Design, synthesis and biological evaluation of novel naphthoquinone derivatives as IDO1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.08.013

Indoleamine 2,3-dioxygenase 1 (IDO1) mediated kynurenine pathway of tryptophan degradation is identified as an appealing and novel target in immunotherapy for the treatment of cancer. In this study, a novel series of naphthoquinone derivatives were synthesized, characterized and evaluated for their inhibitory activities against IDO1, and their structure?activity relationship was investigated. Among them, compounds T16, T44, T47, T49, T53 and T54 displayed potent IDO1 inhibitory activities with IC₅₀ values ranging between 18 and 61 nM, which are more potent than INCB024360 undergoing clinical trial III evaluation. In addition, compounds T28, T44 and T53 decreased the kynurenine levels in rat plasma by 30%–50%. Compounds exhibiting excellent IDO1 inhibitory activities were also evaluated for their inhibitory activities against tryptophan 2,3-dioxygenase (TDO). Of which, compound T28 (IDO1 IC₅₀ = 120 nM) showed promising TDO inhibition (IC₅₀ 72 nM) and was identified as an IDO1/TDO dual inhibitor.

Full text of DOI:10.1016/j.ejmech.2018.08.013

Accepted Manuscript
Design, synthesis and biological evaluation of novel naphthoquinone derivatives as
IDO1 inhibitors
Liangkun Pan, Qiang Zheng, Yu Chen, Rui Yang, Yanyan Yang, Zhongjun Li,
Xiangbao Meng
PII:
S0223-5234(18)30671-8
10.1016/j.ejmech.2018.08.013
EJMECH 10624
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 April 2018
Revised Date: 7 June 2018
Accepted Date: 4 August 2018
Please cite this article as: L. Pan, Q. Zheng, Y. Chen, R. Yang, Y. Yang, Z. Li, X. Meng, Design,
synthesis and biological evaluation of novel naphthoquinone derivatives as IDO1 inhibitors, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.013.
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ACCEPTED MANUSCRIPT
O
O
H
N
H
C
H
C
S
N
IDO1 IC50=26 nM
IDO1 IC50/TDO IC50
Decrease 50.1% of
N
N
N
C
H
C
H
O
T44
O
T16
O
O
R
Y
X
G
SAR
IDO1 IC50=48 nM
R1
N
O
O
O
H
N
H
C
H
C
N
C
S
N
S
N
S
S
60 naphthoquinone
derivatives
N
N
N
C
H
C
H
H
O
O
O
T47
T28
T53
IDO1 IC50=18 nM
IDO1 IC50=120 nM
TDO IC50=73 nM
Decrease 38.9% of rat KYN
IDO1 IC50=18 nM
IDO1 IC50/TDO IC50
Decrease 30.5% of r

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel naphthoquinone
derivatives as IDO1 inhibitors
Liangkun Pan, Qiang Zheng, Yu Chen, Rui Yang, Yanyan Yang, Zhongjun Li, Xiangbao Meng*
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking
University, Beijing 100191, China.
*Corresponding author, Email: xbmeng@bjmu.edu.cn
Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1) mediated kynurenine pathway of tryptophan
degradation is identified as an appealing and novel target in immunotherapy for the treatment
of cancer. In this study, a novel series of naphthoquinone derivatives were synthesized,
characterized and evaluated for their inhibitory activities against IDO1, and their
structure−activity relationship was investigated. Among them, compounds T16, T44, T47,
T49, T53 and T54 displayed potent IDO1 inhibitory activities with IC50 values ranging
between 18 and 61 nM, which are more potent than INCB024360 undergoing clinical trial III
evaluation. In addition, compounds T28, T44 and T53 decreased the kynurenine levels in rat
plasma by 30% to 50%. Compounds exhibiting excellent IDO1 inhibitory activities were also
evaluated for their inhibitory activities against tryptophan 2,3-dioxygenase (TDO). Of which,
compound T28 (IDO1 IC50=120 nM) showed promising TDO inhibition (IC50 72 nM) and
was identified as an IDO1/TDO dual inhibitor.
Keywords:
Indoleamine 2,3-dioxygenase 1; cancer immunotherapy; naphthoquinone derivatives;
tryptophan 2,3-dioxygenase.
1. Introduction
The interactions between immune system and the development of tumors are dynamic and
complex. While the host immune system possesses the capacity of recognizing and destroying
tumor cells, many malignancies can develop the capacity to actively suppress a potentially
effective immune response and create an immunosuppressive microenvironment to evade
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immune destruction with the process of cancer immunoediting [1, 2]. This can be achieved
through several molecular mechanisms, including the recruitment of immunosuppressive cells,
exclusion of T cells, and activation of immunoinhibitory checkpoint pathways [3]. To date,
immunotherapy aiming to breach immune suppressive mechanisms established by tumor cells
has become one of the most promising approaches for cancer therapy[4], following operative
treatment, chemotherapy, radiotherapy and targeted therapy.
A large body of evidence indicates that the enzyme indoleamine 2,3-dioxygenase 1 (IDO1)
plays a critical role in tumor-derived immunosuppression in the tumor microenvironment. It
acts by the accumulation of tryptophan metabolites and the depletion of tryptophan. These
studies suggest that IDO could be a valid therapeutic target in cancer immunotherapy [5-7].
IDO1 is an extrahepatic cytosolic heme-containing dioxygenase that catalyzes the oxidative
cleavage of the C2-C3 double bond of the indole ring in tryptophan to form
N-formyl-kynurenine utilizing molecular oxygen or reactive oxygen species via three
proposed reaction mechanisms [8, 9]. This reaction is known as the initial and rate limiting
step in the kynurenine pathway (KP) of tryptophan catabolism in mammals (Fig. 1). The
generated N-formyl-kynurenine is subsequently metabolized to several active metabolites,
including kynurenine, kynurenic acid, 3-hydroxy-kynurenine, quinolinic acid and
nicotinamide adenine dinucleotide, which are also known to be involved in a number of
neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, and cerebral
ischemia [10].
Figure 1. The kynurenine pathway of tryptophan catabolism in mammals
Many studies have confirmed that IDO1 is suppressed in normal human tissues, including
spleen, gut and lung [11, 12], but can be overexpressed by many types of human tumors in a
constitutive manner by proinflammatory stimuli such as interferon-γ (IFN-γ) and transforming
growth factor-β (TGF-β) [13, 14]. High expression of IDO1 by tumors can lead to the
depletion of tryptophan and the accumulation of high levels of kynurenine and its downstream
metabolites in the tumor microenvironment. Tryptophan shortage can lead to anergy of
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effector T cell via mTORC1 inhibition [15] and GCN2 activation [16], and production of
bioactive kynurenine pathway metabolites can result in promotion of Treg differentiation as a
consequence of activating the AHR (aryl hydrocarbon receptor) [17]. Over expression of
IDO1 also have influence on the conversion of dendritic cell [17] and macrophage [18], the
disability of natural killer cell [19] and the production of the reactive oxygen species [20], and
thus enabling tumor cells to elude the host immune response.
The identification of IDO1 as a promising therapeutic target prompts extensive searches for
its highly active inhibitors. The crystal structures of IDO with the heme-bound ligands
cyanide and 4-phenylimidazole (PIM) were reported in 2006 [21], which significantly
facilitated the structure-based design of IDO1 inhibitors. Until now, several types of IDO1
inhibitors including indole-analogs (1-MT), quinines derivatives (annulin B, exiguamine A,
Aulosirazole), phenyl-imidazole derivatives (NLG919) and hydroxyamidine derivatives
(INCB24360) have been reported through structure-based IDO1 inhibitor design, HTS
strategy and natural product derivatization (Fig. 2). However, only a few compounds,
including indole-analogs d-1MT, hydroxyamidine derivatives INCB24360 and
phenyl-imidazole derivatives NLG919, have entered clinical evaluation. There is still an
urgent need to discover novel and structurally diverse IDO1 inhibitors.
Figure 2. Structures of representative IDO1 inhibitors
The 1,4-naphthoquinone unit is a privileged scaffold that could be introduced into the
structures of potential IDO1 inhibitors [22]. In order to identify more potent IDO1 inhibitors,
we designed and synthesized a series of novel 1,4-naphthoquinone derivatives and studied
their IDO1 inhibitory activities.
2. Results and discussion
2.1. Chemistry
The synthetic pathways for compounds T1-T43 and T48-T50 (Table 1) are depicted in
Scheme 1. The key intermediate 4 was prepared according to the procedure described in the
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literature [23]. Initially, 2,3-dichloronaphthalene-1,4-dione was reacted with potassium
phthalimide by refluxing in anhydrous acetonitrile to afford the 2,3-disubstituted
naphthoquinone, which was then reduced by hydrazine hydrate to the desired intermediate 4.
Subsequently, intermediate 4 was treated with appropriate aromatic aldehyde in the presence
of catalyst sodium pyrosulfite in DMF at 120°C to obtain the corresponding target compound
T1-T43 with high yields. 6,7-Dichloroquinoline-5,8-dione (7) was obtained by the
oxidization of 8-hydroxyquinoline in the presence of oxidant sodium chlorate in concentrated
HCl. Target compounds T48-T50 were synthesized from intermediate 7 following the same
route of T1-T43.
Scheme 1. Reagents and conditions: (a) acetonitrile, reflux, 90%; (b) hydrazine hydrate, water,
65-70°C, 94%; (c) Na2S2O5, DMF, 120°C, 70%-90%; (d) NaH, THF, RT, 56%-90%; (e)
NaClO3, concentrated hydrochloric acid, 30°C, 29%.
The synthetic route for compounds T44-T47 and T51-T54 (Table 1) is depicted in Scheme
2. Firstly, 2,3-dichloronaphthalene-1,4-dione was reacted with 7M ammonia in methanol to
produce monosubstituted intermediate 10. Next, intermediate 10 was reacted with Na2S
through refluxing in a mixed solution of water and ethanol to form sulfureted intermediate,
which was subsequently treated with appropriate aromatic aldehyde in one pot to afford the
desired compounds T44-T47. However, the reaction between intermediate 7 and ammonia
could produce two positional isomers intermediate 11a and 11b. The mixture was used
directly in the condensations with appropriate aromatic aldehyde without further separation.
The pure compounds T51-T54 were separated and purified by silica gel chromatography after
the final step. Besides, T16 and T25 were treated with appropriate benzyl bromide in THF
utilizing NaH as base to give target compounds T55-T60.
Scheme 2. Reagents and conditions: (e) 7 N ammonia, methanol, 35°C, 94%; (f) ( ) NaS2,
ethanol, water, reflux; (ii) acetic acid, ethanol, water, reflux, 62%-80%.
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A total of 60 naphthoquinone derivatives were synthesized (Table 1), 45 of which are
newly synthesized compounds. All of the new compounds were systematically characterized
by melting points, 1H-NMR, 13C-NMR, and HRMS analysis.
Table 1. Structures of naphthoquinone derivatives
2.2 IDO1 Inhibitory Activities
Firstly, 1H-naphtho[2,3-d]imidazole-4,9-dione (T25) was chosen as the lead compound. In
order to find out the effect of R1 on the inhibitory activity of IDO1, different substituents were
introduced to the C-2 of the imidazole ring of T25, including methyl (T26), substituted
phenyl (T1-T15), 4-pyridyl (T16), 3-pyridyl (T17), 2-pyridyl (T18), 5-thiazolyl (T27),
4-thiazolyl (T28), 4-imidazolyl (T29), 4-pyrazolyl (T30), 2-thiazolyl (T31), 3-pyrryl (T32),
2-imidazolyl (T33), 3-pyrazolyl (T34), 2-pyrryl (T35), 2-indolyl (T36), 2-thienyl (T37)
,
4-quinolyl (T42) and pyrimidinyl (T43). All the synthesized compounds were evaluated for
their inhibitory activities against IDO1 in comparison with the positive control INCB024360,
according to the literature protocol [24]. The results were expressed as inhibitory rate at 1 µM
in Table 2 and the IC50 values of some compounds displaying potent inhibitory activity were
also presented. Out of these 31 analogs, compound T16 bearing 4-pyridyl substitution at C-2
of imidazole ring showed the highest inhibitory activity against IDO1 (IC50=48nM) compared
to the positive control INCB24360 (IC50=65 nM), whereas the compound with substituted
phenyl substitution exhibited moderate inhibitory activity. In addition, compound T28 with
4-thiazolyl substitution displayed the best inhibitory activity (IC50=120 nM) among all the
five-membered rings substituted derivatives, followed by compound T33 (IC50=134 nM).
Table 2. Inhibitory rate (at 1µM) and IC50 (nM) values of the compounds T1-18, T27-37 and
T42-43 derivatives against IDO1
Next, compounds T16 and T28 which exhibited the best inhibitory activity in the first
round were chosen as parent compounds for further modification. Different substitutions were
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introduced to the pyridine ring of T16 and the thiazole ring of T28 to search for more potent
IDO1 inhibitors. Ten compounds (T19-T24, T38-T41) were newly synthesized and evaluated
for their inhibitory activity against IDO1 at 1 µM. According to the screening results in Table
3, the inhibitory activity of all the newly synthesized compounds decreased significantly
regardless of substitution and where it was linked, compared with the parent compounds,
indicating that further modification at pyridine and thiazole was detrimental to the inhibitory
activity of T16 and T28, respectively.
Table 3. Inhibitory rate (at 1µM) and IC50 (nM) values of the compounds T19-24, T38-41
derivatives against IDO1
We subsequently tried to modify ring A and ring B of T16 and T28 after identifying
pyridine and thiazole rings as the optimal substitutions at C-2 of imidazole, respectively. By
replacing imidazole with thiazole and (or) replacing C-5 or C-8 of 1,4-naphthoquinone with N
atom, 11 new compounds (T44-54) were synthesized and evaluated for their inhibitory
activities expressed as inhibitory rate (at 1 µM) and IC50 value (Table 4). The screening
results showed that most of the synthesized compounds exhibited potent inhibitory activities
against IDO1. The structure-activity-relationships between T16 and T44 (or T28 and T47,
T49 and T53-54) revealed that the replacements of imidazole with thiazole resulted in an
increase of inhibitory activity against IDO1. On the other hand, the introduction of N atom to
C-5 or C-8 of 1,4-naphthoquinone might not be beneficial to the improvement of inhibitory
activity, judging from the structure-activity-relationships between T16 and T48 (or T44 and
T51-52, T28 and T49), whereas T47 and T53-54 was an exception (Figure 3A and Figure
3B). Among these 11 analogs, the most promising compounds T47 and T53 displayed IC50
values of 18 nM against IDO1, more potent than the positive control INCB024360 and the
parent compound T16, T28. In addition, we also introduced substituted benzyl group to N-1
of imidazole of parent compounds T16 and T25 to take advantage of the large hydrophobic
pocket B in IDO1, leading to another 6 new compounds (T55-T60). However, the inhibitory
activities of all these compounds bearing benzyl substituents at N-1 of imidazole decreased
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dramatically (Table 4), implying that N-1 of imidazole might participate in the interactions
with the heme iron of IDO1.
Table 4. Inhibitory rates (at 1µM) and IC50 (nM) values of the compounds T44-60 against
IDO1
Based on the above inhibitory activity data, a preliminary SAR is summarized in Figure 3.
Figure 3. (A)：Structure and inhibitor activity of T16 derivatives. (B)：Structure and inhibitor
activity of T28 derivatives. (C): Preliminary SAR of synthesized naphthoquinone derivatives.
2.2 TDO Inhibitory Activities
Tryptophan 2,3-dioxygenase (TDO) is another heme containing enzyme mediating the first
and rate limiting step of kynurenine pathway of tryptophan degradation, whose tissue
distribution and regulation are different from IDO1[25-27]. Recent studies reveal the
relationship between TDO and immune escape and implicate TDO in tumor progression,
indicating that compounds displaying IDO/TDO dual inhibitory activity may be more
desirable in cancer immunotherapy [28, 29]. In light of this, compounds exhibiting promising
IDO1 inhibitory activity were tested for their ability to inhibit human TDO, according to the
previously described method [30] (Table 5). To our delight, the most promising compound
T28 (IDO1 IC50=120 nM) displayed an IC50 value of 73 nM against TDO, which is identified
as a potent IDO1/TDO dual inhibitor. Besides, other compounds showed great selectivity
towards IDO, especially for T44 (TDO IC50/IDO IC50=61.5) and T16 (inhibitory rate at 10
µM is 0%). In addition, compounds T55-T60 which were almost inactive to IDO1 also
showed no inhibition against TDO (IC50>100 µM), suggesting the involvement of N-1 of
imidazole in the interaction with the heme iron of both IDO1 and TDO.
Table 5. Inhibitory rates and IC50 (nM) values of the corresponding compounds against TDO
at 10 µM
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2.3 Effect of T28, 44 and 53 on the reduction of kynurenine levels in rat plasma
The reduction of plasma kynurenine levels is often used as a pharmacodynamic marker for
the inhibition of IDO activity. IDO inhibitor T28, T44 and T53 were selected for further
evaluation in vivo. A single intragastric 50 mg/kg dose of corresponding compounds was
administered to SD rats. Blood was harvested from individual rat over 8 h. Kynurenine
concentrations were measured by enzyme-linked immunosorbent assay kit. Reductions of
kynurenine levels by ∼30-50% were observed between 0 and 4 h, and kynurenine levels
returned to the baseline level after 8 h with maximum inhibition observed at 2h or 4h (Table
6). Notably, compound T44 (IDO IC50= 44 nM) was identified as the most promising
compound that could decrease the concentration of kynurenine in rat by 50.1%.
Table 6. T28, T44 and T53 suppresses kynurenine generation in vivoa
2.4 MTT assay
IDO-1 inhibition assay showed that compound T16, T44, T45, T47, T49, T53 and T54
exhibited better IDO-1 inhibitory activity than the positive control INCB024360. The
cytotoxic activity of all these compounds and IDO-1/TDO dual inhibitor T28 were evaluated
against human cervical carcinoma cells (HeLa), breast carcinoma cells (MCF-7), and
embryonic kidney cells (HEK293T) by MTT assay, using doxorubicin as the positive control.
The CC50 values of the compounds were represented in Table 7. All the compounds displayed
an CC50 value of micromole level (most CC50 values were over 20 µM), which was
significantly higher than their IC50 values against IDO-1 (showed in Table 7). These results
reveal that all the promising compounds are not cytotoxic at their effective concentration
against IDO-1 (or TDO), and improve their biological importance as cancer immunotherapy
agents.
Table 7. Cytotoxic activity of the promising compounds
2.5 LDH cytotoxicity assay
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Cell death was evaluated by the quantification of plasma membrane damage which resulted
in the release of lactate dehydrogenase (LDH). The level of LDH released in the cell culture
supernatant was detected by LDH cytotoxicity assay detection kit (Beyotime, China)
following the manufacturer's instructions. The CC50 values of the chosen compounds T16,
T23, T28, T33, T44, T45, T47, T48, T49, T50, T51, T52, T53 and T54 (IDO1 IC50<300 nM)
against HeLa and HEK293T at 24h were presented in Table 7. According to the results, all
these compounds showed no cytotoxity (CC50 >40 µM).
3. Conclusion
In conclusion, 60 naphthoquinone derivatives were successfully synthesized and tested for
their ability to inhibit human IDO1. The most promising compounds T47, T53, T54 and T44
displayed IC50 values of 18 nM, 18 nM, 22 nM and 26 nM, respectively. Further evaluation in
vivo showed that T28, T44 and T53 reduced kynurenine levels in plasma by 30%-50%.
Therefore, dual IDO/TDO inhibitor T28, selective IDO1 inhibitors T44 and T53 were
identified as promising lead compounds and merit further investigation.
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)
using commercial silica gel HSGF254 plates. Column chromatography was performed on
Silica Gel 60 (E. Merck, 230-400 mesh). Melting points were measured on an X-5
micromelting point apparatus and were uncorrected. The 1H-MMR (400 MHz) and 13C-NMR
(100.6 MHz) spectra were recorded with BrukerAM-400 spectrometer in CDCl3 and
DMSO-d6 solution. Chemical shifts were referenced with tetramethylsilane (TMS). The
HR-ESI-MS data were measured on a Bruker Apex IV FTMS.
4.1.1. Procedure for the synthesis of 2,3-Diphthalimido-1,4-naphthoquinone (3)
To a solution of 2,3-dichloro-1,4-naphthoquinone (0.02 mol) in anhydrous acetonitrile (100
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mL), potassium phthalidimide (0.084 mol), which was previously finely powdered and dried
under vacuum, was added. The reaction mixture was refluxed for 3 h, and the hot solution
was filtered. The precipitate was washed successively with cold acetonitrile (200 mL), water
(200 mL) and methanol (200 mL). 2,3-Diphthalimido-1,4-naphthoquinone was dried under
vacuum at 50ºC and isolated as a pale yellow powder (8.02 g) in 90% yield. The
characterization data for compound 3 are in accordance with that reported previously[23].
4.1.2. Procedure for the synthesis of 2,3-Diamino-1,4-naphthoquinone (4)
To a suspension of 2,3-diphthalimido-1,4-naphthoquinone (5.0 mmol) in water (150 mL) was
added a 64% solution in water of hydrazine (20 mL). The solution was stirred for 15 min at
room temperature then heated at 65-70°C for 3h. The reaction mixture was allowed to stand at
room temperature. The precipitate was filtered, and washed with a large volume of water.
Compound 4 was dried overnight under vacuum at 100ºC and isolated as a purple powder
(0.88 g) in 94% yield. The characterization data for compound 4 are in accordance with that
reported by R. Diaz et al [31].
4.1.3. Procedure for the synthesis of 6,7-dichloroquinoline-5,8-dione (7)
Sodium chlorate (0.50 mol) was added over a period of 1 h to a solution of
8-hydroxyquinoline (0.10 mol) in concd HCl (600mL) at 40°C and the reaction mixture
stirred for 2 h before being diluted with water to a total volume of 2 L. The white precipitate
that formed was removed by filtration and discarded. The filtrate was then extracted with
CH2Cl2 (6×250mL), the organic phases were combined, washed with water and concentrated
in vacuo to give a yellow solid. The solid was then recrystallized in MeOH to yield 7 as bright
yellow crystals. The characterization data for compound 7 are in accordance with that
reported previously [32].
4.1.4. Procedure for the synthesis of compound 10, 11a, 11b.
To 2,3-dichloro-1,4-naphthoquinone (1.0 mmol) in 1.0 ml of ethanol was added concentrated
ammonia (7N in MeOH, 4.0 mmol) and the mixture was stirred at 35°C for 3h. The formed
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red precipitate was filtered under suction, washed with distilled water and dried to afford the
desired compound as an orange solid, 90%-94.0%. The characterization data for compound
10,11a and 11b are in accordance with that reported previously[33, 34].
4.1.5. General procedure for the preparation of compounds T1-T43.
A solution of 2,3-diamino-1,4-naphthoquinone (1.0mmol), appropriate aromatic aldehyde (1.0
mmol) with sodium pyrosulfite in DMF was stirred at 120 ºC overnight. On completion of the
reaction monitored by TLC, the solvent was evaporated and the residue was purified by silica
gel chromatography by DCM/MeOH system to afford the final product. If necessary, the
crude product could be recrystallized in methanol or DMSO to afford pure sample.
4.1.5.1. 2-(2-Chlorophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T1)
1
Pale yellow solid; yield 80%; m.p. 252-254 ; H NMR (400 MHz, DMSO-d6) δ 14.37 (s,
1H), 8.10 (dd, J = 6.1, 3.0 Hz, 2H), 7.85 (dd, J = 6.1, 3.0 Hz, 2H), 7.79 (d, J = 7.5Hz, 1H),
7.67 (d, J = 7.5 Hz, 1H), 7.59 (t, J = 7.5 Hz 1H), 7.53 (t, J = 7.5 Hz, 1H); 13C NMR (101 MHz,
DMSO) δ 150.83, 134.39, 133.17, 132.65, 132.56, 132.37, 130.72, 129.09, 127.85, 126.80;
HR-EI-MS：Calcd for C17H9ClN2O2 [M+H]+: 309.0425, found: 309.0416.
4.1.5.2. 2-(3-Chlorophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T2)
1
Brown solid, yield 86%; m.p. >300℃; H NMR (400 MHz, DMSO-d6) δ 14.52 (s, 1H), 8.32
(dd, J = 1.7Hz, 1.2 Hz, 1H), 8.21 (ddd, J = 5.0, 3.5, 1.7 Hz, 1H), 8.13 (dd, J = 5.7, 3.3 Hz, 2H),
7.87 (dd, J = 5.7, 3.3 Hz, 2H), 7.63-7.58 (m, 2H); 13C NMR (101 MHz, DMSO) δ 151.18,
134.32, 134.24, 133.15, 131.36, 130.92, 130.65, 126.78, 126.73, 125.78. HR-ESI-MS: Calcd
for C17H9ClN2O2 [M+H]+: 309.0425, found: 309.0411.
4.1.5.3. 2-(4-Chlorophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T3)
1
Pale yellow solid; yield 80%; m.p. 300-302℃; H NMR (400 MHz, DMSO-d6) δ 14.45 (s,
1H), 8.23 (d, J = 8.3 Hz, 2H), 8.09 (dd, J = 5.7, 3.3 Hz, 2H), 7.84 (dd, J = 5.8, 3.3 Hz, 2H),
7.62 (d, J = 8.4 Hz, 2H). Other characterization data are in accordance with that reported
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previously[35].
4.1.5.4. 2-(2-Fluorophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T4)
1
Pale yellow solid; yield 76%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 14.27 (s, 1H),
8.12 (dd, J = 5.9, 3.0 Hz, 2H), 8.01 (t, J = 7.7 Hz, 2H), 7.87 (dd, J = 5.8, 3.0 Hz, 2H), 7.62 (q,
J = 7.2 Hz, 2H), 7.49-7.34 (m, 2H). Other characterization data are in accordance with that
reported previously[36].
4.1.5.5. 2-(4-Fluorophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T5)
1
Pale yellow solid; yield 80%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 14.37 (s, 1H),
8.28 (t, J = 6.4 Hz, 2H), 8.10 (dd, J = 6.1, 3.3 Hz, 2H), 7.85 (dd, J = 6.1, 3.3 Hz, 2H), 7.40 (t,
J = 9.1 Hz, 1H). Other characterization data are in accordance with that reported
previously[35].
4.1.5.6. 2-(2-Bromophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T6)
1
Pale yellow solid; yield 89%; m.p. 245-247 ; H NMR (400 MHz, DMSO-d6) δ 14.39 (s,
1H), 8.12 (dd, J = 5.7, 3.2 Hz, 2H), 7.91-7.79 (m, 3H), 7.71 (d, J = 7.7 Hz, 1H), 7.57 (t, J =
7.7 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 152.10, 134.43, 133.80,
133.19, 132.67, 132.51, 131.33, 128.27, 126.80, 122.37. HR-ESI-MS: Calcd for C17H9BrN2O2
[M+H]+ :352.9920, found:352.9914.
4.1.5.7. 2-(2-Methoxyphenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T7)
1
Pale yellow solid; yield 90%; m.p. 264-266 ; H NMR (400 MHz, DMSO-d6) δ 13.40 (s,
1H), 8.09 (dd, J = 5.7, 3.3 Hz, 2H), 7.97 (dd, J = 7.7, 1.8 Hz, 1H), 7.84 (dd, J = 5.7, 3.3 Hz,
2H), 7.52 (ddd, J = 8.3, 7.7, 1.8 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 7.11 (td, J = 7.7, 1.0 Hz,
1H), 3.94 (s, 3H). 13C NMR (101 MHz, DMSO) δ 157.38, 150.77, 134.26, 133.23, 132.60,
130.92, 126.71, 121.10, 117.76, 112.29, 56.19. HR-ESI-MS: Calcd for C18H12N2O3 [M+H]+:
305.0921, found: 305.0913.
12

ACCEPTED MANUSCRIPT
4.1.5.8. 2-(3-Methoxyphenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T8)
Pale yellow solid; yield 89%; m.p. 310 ; 1H NMR (400 MHz, DMSO-d6) δ 14.29 (s, 1H),
8.06 (dd, J = 5.7, 3.3 Hz, 2H), 7.86 – 7.70 (m, 4H), 7.41 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 8.2
Hz, 1H), 3.84 (s, 3H). 13C NMR (101 MHz, DMSO) δ 159.98, 152.60, 134.31, 133.18, 130.53,
130.17, 126.72, 119.60, 117.36, 111.67, 55.77. HR-ESI-MS: Calcd for C18H12N2O3 [M+H]+:
305.0921, found:305.0910.
4.1.5.9. 2-(4-Methoxyphenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T9)
1
Reddish brown solid; m.p. 320-322 ; yield 86; H NMR (400 MHz, DMSO-d6) δ 14.16 (s,
1H), 8.19 (d, J = 8.8 Hz, 2H), 8.09 (dd, J = 5.7, 3.3 Hz, 2H), 7.84 (dd, J = 5.7, 3.3 Hz, 2H),
7.10 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H). Other characterization data are in accordance with that
reported previously[35].
4.1.5.10. 3-(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-2-yl)benzonitrile (T10)
Orange solid; yield 74%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.45 (s, 1H), 8.51
(s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.02 (dd, J = 5.7, 3.3 Hz, 2H), 7.90 (d, J = 7.7 Hz, 1H), 7.79
(dd, J = 5.7, 3.3 Hz, 2H), 7.70 (t, J = 7.9 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 150.53,
134.31, 134.07, 133.07, 131.52, 130.73, 130.45, 130.03, 126.72, 118.61, 112.59. HR-ESI-MS:
Calcd for C18H9N3O2 [M+H]+: 300.0768, found: 300.0768.
4.1.5.11. 4-(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-2-yl)benzonitrile (T11)
Yellow solid; yield 80%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.60 (s, 1H), 8.45-
8.22 (m, 2H), 8.19 -7.67 (m, 6H).13C NMR (101 MHz, DMSO) δ 181.15, 154.49, 141.66,
135.48, 134.45, 133.44, 133.29, 132.96, 131.29, 127.81, 127.53, 126.83, 118.89, 112.97.
HR-ESI-MS: Calcd for C18H9N3O2 [M+H]+: 300.0768, found: 300.0766.
4.1.5.12. Methyl 4-(4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-2-yl) benzoate (T12)
1
Brown solid; yield 88%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 14.45 (s, 1H), 8.26
(d, J = 8.0 Hz, 2H), 8.04 (dd, J = 5.7, 3.3 Hz, 2H), 7.98 (d, J = 8.1 Hz, 2H), 7.80 (dd, J = 5.8,
13

ACCEPTED MANUSCRIPT
3.3 Hz, 2H), 3.84 (s, 1H). 13C NMR (101 MHz, DMSO) δ 198.67, 165.96, 151.38, 134.40,
133.14, 131.11, 130.23, 127.49, 127.31, 126.70, 55.36. HR-ESI-MS: Calcd for C19H12N2O4
[M+H]+: 333.0870, found: 333.0893.
4.1.5.13. 4-(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-2-yl)benzoic acid (T13)
1
Yellow solid; yield 94%。m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 13.75 (br s, 1H),
8.33 (d, J = 8.1 Hz, 2H), 8.13-8.00 (m, 4H), 7.84 (dd, J = 5.7, 3.3 Hz, 2H). 13C NMR (101
MHz, DMSO) δ 177.36, 167.18, 151.83, 134.29, 133.23, 132.82, 132.54, 130.25, 127.28,
126.73. HR-ESI-MS: Calcd for C18H10N2O4 [M+H]+: 309.0425, found: 309.0416.
4.1.5.14. 2-(4-Nitrophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T14)
Yellow solid; yield 90%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 8.42 (m, 2H), 8.33
(s, 2H), 8.07 (s, 2H), 7.08 (s, 2H). Other characterization data are in accordance with that
reported previously[37].
4.1.5.15. 2-(3-Nitrophenyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T15)
1
Yellow solid; yield 86%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.58
(d, J=7.9Hz, 2H), 8.25 (dd, J=16.9, 8.7Hz, 3H), 8.05 (s, 2H), 7.91 (d, J=16.2Hz, 1H). 13C
NMR (101 MHz, DMSO) δ 188.82, 177.23, 150.99, 148.80, 148.63, 141.22, 136.97, 134.90,
134.25, 133.24, 133.12, 131.06, 130.92, 128.31, 126.70, 125.15, 125.00, 123.33, 121.63.
HR-ESI-MS: Calcd for C17H9N3O4 [M+H]+: 318.0520, found: 318.0533.
4.1.5.16. 2-(Pyridin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T16)
1
Yellow solid; yield 74%; m.p. >300 ; H NMR (400 MHz, CF3COOD) δ 8.78(d, J=6.4Hz,
2H), 8.55(d, J=6.4Hz, 2H), 8.07 (dd, J=5.0, 3.7Hz, 2H), 7.65 (dd, J=5.1Hz, 3.7Hz, 2H). 13C
NMR (101 MHz, CF3COOD) δ 180.73, 145.94, 144.62, 141.43, 137.99, 134.23, 130.32,
126.71. Other characterization data are in accordance with that reported previously[35].
4.1.5.17. 2-(Pyridin-3-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T17)
14

ACCEPTED MANUSCRIPT
Yellow solid; yield 76%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.61 (s, 1H), 9.38
(d, J = 2.1 Hz, 1H), 8.72 (dd, J = 4.8, 2.1 Hz, 1H), 8.60-8.51 (m, 1H), 8.13 (dd, J = 5.7, 3.4 Hz,
2H), 7.88 (dd, J = 5.7, 3.4Hz, 2H), 7.61 (dd, J = 8.1, 4.8 Hz, 1H). 13C NMR (101 MHz,
DMSO) δ 151.51, 150.43, 148.17, 134.57, 134.38, 133.17, 126.78, 125.17, 124.41.
HR-ESI-MS: Calcd for C16H9N3O2 [M+H]+: 276.0768, found: 276.0758.
4.1.5.18. 2-(Pyridin-2-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T18)
1
Brown solid; yield 70%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 14.69 (br.s, 1H),
8.76 (d, J = 4.8 Hz, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.12 (dd, J = 5.7, 3.4 Hz, 2H), 8.04 (td, J =
7.9, 1.5 Hz, 1H), 7.87 (dd, J = 5.7, 3.4 Hz, 2H), 7.62-7.49 (m, 1H). Other characterization
data are in accordance with that reported previously[37].
4.1.5.19. 2-(3-Fluoropyridin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T19)
Yellow solid; yield 80%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.66 (s, 1H), 8.83
(s, 1H), 8.63 (d, J = 5.0 Hz, 1H), 8.13 (dd, J = 6.2, 3.1 Hz, 2H), 8.06 (t, J = 5.8 Hz, 1H), 7.88
(dd, J = 6.2, 3.1 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 157.16, 154.54, 146.81, 145.90,
140.09, 139.86, 134.49, 133.19, 126.87, 124.33, 123.77. HR-ESI-MS: Calcd for C16H8FN3O2
[M+H]+: 294.0673, found: 294.0664.
4.1.5.20. 2-(2-Methoxypyridin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T20)
Yellow solid; yield 82%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.67 (s, 1H), 8.33
(d, J = 5.3 Hz, 1H), 8.11 (dd, J = 5.7, 3.4 Hz, 2H), 7.86 (dd, J = 5.7, 3.3 Hz, 2H), 7.74 (d, J =
5.3 Hz, 1H), 7.58 (s, 1H), 3.91 (s, 3H). 13C NMR (101 MHz, DMSO) δ 164.76, 150.10,
148.46, 138.92, 134.44, 133.21, 126.81, 114.53, 107.66, 54.00. HR-ESI-MS: Calcd for
C17H11N3O3 [M+H]+: 306.0873, found: 306.0878.
4.1.5.21. Procedure for the synthesis of 2-(2-hydroxypyridin-4-yl)-1H-naphtho[2,3-d] imid-
azole-4,9-dione (T21)
A solution of T20 (1mmol) in DMF (2ml) was treated with lithium chloride (5mmol) and
15

ACCEPTED MANUSCRIPT
pTSA (5mmol), heated at 120 ºC for 30 min, cooled to room temperature, quenched with
water (5ml), and extracted with ethyl acetate (2 × 5ml). The combined extracts were washed
with water (2 × 5ml) and saturated aqueous brine (2 × 5ml), dried (Na2SO4), and concentrated
in vacuo to give the desired products T21 in 99% yield. Pale yellow solid; yield 98%;
m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.61 (s, 1H), 11.82 (s, 1H), 8.12 (dd, J = 5.7,
3.4 Hz, 2H), 7.87 (dd, J = 5.7, 3.3 Hz, 2H), 7.54 (d, J = 6.9 Hz, 1H), 7.20 (s, 1H), 6.94 (d, J =
6.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 162.68, 149.98, 140.30, 136.76, 134.40,
133.20, 126.80, 117.61, 102.92. HR-ESI-MS: Calcd for C16H9N3O3 [M+H]+: 292.0717, found:
292.0702.
4.1.5.22. 2-(3-Chloropyridin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T22)
Brown solid; yield 84%; m.p. 286 ; 1H NMR (400 MHz, DMSO-d6) δ 14.67 (s, 1H), 8.86 (s,
13
1H), 8.71 (d, J = 5.0 Hz, 1H), 8.09 (dd, J = 5.7, 3.4 Hz, 2H), 7.92 – 7.77 (m, 3H). C NMR
(101 MHz, DMSO-d6) δ 150.82, 148.80, 148.19, 135.65, 134.49, 133.14, 129.62, 126.87,
125.69. HR-ESI-MS: Calcd for C16H8ClN3O2 [M+H]+: 310.0378, found: 310.0368.
4.1.5.23. 2-(2-Chloropyridin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T23)
Yellow solid; yield 72%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.86 (s, 1H), 8.61
(d, J = 5.2 Hz, 1H), 8.28 (d, J = 1.4 Hz, 1H), 8.17 (dd, J = 5.2, 1.5 Hz, 1H), 8.14 (dd, J = 5.7,
3.3 Hz, 2H), 7.88 (dd, J = 5.7, 3.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 151.81, 151.41,
148.76, 139.56, 139.27, 134.70, 133.05, 126.74, 120.96, 120.20. HR-ESI-MS: Calcd for
C16H8ClN3O2 [M+H]+: 308.0232, found: 308.0242.
4.1.5.24. 4-(4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-2-yl)pyridine 1-oxide (T24)
To a stirred solution of T16 (5 mmol) in CHCl3 (2 mL) was added 70% m-CPBA (5 mmol),
portion wise at 0 ºC. The resulting mixture was stirred at room temperature for 12 h, at which
time complete consumption of starting material was observed by TLC. The reaction mixture
was diluted with CHCl3, and solid K2CO3 (4mol) was added. The resulting mixture was
stirred for an additional 10 min. The solid was separated by filtration, and the filtrate was
16

ACCEPTED MANUSCRIPT
dried over Na2SO4 and concentrated under reduced pressure to afford the T24. Yellow solid;
yield 60%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.61 (s, 1H), 8.36 (d, J = 6.4 Hz,
2H), 8.16 (d, J = 6.4 Hz, 2H), 8.10 (dd, J = 5.8, 3.3 Hz, 2H), 7.85 (dd, J = 5.8, 3.3 Hz, 2H).
13C NMR (101 MHz, DMSO-d6) δ 177.42, 150.16, 139.76, 134.31, 133.32, 126.73, 125.60,
124.08. HR-ESI-MS: Calcd for C16H9N3O3 [M+H]+: 290.0571, found: 290.0565.
4.1.5.25. 1H-Naphtho[2,3-d]imidazole-4,9-dione (T25)
T25 was synthesized using the approach reported previously [38]. A mixture of 2,3-diamino
-1,4-naphthoquinone (79.7mmol) and 25 mL of formic acid (97%) in distilled water (100mL)
was refluxed with stirring for 5 h. After cooling to room temperature, the pH of the solution
was adjusted to 9 with the addition of 30% ammonium hydroxide. The dark yellow powder
(T25) was obtained in yield of 83% after repeated filtration and washing with distilled water.
The product was recrystallized from DMSO for further purification. Dark yellow solid; yield
83%; m.p. 369 ; 1H NMR (400 MHz, DMSO-d6) δ 14.11 (s, 1H), 8.30 (s, 1H), 8.09 (dd, J =
5.7, 3.3 Hz, 2H), 7.85 (dd, J = 5.7, 3.3 Hz, 2H). Other characterization data are in accordance
with that reported previously[38].
4.1.5.26. 2-Methyl-1H-naphtho[2,3-d]imidazole-4,9-dione (T26)
A solution of 2,3-Diamino-1,4-naphthoquinone (1mmol) in glacial acetic acid (30mL) was
heated at reflux for 7 h. The reaction mixture was cooled and the precipitate obtained was
filtered and washed with acetic acid. The product obtained was dissolved in acetone (5mL)
and applied on PLC by silica gel (Toluene: ethyl acetate:10:1) as reported previously [39].
1
The product was recrystallized from ethanol to give gray crystals, yield 70%; m.p. 350 ; H
NMR (400 MHz, DMSO-d6) δ 13.73 (s, 1H), 8.05 (dd, J = 5.8, 3.1 Hz, 2H), 7.82 (dd, J = 5.8,
3.1 Hz, 2H), 2.45 (s, 3H). Other characterization data are in accordance with that reported
previously[39].
4.1.5.27. 2-(Thiazol-5-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T27)
17

ACCEPTED MANUSCRIPT
1
Brown solid; yield 78%; m.p. >200 ; H NMR (400 MHz, DMSO-d6) δ 14.59 (s, 1H), 9.25
(s, 1H), 8.67 (s, 1H), 8.05 (dd, J = 6.1, 3.0 Hz, 2H), 7.81 (dd, J = 6.1, 3.0 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 157.52, 146.04, 143.62, 134.35, 133.07, 128.33, 126.73.
HR-ESI-MS: Calcd for C14H7N3O2S [M+H]+: 280.0186, found: 280.0179.
4.1.5.28. 2-(Thiazol-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T28)
1
Brown solid; yield 80%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 14.56 (s, 1H), 9.34
(d, J = 1.9 Hz, 1H), 8.58 (d, J = 1.9 Hz, 1H), 8.11 (dd, J = 5.7, 3.4 Hz, 2H), 7.86 (dd, J = 5.8,
3.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.29, 148.54, 145.93, 134.27, 133.25,
126.69, 121.93.
HR-ESI-MS: Calcd for C14H7N3O2S [M+H]+:282.0332, found: 282.0325.
4.1.5.29. 2-(1H-imidazol-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T29)
Reddish brown solid; yield 75%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 8.08 (dd, J
= 5.7, 3.3 Hz, 2H), 7.95 (s, 2H), 7.84 (dd, J = 5.6, 3.3 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 177.22, 149.23, 137.72, 134.14, 133.22, 126.57, 121.04. HR-ESI-MS: Calcd for
C14H8N4O2 [M+H]+:265.0720, found: 265.0713.
4.1.5.30. 2-(1H-pyrazol-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T30)
Brown solid; yield 70%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.01 (s, 1H), 13.29
(s, 1H), 8.36 (s, 2H), 8.09 (dd, J = 5.7, 3.3 Hz, 2H), 7.85 (dd, J = 5.7, 3.3 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 179.01, 175.39, 162.79, 148.50, 143.81, 134.50, 134.07, 133.00,
126.82, 126.33, 111.90. HR-ESI-MS: Calcd for C14H8N4O2 [M+H]+:265.0720, found:
265.0705.
4.1.5.31. 2-(Thiazol-2-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T31)
1
Tan solid; yield 68%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 15.06 (s, 1H), 8.16 –
13
8.08 (m, 3H), 8.06 (d, J = 3.1 Hz, 1H), 7.78 (dd, J = 5.7, 3.3 Hz, 2H). C NMR (101 MHz,
18

ACCEPTED MANUSCRIPT
DMSO-d6) δ 157.28, 147.30, 145.11, 144.93, 134.43, 133.27, 126.81, 124.34. HR-ESI-MS:
Calcd for C14H7N3O2S [M+H]+: 282.0332, found: 282.0324.
4.1.5.32. 2-(1H-pyrrol-3-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T32)
Purple solid; yield 74%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 13.72 (s, 1H), 11.37
(s, 1H), 8.12 – 8.05 (dd, J = 5.8, 3.3 Hz, 2H), 7.83 (dd, J = 5.8, 3.3 Hz, 2H), 7.67 (s, 1H), 6.92
13
(d, J = 2.7 Hz, 1H), 6.81 (s, 1H). C NMR (101 MHz, DMSO-d6) δ 179.18, 175.21, 151.65,
143.97, 134.03, 133.18, 126.69, 126.27, 120.02, 113.42, 107.44. HR-ESI-MS: Calcd for
C15H9N3O2 [M+H]+: 286.0587, found: 286.0582.
4.1.5.33. 2-(1H-imidazol-2-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T33)
Brown solid; yield 80%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 8.11 (dd, J = 5.7, 3.3
Hz, 2H), 7.86 (dd, J = 5.7, 3.3 Hz, 2H), 7.39 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ
177.80, 146.28, 141.23, 138.50, 134.16, 133.59, 126.66, 124.13. HR-ESI-MS: Calcd for
C14H8N4O2 [M+H]+: 265.0720, found: 265.0714.
4.1.5.34. 2-(1H-pyrazol-3-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T34)
Tan solid; yield 65%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.36 (s, 1H), 13.47 (s,
1H), 8.15 – 8.04 (m, 2H), 7.96 (s, 1H), 7.85 (dd, J = 5.7, 3.3 Hz, 2H), 7.02 (s, 1H). 13C NMR
(101 MHz, DMSO-d6) δ 179.12, 175.64, 148.88, 143.88, 142.12, 134.86, 134.29, 133.28,
130.84, 126.79, 105.49. HR-ESI-MS: Calcd for C14H8N4O2 [M+Na]+: 287.0539, found:
287.0525.
4.1.5.35. 2-(1H-pyrrol-2-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T35)
Purple solid; yield 68%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 13.92 (s, 1H), 11.99
(s, 1H), 8.08 (dd, J = 5.7, 3.2 Hz, 2H), 7.83 (dd, J = 5.7, 3.3 Hz, 2H), 7.09 (d, J = 2.0 Hz, 1H),
7.00 (s, 1H), 6.34 – 6.16 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 179.09, 175.34, 148.22,
143.82, 134.16, 133.22, 126.70, 126.38, 122.96, 121.50, 111.24, 110.08. HR-ESI-MS: Calcd
for C15H9N3O2 [M+H]+: 262.0622, found: 262.0633.
19

ACCEPTED MANUSCRIPT
4.1.5.36. 2-(1H-indol-2-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T36)
Tan solid; yield 67%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.48 (s, 1H), 12.15 (s,
1H), 8.13 (dd, J = 5.8, 3.1 Hz, 2H), 7.88 (dd, J = 5.8, 3.1 Hz, 2H), 7.66 (d, J = 8.0 Hz, 1H),
7.49 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.08 (t, J = 7.5 Hz,
1H). 13C NMR (101 MHz, DMSO-d6) δ 181.40, 162.83, 147.63, 137.91, 134.33, 133.28,
128.15, 127.33, 126.72, 123.78, 121.56, 120.41, 112.64, 103.48.133.27, 126.81, 124.34.
HR-ESI-MS: Calcd for C19H11N3O2 [M+H]+: 312.0779, found: 312.0769.
4.1.5.37. 2-(Thiophen-2-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T37)
Yellow solid; yield 68%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.45 (s, 1H), 8.10
(dd, J = 5.6, 3.3 Hz, 2H), 8.03 (d, J = 3.6 Hz, 1H), 7.86 (dd, J = 5.9, 3.4 Hz, 2H), 7.83 (d, J =
5.1 Hz, 1H), 7.26 (t, J = 4.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 148.54, 134.35,
133.19, 132.24, 130.60, 129.03, 128.60, 126.74. Other characterization data are in accordance
with that reported previously[37].
4.1.5.38. 2-(2-Phenylthiazol-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T38)
Tan solid; yield 54%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.55 (s, 1H), 8.54 (s,
1H), 8.28 – 8.01 (m, 4H), 7.86 (s, 2H), 7.57 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
185.50, 168.37, 162.79, 148.39, 145.70, 134.33, 133.29, 132.96, 131.26, 129.70, 127.02,
126.76, 121.77. HR-ESI-MS: Calcd for C20H11N3O2S [M+H]+: 358.0645, found: 358.0643.
4.1.5.39. 2-(2-Methylthiazol-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T39)
Tan solid; yield 58%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.46 (s, 1H), 8.35 (s,
1H), 8.09 (dd, J = 5.7, 3.3 Hz, 2H), 7.85 (dd, J = 5.7, 3.3 Hz, 2H), 2.79 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 178.95, 175.39, 167.42, 148.50, 144.57, 134.26, 133.26, 126.76,
121.55, 19.27. HR-ESI-MS: Calcd for C15H9N3O2S [M+H]+: 296.0488, found: 296.0490.
4.1.5.40. 2-(2-Bromothiazol-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T40)
20

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Tan solid; yield 66%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.65 (s, 1H), 8.55 (s,
1H), 8.11 (dd, J = 5.6, 3.3 Hz, 2H), 7.87 (dd, J = 5.6, 3.3 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 147.22, 145.11, 138.06, 134.33, 133.26, 126.74, 126.17. HR-ESI-MS: Calcd for
C14H6BrN3O2S [M+H]+: 359.9437, found: 359.9439.
4.1.5.41. 2-(2-Chlorothiazol-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T41)
Tan solid; yield 68%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.66 (s, 1H), 8.52 (s,
1H), 8.12 (d, J = 5.1 Hz, 2H), 7.89 (d, J = 5.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ
199.89, 160.87, 152.52, 147.34, 143.50, 134.35, 133.28, 126.75, 124.67. HR-ESI-MS: Calcd
for C14H6ClN3O2S [M+H]+: 315.9942, found: 315.9929.
4.1.5.42. 2-(Quinolin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T42)
1
Brown solid; yield 77%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 14.73 (s, 1H), 9.06
(d, J = 6.3 Hz, 1H), 8.15 – 8.07 (m, 3H), 8.04 (d, J = 4.6 Hz, 1H), 7.90 – 7.80 (m, 3H), 7.76 (t,
13
J = 7.8 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 150.53, 150.45, 148.88, 134.45, 133.40,
133.23, 130.36, 130.00, 128.22, 126.84, 124.85, 121.80. HR-ESI-MS: Calcd for C20H11N3O2
[M+H]+: 326.0924, found: 326.0916.
4.1.5.43. 2-(2-Aminopyrimidin-5-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T43)
Yellow solid; yield 70%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.12 (s, 1H), 8.96
(s, 2H), 8.05 (dd, J = 6.1, 3.0 Hz, 2H), 7.82 (dd, J = 6.1, 3.0 Hz, 2H), 7.28 (s, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 164.15, 163.79, 162.27, 157.28, 150.19, 134.23, 133.12, 126.64,
112.44. HR-ESI-MS: Calcd for C15H9N5O2 [M+H]+: 292.0829, found: 292.0813.
4.1.6. General procedure for the preparation of compounds T44-T47.
2-Amino-3-chloronaphthalene-1,4-dione (4.0mmol) was dissolved in a mixed solution of
water and ethanol (3:1, 16 mL). Na2S (6.25mmol) was added to the solution and the mixture
was refluxed for 2 hours. Appropriate aromatic aldehyde was added and the solution was
refluxed for another 2 hours. On completion of the reaction monitored by TLC, the precipitate
21

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was filtered and purified by silica gel chromatography by DCM/MeOH system to afford the
final product.
4.1.6.1. 2-(2-Aminopyrimidin-5-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T44)
1
Brown solid; yield 72%; m.p. 304-306 ; H NMR (400 MHz, CF3COOD) δ 8.71 (s, 2H),
13
8.49 (s, 2H), 8.10 (d, J=43.6, 2H), 7.66 (s, 2H). C NMR (101 MHz, CF3COOD) δ 181.49,
180.58, 168058, 156.84, 149.90, 146.95, 143.95, 137.44, 134.12, 133.27, 130.15, 129.52,
126.26. Other characterization data are in accordance with that reported previously[40].
4.1.6.2 2-(Pyridin-2-yl)naphtho[2,3-d]thiazole-4,9-dione (T45)
1
Brown solid; yield 62%; m.p. 295-297 ; H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 4.8
Hz, 1H), 8.32 (d, J = 7.9 Hz, 1H), 8.26 – 8.02 (m, 3H), 7.94 (dd, J = 5.7, 3.4 Hz, 2H), 7.77 –
7.55 (m, 1H). Other characterization data are in accordance with that reported previously[40].
4.1.6.3. 2-(4-Nitrophenyl)naphtho[2,3-d]thiazole-4,9-dione (T46)
Pale yellow solid; yield 80% ; m.p. 278-280 ; 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H),
8.59 (d, J = 7.8 Hz, 1H), 8.47 (d, J = 8.3 Hz, 1H), 8.22 (d, J = 7.0 Hz, 1H), 8.16 (d, J = 6.8 Hz,
1H), 8.05 – 7.83 (m, 3H). Other characterization data are in accordance with that reported
previously[41].
4.1.6.4. 2-(Thiazol-4-yl)naphtho[2,3-d]thiazole-4,9-dione (T47)
1
Orange solid; yield 80%; m.p. 294-296 ; H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H),
8.76 (s, 1H), 8.17 (d, J = 23.5 Hz, 2H), 7.93 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ
178.76, 177.77, 168.08, 157.51, 155.40, 148.17, 142.02, 135.12, 134.74, 133.23, 133.05,
127.54, 126.84, 122.67. HR-ESI-MS: Calcd for C14H6N2O2S2 [M+H]+: 298.9943, found:
298.9936.
4.1.7. General procedure for the preparation of compounds T48-T50.
Compounds T48-T50 were synthesized following the same route of compounds T1-T44
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4.1.7.1. 2-(Pyridin-4-yl)-3H-imidazo[4,5-g]quinoline-4,9-dione (T48)
Tan solid; yield 70%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.87 (s, 1H), 9.01 (d, J
= 4.6 Hz, 1H), 8.78 (d, J = 5.1 Hz, 2H), 8.48 (d, J = 7.9 Hz, 1H), 8.16 (d, J = 5.1 Hz, 2H),
7.85 (dd, J = 7.9, 4.8 Hz, 1H). Other characterization data are in accordance with that reported
previously[42].
4.1.7.2. 2-(Thiazol-4-yl)-3H-imidazo[4,5-g]quinoline-4,9-dione (T49)
1
Brown solid; yield 62%; m.p. >300 ; H NMR (400 MHz, DMSO-d6) δ 14.64 (s, 1H), 9.34
(d, J = 1.9 Hz, 1H), 8.99 (d, J = 4.7 Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.45 (d, J = 7.8 Hz, 1H),
7.83 (dd, J = 7.8, 4.7 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 156.39, 153.83, 149.24,
148.68, 145.80, 134.74, 130.43, 127.95, 122.21. HR-ESI-MS: Calcd for C13H6N4O2S [M+H]+:
281.0139, found: 281.0132.
4.1.7.3. 2-(Pyridin-2-yl)-3H-imidazo[4,5-g]quinoline-4,9-dione (T50)
Yellow solid; yield 80%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 14.82 (s, 1H), 8.98
(dd, J = 4.6, 1.6 Hz, 1H), 8.74 (d, J = 4.8 Hz, 1H), 8.44 (dd, J = 7.9, 1.7 Hz, 1H), 8.27 (d, J =
7.9 Hz, 1H), 8.02 (td, J = 7.8, 1.7 Hz, 1H), 7.82 (dd, J = 7.9, 4.7 Hz, 1H), 7.56 (ddd, J = 7.5,
4.8, 1.2 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 153.86, 152.48, 150.00, 149.22, 147.57,
138.17, 134.76, 130.43, 127.96, 125.76, 122.65. HR-ESI-MS: Calcd for C15H8N4O2 [M+H]+:
299.0539, found: 299.0568.
4.1.8. General procedure for the preparation of compounds T51-T54.
The mixture of 11a and 11b (4.0mmol) was dissolved in a mixed solution of water and
ethanol (3:1, 16 mL). Na2S (6.25mmol) was added to the solution and the mixture was
refluxed for 2 hours. Appropriate aromatic aldehyde was added and the solution was refluxed
for another 2 hours. On completion of the reaction monitored by TLC, the precipitate was
filtered and purified by silica gel chromatography by DCM/MeOH system to afford the pure
positional isomer.
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4.1.8.1. 2-(Pyridin-4-yl)thiazolo[5,4-g]quinoline-4,9-dione (T51)
Brown solid; yield 54%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 9.09 (d, J = 4.7 Hz,
1H), 8.86 (d, J = 5.0 Hz, 2H), 8.60 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 5.1 Hz, 2H), 7.94 (dd, J =
8.0, 4.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 178.14, 175.79, 171.19, 155.51, 154.93,
151.59, 149.06, 142.55, 138.70, 134.91, 130.60, 128.37, 121.43. HR-ESI-MS: Calcd for
C15H7N3O2S [M+H]+: 316.0151, found: 316.0148.
4.1.8.2. 2-(Pyridin-4-yl)thiazolo[5,4-g]quinoline-4,9-dione (T52)
Yellow solid; yield 21%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 9.13 (d, J = 4.7 Hz,
1H), 8.88 (d, J = 4.8 Hz, 2H), 8.56 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 4.5 Hz, 2H), 7.95 (dd, J =
5.8, 3.5 Hz, 1H).
4.1.8.3. 2-(Thiazol-4-yl)thiazolo[4,5-g]quinoline-4,9-dione (T53)
Yellow solid; yield 50%。m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 9.37 (d, J = 2.0 Hz,
1H), 9.06 (dd, J = 4.5, 1.8 Hz, 1H), 8.78 (d, J = 2.0 Hz, 1H), 8.56 (dd, J = 7.9, 1.8 Hz, 1H),
7.92 (dd, J = 7.9, 4.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 177.48, 177.01, 168.17,
157.60, 154.84, 154.32, 149.12, 148.07, 142.75, 135.48, 130.43, 128.60, 122.87. HR-ESI-MS:
Calcd for C13H5N3O2S2 [M+H]+: 299.9896, found: 299.9909.
4.1.8.4. 2-(Thiazol-4-yl)thiazolo[5,4-g]quinoline-4,9-dione (T54)
Yellow solid; yield 23%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 9.37 (d, J = 1.8 Hz,
1H), 9.08 (dd, J = 4.5, 1.8 Hz, 1H), 8.79 (d, J = 1.9 Hz, 1H), 8.51 (d, J = 7.9 Hz, 1H), 7.91 (dd,
J = 7.9, 4.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 178.26, 175.99, 168.15, 157.62,
155.77, 154.73, 148.99, 148.10, 141.20, 134.77, 130.58, 128.29, 122.90. HR-ESI-MS: Calcd
for C13H5N3O2S2 [M+H]+: 299.9896, found: 299.9888.
4.1.9. General procedure for the preparation of compounds T55-T60.
To a suspension of the 1H-naphtho[2,3-d]imidazole-4,9-dione derivatives (1mmol) in
24

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acetonitrile (10mL) was added caesium carbonate (1.5mmol) and the corresponding alkyl
bromide (1.05mmol). The reaction mixture was stirred at room temperature for 1-3 hours and
after this time it was concentrated in vacuo. The residue was suspended in a mixture of
CH2Cl2 and sat. NaHCO3. The aqueous layer was extracted with CH2Cl2 (2x30mL). The
combined organic layers were dried over Na2SO4, filtered and the solvent was removed under
reduced pressure. The crude product was purified by flash chromatography
4.1.9.1. 1-(2-Methylbenzyl)-2-(pyridin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T55)
Yellow solid; yield 77%; m.p. >300 ; 1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 6.1 Hz,
2H), 8.17 (dd, J = 7.5, 1.6 Hz, 1H), 8.03 (dd, J = 7.1, 1.8 Hz, 1H), 7.92 – 7.84 (m, 2H), 7.62 –
7.58 (m, 2H), 7.27 (d, J = 7.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H), 6.60
(d, J = 7.7 Hz, 1H), 5.79 (s, 2H), 2.34 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 178.84,
176.25, 152.18, 150.86, 143.67, 135.98, 134.99, 134.81, 134.75, 134.52, 133.95, 133.25,
133.03, 130.78, 127.86, 126.98, 126.75, 124.38, 123.14, 109.99, 48.21, 19.13. HR-ESI-MS:
Calcd for C24H17N3O2 [M+H]+: 402.1213, found: 402.1208.
4.1.9.2. 1-(3-Chlorobenzyl)-2-(pyridin-4-yl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T56)
1
Brown solid; yield 56%; m.p. 178-180 ; H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 5.0
Hz, 2H), 8.14 (d, J = 6.7 Hz, 1H), 8.05 (d, J = 7.0 Hz, 1H), 7.93 – 7.81 (m, 2H), 7.62 (d, J =
4.4 Hz, 2H), 7.38 – 7.30 (m, 2H), 7.27 (s, 1H), 7.05 (d, J = 5.2 Hz, 1H), 5.87 (s, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 178.85, 176.48, 151.89, 150.86, 143.51, 138.91, 135.99,
134.76, 134.47, 133.98, 133.89, 133.33, 132.99, 131.18, 128.19, 126.93, 126.76, 126.55,
125.10, 123.37, 49.16.
HR-ESI-MS: Calcd for C23H14ClN3O2 [M+H]+: 422.0667, found: 422.0664.
4.1.9.3. 1-(3-Chlorobenzyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T57)
Brown solid; yield 82%; m.p. 185-187 ; 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.12
– 8.00 (m, 2H), 7.91 – 7.77 (m, 2H), 7.46 (s, 1H), 7.41 – 7.34 (m, 2H), 7.34 – 7.23 (m, 1H),
5.68 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 178.74, 176.66, 144.34, 144.10, 136.87, 135.04,
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134.12, 133.60, 132.99, 132.89, 131.58, 130.47, 128.96, 127.86, 127.23, 126.62, 125.89,
49.77. HR-ESI-MS: Calcd for C18H11ClN2O2 [M+H]+: 323.0587, found: 323.0587.
4.1.9.4. Methyl 3-((4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-1-yl)methyl)benzoate
(T58)
1
Brown solid; yield 78%; m.p. 226-228 ; H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H),
8.11-8.06 (m, 1H), 8.05-8.01 (m, 1H), 7.98 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.86-7.81 (m,
13
2H), 7.62 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 5.74 (s, 2H), 3.84 (s, 3H). C NMR
(101 MHz, DMSO-d6) δ 178.81, 176.34, 166.35, 146.10, 144.08, 137.58, 134.68, 134.31,
133.14, 133.00, 132.79, 132.12, 130.46, 129.77, 129.19, 128.62, 126.94, 126.62, 52.74, 49.44.
HR-ESI-MS: Calcd for C21H16N2O4 [M+H]+: 317.1068, found: 317.1060.
4.1.9.5. 1-(2-Methylbenzyl)-1H-naphtho[2,3-d]imidazole-4,9-dione (T59)
1
Yellow solid; yield 82%; m.p. 214-216 ; H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H),
8.13 – 8.08 (m, 1H), 8.00 (dd, J = 7.0, 1.9 Hz, 1H), 7.90 – 7.78 (m, 2H), 7.26 (d, J = 7.5 Hz,
1H), 7.20 (t, J = 7.4 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.71 (d, J = 7.6 Hz, 1H), 5.68 (s, 2H),
2.39 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 178.85, 176.30, 146.26, 144.01, 135.66,
135.02, 134.65, 134.30, 133.19, 133.03, 132.51, 130.69, 128.09, 126.94, 126.80, 126.57,
126.13, 47.95, 19.18. HR-ESI-MS: Calcd for C19H14N2O2 [M+H]+: 303.1134, found:
303.1137.
4.1.9.6. 3-((4,9-Dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-1-yl)methyl)benzoic acid
(T60)
1
Yellow solid; yield 80%; m.p. >200 ; H NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 8.56
(s, 1H), 8.07 (dd, J = 20.1, 7.0 Hz, 2H), 7.94 (s, 1H), 7.91 – 7.80 (m, 3H), 7.60 (d, J = 7.7 Hz,
1H), 7.50 (t, J = 7.7 Hz, 1H), 5.74 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 178.80, 176.36,
174.99, 173.54, 167.40, 146.10, 144.09, 137.35, 134.67, 134.30, 133.02, 132.37, 131.67,
129.55, 128.71, 126.95, 126.63, 57.50.
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4.2. Biological evaluations
4.2.1. IDO-1 Inhibition Assay
IDO1 catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of
tryptophan to yield N-formyl-kynurenine. The assays were performed at room temperature as
described in the literature using 20 nM IDO and 2 mM D-Trp in the presence of 20 mM
ascorbate, 3.5 µM methylene blue and 0.2 mg/mL catalase in 50 mM potassium phosphate
buffer (pH 6.5). The initial reaction rates were recorded by continuously following the
absorbance increase at 321 nm due to the formation of N-formyl-kynurenine. The IC50 values
were calculated using nonlinear regression with normalized dose−response fit using Prism
GraphPad software.
4.2.2. TDO Inhibition Assay
The assay was performed by UV absorption using recombinant TDO and L-Tryptophan as
the substrate. The UV absorption signal at 321 nm is correlated with the amount of
N-formylkynurenine reaction product of TDO. All of the reactions were conducted at room
temperature. The 100 µL reaction mixture in TDO Assay Buffer contains 50 nM TDO, the
indicated amount of the inhibitor, 200 µM tryptophan, and the coupled reaction components.
The reaction mixture incubated for 75 min prior to reading the UV absorption signal. For the
negative control (blank), 5 µL of the assay buffer was added instead of the TDO. The initial
reaction rates were recorded by continuously following the absorbance increase at 321 nm
due to the formation of N-formlylkynurenine. The IC50 values were calculated using nonlinear
regression with normalized dose−response fit using Prism GraphPad software.
4.2.3. Kynurenine Generation Suppression Assay
SD rats (250-300g) were chosen to determine the effect of IDO inhibition on plasma
kynurenine. A single intragastric 50 mg/kg dose of corresponding compounds was
administered to 3 rats. Blood was harvested from the rat eye socket at various time points
after dosing over 8 h (0h, 1h, 2h, 3h, 4h, 6h, 8h). The blood sample was centrifuged for 10
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mins at 3000RPM and the supernatant was got. Kynurenine concentrations were measured by
enzyme-linked immunosorbent assay kit.
4.2.4. MTT assay of cell viability
Human cells [human cervical carcinoma cell (HeLa), breast carcinoma cell (MCF-7), and
embryonic kidney cells (HEK293T)] were seeded in each well of 96-well culture plate
(3-4×103 per well). After overnight incubation, solution of compounds in DMSO (10 mM)
was added to the cells. Cells treated with DMSO 1% were used as negative control. After
further incubation for 48 h, MTT solution (5 mg/mL in PBS) was added to each well. After
incubation for 4 h at 37 °C, MTT solution was removed and 150 µL of DMSO was added to
dissolve the crystals formed. Then, absorbance at 570 nm was read using a microplate reader.
The IC50 values were calculated using Prism 5.0.
4.2.5. LDH cytotoxicity assay
Human cells [human cervical carcinoma cell (HeLa) and embryonic kidney cells
(HEK293T)] were seeded in each well of 96-well culture plate (3-4×103 per well). After target
compounds treatment for 24 h in DMEM, the supernatant was got through centrifugation for 5
minutes. The LDH release activity was determined using an LDH cytotoxicity assay kit (Beyotime,
Haimen, Jiangsu, China) according to the manufacturer's instructions. The absorbance was
measured at 490 nm by a microplate reader within 1 h. The cytotoxicity was expressed using the
formula: Cytotoxicity (%) = (OD Sample-OD Control) / (OD Maximum release- OD Control) x 100.
Acknowledgment
We gratefully acknowledge the National Natural Science Foundation of China (NSFC No.
81573272) and Beijing Natural Science Foundation (No. 7162109) for generous financial
supports.
References
[1] H. Li, K.B. Chiappinelli, A.A. Guzzetta, H. Easwaran, R.W.C. Yen, R. Vatapalli, M.J. Topper, J. Luo, R.M.
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