which maintains IDO1
in active state (Fe2+) or environmental effect on the assay system. In general, a good
correlation between these assays confirmed that these tested hydroxyamidine analogues
are potent inhibitors of IDO1 enzyme. MTT assay of the compounds (concentrations of
IC50 and 2 × IC50 values from the enzymatic assay) in MDA-MB-231 cells also
demonstrated no/negligible level of toxicity of the compounds under the tested conditions
(Fig. S7). However, small discrepancy between the IC50 values of compounds 2i and 2k
(as measured by in vitro enzymatic and cellular assay) could be also due to the toxicity of
the dichloro substituted compound 2k over difluoro substituted compound 2i (Fig. S8).
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Table 4: IDO1 enzyme inhibitory activity of the selected compounds in MDA-MB-
231 cells.
Compound IDO1 inhibition in
MDA-MB-231 cella
IC50 (nM)b
131 ± 17
1b
1d
1f
66 ± 8
242 ± 15
125 ± 17
107 ± 12
131 ± 14
110 ± 19
151 ± 17
50 ± 8
1j
1n
2b
2f
2h
2i
71 ± 10
59 ± 4
2k
5lc
aIDO protein expression in MDA-MB-231 cells was induced by human IFN-γ (20 ng/mL).
bIC50 values are the mean of three independent assays.
cReported compound
2.4. Mode of IDO1 enzyme inhibition by the potent hydroxyamidines
The enzyme kinetics of selected compounds was performed and their mode of IDO1
inhibition was calculated from the plots of [S]/V against inhibitor concentrations. Four
compounds from both the series of hydroxyamidines were selected best on their stronger
inhibitory activities against purified IDO1 enzyme. The results showed that compounds
1b, 1n, 2b, 2h, 2i and 2k had competitive modes of IDO1 enzyme inhibition whereas
compounds 1d, 1f and 1j followed uncompetitive modes of IDO1 enzyme inhibition
under the similar experimental conditions (Fig. 2 and S9). [S] and V represent the
substrate concentration and initial rate of the reaction, respectively [32]. It is important to
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8 104
7 104
6 104
5 104
4 104
3 104
2 104
1 104
0
1.2 105
[L-Trp] - 50 uM
[L-Trp] - 100 uM
[L-Trp] - 150 uM
[L-Trp] - 50 uM
[L-Trp] - 100 uM
[L-Trp] - 150 uM
(A)
(C)
1 105
8 104
6 104
4 104
2 104
0
-150
-100
-50
0
50
100
150
200
-150
-100
-50
0
50
100
150
200
[5l] (nM)
[1d] (nM)
8 104
4 104
3.5 104
3 104
2.5 104
2 104
1.5 104
1 104
5000
[L-Trp] - 50 uM
[L-Trp] - 100 uM
[L-Trp] - 150 uM
[L-Trp] - 50 uM
[L-Trp] - 100 uM
[L-Trp] - 150 uM
7 104
6 104
5 104
4 104
3 104
2 104
1 104
0
(D)
(E)
0
-150
-100
-50
0
50
100
150
200
-150
-100
-50
0
50
100
150
200
[2k] (nM)
[2i] (nM)
Fig. 2: Determination of mode of inhibition of the potent compounds. Plot of [S]/V
against concentrations of compounds 5l (A), 1d (B), 2i (C) and 2k (D). Concentration of
L-Trp was varied from 50 to 150 ꢀM. The concentrations of compounds were varied from
25 to 150 nM.
mention that IDO1 is an oxido-reductase enzyme and binding of O2 to the heme-group is
required for the transformation of
the tested compounds followed competitive or uncompetitive modes of enzyme inhibition
with respect to -Trp. However, these compounds may also follow different mode of
L-Trp to N-formylkynurenine. The results showed that
L
inhibition with respect to O2 under the similar experimental conditions. Hence, additional
16

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enzyme kinetics measurement with respect to O2 is required for detailed understanding,
which is beyond the scope of the current study. Compound 5l is a known competitive
inhibitor of hIDO1 enzyme which is in accordance with our experimental results [20].
2.5. Probable mode of interaction of the potent compounds with IDO1 enzyme
With confirmation that potent hydroxyamidines strongly inhibit IDO1 enzyme activity,
we performed molecular docking analyses to investigate the probable mode of interaction
of the potent compounds with the IDO1 enzyme (PDB code: 4PK5) [33]. The docked
structures propose that nitrobenzofurazan derivatives of the substituted hydroxyamidines
may have different modes of interactions with the IDO1 enzyme (Fig. 3 and S10).
Although both the substituted phenyl ring of the N'-hydroxy-benzimidamide and N'-
hydroxy-phenylacetimidamide moieties of the compounds are placed in ‘pocket-A’ of the
IDO1 enzyme, but the N'-hydroxy group may interact differently. For analogous of
compound 1 the N'-hydroxy group interacts with Ser167 residue and pyrrole ring of the
heme-group through hydrogen bonding (Fig. S9A-C). Whereas, for analogous of
compound 2 the N'-hydroxy group interacts with Fe-atom and pyrrole ring of the heme-
group. Hydrophobic amino acids like Phe163, Phe164, Tyr126 and others present in
‘pocket-A’ could be also involved in hydrophobic interaction with the substituted
benzene and phenyl groups. A similar mode of interaction was also observed for the
reported compounds 5l, which is accordance with the reported mode of interaction [20].
Therefore, both hydrogen bonding and hydrophobic interactions play important roles in
stronger binding of the compounds to the active site of IDO1 enzyme. However, there are
no considerable differences in mode of interaction of the compounds from the same
17

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series with the IDO1 enzyme (Fig. 3 and S10). Therefore, in addition to the presence of
nitrobenzofurazan and N′-hydroxyamidine moieties to the compound’s core structure,
volume and electronic properties of the substituents in the benzene ring are also crucial
for stronger binding to the IDO1 enzyme. These docking analyses also support that
substituted hydroxy-2-phenylacetimidamides may act as a mimic of alkylperoxy
transition/intermediate state of the transformation of
IDO1 enzyme.
L-Trp to N-formylkynurenine by
Fig. 3: Predicted mode of interaction of the compounds, 5l (A), 2i (B) and 2k (C) with
the active site of the human IDO1 enzyme (4PK5). The modeled structures were
generated using MoleGro Virtual Docker (MVD), version 6.0. The oxygen and nitrogen
atoms are shown in red and blue, respectively. Residues involved in interactions through
hydrogen bond formation are shown using dashed lines (pink color).
2.6. Inhibitory activities of hydroxyamidines against purified TDO enzyme
Strong IDO1 inhibition proficiency and very good ligand efficiency (LE > 0.4) of the
compounds prompted us to determine their selectivity for IDO1 over TDO enzyme. It is
well documented that TDO enzyme is primarily responsible for the catabolism of
L-Trp in
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liver and maintaining its level through kynurenine pathway. Counter-screening against
TDO enzyme showed that the potent compounds were appreciably inactive (IC50 ≥ 20
ꢀM) against TDO enzyme (Table 5). Tested compounds showed 100 to 1700-fold
stronger IDO1 inhibition in comparison with TDO enzyme, demonstrating their
selectivity for IDO1 enzyme. The most potent compound, 2i (based on the cellular assay)
exhibited over 1550-fold stronger IDO1 inhibitory activity against that of TDO enzyme.
HPLC-based TDO activity assay also showed that these potent compounds have
preferential selectivity for IDO1 over TDO enzyme inhibitions (Table S2).
Table 5: Inhibitory activity of the selected compounds against purified human IDO1
and TDO enzymes
Compound
Mode of hIDO1
Inhibition
hIDO1 inhibition
(IC50 (nM))a
hTDO inhibition
(IC50 (
M))a
Selectivity
ratiob
µ
competitive
uncompetitive
uncompetitive
NMc
63 ± 3
80 ± 6
110 ± 13
25 ± 3
45 ± 5
20 ± 1
21 ± 1
40 ± 2
50 ± 7
47 ± 1
30 ± 2
51 ± 8
43 ± 2
65 ± 12
94 ± 14
19 ± 1
32 ± 2
24 ± 1
1746
312
600
190
210
354
427
511
297
520
338
580
1593
118
820
263
1b
1d
1f
75 ± 5
105 ± 3
100 ± 9
113 ± 7
117 ± 1
92 ± 1
1h
1j
uncompetitive
NMc
1k
1l
NMc
competitive
competitive
NMc
1n
2b
2f
101 ± 5
98 ± 10
127 ± 1
112 ± 7
59 ± 5
NMc
2g
2h
2i
competitive
competitive
NMc
160 ± 12
39 ± 1
2j
competitive
competitive
2k
5ld
91 ± 2
aIC50 values are the mean of three independent assays against purified enzymes.
bSelectivity ratio is calculated as (IC50 value of IDO1)/ (IC50 value of TDO).
cNM = not measured
dReported compound
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In this study nitrobenzofurazan derivatives of the N'-hydroxy-benzimidamides
and N'-hydroxy-phenylacetimidamides were designed as a new class of IDO1 enzyme
inhibitors. Suitable substitutions in the benzene ring of the compounds lead to the
identification of potent compounds with nanomolar (< 100 nM) IDO1 enzyme inhibitory
activities under in vitro conditions. The potency of the selected compounds under cellular
environment (IC50 = 50-242 nM) and no/ negligible level of toxicity at the cellular level
reveal the therapeutic potential of the nitrobenzofurazan derivatives of N′-
hydroxyamidines. Measured IC50 values and molecular modeling studies of the
compounds suggest that the presence of hydroxyamidine and nitrobenzofurazan moieties
and suitable substitution in the benzene ring of the compounds play a critical role in their
stronger IDO1 enzyme inhibitory properties. Until now, only few hydroxyamidine based
compounds have been reported with such stronger IDO1 enzyme inhibition properties
[20]. The inhibition modes of the tested compounds were determined to be both either
competitive or uncompetitive. The potent hydroxyamidine analogues also showed 100 to
1700-fold stronger IDO1 enzyme inhibitory activities in comparison with TDO enzyme,
which catabolizes -Trp through the same mechanistic pathway. The low cytotoxicity and
L
inactivity for TDO enzyme support further development of nitrobenzofurazan derivatives
of hydroxyamidines.
3. Conclusion
In this study, we synthesized a new series of nitrobenzofurazan derivatives of N′-
hydroxyamidines. Most of the tested compounds showed strong inhibitory activities
against purified IDO1 enzyme. The presence of both N'-hydroxyamidine and
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nitrobenzofurazan moieties in the compound’s core structure could be the driving force
for their strong inhibitory activities. Halogen substitutions in the aryl ring were successful
in improving the potency. IDO1 inhibition assay in the interferon-γ-induced MBA-MB-
231 cells showed that the nitrobenzofurazan derivatives of hydroxyamidines 1d, 2i and
2k have minimal toxicity and stronger potencies. These hydroxyamidine derivatives also
exhibited stronger selectivity for IDO1 enzyme over TDO enzyme. These compounds
also could be considered as good molecular probes on the basis of their ligand efficiency
values. Overall, our findings suggest that these nitrobenzofurazan derivatives of the N′-
hydroxyamidines can be a useful structural class of compounds for cancer and other
human diseases.
4. Experimental section
4.1. General information
All reagents were purchased from different commercial sources and used directly without
further purification. Column chromatography was performed using 60―120 mesh silica
gel. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254
(0.25 mm). 1H NMR and 13C NMR were recorded at 400 and 100 MHz respectively, with
Varian AS400 spectrometer and 600 and 151 MHz respectively, with Brucker
spectrometer, using TMS as an internal standard with CDCl3 and DMSO-d6. The
coupling constant (J values) and chemical shifts (δppm) were described in Hertz (Hz) and
parts per million (ppm) respectively. Multiplicities are described as follows: s (singlet), d
(doublet), t (triplet), m (multiplet) and br (broadened). High resolution mass spectra
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(HRMS) were recorded at Agilent Q-TOF mass spectrometer with Z-spray source using
built-in software for analysis of the recorded data.
4.2. General procedure for the synthesis of substituted hydroxyamidines
To a stirring solution of substituted benzonitrile/ phenylacetonitrile (1 mmol) in
anhydrous methanol (20 mL) under N2 atmosphere was added hydroxylamine
hydrochloride (1.2 mmol). Triethylamine (2.5 mmol) was added under continuous
stirring condition and the mixture was then heated under reflux condition for overnight at
60 °C and continued until complete consumption of the starting material (monitored by
TLC) [22, 23]. The excess solvent was then removed under reduced pressure and the
obtained residue was washed successively with water and brine, and extracted with
ethylacetate (3 × 10 mL).The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. This reaction mixture was purified using silica gel
column chromatography with a gradient solvent system of 10-20% ethylacetate to hexane
to obtain the desired product with an average yield of 70-80%.
4.3. General procedure for the synthesis of 2-substituted N'-hydroxy, N- (7 nitro
benzoxadiazole) imidamide
Sodium bicarbonate (3.5 mmol) was added to a stirring solution of 2-substituted N'-
hydroxyimidamide (1.1 mmol) in 20 ml of EtOH/H2O (9:1) and the solution was stirred
for 10 min at room temperature. To this mixture an ethanolic solution of 4-chloro 7-
nitrobenzofurazan (1 mmol) was added drop wise under continuous stirring condition and
the resulting solution was stirred at 50 °C for another 2-4 hours [24]. After completion of
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the reaction (monitored by TLC) the excess solvent was removed under reduced pressure
and the pH adjusted to 1.5 using 1N HCl solution. Then aqueous layer was extracted with
dichloromethane (3 × 20 ml) and the organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The reaction mixture was further purified using
silica gel column chromatography using 20-30% ethylacetate to hexane gradient solvent
system to afford the desired pure product with 60-90% yield.
4.4. Characterization of the synthesized compounds
(Z)-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1a): As yellow
1
solid (85 % yield; mp: 201-203 °C); H NMR (600MHz, CDCl3 + DMSO-d6) δppm 8.50
(d, 1H, J = 12 Hz), 7.70 (d, 1H, J = 12 Hz), 7.46-7.36 (m, 5H), 6.12 (br s, 1H);13C NMR
(151 MHz, CDCl3 + DMSO-d6) δppm 157.7, 154.3, 144.1, 144.0, 134.9, 131.5, 130.6,
129.2, 128.9, 126.9, 106.7; HRMS (ESI) calcd. for C13H9N5O4 [M + H]+ 300.0727, found
300.0725.
(Z)-2-fluoro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1b):
As yellow solid (60 % yield; mp: 134-135 °C); 1H NMR (400MHz, DMSO-d6) δppm 8.55
(d, 1H, J = 8.8 Hz), 7.64-7.62 (m, 1H), 7.47-7.45 (m, 1H), 7.37 (d, 1H, J = 8.4 Hz), 7.23-
7.13 (m, 2H), 6.74 (br, s, 1H); 13C NMR (151 MHz, CDCl3+DMSO-d6) δppm 160.7,
154.2, 153.5, 143.2, 143.1, 134.6, 131.8, 129.7, 128.0, 123.6, 115.7, 115.4, 106.1; HRMS
(ESI) calcd. for C13H8FN5O4 [M + H]+ 318.0633, found 318.0635.
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(Z)-2-chloro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1c):
As yellow solid (70 % yield; mp: 178-180 °C); 1H NMR (600MHz, CDCl3 + DMSO-d6)
δppm 8.45 (d, 1H, J = 12 Hz), 7.48-7.46 (m, 1H), 7.40-7.35 (m, 2H), 7.32-7.27 (m, 2H),
6.24 (br s, 1H); 13C NMR (151 MHz, CDCl3 + DMSO-d6) δppm 161.3, 158.5, 148.0,
147.9, 139.5, 139.3, 136.9, 135.6, 134.8, 134.7, 132.8, 131.0, 110.9; HRMS (ESI) calcd.
for C13H8ClN5O4 [M + H]+ 334.0338, found 334.0340.
(Z)-2-bromo-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1d):
1
As brown solid (60 % yield; mp: 162-164 °C); H NMR (400MHz, CDCl3 + DMSO-d6)
δppm 8.50 (d, 1H, J = 8.4 Hz), 7.61 (d, 1H, J = 7.6 Hz), 7.49-7.42 (m, 1H), 7.37-7.29 (m,
3H), 6.53 (br, s, 1H); 13C NMR (151 MHz, CDCl3 + DMSO-d6) δppm 157.8, 154.0, 143.7,
143.5, 134.9, 133.0, 132.2, 131.4, 130.8, 128.5, 127.2, 121.7, 106.5; HRMS (ESI) calcd.
for C13H8BrN5O4 [M + H]+ 377.9832, found 377.9835.
(Z)-N'-hydroxy-3-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1e):
As yellow solid (75 % yield; mp: 203-205 °C); 1H NMR (400MHz, CDCl3 + DMSO-d6)
δppm 8.38-8.35 (m, 1H), 7.58-7.56 (m, 1H),7.30-7.21 (m, 4H), 6.18 (br, s, 1H), 2.29 (s,
3H); 13C NMR (151 MHz, CDCl3 + DMSO-d6) δppm 157.8, 154.6, 144.2, 135.1, 132.3,
131.3, 130.6, 128.7, 126.9, 106.7, 22.8; HRMS (ESI) calcd. for C14H11N5O4 [M + H]+
314.0884, found 314.0880.
(Z)-3-fluoro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1f): As
yellow solid (75 % yield; mp: 167-169 °C); 1H NMR (600MHz, CDCl3 + DMSO-d6) δppm
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8.54-8.51 (m, 1H), 7.75-7.72 (m, 1H), 7.47-7.39 (m, 3H), 7.35-7.34 (m, 1H), 6.23 (br, s,
1H); 13C NMR (151 MHz, CDCl3 + DMSO-d6) δppm 157.7, 154.3, 143.9, 143.7, 135.0,
131.1, 130.5, 128.8, 128.6, 126.8, 106.5; HRMS (ESI) calcd. for C13H8FN5O4 [M + H]+
318.0633, found 318.0635.
(Z)-3-chloro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1g):
As yellow solid (77 % yield; mp: 210-212 °C);1H NMR (400MHz, CDCl3 + DMSO-d6)
δppm 8.64 (d, 1H, J = 8.8 Hz), 7.87 (s, 1H), 7.77 (d, 1H, J = 7.6 Hz), 7.53-7.51 (m, 2H),
7.47-7.43 (m, 1H), 6.72 (br, s, 1H); 13C NMR (100 MHz, CDCl3 + DMSO-d6) δppm 156.2,
153.8, 143.6, 143.4, 134.8, 133.9, 132.0, 130.6, 129.5, 128.5, 126.7, 124.8, 106.3; HRMS
(ESI) calcd. for C13H8ClN5O4 [M + H]+ 334.0338, found 334.0340.
(Z)-3-bromo-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1h):
As yellow solid (78 % yield; mp: 189-191 °C); 1H NMR (400MHz, CDCl3 + DMSO-d6)
δppm 8.53-8.49 (m, 1H), 7.90-7.87 (m, 1H), 7.69-7.65 (m, 1H), 7.56-7.52(m, 1H),7.42-
7.40 (m, 1H), 7.29-7.24 (m, 1H), 6.40 (br, s, 1H); 13C NMR (151 MHz, CDCl3 + DMSO-
d6) δppm 156.3, 154.0, 134.8, 133.9, 132.5, 130.1, 129.8, 125.5, 106.5; HRMS (ESI) calcd.
for C13H8BrN5O4 [M + H]+ 377.9832, found 377.9828.
(Z)-N'-hydroxy-4-methyl-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1i):
As yellow semi solid (70 % yield); 1H NMR (600MHz, CDCl3 + DMSO-d6) δppm 8.65 (d,
1H, J = 12 Hz), 7.71 (d, 2H, J = 12 Hz), 7.57 (d, 1H, J = 12 Hz), 7.37 (d, 2H, J = 12 Hz),
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13
1.31 (s, 3H); C NMR (151 MHz, CDCl3 + DMSO-d6) δppm157.5, 154.2, 143.8, 143.6,
140.7, 135.8, 128.8, 128.1, 127.5, 126.6, 106.7, 21.0; HRMS (ESI) calcd. for C14H11N5O4
[M + H]+ 314.0884, found 314.0882.
(Z)-4-fluoro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1j): As
yellow solid (70 % yield; mp: 191-193 °C); 1H NMR (600MHz, CDCl3) δppm 8.58 (d, 1H,
J = 8.4 Hz), 7.79-7.76 (m, 2H),7.49 (d, 1H, J = 8.4 Hz), 7.22-7.19 (m, 2H), 5.49 (br, s,
1H);13C NMR (151 MHz, CDCl3) δppm 156.5, 154.1, 144.0, 134.8, 129.1, 129.0, 116.6,
116.5, 106.9; HRMS (ESI) calcd. for C13H8FN5O4 [M + H]+318.0633, found 318.0635.
(Z)-4-chloro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide (1k):
As orange solid (50 % yield; mp: 200-202 °C); 1H NMR (600MHz, CDCl3 + MeOD-d4+
DMSO-d6) δppm 8.56 -8.54 (m, 1H), 7.74-7.72 (m, 2H), 7.44-7.41 (m, 1H), 7.38-7.36 (m,
13
2H), 6.68 (br, s, 1H); C NMR (151 MHz, CDCl3 + MeOD-d4 + DMSO-d6) δppm 156.8,
154.2, 144.0, 143.8, 136.8, 135.3, 129.1, 128.7, 128.3, 106.7; HRMS (ESI) calcd. for
C13H8ClN5O4 [M + H]+334.0338, found 334.0338.
(Z)-4-bromo-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzimidamide
(1l):
As orange solid (65 % yield; mp: 213-215 °C); 1H NMR (400MHz, CDCl3 + DMSO-d6)
δppm 8.61-8.59 (m, 1H), 7.73-7.71 (m, 2H), 7.56-7.54 (m, 2H), 7.44-7.42 (m, 1H), 6.98
(br, s, 1H); 13C NMR (100 MHz, CDCl3 + DMSO-d6) δppm 156.6, 154.0, 143.7, 143.5,
135.4, 131.3, 129.5, 128.5, 128.3, 124.7, 106.6; HRMS (ESI) calcd. for C13H8BrN5O4 [M
+ H]+ 379.9813, found 379.9848.
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(Z)-3-chloro-4-fluoro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
1
yl)benzimidamide (1m): As yellow solid (50 % yield; mp: 251-253 °C); H NMR
(600MHz, MeOD-d4) δppm 8.43 (d, 2H, J = 8 Hz), 7.42 (s, 1H), 6.08 (d, 2H, J = 8 Hz); 13C
NMR (151 MHz, MeOD-d4+ DMSO-d6) δppm 148.5, 146.5, 139.3, 114.7, 112.1; HRMS
(ESI) calcd. for C13H7ClFN5O4 [M + H]+ 351.0171 found 351.0168.
(Z)-3-bromo-4-fluoro-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
1
yl)benzimidamide (1n): As brown solid (60 %; yield; mp: 112-113 °C); H NMR
(600MHz, CDCl3 + DMSO-d6) δppm 8.55 (d, 1H, J = 12 Hz), 8.03-8.01 (m, 1H), 7.95 (s,
13
1H), 7.75-7.71 (m, 1H), 7.44 (d, 1H, J = 12 Hz), 7.21-7.17 (m, 1H), 6.20 (br s, 1H); C
NMR (100 MHz, MeOD-d4 + CDCl3 + DMSO-d6) δppm 160.9, 159.2, 155.5, 153.7, 143.5,
143.4, 134.9, 131.7, 128.4, 127.9, 116.2, 116.0, 106.3; HRMS (ESI) calcd. for
C13H7BrFN5O4 [M + H]+ 397.9719, found 397.9752 .
(Z)-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-2-phenylacetimidamide (2a):
As red semi solid (90% yield); 1H NMR (400MHz, CDCl3 + DMSO-d6) δppm8.59 (d, 1H,
J = 8 Hz), 7.45 (d, 1H, J = 8 Hz), 7.41-7.34 (m, 5H), 5.10 (br, s, 1H), 3.66 (s, 2H);13C
NMR (151 MHz, CDCl3 + DMSO-d6) δppm 158.5, 154.4, 144.2, 144.0, 135.1, 134.9,
129.2, 129.0, 127.8, 106.7, 37.2; HRMS (ESI) calcd. for C14H11N5O4 [M + H]+ 314.0884,
found 314.0909.
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(Z)-2-(2-fluorophenyl)-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)acetimidamide (2b): As yellow solid (85 % yield; mp:148-150 °C);1H NMR (600MHz,
CDCl3 + DMSO-d6) δppm 8.48-8.45 (m, 1H), 7.29-7.25 (m, 2H), 7.20-7.18 (m, 1H), 7.06-
7.04 (m, 1H), 7.01-6.99 (m, 1H), 5.69 (br, s, 1H), 3.57 (s, 2H); 13C NMR (100 MHz,
CDCl3 + DMSO-d6) δppm 157.9, 154.4, 144.2, 144.0, 135.2, 131.1, 129.6, 129.6, 127.2,
124.8, 119.7, 115.6, 106.6, 30.5; HRMS (ESI) calcd. for C14H10FN5O4 [M + H]+
332.0790, found 332.0789.
(Z)-2-(3-fluorophenyl)-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)acetimidamide (2c): As yellow solid (70 % yield; mp:140-142 °C);1H NMR (600MHz,
CDCl3 + DMSO-d6) δppm8.61-8.54 (m, 1H), 7.38-7.31 (m, 3H), 7.20-6.93 (m, 2H), 5.84
13
(s, 1H), 3.55 (s, 2H); C NMR (151 MHz, CDCl3 + DMSO-d6) δppm163.9, 162.3, 158.3,
154.5, 144.2, 144.0, 137.8, 135.2, 130.5, 124.7, 116.1, 114.6, 106.6, 36.9; HRMS (ESI)
calcd. for C14H10FN5O4 [M + H]+ 332.0790, found 332.0791.
(Z)-2-(3-chlorophenyl)-N'-hydroxy-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
1
yl)acetimidamide (2d): As orange solid (87% yield; mp: 152-154 °C); H NMR
(400MHz, CDCl3 + DMSO-d6) δppm 8.59 (d, 1H, J = 8 Hz), 7.44 (d, 1H, J = 8 Hz), 7.36-
7.32 (m, 3H), 7.26-7.24 (m, 1H), 5.14 (br, s, 1H), 3.64 (s, 2H);13C NMR (151 MHz,
CDCl3 + DMSO-d6) δppm 158.0, 154.0, 143.5, 143.4, 137.3, 134.8, 129.5, 128.4, 126.8,
126.6, 106.0, 36.1; HRMS (ESI) calcd. for C14H10ClN5O4 [M + H]+348.0494, found
348.0525 .
28