Discovery of novel indoleamine 2,3-dioxygenase 1 (IDO1) and histone deacetylase 1 (HDAC1) dual inhibitors derived from the natural product saprorthoquinone

Article abstract of DOI:10.3390/molecules25194494

The discovery of IDO1 and HDAC1 dual inhibitors may provide a novel strategy for cancer treatment by taking advantages of both immunotherapeutic and epigenetic drugs. In this paper, saprorthoquinone (1) and 13 of its analogues from Salvia prionitis Hance were investigated for their SAR against IDO1, the results demonstrated the ortho-quinone was a key pharmacophore. Then a series of IDO1 and HDAC dual inhibitors connected by appropriate linkers were designed, synthesized, and evaluated from the hit compound saprorthoquinone (1). Among them, compound 33d showed balanced activity against both IDO1 (IC50 = 0.73 μM) and HDAC1 (IC50 = 0.46 μM). Importantly, the structure of 33d suggested that an ortho-quinone pharmacophore and a N-(2- aminophenyl) amide pharmacophore were necessary for the IDO inhibition and HDAC inhibition respectively. Meanwhile, these two pharmacophore groups should be combined by a pentane linker. Moreover, the binding modes of 33d to the enzyme active site showed that the hydrogen bond with Leu234 of IDO1 appeared to confer increased potency to this class of inhibitors, which may explain the higher activity of 33d. This study provides a new strategy for future IDO1/HDAC dual inhibitors with synergistic antitumor activity started from lead compound 33d.

Full text of DOI:10.3390/molecules25194494

Article
Discovery of Novel Indoleamine 2,3-Dioxygenase 1
(IDO1) and Histone Deacetylase 1 (HDAC1)
Dual Inhibitors Derived from the Natural
Product Saprorthoquinone
Yang Lin 1, Heyanhao Zhang 1, Tong Niu 1, Mei-Lin Tang 1,2,* and Jun Chang 1,*
1
School of Pharmacy, Human Phenome Institute, Fudan University, Shanghai 201203, China;
14211030004@fudan.edu.cn (Y.L.); 17211030076@fudan.edu.cn (H.Z.); 18211030065@fudan.edu.cn (T.N.)
2
State Key Laboratory of Molecular Engineering and Institutes of Biomedical Sciences, Fudan University,
Shanghai 200433, China
* Correspondence: tangmeilin@fudan.edu.cn (M.-L.T.); jchang@fudan.edu.cn (J.C.);
Tel./Fax: +86-21-5198-0101 (M.-L.T. & J.C.)
Received: 4 August 2020; Accepted: 28 September 2020; Published: 30 September 2020
Abstract: The discovery of IDO1 and HDAC1 dual inhibitors may provide a novel strategy for
cancer treatment by taking advantages of both immunotherapeutic and epigenetic drugs. In this
paper, saprorthoquinone (1) and 13 of its analogues from Salvia prionitis Hance were investigated
for their SAR against IDO1, the results demonstrated the ortho-quinone was a key pharmacophore.
Then a series of IDO1 and HDAC dual inhibitors connected by appropriate linkers were designed,
synthesized, and evaluated from the hit compound saprorthoquinone (1). Among them, compound
33d showed balanced activity against both IDO1 (IC50 = 0.73 μM) and HDAC1 (IC50 = 0.46 μM).
Importantly, the structure of 33d suggested that an ortho-quinone pharmacophore and a N-(2-
aminophenyl) amide pharmacophore were necessary for the IDO inhibition and HDAC inhibition
respectively. Meanwhile, these two pharmacophore groups should be combined by a pentane
linker. Moreover, the binding modes of 33d to the enzyme active site showed that the hydrogen
bond with Leu234 of IDO1 appeared to confer increased potency to this class of inhibitors, which
may explain the higher activity of 33d. This study provides a new strategy for future IDO1/HDAC
dual inhibitors with synergistic antitumor activity started from lead compound 33d.
Keywords: saprorthoquinone; structural modification; IDO1; HDAC; dual inhibitors; antitumor
1. Introduction
Cancer is one of the most important factors have been seriously threatening human’s life and
health. According to the GLOBOCAN 2018 estimates of cancer incidence and mortality produced by
the International Agency for Research on Cancer, there were an estimated 18.1 million new cases of
cancer and 9.6 million deaths from cancer worldwide in 2018 [1]. Traditional medications for cancer
patients through targeting the tumor cells directly, showed a series of disadvantages such as low
efficacy, severe side effects, muti-drug resistance, as well as significant individual differences [2]. On
the contrary, cancer immunotherapy, which is one of the most promising treatments, has been an
alternative fundamental strategy of cancer treatment by activating T cells to enhance patients’ native
immune response [3]. The immune checkpoint inhibitors cytotoxic T-lymphocyte-associated protein
4 (CTLA-4) antibody ipilimumab and programmed death (PD-1) antibodies pembrolizumab and
nivolumab were approved by FDA [4]. However, these biologic therapies exhibited inevitable
Molecules 2020, 25, 4494; doi:10.3390/molecules25194494
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weaknesses, such as risk of infection, high cost, and the requirement for intravenous injections [5].
By contrast, small-molecules for cancer immunotherapy with significant advantages, such as oral
administration, access to intracellular targets, and greater drug exposure within the tumor
microenvironment, which not only serves as a supplement for the monoclonal antibody, but also
plays a synergistic effect, having important clinical and economic value [6]. Therefore, there is an
urgent demand for developing small molecules to enhance the immune system and fight against
cancer.
IDO1 is an intracellular heme-containing enzyme that catalyzes the oxidation of L-tryptophan
(Trp) to N-formyl-L-kynurenine (Kyn) [7]. This enzyme is overexpressed in the microenvironment of
several tumor types including carcinomas of the endometrium, cervix, kidney, lung, and colon [8]. In
detail, IDO1 activity has a dual effect on the immune tolerogenic environment, including a depletion
of Trp storages and an accumulation of Kyn products [9]. By co-opting IDO1 activity, tumor cells can
mask their rapid cellular growth effectively and evade the host immune response [10]. Given the
important role in tumor immune escape, IDO1 represents a valuable therapeutic target in cancer
immunotherapy. Unfortunately, single treatments of IDO inhibitors showed limited antitumor
activity in pre-clinical models. IDO inhibitor has been shown to behave synergistically when used in
combination with therapies which target immune checkpoints [11]. Though the synergistic effects
observed by the combination of IDO1 inhibitors with other therapies, drug combination strategies
are always limited by complex pharmacokinetics and drug−drug interactions [12]. To overcome the
problem, designing a single agent that simultaneously targets two or more synergistic mechanisms
has attracted great interests [13].
Histone deacetylases (HDACs) are a family of enzymes, which catalyze the deacetylation of the
lysine residues at the amino terminal of histones [14]. DNA deregulation and post-translational
modifications of the histone tails is a characteristic feature of mammalian cancer. It had been seen
that histone deacetylation causes tumor invasion and metastasis [15]. Studies resulted that HDAC
inhibitors enhance the anticancer effectiveness of already present therapeutically and would be very
influent in the co-activity together with other anticancer treatment approaches [16]. Several HDAC
inhibitors, such as SAHA and chidamide, have been approved for the treatment of various
hematological malignancies (Figure 1) [17]. Recent studies showed that HDACs could also improve
tumor recognition and reverse immune suppression via various mechanisms [18]. Therefore, the
discovery of dual IDO1 and HDAC inhibitors may provide a novel strategy for cancer treatment by
taking advantages of both immunotherapeutic and epigenetic drugs.
Figure 1. Chemical structures of IDO1 inhibitors and HDAC inhibitors, and design strategy of IDO1
and HDAC dual inhibitors.

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Traditional Chinese Medicine (TCM) has evolved over thousands of years and is an invaluable
source of inspiration for drug discovery, which led us to isolate its active components as lead
compounds for drug development [19]. Some of the best IDO1 inhibitors are natural products that
contain an oligocyclicquinone scaffold [20]. Saprorthoquinone was isolated from a traditional
Chinese medicine Salvia prionitis Hance, and more than 40 diterpenoidal compounds have been
isolated from this plant [21]. In 1999, Zhang reported that saprorthoquinone and its analogues
showcased potent cytotoxicity [22]. Among them a novel compound named salvicine possessed a
significant activity against malignant tumors, especially solid tumors such as lung cancer by
inhibiting the topoisomerase II [23]. Furthermore, the preliminary structure-activity relationship of
saprorthoquinone analogues was discussed by Deng in 2010 [24]. These compounds drawn our great
interest due to their structural diversity. Consequently, we hope to obtain new breakthroughs by
biological screening of these natural products to find a privileged scaffold different from those existed
IDO1 inhibitors, which is valuable for further drug discovery and development.
Recently, we screened the IDO1 inhibitory activities of saprorthoquinone (1) and its analogues
(Table 1). Surprisingly we found that ortho-quinones showed significantly better inhibitory activities
than para-quinones among these compounds, while para-quinones were usually established as potent
IDO1 inhibitors [20]. Among these compounds, 1 has both good activity and structural plasticity. To
elucidate the binding mode of compound 1 to IDO1, we performed molecular docking. Predicted
binding mode demonstrate that the carbonyl group of quinone scaffold formed hydrogen bonding
interaction with Ala264 and coordination bonding interaction with Fe2+ of heme. The quinone scaffold
of compound 1 fitted into one of IDO1 hydrophobic pockets (pocket A), and its side chain extended
toward the direction of the other hydrophobic pocket (pocket B) that was mainly composed of
Phe226, Phe227, Arg231 and Ile354 (Figure 2).
Table 1. Structures and IDO1 inhibitory activities of saprorthoquinone and its analogues.
IDO1 Inhibition
@ 10 μM (%)
IDO1 Inhibition
@ 10 μM (%)
Compds.
1
Structure
Compds.
10
Structure
85%
65%
81%
2%
2%
4
5
11
12
13%
6
7
8
66%
86%
73%
13
14
15
6%
30%
3%
9
61%
16
3%
Control
Epacadostat
100%

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Figure 2. Modeling structure of 1-IDO1 complex. Hydrogen bonds are labeled as red dashed lines,
and coordination bonds are labeled as orange dashed lines. The figure was generated using PyMol.
Based on above work, we used saprorthoquinone (1) as a hit compound and tried to find more
effective IDO1 and HDAC dual inhibitors through modification of the side chain of the hit compound
by a pharmacophore fusion strategy. Meanwhile, the zinc binding functional group N-(2-
aminophenyl) amide (Chidamide as a template) is essential for HDAC inhibition can be introduced
on the IDO heme binding scaffold directly or via a proper spacer (Figure 1). As a result, a series of
novel IDO1 and HDAC dual targeting molecules were designed, synthesized, and assayed.
2. Results and Discussion
2.1. IDO1 Inhibitory Activities of Saprorthoquinone and Its Analogues Isolated from Salvia Prionitis Hance
Some of the best IDO1 inhibitors are natural products that contain an oligocyclic quinone
scaffold [20]. Saprorthoquinone was isolated from a traditional Chinese medicine Salvia prionitis
Hance, and more than 40 diterpenoid compounds have been isolated from this plant [21]. Recently,
we screened the IDO1 inhibitory activities of saprorthoquinone (1) and its analogues, and the IDO1
inhibitor epacadostat (INCB024360) was chosen as positive controls (Table 1). Surprisingly we found
that ortho-quinones showed significantly better inhibitory activities than para-quinones among these
compounds, while para-quinones were usually established as potent IDO1 inhibitors. Among them,
ortho-quinone analogues 1, 4–9 exhibited over 60% IDO1 inhibitory activity in 10 μM. On the contrary,
para-quinones analogues 10–16 exhibited lower than 30% IDO1 inhibitory activity in 10 μM. Some of
them (10, 11, 15, and 16) almost lost their activity. This result demonstrated that the ortho-quinone is
the key pharmacophore in response to IDO1. Particularly, compound 1 and 7 exhibited the most
potent IDO1 inhibitory activity (85% and 86%) at the concentration of 10 μM, which used for further
drug discovery and development.
2.2. Novel Saprorthoquinone Analogues with Different Side Chain as Linker
Compound 1 with potent IDO1 inhibitory was chosen for structure-activity relationship study,
in which the key pharmacophore ortho-quinone of 1 was maintained, and the side chain was
transformed into three kinds of functional groups, such as amino group, phenylamino group and
sulfonyl group.
The key intermediate 18 was synthesized by the method reported in the literature (Scheme 1)
[22]. Starting from the key intermediate, a simple amidation product 19 was prepared from an acid
chloride. Then we used condensation reaction to get compounds 21a–g, which allows for the rapid
and versatile generation of different groups in the side chain, in order to allow for the penetration in

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the pocket B of IDO1 active site. Furthermore, an unstable compound 22 was obtained by reduction
reaction, and then we synthesized stable sulfonyl products 24a–c.
Scheme 1. Reagents and conditions: (a) (I) m-CPBA, DCM, rt; (II) 8% perchloric acid, THF/H2O, rt, 92%;
(b) PCC, H2O, DCM, rt, 47%; (c) (I) SOCl2, DCM, reflux; (II) NH3·H2O, DCM, 0 °C, 40%; (d) HATU,
DIPEA, dry DMF, rt, 26–85%; (e) LiAlH4, dry THF, 0 °C, 53%; (f) dry DCM, 0 °C, 18–40%.
The activity of the first batch of products were assayed against IDO1 and A549 cell line with 1
and NLG-919 as the positive control and the results were shown in Table 2. Basically, when the side
chain was replaced by a large group (phenyl), the IDO1 inhibitory activity was generally reduced
(e.g., 21a–g). On the contrary, if the side chain was replaced by a small group, the activity of IDO1
inhibitory could be increased. Sulfonyl group product 24a showcased better IDO1 inhibitory activity
than hit compound 1 (IC50 = 0.72 μM vs. IC50 = 1.76 μM). Amidated product 19 with a simple side
chain gave the best enzymatic inhibitory activity (IC50 = 0.51 μM).
Table 2. IDO1 inhibitory and antiproliferative activities of the target compounds.
Compound IDO1 IC50 (μM) a or % Inhibition at 10 μM A549 IC50 (μM)
1
17
1.76 ± 0.27 (85%)
0.74 ± 0.15 (90%)
0.99 ± 0.24 (86%)
0.51 ± 0.04 (78%)
1.33 ± 0.01 (80%)
64%
9.97
12.84
>20
18
19
14.48
13.07
14.83
7.49
21a
21b
21c
64%
21d
21e
21f
65%
9.31
66%
>20
79%
>20
21g
24a
24b
24c
74%
12.17
11.82
15.54
11.05
NT b
0.72 ± 0.13 (91%)
87%
86%
NLG-919
0.10 ± 0.02
a IC50 values are the mean of at least two independent assays, presented as mean ± SD. b NT means not tested.

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On the other hand, observation of cytotoxicities presented for these compounds couldn’t infer
clear structure-activity relationship. Probably as reported in the literature, the cytotoxicities might be
related to the ability of quinone carbonyl to accept one or two electrons to form the corresponding
radical anion or dianion species [24].
2.3. IDO1 and HDAC Dual Inhibitors Derived from Natural Product Saprorthoquinone
The zinc binding functional group N-(2-aminophenyl) amide is essential for HDAC inhibition
can be introduced on the IDO1 heme binding scaffold directly or via a proper spacer (Scheme 2). As
a result, a series of novel IDO1 and HDAC dual target molecules were designed, synthesized, and
assayed. Compounds 27a to 27c were prepared to observe the effect of substituents on the phenyl
ring, and 33a to 33d were used to observe the effect of the length of linker. Compounds 33a–d were
condensed by 32a–d and 18 using EDCI as a condensing agent, because HATU is more advantageous
for the condensation of an amine group on the phenyl ring. All products were purified by column
chromatography.
Scheme 2. Reagents and conditions: (a) (Boc)2O, TEA, DCM, 0 °C, 66–76%; (b) (I) HATU, DIPEA, dry
DMF, rt; (II) TFA, DCM, 0 °C, 10–27%; (c) Fmoc-Cl, NaHCO3, DCM, rt, 36%; (d) TFA, DCM, 0 °C; (e)
(I) HATU, DIPEA, dry DMF, rt; (II) DEA, DCM, rt, 4%; (f) HATU, DIPEA, dry DMF, rt, 41–84%; (g)
18, EDCI, DMAP, DIPEA, dry DMF, rt, 9–25%.
The activities of the second batch of products were assayed against IDO1, HDAC1 and A549 cell
line were shown in Table 3. NLG-919 and SAHA were used as the positive controls. The results
showed that the ortho-substitution on the phenyl ring in the terminal amide group 27a might have a
positive effect on inhibitory activity of IDO1 (IC50 = 0.72 μM), the substitution of fluorine atoms at
positions 4 (27b, 84% inhibiton @ 10 μM) and 5 (27c, 56% inhibiton @ 10 μM) confirmed the adverse
effects of meta and para-substitution on IDO1 inhibition. Additionally, these three compounds (27a–
c) have no inhibitory activity on HDAC1, due to their unsuitable linker. By contrast, the order of
influence of carbon chain length on HDAC1 inhibition was five carbons (33d, IC50 = 0.46 μM) > three
carbons (33b, IC50 = 34.87 μM) ≈ four carbons (33c, IC50 = 39.48 μM) > two carbons (33a, IC50 > 100 μM),
compound 33d was the most potent HDAC1 inhibitor. Notably, improvement of the IDO1 inhibitory
was observed for these compounds with a long linker. However, compounds 33a–d had reduced
activity on the solid tumor cell line A549 may be due to the fact that IDO1 inhibitors do not destroy

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tumor cells directly, which may provide an indirect evidence for the increase of its inhibitory activity
of IDO1. In vivo antitumor activity of compound 33d is in the process experiment.
Table 3. HDAC1, IDO1 inhibitory and antiproliferative activities of the target compounds.
Compd. IDO1 IC50 (μM) a or % Inhibition at 10 μM HDAC1 IC50 (μM) a A549 IC50 (μM)
27a
27b
0.72 ± 0.10 (88%)
84%
>100
>100
17.75
9.51
>20
27c
56%
>100
33a
89%
>100
>20
33b
0.58 ± 0.11 (80%)
0.55 ± 0.02 (90%)
0.71 ± 0.03 (82%)
0.10 ± 0.02 (85%)
NT b
34.87 ± 0.75
39.48 ± 0.74
0.46 ± 0.07
NT b
19.06
>20
33c
33d
>20
NLG-919
SAHA
NT b
NT b
0.015 ± 0.00012
a IC50 values are the mean of at least two independent assays, presented as mean ± SD. b NT means not tested.
Besides, we have evaluated the toxicity of all the compounds on a non-tumor cell (ECs, primary
endothelial cells). The cytotoxic concentration 50 (CC50) of all the compounds were over 200 μM. The
results demonstrated the compounds have good safety.
2.4. Molecular Modeling Studies of Promising Compound 1, 19 and 33d
To gain insight into the molecular determinants that modulate the inhibitory activity of our
compounds, molecular modeling studies were performed based on the X-ray cocrystal structure of
IDO1 complex and HDAC1 complex. Protein structures were reconstructed by SWISS-MODEL
homology-modelling server [25] using IDO1 (PDB code: 5EK2) and HDAC1 (PDB code: 4BKX) as
templates. Compound 1, 19 and 33d were selected as the ligands for the molecular docking. Since the
high flexibility of side chain and backbone of IDO1 protein, classical semi-flexible docking method
often poses a problem for IDO1 docking. To overcome difficulty mentioned above, after docking by
AutoDock software, best poses of 33d-protein complexes with dominant conformation underwent
QM/MM simulations [26].
Docking poses of the most promising compounds (19 and 33d) and human IDO1 are reported
in Figure 3A,B. Docking studies showing that these analogues of saprorthoquinone had similar
binding modes to compound 1 (Figure 2). The quinone scaffold of both 19 and 33d was
accommodated in the hydrophobic pocket A (Mainly composed of Tyr126, Phe163, Phe164, Gly262,
and Ala264), while the carbonyl group of quinone scaffold was able to form a strong coordination
bond with the heme group. The carbonyl group of quinone scaffold could also formed hydrogen
bonding interactions with Ala264, which is same as the binding mode of 1. The NH group on the
amide or terminal amine group could form a hydrogen bond with the oxygen atom on heme, Gly262
or Leu234, which is different from the predicted binding mode of compound 1. Probably for this
reason, compounds 19 and 33d could bind to pocket A more strongly and had higher inhibitory
activities against IDO1. However, none of these compounds had a good combination with amino-
acid residue of pocket B (Phe226, Arg231 and Ile354). The linker in the structure may be very
important for occupying of pocket B, and further optimization of such inhibitors in ongoing in our
lab.
For the binding mode of 33d with HDAC1, main interactions were involved in the zinc binding
group (ZBG) (Figure 3C). Docking studies were carried out using the same method as above. Besides
chelating with Zn2+, the N-(2-aminophenyl) amide group was engaged with a hydrogen bonding
network with His141 and Tyr303. The amide NH group formed hydrogen bonding interaction with
the phenolic hydroxyl group of Tyr303, and the carbonyl group of ZBG formed hydrogen bond with
the nitrogen atom in the imidazole of His141.
To evaluate the dynamics stabilities of the complexes of compound 33d with IDO1 and HDAC1,
GROMACS software [27] with charm 36 forcefield [28] was applied to conduct the simulations.
Complexes and proteins in the apo form (no ligand bound) were simulated under the temperature of

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310 K and pressure of 1 bar for 100 ns. Sodium or chloride ions were added to the water-protein
systems to preserve electroneutrality.
Figure 3. Modeling structure of ligand-protein complexes. (A,B) For compound 19 and 33d with
IDO1. (C) For compound 33d with HDAC1. Hydrogen bonds are labeled as red dashed lines, and
coordination bonds are labeled as orange dashed lines. The figure was generated using PyMol.
All models went stable in the later stage of the simulations. The backbone atomic RMSD values
of IDO1 and HDAC1 proteins in apo and bounding forms with 33d were 0.292 ± 0.013 nm (IDO1 in
apo), 0.233 ± 0.012 nm (IDO1 bounding form), 0.398 ± 0.022 nm (HDAC1 in apo) and 0.402 ± 0.014 nm
(HDAC1 bounding form) during the last 30 ns of simulation, respectively (Figure 4A,C). These results
indicated that the presence of 33d bound to IDO1 and HDAC1 influenced the backbone atomic RMSD.

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The ligand-bound proteins exhibited the similar or less mobility than proteins in apo state under the
same simulation conditions.
Figure 4. Dynamic simulations of 33d bound with IDO1 or HDAC1. RMSD analysis of IDO1 (A) and
HDAC1 (C) in apo or bounding form. RMSF analysis of IDO1 (B) and HDAC1 (D) in apo or bounding
form.
Similar RMSF distributions were observed for the IDO1 and HDAC1 in apo/bounding forms,
respectively (Figure 4B,D). The greatest fluctuations observed in the terminals of IDO1/HDAC1 and
regions (residues 203–212 and 265–274) of HDAC1. Residues in Pocket A and Pocket B of IDO1,
including Tyr126, Phe163, Phe164, Phe226, Arg231, Gly262 and Ala264, were quite stable. Residues
in active sites of HDAC1, such as His140, His141 and Tyr303, were stable too. Overall, the RMSF
analysis indicated that the complex and protein exhibited similar dynamics during the MD
simulations.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
All starting materials were commercially available and used without further purification unless
otherwise stated. TLC analysis was performed using pre-coated glass plates. Column
chromatography was performed using silica gel (300–400 mesh) or octadecylsilyl silica gel (75 μm)
for separation and purification. 1H-NMR and 13C-NMR spectra were recorded on DRX-400
spectrometer (Bruker, Billerica, MA, USA) with CDCl3, CD3OD, acetone-d6 or DMSO-d6 as solvents.
Resonances (δ) are given in parts per million relatives to tetramethylsilane or a residual solvent peak
(CDCl3: 1H: δ = 7.26 ppm, 13C: δ = 77.00 ppm; CD3OD: 1H: δ = 3.31 ppm, 13C: δ = 49.00 ppm; acetone-d6:
1H: δ = 2.05 ppm, 13C: δ = 206.26 ppm, 29.84 ppm; DMSO-d6: 1H: δ = 2.50 ppm, 13C: δ = 39.50 ppm). Data
are reported as follows: chemical shift; multiplicity (s = singlet, d = doublet, t = triplet,q = quartet, m
= multiplet); coupling constants (Hz); and integration. HRMS were obtained on an Triple TOF®® 5600+
(AB Sciex, Foster City, CA, USA). More spectral information can be found in Supplementary
Materials.

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3.1.2. Synthesis of 8-(3,4-Dihydroxy-4-methylpentyl)-3-isopropyl-7-methylnaphthalene-1,2-dione
(17)
To a stirred solution of m-chloroperbenzoic acid (1.45 g, 8.40 mmol) in dichloromethane (24 mL)
held at 0 °C was added a solution of compound 1 (2 g, 6.75 mmol) dissolved in dichloromethane (20
mL) during a period of 30 min. The mixture was stirred overnight at room temperature, washed with
saturated solution of NaHCO3 and dried over anhydrous MgSO4. The solvent was concentrated in
vacuo and dissolved in tetrahydrofuran (35 mL) was added 6.7 mL of water. The solution was stirred
and 4 mL of 8% perchloric acid was added. After stirring for 6 h under N2 at room temperature,
saturated solution of NaCl was added and the mixture was extracted several times with ethyl acetate.
The organic phase was washed with dilute sodium bicarbonate and dried over anhydrous MgSO4,
evaporated under reduced pressure and purified by silica gel column chromatography using
petroleum ether/ethyl acetate mixture (4:1 to 1:1) as eluent to afford 17 as an orange solid in 92% yield
(2.05 g). 1H-NMR (400 MHz, CDCl3) δ 7.38 (d, J = 7.6 Hz, 1H, Ar-H), 7.10 (s, 1H, Ar-H), 7.09 (d, J = 7.6
Hz, 1H, Ar-H), 3.36–3.27 (m, 1H, CH of hydroxymethine), 3.15–3.05 (m, 1H, one of Ar-CH2 protons),
3.05–2.99 (m, 1H, CH of isopropyl group), 2.98–2.93 (m, 1H, one of Ar-CH2 protons), 2.39 (s, 3H, Ar-
CH3), 1.90–1.78 (m, 1H, one of CH2CO protons), 1.75–1.64 (m, 1H, one of CH2CO protons), 1.33 (s, 3H,
CH3), 1.28 (s, 3H, CH3), 1.18 (d, J = 6.9 Hz, 3H, CH3 of isopropyl group), 1.17 (d, J = 6.9 Hz, 3H, CH3 of
isopropyl group). ESI-MS m/z: 353.2 [M + Na]+.
3.1.3. Synthesis of 3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanoic acid
(18)
Compound 17 (505.2 mg, 1.53 mmol) was dissolved in dichloromethane (5 mL),
pyridiniumchlorochromate (3.5 g, 16.24 mmol) was added and 1.5 mL of water was added dropwise.
The mixture was stirred overnight at room temperature. The crude material was filtered through
kieselguhr and purified by silica gel column chromatography using petroleum ether/ethyl acetate
1
mixture (3:1 to 1:2) as eluent to afford 18 as an orange solid in 47% yield (204.9 mg). H-NMR (400
MHz, CDCl3) δ 7.38 (d, J = 7.6 Hz, 1H, Ar-H), 7.09 (s, 1H, Ar-H), 7.08 (d, J = 7.6 Hz, 1H, Ar-H), 3.35 (t,
J = 7.7 Hz, 2H, Ar-CH2), 3.00 (septet, J = 6.9 Hz, 1H, CH), 2.61 (t, J = 7.7 Hz, 2H, CH2CO), 2.39 (s, 3H,
Ar-CH3), 1.15 (d, J = 6.9 Hz, 6H, 2CH3). ESI-MS m/z: 287.0 [M + H]+.
3.1.4. Synthesis of 3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydro- naphthalen-1-yl)propanamide
(19)
To a solution of compound 18 (100.0 mg, 0.35 mmol) in dichloromethane (5 mL) held at 0 °C was
added thionylchloride (100 μL) and 1H-benzotriazole (42.9 mg, 0.36 mmol), and the mixture was
stirred at reflux temperature for 2 h. The solvent was concentrated in vacuo and dissolved in
dichloromethane (1 mL). Ammonium hydroxide solution was added at 0 °C and the mixture was
stirred at room temperature for 1 h. Water was added and the mixture was extracted several times
with DCM. The organic phase was dried over anhydrous MgSO4, evaporated under reduced pressure
and purified by octadecylsilyl silica gel column chromatography using methanol/water (2:3) as eluent
to afford 19 as an orange solid in 40% yield (40.0 mg). 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.1 Hz,
1H, Ar-H), 7.12 (d, J = 7.1 Hz, 2H, Ar-H), 6.39 (s, 1H, one of CONH2 protons), 5.45 (s, 1H, one of
CONH2 protons), 3.33 (m, 2H, Ar-CH2), 3.03 (septet, J = 5.9 Hz, 1H, CH), 2.43 (s, 3H, Ar-CH3), 2.39 (m,
2H, CH2CO), 1.18 (d, J = 5.9 Hz, 6H, 2CH3). 13C NMR (100 MHz, CDCl3) δ 182.67, 181.09, 174.87, 146.50,
144.98, 140.32, 140.26, 137.47, 135.31, 128.87, 128.14, 35.27, 27.88, 26.99, 21.48, 19.74. ESI-MS m/z: 308.0
+
[M + Na]+. HRESI-MS calcd. for C17H20NO3 ([M + H]+) 286.1438, found 286.1440.
3.1.5. General Procedure A: Synthesis of Compounds 21a–21g and 31a–31d
Compound 18 or 30a–30d was dissolved in 1 mL of dry N,N-dimethylformamide, 2-(7-aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N-ethyl-diisopropylamine
(DIPEA) were added. After stirring uniformly, amine 20a–20g or 26a was added. The mixture was
stirred overnight at room temperature. dichloromethane was added and the mixture was washed

Molecules 2020, 25, 4494
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with saturated solution of NaHCO3,the organic phase was purified by column chromatography to
afford 21a–21g or 31a–31d.
N-(3-Fluorophenyl)-3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamide
(21a):
Compound 18 (40.1 mg, 0.14 mmol), HATU (64.6 mg, 0.17 mmol), DIPEA (45.3 μL, 0.26 mmol) and
20a (16.3 μL, 0.17 mmol) were used in general procedure A. The crude product was purified by
octadecylsilyl silica gel column chromatography using methanol/water (3:1) as eluent to afford 21a
as an orange solid in 72% yield (38.4 mg). 1H-NMR (400 MHz, CDCl3) δ 8.42 (s, 1H, CONH), 7.64 (d, J
= 11.3 Hz, 1H, Ar-H of phenyl group), 7.44 (d, J = 7.5 Hz, 1H, Ar-H of naphthoquinone), 7.31–7.22 (m,
2H, Ar-H of phenyl group), 7.15–7.10 (m, 2H, Ar-H of naphthoquinone), 6.88–6.71 (m, 1H, Ar-H of
phenyl group), 3.42–3.32 (m, 2H, Ar-CH2), 3.03 (septet, J = 6.9 Hz, 1H, CH), 2.55–2.48 (m, 2H, CH2CO),
2.45 (s, 3H, Ar-CH3), 1.19 (d, J = 6.9 Hz, 6H, 2CH3).13C-NMR (100 MHz, CDCl3) δ 182.76, 181.04, 170.93,
162.99 (d, J = 244.3 Hz), 146.31, 144.96, 140.38, 140.32, 139.74 (d, J = 10.9 Hz), 137.64, 135.40, 129.94 (d,
J = 9.3 Hz), 129.02, 128.11, 115.11, 110.71 (d, J = 21.3 Hz), 107.29 (d, J = 26.4 Hz), 36.96, 27.83, 27.06,
+
21.45, 19.75. ESI-MS m/z: 380.0 [M + H]+. HRESI-MS calcd. for C23H23FNO3 ([M + H]+) 380.1656, found
380.1659.
N-(4-Fluorophenyl)-3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamide
(21b):
Compound 18 (28.6 mg, 0.10 mmol), HATU (49.4 mg, 0.13 mmol), DIPEA (34.8 μL, 0.20 mmol) and
20b (12.3 μL, 0.13 mmol) were used in general procedure A. The crude product was purified by
octadecylsilyl silica gel column chromatography using methanol/water (3:1) as eluent to afford 21b
as an orange solid in 53% yield (20.1 mg). 1H-NMR (400 MHz, CDCl3) δ8.25 (s, 1H, CONH), 7.63–7.56
(m, 2H, Ar-H of phenyl group), 7.43 (d, J = 7.7 Hz, 1H, Ar-H of naphthoquinone), 7.12 (s, 1H, Ar-H of
naphthoquinone), 7.12 (d, J = 7.7 Hz, 1H, Ar-H of naphthoquinone), 7.06–6.99 (m, 2H, Ar-H of phenyl
group), 3.43–3.34 (m, 2H, Ar-CH2), 3.03 (septet, J = 6.9 Hz, 1H, CH), 2.55–2.45 (m, 2H, CH2CO), 2.45
(s, 3H, Ar-CH3), 1.19 (d, J = 6.9 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ 182.82, 181.08, 170.70,
159.30 (d, J = 243.3 Hz), 146.37, 145.02, 140.36, 140.24, 137.59, 135.41, 134.17, 128.95, 128.15, 121.75 (d,
J = 7.8 Hz), 115.51 (d, J = 22.4 Hz), 36.88, 27.97, 27.08, 21.44, 19.71. ESI-MS m/z: 380.0 [M + H]+. HRESI-
+
MS calcd. for C23H23FNO3 ([M + H]+) 380.1656, found 380.1660.
3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)-N-(4-(trifluoromethyl)phenyl)propanamide
(21c): Compound 18 (28.6 mg, 0.10 mmol), HATU (49.4 mg, 0.13 mmol), DIPEA (34.8 μL, 0.20 mmol)
and 20c (20.9 mg, 0.13 mmol) were used in general procedure A. The crude product was purified by
octadecylsilyl silica gel column chromatography using methanol/water (3:1) as eluent to afford 21c
as an orange solid in 85% yield (36.5 mg). 1H-NMR (400 MHz, CDCl3) δ 8.58 (s, 1H, CONH), 7.79 (d,
J = 8.3 Hz, 2H, Ar-H of phenyl group), 7.59 (d, J = 8.3 Hz, 2H, Ar-H of phenyl group), 7.44 (d, J = 7.6
Hz, 1H, Ar-H of naphthoquinone), 7.14 (d, J = 7.6 Hz, 1H, Ar-H of naphthoquinone), 7.13 (s, 1H, Ar-
H of naphthoquinone), 3.43–3.34 (m, 2H, Ar-CH2), 3.03 (septet, J = 6.8 Hz, 1H, CH), 2.57–2.48 (m, 2H,
CH2CO), 2.45 (s, 3H, Ar-CH3), 1.19 (d, J = 6.8 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ 182.82,
181.05, 171.16, 146.26, 144.97, 141.36, 140.36, 137.70, 135.43, 129.07, 128.10, 126.15 (q, J = 3.4 Hz), 125.69
(q, J = 32.1 Hz), 122.84, 119.42, 36.95, 27.76, 27.07, 21.44, 19.74. ESI-MS m/z: 430.0 [M + H]+. HRESI-MS
+
calcd. for C24H23F3NO3 ([M + H]+) 430.1625, found 430.1625.
N-(3-Bromo-4-fluorophenyl)-3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamide
(21d): Compound 18 (40.1 mg, 0.14 mmol), HATU (64.6 mg, 0.17 mmol), DIPEA (45.3 μL, 0.26 mmol)
and 20d (32.1 mg, 0.17 mmol) were used in general procedure A. The crude product was purified by
octadecylsilyl silica gel column chromatography using methanol/water (4:1) as eluent to afford 21d
as an orange solid in 53% yield (34.0 mg).1H-NMR (400 MHz, CDCl3) δ 8.33 (s, 1H, CONH), 8.02–7.95
(m, 1H, Ar-H of phenyl group), 7.56–7.49 (m, 1H, Ar-H of phenyl group), 7.45 (d, J = 7.7 Hz, 1H, Ar-
H of naphthoquinone), 7.14 (d, J = 7.7 Hz, 1H, Ar-H of naphthoquinone), 7.13 (s, 1H, Ar-H of
naphthoquinone), 7.12–7.05 (m, 1H, Ar-H of phenyl group), 3.41–3.33 (m, 2H, Ar-CH2), 3.04 (septet, J
= 6.9 Hz, 1H, CH), 2.52–2.47 (m, 2H, CH2CO), 2.45 (s, 3H, Ar-CH3), 1.19 (d, J = 6.9 Hz, 6H, 2CH3). 13C-
NMR (100 MHz, DMSO-d6) δ 182.11, 180.85, 171.27, 154.46 (d, J = 242.1 Hz), 145.79, 144.13, 140.46,
140.44, 137.16, 135.15, 134.20 (d, J = 12.4 Hz), 129.33, 128.96, 123.68, 120.44 (d, J = 6.7 Hz), 117.14 (d, J =

Molecules 2020, 25, 4494
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22.8 Hz), 107.95 (d, J = 21.6 Hz), 35.47, 26.96, 25.74, 21.79, 19.84. ESI-MS m/z: 458.8 [M + H]+. HRESI-
+
MS calcd. for C23H22BrFNO3 ([M + H]+) 458.0762, found 458.0765.
3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)-N-(4-methoxyphenyl)propanamide
(21e):
Compound 18 (28.6 mg, 0.10 mmol), HATU (49.4 mg, 0.13 mmol), DIPEA (34.8 μL, 0.20 mmol) and
20e (16.0 mg, 0.13 mmol) were used in general procedure A. The crude product was purified by
octadecylsilyl silica gel column chromatography using methanol/water (3:1) as eluent to afford 21e
as an orange solid in 26% yield (10.3 mg).1H-NMR (400 MHz, CDCl3) δ 8.06 (s, 1H, CONH), 7.52 (d, J
= 8.8 Hz, 2H, Ar-H of phenyl group), 7.43 (d, J = 7.7 Hz, 1H, Ar-H of naphthoquinone), 7.13 (s, 1H,
Ar-H of naphthoquinone), 7.12 (d, J = 7.7 Hz, 1H, Ar-H of naphthoquinone), 6.88 (d, J = 8.8 Hz, 2H,
Ar-H of phenyl group), 3.80 (s, 3H, OCH3), 3.44–3.35 (m, 2H, Ar-CH2), 3.04 (septet, J = 6.9 Hz, 1H,
CH), 2.52–2.47 (m, 2H, CH2CO), 2.45 (s, 3H, Ar-CH3), 1.19 (d, J = 6.9 Hz, 6H, 2CH3). 13C-NMR (100
MHz, CDCl3) δ 182.75, 181.11, 170.51, 156.34, 146.52, 145.00, 140.41, 140.25, 137.54, 135.36, 131.26,
128.90, 128.16, 121.85, 114.11, 55.49, 36.90, 28.11, 27.05, 21.47, 19.78. ESI-MS m/z: 392.0 [M + H]+. HRESI-
+
MS calcd. for C24H26NO4 ([M + H]+) 392.1856, found 392.1861.
N-((1H-indol-3-yl)methyl)-3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamide
(21f): Compound 18 (28.6 mg, 0.10 mmol), HATU (49.4 mg, 0.13 mmol), DIPEA (34.8 μL, 0.20 mmol)
and 20f (19.0 mg, 0.13 mmol) were used in general procedure A. The crude product was purified by
flash chromatography on silica gel then octadecylsilyl silica gel column chromatography using
methanol/water (3:1) as eluent to afford 21f as an orange solid in 52% yield (21.5 mg). 1H-NMR (400
MHz, CDCl3) δ 8.36 (s, 1H, CONH), 7.65 (d, J = 7.8 Hz, 1H, Ar-H of indole), 7.37 (d, J = 7.8 Hz, 1H, Ar-
H of naphthoquinone), 7.23–7.01 (m, 5H, Ar-H, 3H of indole and 2H of naphthoquinone), 6.45–6.36
(m, 1H, Ar-H of indole), 4.67 (d, J = 5.3 Hz, 2H, CH2 by indole), 3.34–3.26 (m, 2H, CH2
bynaphthoquinone), 2.99 (septet, J = 6.9Hz, 1H, CH), 2.42 (s, 3H, Ar-CH3), 2.41–2.31 (m, 2H, CH2CO),
1.15 (d, J = 6.9 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ 182.39, 181.08, 172.29, 146.66, 144.79,
140.50, 140.37, 137.35, 136.41, 135.12, 128.76, 128.13, 126.59, 123.27, 122.30, 119.76, 118.99, 112.94,
111.31, 35.67, 35.19, 27.79, 26.95, 21.47, 19.84. ESI-MS m/z: 415.2 [M + H]+. HRESI-MS calcd. for
+
C26H27N2O3 ([M + H]+) 415.2016, found 415.2026.
3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)-N-((1-methyl-1H-indol-3-yl)methyl)-
propanamide (21g): Compound 18 (28.6 mg, 0.10 mmol), HATU (49.4 mg, 0.13 mmol), DIPEA (34.8 μL,
0.20 mmol) and 20g (20.8 mg, 0.13 mmol) were used in general procedure A. The crude product was
purified by flash chromatography on silica gel then octadecylsilyl silica gel column chromatography
using methanol/water (4:1) as eluent to afford 21g as an orange solid in 44% yield (19.0 mg). 1H-NMR
(400 MHz, CDCl3) δ 7.65 (d, J = 7.9 Hz, 1H, Ar-H of indole), 7.37 (d, J = 7.6 Hz, 1H, Ar-H of
naphthoquinone), 7.32–7.04 (m, 6H, CONH and 3Ar-H of indole and 2Ar-H of naphthoquinone),
6.40–6.29 (m, 1H, Ar-H of indole), 4.65 (d, J = 5.2 Hz, 2H, CH2 by indole), 3.77 (s, 3H, NCH3), 3.34–3.26
(m, 2H, CH2 by naphthoquinone), 3.00 (septet, J = 6.9 Hz, 1H, CH), 2.43 (s, 3H, Ar-CH3), 2.39–2.31 (m,
2H, CH2CO), 1.15 (d, J = 6.9 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3)δ 182.42, 181.08, 172.17, 146.65,
144.81, 140.48, 140.28, 137.32, 137.14, 135.13, 128.73, 128.16, 127.93, 127.06, 121.89, 119.33, 119.12,
111.44, 109.32, 35.73, 35.03, 32.73, 27.82, 26.95, 21.47, 19.84. ESI-MS m/z: 429.2 [M + H]+. HRESI-MS
+
calcd. for C27H29N2O3 ([M + H]+) 429.2173, found 429.2175.
tert-Butyl-(3-((2-((tert-butoxycarbonyl)amino)phenyl)amino)-3-oxopropyl)carbamate (31a): Compound 30a
(39.7 mg, 0.21 mmol), HATU (95.1 mg, 0.25 mmol), DIPEA (73.2 μL, 0.42 mmol) and 26a (52.0 mg,
0.25 mmol) were used in general procedure A. The crude product was purified by silica gel column
chromatography using petroleum ether/ethyl acetate mixture (3:1 to 1:1) as eluent to afford 31a as a
colorless oil in 84% yield (66.5 mg). 1H-NMR (400 MHz, CDCl3) δ8.59 (s, 1H,Ph-NHCO), 7.54–7.44 (m,
1H, Ar-H), 7.38 (d, J = 7.0 Hz, 1H, Ar-H), 7.25 (s, 1H, Ph-NHCO), 7.21–7.06 (m, 2H, Ar-H), 5.32 (s, 1H,
CONH), 3.58–3.29 (m, 2H, NH-CH2), 2.64–2.36 (m, 2H, CH2CO), 1.51 (s, 9H, 3CH3), 1.43 (s, 9H,
3CH3).ESI-MS m/z: 402.2 [M + Na]+.
tert-Butyl-(2-(4-((tert-butoxycarbonyl)amino)butanamido)phenyl)carbamate (31b): Compound 30b (99.5
mg, 0.49 mmol), HATU (224.2 mg, 0.59 mmol), DIPEA (170.7 μL, 0.98 mmol) and 26a (122.8 mg, 0.59

Molecules 2020, 25, 4494
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mmol) were used in general procedure A. The crude product was purified by octadecylsilyl silica gel
column chromatography using methanol/water (3:2) as eluent to afford 31b as a colorless oil in 41%
1
yield (79.2 mg). H-NMR (400 MHz, CDCl3) δ8.98 (s, 1H,Ph-NHCO), 7.57 (d, J = 7.0 Hz, 1H, Ar-H),
7.47 (s, 1H,Ph-NHCO), 7.35 (d, J = 7.0 Hz, 1H, Ar-H), 7.15 (t, J = 7.0 Hz, 1H, Ar-H), 7.07 (t, J = 7.0 Hz,
1H, Ar-H), 5.03 (s, 1H, CONH), 3.21–3.08 (m, 2H, NH-CH2), 2.37–2.26 (m, 2H, CH2CO), 1.87–1.75 (m,
2H, CH2), 1.50 (s, 9H, 3CH3), 1.45 (s, 9H, 3CH3). m/z: 416.2 [M + Na]+.
tert-Butyl(2-(5-((tert-butoxycarbonyl)amino)pentanamido)phenyl)carbamate (31c): Compound 30c (99.9
mg, 0.46 mmol), HATU (209.1 mg, 0.55 mmol), DIPEA (160.2 μL, 0.92 mmol) and 26a (114.5 mg, 0.55
mmol) were used in general procedure A. The crude product was purified by octadecylsilyl silica gel
column chromatography using methanol/water (3:2) as eluent to afford 31c as a colorless oil in 53%
yield (99.0 mg).1H-NMR (400 MHz, CDCl3) δ 8.74 (s, 1H,Ph-NHCO), 7.50 (d, J = 7.5 Hz, 1H, Ar-H),
7.44 (s, 1H,Ph-NHCO), 7.30 (d, J = 7.5 Hz, 1H, Ar-H), 7.14 (t, J = 7.5 Hz, 1H, Ar-H), 7.06 (t, J = 7.5 Hz,
1H, Ar-H), 4.86 (s, 1H, CONH), 3.10–2.99 (m, 2H, NH-CH2), 2.36–2.21 (m, 2H, CH2CO), 1.70–1.56 (m,
2H, CH2), 1.50 (s, 9H, 3CH3), 1.47–1.33 (m, 11H, CH2 and 3CH3). ESI-MS m/z: 430.2 [M + Na]+.
tert-Butyl(2-(6-((tert-butoxycarbonyl)amino)hexanamido)phenyl)carbamate (31d): Compound 30d (99.4
mg, 0.43 mmol), HATU (178.7 mg, 0.47 mmol), DIPEA (149.8 μL, 0.86 mmol) and 26a (97.8 mg, 0.47
mmol) were used in general procedure A. The crude product was purified by octadecylsilyl silica gel
column chromatography using methanol/water (3:2) as eluent to afford 31d as a colorless oil in 50%
yield (91.2 mg). 1H-NMR (400 MHz, CDCl3) δ 8.61 (s, 1H,Ph-NHCO), 7.45 (d, J = 7.3 Hz, 1H, Ar-H),
7.36 (d, J = 7.3 Hz, 1H, Ar-H), 7.35 (s, 1H,Ph-NHCO), 7.14 (t, J = 7.3 Hz, 1H, Ar-H), 7.08 (t, J = 7.3 Hz,
1H, Ar-H), 4.76 (s, 1H, CONH), 3.17–2.92 (m, 2H, NH-CH2), 2.46–2.16 (m, 2H, CH2CO), 1.75–1.57 (m,
2H, CH2), 1.51 (s, 9H, 3CH3), 1.47–1.37 (m, 11H, CH2 and 3CH3), 1.36–1.24 (m, 2H, CH2). ESI-MS m/z:
444.2 [M + Na]+.
3.1.6. Synthesis of 8-(3-Hydroxypropyl)-3-isopropyl-7-methylnaphthalene-1,2-dione (22)
Compound 18 (80.1 mg, 0.28 mmol) was dissolved in 4 mL of dry tetrahydrofuran, and a solution
of 1M lithium aluminum hydride in tetrahydrofuran (560 μL, 0.56 mmol) was added portionwise
over a period of 30 min. After refluxing for 1 h, 24 μL of water was added dropwise, followed by an
equal volume of 15% sodium hydroxide and 3 times the amount of water. The crude material was
filtered through kieselguhr and purified by silica gel column chromatography using petroleum
ether/ethyl acetate mixture (3:1 to 1:1) as eluent to afford 22 as an orange oil (40.7mg, 53% yield). 1H-
NMR (400 MHz, CDCl3) δ 7.36 (d, J = 7.6 Hz, 1H, Ar-H), 7.08 (s, 1H, Ar-H), 7.05 (d, J = 7.6 Hz, 1H, Ar-
H), 3.79 (t, J = 6.1 Hz, 2H, OH-CH2), 3.14–3.07 (m, 2H, Ar-CH2), 3.01 (septet, J = 6.9 Hz, 1H, CH), 2.38
(s, 3H, Ar-CH3), 1.80–1.71 (m, 2H, CH2), 1.15 (d, J = 6.9 Hz, 6H, 2CH3). ESI-MS m/z: 295.2 [M + Na]+.
3.1.7. General Procedure B: Synthesis of Compounds 24a–24c
To a solution of compound 22 (32.7 mg, 0.12 mmol) in dry dichloromethane (5 mL) held at 0 °C
was added dry triethylamine (0.1 mL, 0.74 mmol) and 23a–23c. The mixture was stirred 2 h at room
temperature. Saturated solution of NaHCO3 was added and the mixture was extracted several times
with dichloromethane. The organic phase was dried over anhydrous MgSO4, evaporated under
reduced pressure and purified by octadecylsilyl silica gel column chromatography using
methanol/water (7:3) as eluent to afford compounds 24a–24c.
3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propyl methanesulfonate (24a): Compound
23a (53.8 mg, 0.47 mmol) were used in general procedure B to afford 24a as an orange solid in 38%
yield (16.2 mg). 1H-NMR (400 MHz, CDCl3) δ 7.40 (d, J = 7.6 Hz, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 7.10
(d, J = 7.6 Hz, 1H, Ar-H), 4.43 (t, J = 6.1 Hz, 2H, CH2OSO2), 3.18–3.10 (m, 2H, Ar-CH2), 3.07 (s, 3H,
SO2CH3), 3.01 (septet, J = 6.8 Hz, 1H, CH), 2.39 (s, 3H, Ar-CH3), 2.02–1.91 (m, 2H, CH2), 1.17 (d, J = 6.8
Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ 182.45, 181.18, 146.44, 144.92, 140.22, 140.13, 137.16,
135.16, 128.67, 128.35, 70.46, 37.38, 28.28, 26.95, 26.48, 21.48, 19.78. ESI-MS m/z: 373.0 [M + Na]+. HRESI-
MS calcd. for C18H22NaO5S+ ([M + Na]+) 373.1080, found 373.1086.

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3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propyl ethanesulfonate (24b): Compound
23b (46.3 mg, 0.36 mmol) were used in general procedure B to afford 24b as an orange solid in 18%
yield (8.0 mg). 1H-NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.6 Hz, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 7.10 (d,
J = 7.6 Hz, 1H, Ar-H), 4.43 (t, J = 6.2 Hz, 2H, CH2OSO2), 3.19 (q, J = 7.4 Hz, 2H, SO2CH2), 3.16–3.11 (m,
2H, Ar-CH2), 3.03 (septet, J = 6.9 Hz, 1H, CH), 2.40 (s, 3H, Ar-CH3), 2.02–1.92 (m, 2H, CH2), 1.46 (t, J =
7.4 Hz, 3H, CH3), 1.17 (d, J = 6.9 Hz, 6H, 2CH3 of isopropyl group). 13C-NMR (100 MHz, CDCl3)
δ182.41, 181.18, 146.49, 144.88, 140.24, 140.13, 137.14, 135.13, 128.66, 128.33, 70.13, 44.88, 28.41, 26.94,
26.51, 21.48, 19.77, 8.24. ESI-MS m/z: 387.0 [M + Na]+. HRESI-MS calcd. for C19H24NaO5S+ ([M + Na]+)
387.1237, found 387.1241.
3-(6-Isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propyl sulfamate (24c): Compound 23c (32.7
mg, 0.12 mmol) were used in general procedure B to afford 24c as an orange solid in 40% yield (17.0
mg). 1H-NMR (400 MHz, CD3OD) δ 7.48 (d, J = 7.9 Hz, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.24 (d, J = 7.9
Hz, 1H, Ar-H), 4.28 (t, J = 6.3 Hz, 2H, CH2OSO2), 3.24–3.11 (m, 2H, Ar-CH2), 2.96 (septet, J = 6.9 Hz,
1H, CH), 2.41 (s, 3H, Ar-CH3), 2.00–1.84 (m, 2H, CH2), 1.18 (d, J = 6.9 Hz, 6H, 2CH3).13C-NMR (100
MHz, acetone-d6) δ 182.41, 181.01, 146.03, 144.42, 140.02, 139.90, 136.82, 135.32, 128.73, 69.78, 28.04,
26.87, 26.10, 20.82, 18.85. ESI-MS m/z: 374.0 [M + Na]+. HRESI-MS calcd. for C17H21NNaO5S+ ([M +
Na]+) 374.1033, found 374.1038.
3.1.8. General Procedure C: Synthesis of Compounds 26a–26b
The reactant was dissolved in 6 mL of dichloromethane, and 0.1 mL of triethylamine was added.
Di-tert-butyl dicarbonate was added at 0 °C and the mixture was stirred overnight at room
temperature. The mixture was washed with water to give the crude product, which was used in the
next reaction directly.
tert-Butyl (2-aminophenyl)carbamate (26a): Compound 25a (198.8 mg, 1.84 mmol) was used in general
procedure C to afford 26a as a light yellow oil in 76% yield (292.5 mg). ESI-MS m/z: 231.0 [M + Na]+.
tert-Butyl (2-amino-4-fluorophenyl)carbamate (26b): Compound 25b (99.6 mg, 0.79 mmol) was used in
general procedure C to afford 26b as a light yellow oil in 66% yield (117.6 mg). ESI-MS m/z: 249.0 [M
+ Na]+.
3.1.9. General Procedure D: Synthesis of Compounds 27a–27b
Compound 18 was dissolved in 1 mL of dry N,N-dimethylformamide, HATU and DIPEA were
added. After stirring uniformly, amine 26a–26b was added. The mixture was stirred overnight at
room temperature. Dichloromethane was added and the mixture was washed with saturated solution
of NaHCO3, the organic phase was purified by octadecylsilyl silica gel column chromatography using
methanol/water as eluent. The solvent was concentrated in vacuo and dissolved in 2.5 mL
dichloromethane, 1 mL of trifluoroacetic acid was added at 0 °C and the mixture was stirred 1 h at
room temperature. Saturated solution of NaHCO3 was added and the mixture was extracted several
times with dichloromethane. The organic phase was dried over anhydrous MgSO4, evaporated under
reduced pressure to afford compound 27a–27b.
N-(2-Aminophenyl)-3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamide
(27a):
Compound 18 (40.1 mg, 0.14 mmol), HATU (64.6 mg, 0.17 mmol), DIPEA (45.3 μL, 0.26 mmol) and
26a (33.3 mg, 0.16 mmol) were used in general procedure D. The crude product was purified by
octadecylsilyl silica gel column chromatography using methanol/water (4:1) as eluent. Finally
1
affording 27a as an orange solid in 27% yield (14.1 mg). H-NMR (400 MHz, CDCl3) δ 8.04 (s, 1H,
CONH), 7.43 (d, J = 7.6 Hz, 1H, Ar-H of phenyl group), 7.31 (d, J = 7.9 Hz, 1H, Ar-H of
naphthoquinone), 7.12 (s, 1H, Ar-H of naphthoquinone), 7.12 (d, J = 7.9 Hz, 1H, Ar-H of
naphthoquinone), 7.05 (t, J = 7.6 Hz, 1H, Ar-H of phenyl group), 6.84–6.75 (m, 2H, Ar-H of phenyl
group), 3.49–3.37 (m, 2H, Ar-CH2), 3.02 (septet, J = 6.8 Hz, 1H, CH), 2.61–2.49 (m, 2H, CH2CO), 2.44
(s, 3H, Ar-CH3), 1.18 (d, J = 6.8 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ 182.75, 181.04, 171.18,
146.44, 144.99, 140.59, 140.39, 140.20, 137.51, 135.37, 128.91, 128.22, 126.95, 125.27, 124.31, 119.30,

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+
117.83, 36.27, 27.98, 27.05, 21.47, 19.82. ESI-MS m/z: 377.2 [M + H]+. HRESI-MS calcd. for C23H25N2O3
([M + H]+) 377.1860, found 377.1865.
N-(2-Amino-5-fluorophenyl)-3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamide
(27b): Compound 18 (85.8 mg, 0.30 mmol), HATU (136.8 mg, 0.36 mmol), DIPEA (104.5 μL, 0.60
mmol) and 26b (81.4 mg, 0.36 mmol) were used in general procedure D. The crude product was
purified by octadecylsilyl silica gel column chromatography using methanol/water (4:1) as eluent.
Finally affording 27b as an orange solid in 10% yield (12.0 mg). 1H-NMR (400 MHz, CDCl3) δ 8.18 (s,
1H, CONH), 7.48–7.38 (m, 2H, Ar-H, 1H of phenyl group and 1H of naphthoquinone), 7.13 (d, J = 6.6
Hz, 1H, Ar-H of naphthoquinone), 7.12 (s, 1H, Ar-H of naphthoquinone), 6.79–6.72 (m, 2H, Ar-H of
phenyl group), 3.46–3.34 (m, 2H, Ar-CH2), 3.02 (septet, J = 6.7 Hz, 1H, CH), 2.60–2.50 (m, 2H, CH2CO),
2.45 (s, 3H, Ar-CH3), 1.18 (d, J = 6.7 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ 182.84, 180.88, 171.03,
162.50 (d, J = 266.9 Hz), 146.36, 145.00, 140.34, 140.22, 137.64, 135.43, 134.77, 129.01, 128.16, 126.39 (d,
J = 10.1 Hz), 118.83 (d, J = 8.6 Hz), 112.45 (d, J = 22.4 Hz), 110.83 (d, J = 26.0 Hz), 36.57, 28.02, 27.05,
+
21.48, 19.84. ESI-MS m/z: 395.2 [M + H]+. HRESI-MS calcd. for C23H24FN2O3 ([M + H]+) 395.1765, found
395.1775.
3.1.10. Synthesis of (9H-fluoren-9-yl) methyl tert-butyl (4-fluoro-1,2-phenylene)dicarbamate (28)
Compound 26b (169.6 mg, 0.75 mmol) was dissolved in 3 mL of dichloromethane, and 6 mL of
saturated solution of NaHCO3 was added. The mixture was stirred and (9H-fluoren-9-
ylmethoxy)carbonyl chloride (271.6 mg, 1.05 mmol) was added. 3 h later, the mixture was washed by
water and was extracted several times with dichloromethane. The organic phase was dried over
anhydrous MgSO4, and purified by octadecylsilyl silica gel column chromatography using
methanol/water (1:1) as eluent to afford 28 as a brown oil in 36% yield (121.1 mg). ESI-MS m/z: 471.0
[M + Na]+.
3.1.11. General Procedure E: Synthesis of Compounds 29 and 32a–32d
Compound 28 or 31a–31d was dissolved in 2.5 mL dichloromethane, 1 mL of trifluoroacetic acid
was added at 0 °C and the mixture was stirred 1 h at room temperature. Saturated solution of
NaHCO3 was added and the mixture was extracted several times with dichloromethane. The organic
phase was dried over anhydrous MgSO4, evaporated under reduced pressure to afford compound 29
and 32a–32d, which was used in the next reaction directly.
3.1.12. Synthesis of N-(2-amino-4-fluorophenyl)-3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-
dihydronaphthalen-1-yl)propanamide (27c)
Compound 18 (60.1 mg, 0.21 mmol) was dissolved in 1 mL of dry N,N-dimethylformamide,
HATU (95.1 mg, 0.25 mmol) and DIPEA (73.2 μL, 0.42 mmol) were added. After stirring uniformly,
29 (88.5 mg, 0.25 mmol) was added. The mixture was stirred overnight at room temperature.
Dichloromethane was added and the mixture was washed with saturated solution of NaHCO3, the
organic phase was purified by silica gel column chromatography using petroleum ether/ethyl acetate
mixture (4:1 to 1:1) as eluent. The solvent was concentrated in vacuo and dissolved in 2 mL
dichloromethane, 2 mL diethylamine was added and the mixture was stirred 1 h at room
temperature. The mixture was evaporated under reduced pressure and purified by octadecylsilyl
silica gel column chromatography using methanol/water (3:2) as eluent to afford 27c as an orange
solid in 4% Yield (4.3 mg). 1H-NMR (400 MHz, CDCl3) δ 8.14 (s, 1H, CONH), 7.44 (d, J = 7.7 Hz, 1H,
Ar-H of naphthoquinone), 7.26–7.22 (m, 1H, Ar-H of phenyl group), 7.13 (s, 1H, Ar-H of
naphthoquinone), 7.13 (d, J = 7.7 Hz, 1H, Ar-H of naphthoquinone), 6.63–6.51 (m, 2H, Ar-H of phenyl
group), 3.46–3.38 (m, 2H, Ar-CH2), 3.02 (septet, J = 6.8 Hz, 1H, CH), 2.60–2.52 (m, 2H, CH2CO), 2.45
(s, 3H, Ar-CH3), 1.18 (d, J = 6.8 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ182.83, 181.02, 171.48,
160.55 (d, J = 264.4 Hz), 146.38, 145.04, 142.93 (d, J = 12.2 Hz), 140.34, 140.23, 137.63, 135.44, 129.00,
128.16, 127.27 (d, J = 10.1 Hz), 119.65, 105.50 (d, J = 22.6 Hz), 103.91 (d, J = 25.6 Hz), 36.18, 28.14, 27.06,
+
21.48, 19.82. ESI-MS m/z: 395.0 [M + H]+. HRESI-MS calcd. for C23H24FN2O3 ([M + H]+) 395.1765, found
395.1769.

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3.1.13. General Procedure F: Synthesis of Compounds 33a–33d
Compound 18 was dissolved in
1
mL of dry N,N-dimethylformamide, 3-(3-
dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine
(DMAP) and DIPEA were added. After stirring uniformly, amine 32a–32d was added. The mixture
was stirred overnight at room temperature. Dichloromethane was added and the mixture was
washed with saturated solution of NaHCO3,the organic phase was purified by column
chromatography to afford 33a–33d.
N-(2-aminophenyl)-3-(3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamido)-
propanamide (33a): Compound 18 (28.6 mg, 0.10 mmol), EDCI (23.0 mg, 0.12 mmol), DMAP (1 mg),
DIPEA (104.5 μL, 0.60 mmol) and 32a were used in general procedure F. The crude product was
purified by flash chromatography on silica gel then octadecylsilyl silica gel column chromatography
using methanol/water (3:2) as eluent to afford 33a as an orange solid in 12% yield (5.7 mg).1H-NMR
(400 MHz, CDCl3) δ 8.21 (s, 1H, Ph-NHCO), 7.38 (d, J = 7.4 Hz, 1H, Ar-H of phenyl group), 7.24 (d, J
= 7.8 Hz, 1H, Ar-H of naphthoquinone), 7.09 (s, 1H, Ar-H of naphthoquinone), 7.08 (d, J = 7.8 Hz, 1H,
Ar-H of naphthoquinone), 7.02 (t, J = 7.4 Hz, 1H, Ar-H of phenyl group), 6.90–6.83 (m, 1H, Ar-H of
phenyl group), 6.81–6.70 (m, 2H, CONH and Ar-H of phenyl group), 3.71–3.59 (m, 2H, NH-CH2),
3.36–3.23 (m, 2H, Ar-CH2), 3.00 (septet, J = 6.2 Hz, 1H, CH), 2.71–2.61 (m, 2H, CH2CO), 2.38 (s, 3H,
Ar-CH3), 2.37–2.29 (m, 2H, CH2CO), 1.17 (d, J = 6.2 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ
182.55, 181.20, 172.97, 170.37, 146.53, 144.77, 140.88, 140.55, 140.32, 137.32, 135.11, 128.82, 128.34,
127.10, 125.58, 124.07, 119.12, 117.82, 36.66, 35.79, 35.40, 27.11, 26.96, 21.48, 19.76. ESI-MS m/z: 448.2
+
[M + H]+. HRESI-MS calcd. for C26H30N3O4 ([M + H]+) 448.2231, found 448.2238.
N-(2-Aminophenyl)-4-(3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamido)-
butanamide (33b): Compound 18 (51.5 mg, 0.18 mmol), EDCI (38.3 mg, 0.20 mmol), DMAP (1.7 mg),
DIPEA (188.1 μL, 1.08 mmol) and 32b were used in general procedure F. The crude product was
purified by flash chromatography on silica gel then octadecylsilyl silica gel column chromatography
using methanol/water (7:3) as eluent to afford 33b as an orange solid in 11% yield (10.2 mg). 1H-NMR
(400 MHz, CDCl3) δ 8.65 (s, 1H, Ph-NHCO), 7.42 (d, J = 7.5 Hz, 1H, Ar-H of phenyl group), 7.37 (d, J
= 7.8 Hz, 1H, Ar-H of naphthoquinone), 7.12 (s, 1H, Ar-H of naphthoquinone), 7.11 (d, J = 7.8 Hz, 1H,
Ar-H of naphthoquinone), 7.02 (t, J = 7.5 Hz, 1H, Ar-H of phenyl group), 6.82–6.70 (m, 3H, CONH
and 2Ar-H of phenyl group), 3.53–3.44 (m, 2H, NH-CH2), 3.33–3.24 (m, 2H, Ar-CH2), 3.02 (septet, J =
6.9 Hz, 1H, CH), 2.51–2.45 (m, 2H, CH2CO), 2.41 (s, 3H, Ar-CH3), 2.39–2.32 (m, 2H, CH2CO), 2.03–1.93
(m, 2H, CH2), 1.17 (d, J = 6.9 Hz, 6H, 2CH3). 13C-NMR (100 MHz, CDCl3) δ 182.75, 181.06, 173.78,
171.45, 146.58, 144.93, 140.58, 140.39, 140.33, 137.56, 135.34, 128.94, 128.10, 126.62, 125.16, 124.54,
118.96, 117.64, 38.40, 35.83, 33.96, 28.07, 27.04, 26.79, 21.46, 19.75. ESI-MS m/z: 462.2 [M + H]+. HRESI-
+
MS calcd. for C27H32N3O4 ([M + H]+) 462.2387, found 462.2398.
N-(2-Aminophenyl)-5-(3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamido)-
pentanamide (33c): Compound 18 (57.2 mg, 0.20 mmol), EDCI (46.0 mg, 0.24 mmol), DMAP (1.9 mg),
DIPEA (209.0 μL, 1.20 mmol) and 32c were used in general procedure F. The crude product was
purified by flash chromatography on silica gel then octadecylsilyl silica gel column chromatography
using methanol/water (7:3) as eluent to afford 33c as an orange solid in 9% yield (10.6 mg).1H-NMR
(400 MHz, CDCl3) δ 7.83 (s, 1H, Ph-NHCO), 7.40 (d, J = 7.4 Hz, 1H, Ar-H of naphthoquinone), 7.11 (s,
1H, Ar-H of naphthoquinone), 7.09 (d, J = 7.4 Hz, 1H, Ar-H of naphthoquinone), 7.03 (t, J = 7.3 Hz,
1H, Ar-H of phenyl group), 6.83–6.53 (m, 3H, CONH and 2Ar-H of phenyl group), 3.43–3.36 (m, 2H,
NH-CH2), 3.32–3.23 (m, 2H, Ar-CH2), 3.01 (septet, J = 6.8 Hz, 1H, CH), 2.56–2.47 (m, 2H, CH2CO), 2.38
(s, 3H, Ar-CH3), 2.36–2.29 (m, 2H, CH2CO), 1.89–1.79 (m, 2H, CH2), 1.74–1.63 (m, 2H, CH2), 1.17 (d, J
= 6.8 Hz, 6H, 2CH3). 13C-NMR (100 MHz, DMSO-d6) δ 182.01, 180.86, 171.76, 171.48, 146.30, 144.02,
142.30, 140.53, 140.48, 137.06, 135.08, 129.19, 128.83, 126.12, 125.68, 124.02, 116.60, 116.33, 38.77, 35.89,
34.66, 29.28, 26.94, 26.16, 23.27, 21.79, 19.91. ESI-MS m/z: 476.2 [M + H]+. HRESI-MS calcd. for
+
C28H34N3O4 ([M + H]+) 476.2544, found 476.2548.

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N-(2-Aminophenyl)-6-(3-(6-isopropyl-2-methyl-7,8-dioxo-7,8-dihydronaphthalen-1-yl)propanamido)
hexanamide (33d): Compound 18 (57.2 mg, 0.20 mmol), EDCI (46.0 mg, 0.24 mmol), DMAP (1.9 mg),
DIPEA (209.0 μL, 1.20 mmol) and 32d were used in general procedure F. The crude product was
purified by flash chromatography on silica gel then octadecylsilyl silica gel column chromatography
using methanol/water (7:3) as eluent to afford 33d as an orange solid in 25% yield (25.7 mg).1H-NMR
(400 MHz, CDCl3) δ 7.97 (s, 1H, Ph-NHCO), 7.39 (d, J = 7.5 Hz, 1H, Ar-H of phenyl group), 7.19 (d, J
= 7.6 Hz, 1H, Ar-H of naphthoquinone), 7.11 (s, 1H, Ar-H of naphthoquinone), 7.09 (d, J = 7.6 Hz, 1H,
Ar-H of naphthoquinone), 7.00 (t, J = 7.5 Hz, 1H, Ar-H of phenyl group), 6.80–6.57 (m, 3H, CONH
and 2Ar-H of phenyl group). 3.37–3.30 (m, 2H, NH-CH2), 3.30–3.21 (m, 2H, Ar-CH2), 3.00 (septet, J =
6.8 Hz, 1H, CH), 2.49–2.43 (m, 2H, CH2CO), 2.39 (s, 3H, Ar-CH3), 2.34–2.27 (m, 2H, CH2CO), 1.85–1.74
(m, 2H, CH2), 1.67–1.57 (m, 2H, CH2), 1.56–1.46 (m, 2H, CH2), 1.17 (d, J = 6.8 Hz, 6H, 2CH3). 13C- NMR
(100 MHz, CDCl3) δ 182.59, 181.24, 172.62, 171.96, 146.84, 144.80, 140.90, 140.61, 140.49, 137.42, 135.17,
128.85, 128.12, 126.90, 125.29, 124.51, 119.21, 118.03, 39.06, 36.88, 35.79, 29.06, 28.08, 26.99, 26.22, 25.28,
+
21.47, 19.72. ESI-MS m/z: 490.2 [M + H]+. HRESI-MS calcd. for C29H36N3O4 ([M + H]+) 490.2700, found
490.2706.
3.2. Biology
3.2.1. IDO1 Enzymatic Assay
Materials
IDO-1 (CP produced, Lot. No. 20160706); NLG-919 (Selleckchem, Houston, TX, USA. Cat. No.
S7111); Catalase (Sigma, St. Louis, MO, USA. Cat. No. C9322-5G); L-tryptophan (Sigma, St. Louis,
MO, USA. Cat. No. 93659-10G); ascorbate (Sigma, St. Louis, MO, USA. Cat. No. 11140-250G);
methylene blue (Sigma, St. Louis, MO, USA. Cat. No. M9140-100G); DMSO (Sigma, St. Louis, MO,
USA. Cat. No. D2650); 96-well plate (Corning Inc., Corning, NY, USA. Cat. No. 3635).
Experimental Methods
(1) Prepare Compound
(1) Prepare 100 compound of the final desired highest inhibitor concentration in reaction by
100% DMSO. For example, if compounds test at 10 μM, then prepare 1 mM of compound solution in
this step. Then dilute compound with DMSO in 3-fold mode to obtain different concentrations. Mark
as source plate. (2) Add 100 μL of 100% DMSO to column 1 and column 12 for background control
and no compound high control respectively.
(2) Prepare Assay Plate
Transfer 2 μL of each well from the 96-well source plate to a 96-well assay plate.
(3) Enzyme Reaction
1. Prepare 2 enzyme solution. Add IDO-1 in assay buffer.
2. Prepare 2 substrate solution. Add L-tryptophan, ascorbate, methylene blue, catalase in the
assay buffer.
3. Assay plate already contains 2 μL of compound in 100% DMSO.
4. Transfer 2 enzyme solution to the assay plate. Add 100 μL of 2 enzyme solution to each
well of the 96-well assay plate.
5. Incubate at room temperature for 10 min.
Transfer 2 ×
1.  substrate solution to each well of the 96-well assay plate.
2. Read OD321 kinetically by Spectramax (Molecular Devices, LLC, San Jose, CA, USA).
(4) Curve Fitting

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1. Copy data from Spectramax program. Percent inhibition = (max-conversion)/(max − min) ×
100. “max” stands for high control; “min” stands for low control.
2. Fit the data in XL-Fit to obtain IC50 values. Equation used is: Y = Bottom + (Top − Bottom)/(1
+ 10^((LogIC50 − X) × Hill Slope)
3.2.2. HDAC Enzymatic Assay
Materials
HDAC1 (BPS Bioscience Inc., San Diego, CA, USA. Cat. No. 50051); DMSO (Sigma, St. Louis,
MO, USA. Cat. No. D2650); 84-well plate (Perkin Elmer Inc., Waltham, MA, USA. Cat. No. 6007279)
Experimental Methods
(1) Prepare 1 assay buffer (modified Tris Buffer).
(2) Compound serial dilution: Transfer compounds to assay plate by Echo in 100% DMSO. The final
fraction of DMSO is 1%.
(3) Prepare enzyme solution in 1 assay buffer.
(4) Add trypsin and Ac-peptide substrate in 1 assay buffer to make the substrate solution.
(5) Transfer 15 μL of enzyme solution to assay plate or for low control transfer 15 μL of 1× assay
buffer.
(6) Incubate at room temperature for 15 min.
(7) Add 10 μL of substrate solution to each well to start reaction.
(8) Incubate at room temperature for 60 min.
(9) Read the plate on Synergy MX with excitation at 355 nm and emission at 460 nm.
(10) Curve fitting: Fit the data in Excel to obtain inhibition values using Equation (1).
Inh% = (Max − Signal)/(Max − Min) × 100
(1)
(2)
Fit the data in XL-Fit to obtain IC50 values using Equation (2);
Y = Bottom + (Top − Bottom)/(1 + 10^((LogIC50 − X) × Hill Slope))
Y is %inhibition and X is compound concentration.
3.2.3. Anti-Proliferation Assay in A549 Cell Line
Cell viability was measured by the CellTiter-Glo Luminescent (CTG) assay. A549 cells (ATCC,
Cat. No.CCL-185) were seeded (2000 cells/well) in 96-well plates and cells were left to incubate at 37
°C for 24 h. After 24 h, prepare compounds with 5-fold dilution in DMSO to the compound plate
(200-fold of the final concentration) and transfer 5 μL from the compound plate to 95 μL intermediate
medium in the intermediate plate (10-fold of the final concentration). Then transfer 10 μL from the
intermediate plate to the cell plates. The final DMSO is 0.5%. After continuing to incubate for 48 h,
add 50 μL CTG reagent to each well and read luminescence according to CTG product manual.
3.2.4. Anti-Proliferation Assay in Primary Endothelial Cells (ECs)
Primary endothelial cells (ECs) were isolated from male rats and cultured in DMEM
supplemented with 10% FBS, 100 units/mL penicillin-streptomycin at 37 °C in a humidified
atmosphere under 5% CO2. ECs were plated in a 96-well plate at a density of 2 × 104 cells/well. After
serum starved overnight, ECs were exposed to a gradient concentration of 200 μM for 24 h. Cell
viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. The results were expressed as fold
changes by normalizing the data to the control values.
3.3. Molecular Docking
Protein structures were reconstructed by SWISS-MODEL homology-modelling server [25] using
IDO1 and HDAC1 as templates. Then the AutoDock 4.2 software with the Lamarckian genetic

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algorithm was used for the molecular docking to predict the binding modes. Crystal structure of
human IDO1 in complex (PDB code: 5EK2) and HDAC1 (PDB code: 4BKX) were retrieved from
Protein Data Bank (www.rcsb.org).
The ADT and grid calculations were carried out using grid box dimensions of 60 × 60 × 60 Å with
0.375 Å spacing followed by the space creations around the binding site of IDO1 protein.
Pretreatment of the compounds and the IDO receptor structure for docking was carried out with the
AutoDockTools program suite (mgltools.scripps.edu). Default parameters were used and one
hundred independent docking runs were conducted for each compound.
The docking study of HDAC1 and 33d was carried out using the same method as described
above.
3.4. QM/MM Simulation
After docking, best poses of 33d-protein complexes with dominant conformation underwent
QM/MM simulations by GROMACS + MOPAC + RM1 packages as mentioned by Gonçalves and
colleagues [26], and the procedure adopted to this task is described in Supporting Information.
System of simulation was set up using charmm27 forcefield and box of TIP3P water molecules. The
selection and characterization of the quantum atoms were performed by the visualization of the
docking system with the PyMOL 2.2 software. Regarding IDO1, select ligand, heme and the
neighboring residues (Tyr126, Phe164, Phe226, Arg231, Gly262 and Ala264) as the quantum atoms.
Regarding HDAC1, select ligand, znic and the neighboring residues (His140, His141 and Tyr303) as
the quantum atoms. The link atoms (LA) were set between the α and β carbon atoms of each amino
acid cited above. These atoms constituted the QM region of the system, and the remaining atoms of
the protein, the counter ions and the solvent molecules constituted the MM region. The condition for
the QM/MM simulation was the frame of the classic simulation. All these steps were performed at
310 K and 1 bar of pressure and the integration step was 1 fs or half of the integration step used in
the classic simulation, in order to make possible to deal with the bond vibrations between C and H,
usually unnoticeable at 2 fs. After 20ns of QM/MM simulation, poses at the last frames in the
simulations were selected for the analysis and visualization by PyMol software.
3.5. Molecular Dynamics Simulation
Molecular dynamics (MD) simulations of compound 33d along with IDO1 and HDAC1, using
the GROMACS 5.1.2 software [27] employed to track the motions of individual atoms. The ACPYPE
server was used to generate the topology and force field parameters for ligand [28]. Each ligand-
target complex was soaked in a dodecahedral box of TIP3P water molecules with a margin of at least
10 Å from the complex surface. Sodium and chloride counter ions were added to preserve
electroneutrality. Each complex was subjected to energy minimization with 50,000 steps of steepest
descent minimization. Afterwards, two MD equilibration phases were simulated. Firstly, a 500-ps
step under the NVT ensemble was applied, using an integration time step of 2.0 fs at a temperature
of 310 K. Another 500 ps NPT equilibration step was then executed at 1 bar pressure to equilibrate
the system. The MD production phase was subsequently conducted using the NPT ensemble at 310
K and the V-rescale temperature coupling algorithm. The pressure in the system was adjusted to 1
atm under isotropic molecule-based scaling using the Parrinello-Rahman pressure coupling method.
Long-range electrostatics was treated with the particle mesh Ewald (PME) algorithm. Finally,
molecular dynamics simulation was performed at a timescale of 100 ns and in 50,000,000 steps. The
MD trajectories were analyzed by plotting the root mean square deviation (RMSD) for each frame
against time, as well as the root mean square fluctuation (RMSF) of the protein backbone, and the
hydrogenbonding interactions between the target and the ligand were also recorded. The g_mmpbsa
procedure was applied to analyze the binding energy during the last 30 ns of the MD simulation.
Data were visualized using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA).
4. Conclusions
A kind of ortho-quinone compounds from natural product were firstly reported to have better
IDO1 inhibition activity than para-quinones. To discover the IDO1 and HDAC dual inhibitors for

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cancer treatment by taking advantages of immunotherapeutic and epigenetic drugs,
saprorthoquinone was chosen as a hit compound through a pharmacophore fusion strategy. A more
effective IDO1 and HDAC dual inhibitor 33d was obtained, which showed balanced activity against
both IDO1 (IC50 = 0.73 μM) and HDAC1 (IC50 = 0.46 μM). Importantly, structure-activity relationship
studies revealed that an ortho-quinone pharmacophore group was necessary for the IDO inhibition
and a N-(2-aminophenyl) amide pharmacophore group was necessary for the HDAC inhibition,
which combined by an appropriate five carbons linker. Moreover, the binding modes of these
inhibitors to the enzyme active site were studied by molecular docking, showed that the coordination
bonding interaction and hydrogen bonding interaction were the major interaction between potent
inhibitors and IDO1. The hydrogen bond with Gly262 and Leu234 of IDO1 appeared to confer
increased potency to this class of inhibitors which explained the higher activity of both 19 and 33d.
This study provides a new strategy for future design of IDO1/HDAC dual inhibitors as antitumor
agents. Structural optimization of lead compound 33d remains to be further investigated in our lab.
Supplementary Materials: The following are available online. Supporting information including the spectra of
1H, 13C-NMR, compilation of GROMACS + MOPAC + RM1 and general procedure of input file set-up in QM/MM
Simulation, are available online.
Author Contributions: J.C. directed this project, Y.L., H.Z., T.N. carried out the experiments, M.-L.T. checked
and analyzed the data and revised the paper. All authors have read and agreed to the published version of the
manuscript.
Funding: This project was supported financially by the National Natural Science Foundation of China
(81573268), Science and Technology Commission of Shanghai Municipality (18431900600), and the National
Science and Technology Major Project of China (2018ZX09711002-002-016).
Conflicts of Interest: The authors declare no conflict of interest.
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