Optimization of WZ4003 as NUAK inhibitors against human colorectal cancer

Article abstract of DOI:10.1016/j.ejmech.2020.113080

NUAK, the member of AMPK (AMP-activated protein kinase) family of protein kinases, is phosphorylated and activated by the LKB1 (liver kinase B1) tumor suppressor protein kinase. Recent work has indicated that NUAK1 is a key component of the antioxidant stress response pathway, and the inhibition of NUAK1 will suppress the growth and survival of colorectal tumors. As a promising target for anticancer drugs, few inhibitors of NUAK were developed. With this goal in mind, based on NUAK inhibitor WZ4003, a series of derivatives has been synthesized and evaluated for anticancer activity. Compound 9q, a derivative of WZ4003 by removing a methoxy group, was found to be the most potential one with stronger inhibitory against NUAK1/2 enzyme activity, tumor cell proliferation and inducing apoptosis of tumor cells. By in vivo efficacy evaluations of colorectal SW480 xenografts, 9q suppresses tumor growth more effectively with an excellent safety profile in vivo and is therefore seen as a suitable candidate for further investigation.

Full text of DOI:10.1016/j.ejmech.2020.113080

Journal Pre-proof
Optimization of WZ4003 as NUAK Inhibitors against Human Colorectal Cancer
Huali Yang, Xiaobing Wang, Cheng Wang, Fucheng Yin, Lailiang Qu, Cunjian Shi,
Jinhua Zhao, Shang Li, Limei Ji, Wan Peng, Heng Luo, Maosheng Cheng, Lingyi
Kong
PII:
S0223-5234(20)31052-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.113080
EJMECH 113080
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 September 2020
Revised Date: 16 November 2020
Accepted Date: 1 December 2020
Please cite this article as: H. Yang, X. Wang, C. Wang, F. Yin, L. Qu, C. Shi, J. Zhao, S. Li, L.
Ji, W. Peng, H. Luo, M. Cheng, L. Kong, Optimization of WZ4003 as NUAK Inhibitors against
Human Colorectal Cancer, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.113080.
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Optimization of WZ4003 as NUAK Inhibitors against Human
Colorectal Cancer
ab
a
a
a
a
Huali Yang †, Xiaobing Wang †, Cheng Wang , Fucheng Yin , Lailiang Qu ,
a
a
a
a
a
a
Cunjian Shi , Jinhua Zhao , Shang Li , Limei Ji , Wan Peng , Heng Luo ,
b
ab
Maosheng Cheng * and Lingyi Kong *
a
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, China.
b
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, Shenyang 110016, China.
*
Corresponding
Authors.
Tel/Fax:
+86-25-83271405;
E-mail:
mscheng@syphu.edu.cn (Maosheng Cheng); cpu_lykong@126.com (Lingyi Kong).
†
Huali Yang and Xiaobing Wang contributed equally to this work.
1

ABSTRACT
NUAK, the member of AMPK (AMP-activated protein kinase) family of protein
kinases, is phosphorylated and activated by the LKB1 (liver kinase B1) tumor
suppressor protein kinase. Recent work has indicated that NUAK1 is a key
component of the antioxidant stress response pathway, and the inhibition of
NUAK1 will suppress the growth and survival of colorectal tumors. As a promising
target for anticancer drugs, few inhibitors of NUAK were developed. With this goal
in mind, based on NUAK inhibitor WZ4003, a series of derivatives has been
synthesized and evaluated for anticancer activity. Compound 9q, a derivative of
WZ4003 by removing a methoxy group, was found to be the most potential one
with stronger inhibitory against NUAK1/2 enzyme activity, tumor cell proliferation
and inducing apoptosis of tumor cells. By in vivo efficacy evaluations of colorectal
SW480 xenografts, 9q suppresses tumor growth more effectively with an excellent
safety profile in vivo and is therefore seen as a suitable candidate for further
investigation.
Keywords: AMP-activated protein kinase (AMPK), NUAK, WZ4003 derivatives,
Colorectal Cancer.
2

1
. INTRODUCTION
NUAK1 [ARK5 (AMPK-related kinase 5)] and NUAK2 [SNARK
(SNF1/AMPK-related kinase)] are members of the AMPK (AMP-activated protein
kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1)
tumor suppressor protein kinase [1-2]. NUAK isoforms are ubiquitously expressed
and possess an N-terminal kinase domain (residues 55–306, NUAK1), followed by a
non-catalytic C-terminal region (residues 307–661, NUAK1) [2-3]. Recent studies
suggest that they hint at roles in regulating multiple processes, such as cancer cell
invasion, survival, senescence, and proliferation [4-9]. Collectively, one study points
that NUAK1, a key component in the antioxidant defense response, is highly
expressed in human colorectal cancer, revealing its important role [10]. The data
suggests that temporarily inhibiting NUAK1 to destroy the antioxidant defense
mechanisms of cancer cells may be a safe and effective treatment for colorectal cancer.
In fact, as early as 2004, Kusakai et al. suggested that high expression of NUAK1 was
involved in tumor progression of colon cancer clinically [11]. Therefore, targeting
NUAK is one promising approach for cancer treatments.
Although considerable pharmacological works have shown that the importance
of NUAK in cancer progression, studies on synthesis and SAR (Structure Affect
Relationship) of NUAK inhibitors still remain to be investigated. To date, only few
NUAK inhibitors have been reported as shown in Figure 1. In 2014, university of
Dundee reported two NUAK inhibitors namely WZ4003 and HTH-01-015 [3].
WZ4003 inhibits both NUAK1 and NUAK2, whereas HTH-01-015 inhibits NUAK1
with >100-fold higher potency than NUAK2. In tumor cells with high NUAK
expression, they can inhibit the phosphorylation of MYPT1 by inhibiting NUAK, and
it is consistent with NUAK cell knockout. In addition, XMD-17-51, XMD-18-42
(NUAK1 inhibitors, poor selectivity) and HTH-02-006 (semispecific NUAK2
inhibitor) have also been found and applied to the study of NUAK mechanism [3, 12].
In a number of protein kinase inhibitory activity tests, WZ4003 showed excellent
specificity, with no significant inhibitory effect on any kinases except NUAK, and
3

there are no detailed SAR studies on WZ4003. Therefore, WZ4003 drew our attention
to the possibilities of further modification.
Figure 1. The NUAK inhibitors.
With this in mind, a series of derivatives 9a-s has been designed and synthesized
for the modification of N-methyl piperazine ring (A Ring) and methoxybenzene ring
(B Ring) on the base of WZ4003 (Figure 2). The optimized products with superior
NUAK inhibition and anti-tumor activities are desired to obtain as promising
candidates for anticancer drugs.
Figure 2. Design of the target derivatives based on WZ4003.
2
. RESULTS AND DISCUSSION
2
.1. Chemistry
The synthetic route of the target compounds 9a-s was illustrated in Scheme 1
[
12-15]. First, the reaction between p-fluoronitrobenzene compounds 1a-c and the
4

corresponding amines led to the amine compounds 2a-r as a yellow solid. The
subsequent reduction of nitro group with Zn powder and ammonium chloride
provided amino compounds 3a-r. On the other side, intermediates 6a-b were also
formed by the mixture of pyrimidine reactants and 3-nitrophenol for 2 hours under 60
ºC. Subsequently, the mixture of intermediates 3a-r and 6a-b in 2-BuOH were added
TFA and heated to obtain 7a-s. At room temperature, intermediates 7a-s was reduced
in the presence of Zn powder and ammonium chloride yielded 8a-s. Finally, target
compounds 9a-s were successfully obtained via the reaction between 8a-s and
propionyl chloride in CH Cl under an ice bath at 0 ºC. The structures of all the target
2
2
1
13
compounds 9a-s were identified by H NMR, C NMR and high-resolution mass
spectrometry (HRMS).
Scheme 1. Synthesis of compounds 9a-s. Reagents and conditions: (i) K CO , DMF,
2
3
amine, r.t. or 70 ºC; (ii) Zn powder, NH Cl, THF/MeOH (3:2), r.t.; (iii) K CO , DMF,
4
2
3
6
2
2
0
ºC; (iv) 2-BuOH, TFA, 100 ºC; (v) propionyl chloride, CH Cl , 0 ºC.
2
2
.2. Biological Assays
.2.1. In Vitro NUAK1/2 Activity and MTT assay for cell viability
5

To evaluate the enzyme inhibitory potential of the target compounds 9a-s
towards NUAK, a well established ADP-Glo™ Assay was used (Table 1) [16]. In
order to confirm the suitability of the selected assay system, the inhibitory activity of
positive controls WZ4003 (NUAK1 and NUAK2 inhibitor) and HTH-01-015 (only
NUAK1 inhibitor) at 10 μM towards NUAK1/2 were evaluated. The results showed
that both of them had a high inhibitory effect on NUAK1 enzyme activity, and
HTH-01-015 (NUAK2 inhibition ratio: 15.59 ± 1.67 %) had a weak inhibitory effect
on NUAK2 unlike WZ4003 (NUAK2 inhibition ratio %: 84.85 ± 2.92), which was
consistent with reported data [3].
First, by comparing 9a-h and 9i-p, it is not difficult to find that the absence of R2
methoxy group is more conducive to the inhibition of NUAK enzyme activity of the
target compounds when R substituent is the same. In addition, compared with R
1
2
methoxy substitution compound WZ4003 and R methoxy deletion compound 9q, the
2
inhibitory activity of R halogen substitution compound 9s (NUAK1/2 inhibition
2
ratio %: 71.54 ± 7.46, 69.11 ± 6.69, respectively) against NUAK also decreased
slightly. Secondly, the NUAK inhibitory activity of the piperazine ring modified
derivative 9a-h was significantly decreased or even lost, which strongly demonstrated
the necessity of piperazine ring structure. Among them, when R piperazine ring is
1
replaced by chain alkane, the activity results of 9a-c and 9i-k indicated the inhibition
rule: dimethylamine >diethylamine >dipropylamine. Consistent with the above two
rules, compound 9q, keeping piperazine ring and missing a methoxy group based on
WZ4003, demonstrated a remarkable capability to inhibit NUAK activity (NUAK1/2
inhibition ratio %: 92.90 ± 2.53, 82.55 ± 2.27, respectively).
Table 1. In vitro enzyme activity and cell growth inhibitory effects of compounds.
a
Compd
Inhibition ratio ± SD (%)
R1
R2
R3
NUAK1
NUAK2
U2OS
9
a
OCH
3
Cl
80.56 ± 2.60 14.99 ± 8.69
4.83 ± 4.26
8.69 ± 3.69
b
9
b
OCH
3
Cl
10.76 ± 7.22
N
N
6

b
b
b
9
c
OCH
3
Cl
N
N
33.82 ± 5.42
9
d
OCH
OCH
OCH
OCH
OCH
H
3
3
3
3
3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
25.77 ± 9.31
65.57 ± 7.68
13.90 ± 5.96
9.20 ± 5.18
5.08 ±4.15
N
18.50 ± 2.04
9.70 ± 1.07
12.93 ± 1.59
20.92 ± 2.36
10.75 ± 1.21
9
e
8.45 ± 3.49
b
9
f
N
b
9
g
N
b
9
h
N
9
i
72.60 ±8.19
54.41 ±6.94
9.30 ±7.59
31.71 ± 2.85 60.26 ± 0.72
9
j
H
21.23 ± 4.40 38.79 ± 0.94
N
b
9
k
H
N
23.86 ± 1.24
38.48 ± 2.45
b
b
9
l
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
N
N
9
m
70.92± 6.12
43.47 ± 0.51 61.26 ± 0.91
9
n
86.00 ± 5.18 72.33 ± 3.22 48.43 ± 1.06
13.40 ± 6.32 24.81 ± 4.18 44.19 ± 3.25
86.53 ± 2.57 53.36 ± 1.70 48.70 ± 2.52
92.90 ± 2.53 82.55 ± 2.27 67.16 ± 1.65
85.62 ± 1.88 48.75 ±12.33 24.01 ± 2.45
71.54 ± 7.46 69.11 ± 6.69 34.80 ± 1.71
9
o
p
q
9
9
9
r
9
s
Cl
WZ4003
85.84 ± 4.21 84.85 ± 2.92 47.29 ± 2.56
78.29 ± 5.59 15.59 ± 1.67 38.62 ± 4.46
HTH-01-015
a
SD: standard deviation. All experiments were tested at 10 μM.
Inactive at 10 μM.
b
U2OS cells with NUAK1 overexpression have been used in many
pharmacological studies related to NUAK [3, 10, 17]. The cytotoxic activities of the
7

target compounds at 10 μM were evaluated in U2Os cells by the MTT assay. It is
more confident that the inhibition of the target products for U2OS consisted very
much with the results of enzyme activity detection. As shown in Table 1, R2
methoxy-deficient derivatives 9i-p do show significant cytotoxicity compared to R2
methoxy-substituted products 9a-h. Among R methoxy-deficient derivatives, the
2
candidate compound 9q produced 67.16 ± 1.65 % inhibition of U2OS at 10 μM, while
WZ4003 showed 47.29 ± 2.56 % inhibition.
A recent work has shown that the expression level of NUAK1 in human
colorectal cancer is associated with more aggressive disease and reduced overall
survival [10]. AMPK-related kinase NUAK1 as a key component of the antioxidant
stress response pathway in this signaling pathway has been found to be particularly
needed by cancer cells in colorectal cancer. The evidence suggested that antioxidant
defense mechanisms were destroyed by moderate inhibition of NUAK1, which may
be a safe and effective treatment for colorectal cancer. In this work, HCT116 cells and
SW480 cells growth tests for 1-5 days were made after treatment with different
compounds at 10 μM. In U2OS cells and colorectal cancer cells, 9q-treating cells
showed weaker survival than the premodified compound WZ4003-treating cells with
the extension of administration timing, indicating its outstanding role in controlling
cell growth in vitro (Figure 3).
Figure 3. Cell viability of three cell lines for 1-5 days after treatment with different
compounds at 10 μM. Data are presented as mean ± SD; n = 3.
2
.2.2. Proliferation assay
Colony formation assays and 5-ethynyl-2’-deoxyuridine (EdU) assays were
adopted to analyze cell proliferation. For the colony formation assay, colony forming
8

ability of cells was evidenced through crystal violet stained microscope images.
Figure 4 showed that the clonogenicity of three cancer cell lines, when treated with
9
q, was reduced in a dosedependent manner, compared with untreated cells.
Figure 4. Effect of 9q on cell proliferation of U2OS, HCT116 and SW480 cells in
colony formation assay. Three cancer cell lines were treated with different
concentrations of 9q and the control WZ4003 for 14 days, and colony formation was
assessed by staining with crystal violet.
Next, we use the EdU assays based on the click chemistry to further verify the
effect of drugs on cell proliferation. EdU as a terminal alkyne-containing nucleoside
analog of thymidine, is incorporated in to DNA during the active DNA synthesis
[
18-19]. Correspondingly, test results also revealed that the cell growth of U2OS,
HCT116 and SW480 cells was suppressed by 9q (Figure 5). This indicated that
methoxy-deficient derivative 9q can inhibit the proliferation of colorectal cancer cells
more effectively than WZ4003.
9

Figure 5. Effect of 9q on cell proliferation of U2OS, HCT116 and SW480 cells in
EdU assay. Three cancer cell lines were incubated with various concentrations of 9q
and the control WZ4003 for 48 h, EdU staining was then performed and the cells were
observed by ImageXpress® Micro Confocal. Blue cells were counted as DAPI
positive cells; red cells were counted as EdU-positive cells.
2
.2.3. Induces cell cycle arrest in G2/M phase
We further analyzed the effect of 9q on cell cycle progression to obtain more
information about the anti-proliferative mechanism. Three cancer cell lines were
treated with 9q and WZ4003 at 10 μM for 48 h, and the proportion of cells was
assessed by flow cytometric analysis after PI staining. In Figure 6, we observed that
the proportion of cells in G2/M phase was larger, and the proportion of cells in G1, S
phase was lower in the 9q-treating group compared with the control group. This
phenomenon was more significant in U2OS cells than in HCT116 and SW480 cells.
However, at the same concentration, WZ4003 had little effect on the repartition of cell
populations.
1
0

Figure 6. Flow cytometry images of the cell cycle in U2OS, HCT116 and SW480
cells. Columns showing the percentage of cells in G0/G1, S and G2/M phase of the
cell cycle.
2
.2.4. Promotes ROS production in cells
Tumor cells tend to be highly sensitive to ROS and raising appropriate ROS
levels is becoming a viable strategy for cancer treatment [20, 21]. The effects of 9q
and WZ4003 on ROS production in U2OS cells and multiple colorectal cancer cell
lines was evaluated (Figure 7). In our experiments, the results showed that at the
concentration of 10 μM, 9q can indeed strongly improve the ROS level in cells. The
1
1

possible reason analyzed was the inhibition of NUAK1 as a key facilitator of the
adaptive antioxidant response.
Figure 7. Flow cytometer detection of the ROS levels in U2OS (A), HCT116 (B) and
SW480 (C) cells after the treatment with 9q and WZ4003 at 10 μM for 48 h. Top,
representative Flow cytometer graph; bottom, mean ± SEM fluorescence intensity of
compound-treated relative to vehicle-treated control cells from 3 independent
experiments.
2
.2.5. Promotes apoptosis in cells
According to the above-mentioned ROS level analysis, we suppose that 9q might
induce the apoptosis of cancer cells. Therefore, Annexin V-FITC and propidium
iodide (PI) staining were carried out and the percentages of apoptotic cells were tested
using flow cytometry assay. Three cancer cell lines were incubated with different
compound at the concentration of 10 μM for 48 h. As shown in Figure 8, the
percentages of apoptotic cells in colorectal cancer cells were strikingly elevated from
the control group, especially in HCT116 cells. However, U2OS cells were
comparatively resistant to NUAK inhibitor, consistent with some reports showing that
U2OS cells were refractory to NUAK1 depletion [7, 10]. The data suggested that the
inhibition of NUAK1 by 9q was sufficient to drive apoptosis in colorectal cancer
cells.
1
2

Figure 8. Flow cytometer detection of cell apoptosis in U2OS (A), HCT116 (B) and
SW480 (C) cells. The cells were incubated with compound at 10 μM for 48 h and
stained with AnnexinV and PI double staining.
2
.2.6. Inhibits the growth of xenograft tumors
To evaluate the anti-tumor capacity of 9q in vivo, human colorectal cancer
SW480 xenograft model in nude mice was established. When the volume of solid
3
tumors reached 100 mm , the nude mice were randomly divided into four groups with
six mice. Over an 18-day period, compound 9q was intraperitoneally administered at
1
0 or 20 mg/kg every two days. Moreover, the positive control compound oxaliplatin
(Oxa) was dosed with a vehicle at 10 mg/kg intraperitoneally [22, 23]. As shown in
Figure 9A, B, C, 9q could significantly inhibit the growth of tumor by comparing the
tumor volume and tumor weight in different groups. With the increase of
administration days, the tumor volume of mice in 9q and Oxa groups gradually gaps
away from the control group (Figure 9D). Furthermore, 9q treated mice exhibited no
body weight loss throughout the experiment, whereas the Oxa treated group showed a
1
3

body weight loss (Figure 9E). Pathologically, no obvious morphological changes
were observed in the organs of the tumor-bearing mice that were treated with 9q,
whereas lesions of the liver were detected in the Oxa treatment group (Figure 9F). All
these results revealed that 9q exerted antitumor activity with less toxicity in vivo. To
further confirm the tumor inhibition mechanism of 9q, we examined the expression of
Ki67 in the tumor tissue. Immunohistochemistry analysis demonstrated that the
expression of Ki67 was reduced after 9q treatment ((Figure 9G), indicating that 9q
suppressed the proliferation of tumor cells. Collectively, the efficacious ability of 9q
to inhibit tumor growth suggested that methoxy-deficient derivative 9q is a promising
agent with less toxicity in vivo for the treatment of malignant cancers.
1
4

Figure 9. In vivo antitumor activity of 9q in mice bearing the SW480 xenograft. (A, B)
Images of SW480 tumor-bearing mice and tumor morphology after treated with 9q
1
5

(10 and 20 mg/kg) or oxaliplatin (10 mg/kg). (C) Tumor weights measured after 18
days of 9q or vehicle treatment. (D, E) Changes in tumor volume and mouse body
weight measured one times per two days during treatment. Data represent the means ±
SD (n = 6), **P < 0.01, compared with control group. (F) Hearts, livers, spleen, lungs
and kidneys were harvested and sectioned for HE staining. Original magnification: ×
4
00. (G) Histopathology of xenograft tumors stained with HE and Ki67. Original
magnification: × 400.
3
. CONCLUSIONS
In summary, with NUAK inhibitor WZ4003 as the parent compound, we
synthesized a series of novel derivatives and evaluated their abilities of NUAK
inhibition and anti-tumor activities. By the enzyme activity detection and MTT assay,
we found the necessity of N-methyl piperazine ring and the redundancy of methoxy
on the benzene ring. Among them, derivative 9q, deleting a methoxy group based on
WZ4003, showed the strongest NUAK1/2 inhibition activity and antiproliferative
activity against human osteosarcoma and colon cancer cell lines. In addition, 9q can
induce cell cycle arrest in G2/M phase, significantly improve ROS levels in tumor
cells and promote apoptosis. In vivo efficacy evaluation of 9q was investigated using
a human colorectal cancer SW480 xenograft model. 9q exhibited potent anti-tumor
activity without obvious toxic effect. Thus, methoxy-deficient 9q based on WZ4003
has the potential to be a suitable candidate for the development of anti-tumor drugs.
4
. EXPERIMENTAL SECTION
4
.1. Chemistry
All chemicals (reagent grade) used were purchased from Sino pharm Chemical
Reagent Co., Ltd. (China). Reaction progress was monitored using analytical thin
layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang
Chemical Plant, Qing-Dao, China) plates and the spots were detected under UV light
(254 nm). Column chromatography was performed on silica gel (90-150 μm; Qingdao
1
13
Marine Chemical Inc.). H NMR and C NMR spectra were measured on a Bruker
ACF-500 spectrometer at 25°C and referenced to TMS. Chemical shifts are reported
1
6

in ppm (δ) using the residual solvent line as internal standard. Splitting patterns are
designed as s, singlet; d, doublet; t, triplet; m, multiplet. Mass spectra were obtained
on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS) and a
Mariner ESI-TOF spectrometer (HRESIMS), respectively. High performance liquid
chromatography (HPLC) was performed for purity checking using Agilent 1100 series
2
HPLC on a Shimadzu XDB-C18 (5 μm, 4.6 × 150 mm ) column. All the final
compounds were found to be pure up to 95% or higher.
4
.1.1 General procedures for the preparation of compound 2a-r
To a solution of 1a-c (3.0 mmol, 1 eq) in DMF (10 mL) was added potassium
carbonate (4.5 mmol, 1.5 eq) and amine (3.0 mmol, 1 eq). The resulting mixture was
stirred at room temperature or 70 ºC for 24 h. The reaction mixture was then diluted
with water (50 mL) and extracted with ethyl acetate (3×30 mL). The yellow extract
was washed with brine (20 mL), dried over anhydrous sodium sulfate and evaporated
to dryness to obtain a crude product. The crude was purified by flash chromatography
with 98:2 (v/v) dichloromethane - methanol.
4
.1.1.1. 3-methoxy-N,N-dimethyl-4-nitroaniline (2a, CAS: 14703-82-3)
To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a, CAS: 448-19-1) in
DMF was added potassium carbonate and dimethylamine. The resulting mixture was
1
stirred at 70 ºC for 24 h. Yellow solid, 71.9% yield; H NMR (500 MHz, DMSO-d ) δ
6
7
1
4
.90 (dd, J = 9.4, 1.3 Hz, 1H), 6.34 (ddd, J = 9.4, 2.6, 1.3 Hz, 1H), 6.18 (d, J = 2.6 Hz,
H), 3.90 (s, 3H), 3.07 (s, 6H).
.1.1.2. N, N-diethyl-3-methoxy-4-nitroaniline (2b, CAS: 885057-34-1)
To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a) in DMF was added
potassium carbonate and diethylamine. The resulting mixture was stirred at 70 ºC for
1
2
1
7
4
4 h. Yellow solid, 75.4% yield; H NMR (500 MHz, DMSO-d ) δ 7.90 (d, J = 9.5 Hz,
6
H), 6.35 (dd, J = 9.5, 2.6 Hz, 1H), 6.19 (d, J = 2.5 Hz, 1H), 3.90 (s, 3H), 3.47 (q, J =
.1 Hz, 4H), 1.15 (t, J = 7.1 Hz, 6H).
.1.1.3. 3-methoxy-4-nitro-N,N-dipropylaniline (2c)
17

To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a) in DMF was added
potassium carbonate and dipropylamine. The resulting mixture was stirred at 70 ºC for
1
2
4 h. Yellow solid, 81.3% yield; H NMR (500 MHz, DMSO-d ) δ 7.88 (d, J = 9.5 Hz,
6
1
H), 6.35 (dd, J = 9.5, 2.6 Hz, 1H), 6.18 (d, J = 2.6 Hz, 1H), 3.89 (s, 3H), 3.43-3.33
(m, 4H), 1.65-1.52 (m, 4H), 0.91 (t, J = 7.4 Hz, 6H).
4
.1.1.4. 1-(3-methoxy-4-nitrophenyl)pyrrolidine (2d, CAS: 339234-68-3)
To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a) in DMF was added
potassium carbonate and pyrrolidine. The resulting mixture was stirred at room
1
temperature for 24 h. Yellow solid, 72.5% yield; H NMR (500 MHz, DMSO-d ) δ
6
7
3
4
.91 (d, J = 9.3 Hz, 1H), 6.21 (dt, J = 9.5, 1.6 Hz, 1H), 6.09 (s, 1H), 3.90 (s, 3H),
.40-3.36 (m, 4H), 2.02-1.95 (m, 4H).
.1.1.5. 4-(3-methoxy-4-nitrophenyl)morpholine (2e, CAS: 6950-88-5)
To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a) in DMF was added
potassium carbonate and morpholine. The resulting mixture was stirred at room
1
temperature for 24 h. Yellow solid, 86.3% yield; H NMR (500 MHz, DMSO-d ) δ 7.90
6
(d, J = 9.4 Hz, 1H), 6.60 (dd, J = 9.4, 2.5 Hz, 1H), 6.55 (d, J = 2.5 Hz, 1H), 3.91 (s,
3
H), 3.73 (t, J = 4.9 Hz, 4H), 3.40 (t, J = 5.0 Hz, 4H).
4
.1.1.6. 1-(3-methoxy-4-nitrophenyl)piperidine (2f, CAS:352651-56-0)
To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a) in DMF was added
potassium carbonate and piperidine. The resulting mixture was stirred at room
1
temperature for 24 h. Yellow solid, 71.8% yield; H NMR (500 MHz, DMSO-d ) δ 7.87
6
(d, J = 9.4 Hz, 1H), 6.57 (dd, J = 9.5, 2.6 Hz, 1H), 6.47 (d, J = 2.5 Hz, 1H), 3.90 (s,
3
H), 3.52-3.45 (m, 4H), 1.65-1.60 (m, 2H), 1.58 (m, 4H).
4
.1.1.7. 1-(3-methoxy-4-nitrophenyl)-4-methylpiperidine (2g, CAS: 1416351-93-3)
To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a) in DMF was added
potassium carbonate and 4-methylpiperidine. The resulting mixture was stirred at
1
room temperature for 24 h. Yellow solid, 65.8% yield; H NMR (500 MHz, DMSO-d )
6
δ 7.87 (d, J = 9.4 Hz, 1H), 6.57 (dd, J = 9.5, 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H),
4
.08 – 3.98 (m, 2H), 3.90 (s, 3H), 2.93 (td, J = 12.8, 2.6 Hz, 2H), 1.70 (d, J = 13.2 Hz,
2
H), 1.67-1.60 (m, 1H), 1.14 (qd, J = 12.5, 3.9 Hz, 2H), 0.92 (d, J = 6.4 Hz, 3H).
1
8

4
.1.1.8. 4-methoxy-1-(3-methoxy-4-nitrophenyl)piperidine (2h, CAS:2428383-11-1)
To a solution of 4-fluoro-2-methoxy-1-nitrobenzene (1a) in DMF was added
potassium carbonate and 4-methoxypiperidine. The resulting mixture was stirred at
1
room temperature for 24 h. Yellow solid, 81.4% yield; H NMR (500 MHz, DMSO-d6)
δ 7.88 (d, J = 9.4 Hz, 1H), 6.59 (dd, J = 9.5, 2.5 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H),
3
.90 (s, 3H), 3.78-3.74 (m, 2H), 3.45 (tt, J = 8.0, 3.8 Hz, 1H), 3.28 (s, 3H), 3.24 (ddd,
J = 13.1, 9.2, 3.3 Hz, 2H), 1.93-1.89 (m, 2H), 1.51-1.46 (m, 2H).
4
.1.1.9. N,N-dimethyl-4-nitroaniline (2i, CAS: 100-23-2)
To a solution of 1-fluoro-4-nitrobenzene (1b, CAS: 350-46-9) in DMF was
added potassium carbonate and dimethylamine. The resulting mixture was stirred at
1
7
0 ºC for 24 h. Yellow solid, 61.4% yield; H NMR (500 MHz, DMSO-d ) δ 8.05 (d, J
6
=
9.4 Hz, 2H), 6.77 (d, J = 9.5 Hz, 2H), 3.09 (s, 6H).
4
.1.1.10. N,N-diethyl-4-nitroaniline (2j, CAS: 2216-15-1)
To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and diethylamine. The resulting mixture was stirred at 70 ºC for 24 h.
1
Yellow solid, 73.1% yield; H NMR (500 MHz, Chloroform-d) δ 8.00 (d, J = 9.5 Hz,
2
H), 6.70 (d, J = 9.6 Hz, 2H), 3.44 (q, J = 7.1 Hz, 4H), 1.11 (t, J = 7.2 Hz, 6H).
4
.1.1.11. 4-nitro-N,N-dipropylaniline (2k, CAS: 49645-18-3)
To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and dipropylamine. The resulting mixture was stirred at 70 ºC for 24 h.
1
Yellow solid, 77.8% yield; H NMR (500 MHz, DMSO-d ) δ 8.01 (d, J = 9.4 Hz, 2H),
6
6
.75 (d, J = 9.5 Hz, 2H), 3.40-3.35 (m, 4H), 1.57 (h, J = 7.4 Hz, 4H), 0.90 (t, J = 7.4
Hz, 6H).
4
.1.1.12. 1-(4-nitrophenyl)pyrrolidine (2l, CAS: 10220-22-1)
To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and pyrrolidine. The resulting mixture was stirred at room temperature for
1
2
2
4
4 h. Yellow solid, 86.3% yield; H NMR (500 MHz, DMSO-d ) δ 8.05 (d, J = 9.4 Hz,
6
H), 6.62 (d, J = 9.3 Hz, 2H), 3.42-3.34 (m, 4H), 2.04-1.93 (m, 4H).
.1.1.13. 4-(4-nitrophenyl)morpholine (2m, CAS: 10389-51-2)
1
9

To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and morpholine. The resulting mixture was stirred at room temperature for
1
2
2
4
4 h. Yellow solid, 81.4% yield; H NMR (500 MHz, DMSO-d ) δ 8.05 (d, J = 9.5 Hz,
6
H), 7.00 (d, J = 9.5 Hz, 2H), 3.77-3.69 (m, 4H), 3.43-3.37 (m, 4H).
.1.1.14. 1-(4-nitrophenyl)piperidine (2n, CAS: 6574-15-8)
To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and piperidine. The resulting mixture was stirred at room temperature for
1
2
2
4
4
4 h. Yellow solid, 89.3% yield; H NMR (500 MHz, DMSO-d ) δ 7.97 (d, J = 9.5 Hz,
6
H), 6.89 (d, J = 9.7 Hz, 2H), 3.45-3.38 (m, 4H), 1.59-1.55 (m, 2H), 1.55-1.51 (m,
H).
.1.1.15. 4-methyl-1-(4-nitrophenyl)piperidine (2o, CAS: 78019-77-9)
To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and 4-methylpiperidine. The resulting mixture was stirred at room
1
temperature for 24 h. Yellow solid 79.8% yield; H NMR (500 MHz, DMSO-d ) δ
6
8
.02 (d, J = 9.3 Hz, 2H), 6.99 (d, J = 9.3 Hz, 2H), 4.03 (d, J = 13.2 Hz, 2H), 2.96 (t, J
12.9 Hz, 2H), 1.75-1.59 (m, 3H), 1.19-1.06 (m, 2H), 0.92 (d, J = 6.5 Hz, 3H).
=
4
.1.1.16. 4-methoxy-1-(4-nitrophenyl)piperidine (2p, CAS: 1268153-91-8)
To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and 4-methoxypiperidine. The resulting mixture was stirred at room
1
temperature for 24 h. Yellow solid, 88.9% yield; H NMR (500 MHz, DMSO-d ) δ
6
8
3
2
4
.03 (d, J = 9.5 Hz, 2H), 7.02 (d, J = 9.5 Hz, 2H), 3.78-3.74 (m, 2H), 3.46 (tt, J = 8.0,
.8 Hz, 1H), 3.29-3.23 (m, 5H), 1.95-1.87 (m, 2H), 1.48 (dtd, J = 12.7, 8.6, 3.8 Hz,
H).
.1.1.17. 1-methyl-4-(4-nitrophenyl)piperazine (2q, CAS: 16155-03-6)
To a solution of 1-fluoro-4-nitrobenzene (1b) in DMF was added potassium
carbonate and 1-methylpiperazine. The resulting mixture was stirred at room
1
temperature for 24 h. Yellow solid, 76.3% yield; H NMR (500 MHz, DMSO-d ) δ
6
8
.05 (d, J = 9.5 Hz, 2H), 7.03 (d, J = 9.5 Hz, 2H), 3.48-3.40 (m, 4H), 2.42 (t, J = 5.2
Hz, 4H), 2.22 (s, 3H).
.1.1.18. 1-(3-chloro-4-nitrophenyl)-4-methylpiperazine (2r, CAS: 1059705-52-0)
4
2
0

To a solution of 2-chloro-4-fluoro-1-nitrobenzene (1c, CAS: 2106-50-5) in DMF
was added potassium carbonate and 1-methylpiperazine. The resulting mixture was
1
stirred at room temperature for 24 h. Yellow solid, 61.9% yield; H NMR (500 MHz,
DMSO-d ) δ 8.01 (d, J = 9.4 Hz, 1H), 7.10 (d, J = 2.7 Hz, 1H), 6.99 (dd, J = 9.5, 2.8
6
Hz, 1H), 3.44 (t, J = 5.2 Hz, 4H), 2.41 (t, J = 5.1 Hz, 4H), 2.21 (s, 3H).
4
.1.2 General procedures for the preparation of compound 3a-r
Nitro compound 2a-r (3.0 mmol, 1 eq) was dissolved in a mixture of THF (9 mL)
and MeOH (6 mL) at 0 ºC. Subsequently, zinc powder (15.0 mmol, 5 eq), followed by
ammonium chloride (15.0 mmol, 5 eq) were added and the reaction mixture was
stirred at room temperature for 18 h. After completion of the reaction, the resulting
mixture was filtered through Celite and the filtrate was concentrated in vacuo. The
crude was purified by flash chromatography with 97:3 (v/v) dichloromethane -
methanol.
1
1
4
.1.2.1. 3-methoxy-N ,N -dimethylbenzene-1,4-diamine (3a, CAS: 7474-82-0)
1
Dark brown oil, 81.4% yield; H NMR (500 MHz, DMSO-d ) δ 6.51 (d, J = 8.3
6
Hz, 1H), 6.35 (d, J = 2.6 Hz, 1H), 6.16 (dd, J = 8.4, 2.6 Hz, 1H), 4.06 (s, 2H), 3.74 (s,
3
H), 2.73 (s, 6H).
1
1
4
.1.2.2 N ,N -diethyl-3-methoxybenzene-1,4-diamine (3b, CAS: 2165-85-7)
1
Dark brown oil, 78.9% yield; H NMR (500 MHz, DMSO-d ) δ 6.50 (d, J = 8.3
6
Hz, 1H), 6.32 (d, J = 2.5 Hz, 1H), 6.16 (dd, J = 8.4, 2.6 Hz, 1H), 4.05 (s, 2H), 3.72 (s,
3
H), 3.13 (q, J = 7.0 Hz, 4H), 0.99 (t, J = 7.0 Hz, 6H).
1
1
4
.1.2.3. 3-methoxy-N ,N -dipropylbenzene-1,4-diamine (3c, CAS: 137856-11-2)
1
Dark brown oil, 63.6% yield; H NMR (500 MHz, DMSO-d ) δ 6.49 (d, J = 8.4
6
Hz, 1H), 6.27 (d, J = 2.6 Hz, 1H), 6.12 (dd, J = 8.4, 2.6 Hz, 1H), 4.02 (s, 2H), 3.72 (s,
3
H), 3.12-2.97 (m, 4H), 1.50-1.35 (m, 4H), 0.85 (t, J = 7.4 Hz, 6H).
4
.1.2.4. 2-methoxy-4-(pyrrolidin-1-yl)aniline (3d, CAS: 143525-62-6)
1
Dark brown oil, 81.4% yield; H NMR (500 MHz, DMSO-d ) δ 6.52 (d, J = 8.3
6
Hz, 1H), 6.14 (d, J = 2.5 Hz, 1H), 5.95 (dd, J = 8.3, 2.5 Hz, 1H), 3.91 (s, 2H), 3.74 (s,
3
H), 3.15-3.08 (m, 4H), 1.93-1.86 (m, 4H).
4
.1.2.5. 2-methoxy-4-morpholinoaniline (3e, CAS: 209960-91-8)
2
1

1
Dark brown oil, 75.5% yield; H NMR (500 MHz, DMSO-d ) δ 6.53 (d, J = 8.3
6
Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H), 6.29 (dd, J = 8.3, 2.5 Hz, 1H), 4.23 (s, 2H), 3.74 (s,
3
H), 3.70 (t, J = 4.7 Hz, 4H), 2.91 (t, J = 4.7 Hz, 4H).
4
.1.2.6. 2-methoxy-4-(piperidin-1-yl)aniline (3f, CAS: 1340334-65-7)
1
Dark brown oil, 78.1% yield; H NMR (500 MHz, DMSO-d ) δ 6.50 (d, J = 8.3
6
Hz, 1H), 6.47 (d, J = 2.5 Hz, 1H), 6.28 (dd, J = 8.3, 2.5 Hz, 1H), 4.21 (s, 2H), 3.73 (s,
3
H), 2.89 (t, J = 5.4 Hz, 4H), 1.61 (p, J = 5.7 Hz, 4H), 1.46 (p, J = 5.9 Hz, 2H).
4
.1.2.7. 2-methoxy-4-(4-methylpiperidin-1-yl)aniline (3g, CAS: 1343635-28-8)
1
Dark brown oil, 71.3% yield; H NMR (500 MHz, DMSO-d ) δ 6.50 (d, J = 8.4
6
Hz, 1H), 6.47 (d, J = 2.5 Hz, 1H), 6.28 (dd, J = 8.4, 2.5 Hz, 1H), 4.19 (s, 2H), 3.73 (s,
3
1
4
H), 3.33 (s, 2H), 2.47 (t, J = 12.6 Hz, 2H), 1.66 (d, J = 12.2 Hz, 2H), 1.43-1.37 (m,
H), 1.24 (qd, J = 12.2, 3.9 Hz, 2H), 0.93 (d, J = 6.4 Hz, 3H).
.1.2.8. 2-methoxy-4-(4-methoxypiperidin-1-yl)aniline (3h, CAS: 1021441-14-4)
1
Dark brown oil, 69.9% yield; H NMR (600 MHz, DMSO-d ) δ 6.51 (d, J = 8.3
6
Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H), 6.30 (dd, J = 8.3, 2.5 Hz, 1H), 4.21 (s, 2H), 3.74 (s,
3
1
4
H), 3.30-3.18 (m, 6H), 2.67 (ddd, J = 12.4, 9.8, 3.0 Hz, 2H), 1.96-1.89 (m, 2H),
.56-1.50 (m, 2H).
1
1
.1.2.9. N ,N -dimethylbenzene-1,4-diamine (3i, CAS: 99-98-9)
1
Dark brown oil, 88.1% yield; H NMR (500 MHz, DMSO-d ) δ 6.56 (d, J = 7.9
6
Hz, 2H), 6.49 (d, J = 6.9 Hz, 2H), 2.68 (s, 6H).
1
1
4
.1.2.10. N ,N -diethylbenzene-1,4-diamine (3j, CAS: 93-05-0)
1
Dark brown oil, 68.4% yield; H NMR (500 MHz, DMSO-d ) δ 6.55 (d, J = 8.9
6
Hz, 2H), 6.48 (d, J = 8.6 Hz, 2H), 4.40 (s, 2H), 3.08 (q, J = 7.0 Hz, 4H), 0.96 (t, J =
7
.1 Hz, 6H).
1
1
4
.1.2.11. N ,N -dipropylbenzene-1,4-diamine (3k, CAS: 105293-89-8)
1
Dark brown oil, 75.6% yield; H NMR (500 MHz, DMSO-d ) δ 6.50 (d, J = 8.6
6
Hz, 2H), 6.46 (d, J = 8.4 Hz, 2H), 4.36 (s, 2H), 2.99 (t, J = 7.4 Hz, 4H), 1.41 (h, J =
7
.4 Hz, 4H), 0.83 (t, J = 7.4 Hz, 6H).
4
.1.2.12. 4-(pyrrolidin-1-yl)aniline (3l, CAS: 2632-65-7)
2
2

1
Dark brown oil, 91.2% yield; H NMR (500 MHz, DMSO-d ) δ 6.49 (s, 2H),
6
6
.35 (s, 2H), 4.26 (s, 2H), 3.07 (s, 4H), 1.97-1.80 (m, 4H).
4
.1.2.13. 4-morpholinoaniline (3m, CAS: 2524-67-6)
1
Dark brown oil, 85.6% yield; H NMR (500 MHz, DMSO-d ) δ 6.68 (d, J = 8.7
6
Hz, 2H), 6.50 (d, J = 8.6 Hz, 2H), 4.56 (s, 2H), 3.73-3.61 (m, 4H), 2.93-2.81 (m, 4H).
4
.1.2.14. 4-(piperidin-1-yl)aniline (3n, CAS: 2359-60-6)
1
Dark brown oil, 83.1% yield; H NMR (500 MHz, DMSO-d ) δ 6.64 (d, J = 8.7
6
Hz, 2H), 6.46 (d, J = 8.8 Hz, 2H), 4.50 (s, 2H), 2.83-2.80 (m, 4H), 1.58-1.54 (m, 4H),
1
.42 (p, J = 5.8 Hz, 2H).
4
.1.2.15. 4-(4-methylpiperidin-1-yl)aniline (3o, CAS: 342013-25-6)
1
Dark brown oil, 89.3% yield; H NMR (500 MHz, DMSO-d ) δ 6.67 (d, J = 8.7
6
Hz, 2H), 6.47 (d, J = 8.7 Hz, 2H), 4.55 (s, 2H), 3.29 (dt, J = 11.5, 2.7 Hz, 2H), 2.44
td, J = 11.9, 2.5 Hz, 2H), 1.65 (d, J = 12.8 Hz, 2H), 1.42-1,35 (m, 1H), 1.23 (qd, J =
(
1
2.1, 3.8 Hz, 2H), 0.92 (d, J = 6.5 Hz, 3H).
4
.1.2.16. 4-(4-methoxypiperidin-1-yl)aniline (3p, CAS: 1018635-74-9)
1
Dark brown oil, 86.1% yield; H NMR (500 MHz, DMSO-d ) δ 6.68 (d, J = 8.7
6
Hz, 2H), 6.47 (d, J = 8.7 Hz, 2H), 4.55 (s, 2H), 3.27-3.21 (m, 4H), 3.20-3.14 (m, 2H),
.62 (ddd, J = 12.4, 9.9, 3.0 Hz, 2H), 1.94-1.87 (m, 2H), 1.50 (dtd, J = 12.9, 9.4, 3.7
Hz, 2H).
2
4
.1.2.17. 4-(4-methylpiperazin-1-yl)aniline (3q, CAS: 16153-81-4)
1
Dark brown oil, 78.4% yield; H NMR (500 MHz, DMSO-d ) δ 6.67 (d, J = 8.7
6
Hz, 2H), 6.48 (d, J = 8.7 Hz, 2H), 4.55 (s, 2H), 2.88 (t, J = 5.0 Hz, 4H), 2.41 (t, J =
5
.0 Hz, 4H), 2.19 (s, 3H).
4
.1.2.18. 2-chloro-4-(4-methylpiperazin-1-yl)aniline (3r, CAS: 1124330-00-2)
1
Dark brown oil, 80.2% yield; H NMR (500 MHz, DMSO-d ) δ 6.76 (d, J = 2.4
6
Hz, 1H), 6.73-6.71 (m, 2H), 4.78 (s, 2H), 2.92 (t, J = 4.9 Hz, 4H), 2.41 (t, J = 4.9 Hz,
4
H), 2.19 (s, 3H).
4
.1.3 General procedures for the preparation of compound 6a-b
To a solution of 3-nitrophenol (CAS: 554-84-7) (14.3 mmol, 1 eq) in DMF (40
mL) was added potassium carbonate (28.6 mmol, 2 eq) and 2, 4,
2
3

5
-trichloropyrimidine (CAS: 5750-76-5) or 2, 4-dichloropyrimidine (CAS: 3934-20-1)
(14.3 mmol, 1 eq). The reaction was heated to 60 ºC for 2 hours. The reaction liquid
was cooled to room temperature and then added to the ice water while stirring. The
mixture was filtered, and the solid was washed three times with ethyl acetate. The
white solid was obtained, which was used without further purification.
4
.1.3.1. 2, 5-dichloro-4-(3-nitrophenoxy)pyrimidine (6a, CAS: 76661-24-0)
1
White solid, 86.9% yield; H NMR (500 MHz, DMSO-d ) δ 8.89 (s, 1H), 8.30 (t,
6
J = 2.2 Hz, 1H), 8.23 (ddd, J = 8.0, 2.2, 1.2 Hz, 1H), 7.85 (ddd, J = 8.2, 2.3, 1.2 Hz,
1
H), 7.82 (t, J = 8.1 Hz, 1H).
4
.1.3.2. 2-chloro-4-(3-nitrophenoxy)pyrimidine (6b, CAS: 1282669-97-7)
1
White solid, 87.4% yield; H NMR (500 MHz, DMSO-d ) δ 8.69 (d, J = 5.7 Hz,
6
1
H), 8.25-8.17 (m, 2H), 7.81 (m, 2H), 7.31 (d, J = 5.7 Hz, 1H).
4
.1.4 General procedures for the preparation of compound 7a-s
To a solution of 3a-r (1.50 mmol, 1 eq) and 6a-b (1.50 mmol, 1 eq) in 2-BuOH
(8 mL) were added TFA (2 mL). The slurry was heated to 100 ºC for 2-3 hours. The
reaction mixture was allowed to cool to room temperature and was neutralized with a
saturated aqueous sodium bicarbonate solution. The mixture was then extracted with
ethyl acetate (50 mL) three times. The crude was purified by flash chromatography
with 25:1 (v/v) dichloromethane - methanol.
1
4
4
4
.1.4.1.N -(5-chloro-4-(3-nitrophenoxy)pyrimidin-2-yl)-2-methoxy-N ,N -dimethylben
zene-1,4-diamine (7a)
To a solution of 3a and 6a in 2-BuOH were added TFA. Orange solid, 69.8%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.35 (s, 1H), 8.30 (s, 1H), 8.17-8.14 (m, 2H),
6
7
.75-7.73 (m, 2H), 7.01 (s, 1H), 6.24 (s, 1H), 5.95 (s, 1H), 3.69 (s, 3H), 2.83 (s, 6H).
1
4
4
4
.1.4.2.N -(5-chloro-4-(3-nitrophenoxy)pyrimidin-2-yl)-N ,N -diethyl-2-methoxybenze
ne-1,4-diamine (7b)
To a solution of 3b and 6a in 2-BuOH were added TFA. Orange solid, 69.3%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.34 (s, 1H), 8.27 (s, 1H), 8.18 (t, J = 2.2 Hz,
6
1
H), 8.14 (d, J = 7.6 Hz, 1H), 7.77-7.71 (m, 2H), 6.97 (s, 1H), 6.15 (s, 1H), 5.89 (s,
H), 3.67 (s, 3H), 3.26 (q, J = 7.2 Hz, 4H), 1.04 (t, J = 7.0 Hz, 6H).
1
2
4

1
4
4
4
.1.4.3.N -(5-chloro-4-(3-nitrophenoxy)pyrimidin-2-yl)-2-methoxy-N ,N -dipropylben
zene-1,4-diamine (7c)
To a solution of 3c and 6a in 2-BuOH were added TFA. Orange solid, 52.7%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.33 (s, 1H), 8.27 (s, 1H), 8.19 (t, J = 2.1 Hz,
6
1
1
6
4
H), 8.13 (d, J = 7.3 Hz, 1H), 7.77-7.70 (m, 2H), 6.95 (s, 1H), 6.11 (s, 1H), 5.81 (s,
H), 3.67 (s, 3H), 3.16 (t, J = 7.4 Hz, 4H), 1.48 (q, J = 7.6 Hz, 4H), 0.88 (t, J = 7.3 Hz,
H).
.1.4.4.5-chloro-N-(2-methoxy-4-(pyrrolidin-1-yl)phenyl)-4-(3-nitrophenoxy)pyrimidi
n-2-amine (7d)
To a solution of 3d and 6a in 2-BuOH were added TFA. Orange solid, 73.80%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.33 (s, 1H), 8.28 (s, 1H), 8.19-8.11 (m, 2H),
6
7
4
4
.79-7.69 (m, 2H), 6.97 (s, 1H), 6.03 (s, 1H), 5.77 (s, 1H), 3.67 (s, 3H), 3.20-3.11 (m,
H), 1.95 – 1.91 (m, 4H).
.1.4.5.5-chloro-N-(2-methoxy-4-morpholinophenyl)-4-(3-nitrophenoxy)pyrimidin-2-a
mine (7e)
To a solution of 3e and 6a in 2-BuOH were added TFA. Orange solid, 75.2%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.38 (s, 1H), 8.34 (s, 1H), 8.19-8.16 (m, 2H),
6
7
4
4
.81-7.74 (m, 2H), 7.10 (s, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.16 (s, 1H), 3.75-3.72 (m,
H), 3.71 (s, 3H), 3.02 (t, J = 4.8 Hz, 4H).
.1.4.6.5-chloro-N-(2-methoxy-4-(piperidin-1-yl)phenyl)-4-(3-nitrophenoxy)pyrimidin
-
2-amine (7f)
To a solution of 3f and 6a in 2-BuOH were added TFA. Orange solid, 68.4%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.38 (s, 1H), 8.30 (s, 1H), 8.21 – 8.15 (m, 2H),
6
7
5
4
.80-7.72 (m, 2H), 7.07 (s, 1H), 6.47 (s, 1H), 6.14 (s, 1H), 3.70 (s, 3H), 3.03 (t, J =
.3 Hz, 4H), 1.61 (p, J = 5.9 Hz, 4H), 1.55-1.49 (m, 2H).
.1.4.7.5-chloro-N-(2-methoxy-4-(4-methylpiperidin-1-yl)phenyl)-4-(3-nitrophenoxy)p
yrimidin-2-amine (7g)
To a solution of 3g and 6a in 2-BuOH were added TFA. Orange solid, 71.5%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.37 (s, 1H), 8.29 (s, 1H), 8.18-8.15 (m, 2H),
6
7
.76-7.73 (m, 2H), 7.05 (s, 1H), 6.46 (d, J = 2.6 Hz, 1H), 6.13 (s, 1H), 3.69 (s, 3H),
2
5

3
1
4
.54 (d, J = 11.9 Hz, 2H), 2.58-2.54 (m, 2H), 1.71-1.67 (m, 2H), 1.51-1.44 (m, 1H),
.25-1.20 (m, 2H), 0.94 (s, 3H).
.14.8.5-chloro-N-(2-methoxy-4-(4-methoxypiperidin-1-yl)phenyl)-4-(3-nitrophenoxy)
pyrimidin-2-amine (7h)
To a solution of 3h and 6a in 2-BuOH were added TFA. Orange solid, 66.3%
1
yield; H NMR (500 MHz, DMSO-d ) δ 8.37 (s, 1H), 8.30 (s, 1H), 8.19-8.15 (m, 2H),
6
7
2
.78-7.72 (m, 2H), 7.07 (s, 1H), 6.48 (s, 1H), 6.14 (s, 1H), 3.70 (s, 3H), 3.41-3.35 (m,
H), 3.33-3.29 (m, 1H), 3.27 (s, 3H), 2.79 (ddd, J = 12.9, 9.9, 3.0 Hz, 2H), 1.95-1.88
(m, 2H), 1.50 (dt, J = 12.8, 4.6 Hz, 2H).
1
4
4
4
.1.4.9.N -(5-chloro-4-(3-nitrophenoxy)pyrimidin-2-yl)-N ,N -dimethylbenzene-1,4-di
amine (7i)
To a solution of 3i and 6a in 2-BuOH were added TFA. Orange solid, 56.1%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.45 (s, 1H), 8.41 (s, 1H), 8.26 (t, J = 2.1 Hz,
6
1
H), 8.22 (dt, J = 7.1, 2.1 Hz, 1H), 7.82-7.77 (m, 2H), 7.10 (s, 2H), 6.42 (s, 2H), 2.77
s, 6H).
.1.4.10.N -(5-chloro-4-(3-nitrophenoxy)pyrimidin-2-yl)-N ,N -diethylbenzene-1,4-di
amine (7j)
(
1
4
4
4
To a solution of 3j and 6a in 2-BuOH were added TFA. Orange solid, 70.2%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.43 (s, 1H), 8.40 (s, 1H), 8.27 (s, 1H), 8.21
6
(dt, J = 7.1, 2.1 Hz, 1H), 7.85-7.76 (m, 2H), 7.04 (s, 2H), 6.33 (s, 2H), 3.21 (q, J = 7.0
Hz, 4H), 1.01 (t, J = 7.0 Hz, 6H).
1
4
4
4
.1.4.11.N -(5-chloro-4-(3-nitrophenoxy)pyrimidin-2-yl)-N ,N -dipropylbenzene-1,4-d
iamine (7k)
To a solution of 3k and 6a in 2-BuOH were added TFA. Orange solid, 74.9%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.43 (s, 1H), 8.39 (s, 1H), 8.24-8.17 (m, 2H),
6
7
.80-7.77 (m, 2H), 7.00 (s, 2H), 6.26 (s, 2H), 3.11 (t, J = 7.9 Hz, 4H), 1.44 (q, J = 7.4
Hz, 4H), 0.85 (t, J = 7.4 Hz, 6H).
.1.4.12.5-chloro-4-(3-nitrophenoxy)-N-(4-(pyrrolidin-1-yl)phenyl)pyrimidin-2-amine
7l)
4
(
2
6

To a solution of 3l and 6a in 2-BuOH were added TFA. Orange solid, 65.7%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.39 (s, 1H), 8.39 (s, 1H), 8.24 (s, 1H),
6
8
4
4
.23-8.19 (m, 1H), 7.83-7.77 (m, 2H), 7.08 (s, 2H), 6.22 (s, 2H), 3.10 (d, J = 6.4 Hz,
H), 1.94-1.88 (m, 4H).
.1.4.13.5-chloro-N-(4-morpholinophenyl)-4-(3-nitrophenoxy)pyrimidin-2-amine
(7m)
To a solution of 3m and 6a in 2-BuOH were added TFA. Orange solid, 80.4%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.56 (s, 1H), 8.45 (s, 1H), 8.31-8.18 (m, 2H),
6
7
4
4
.85-7.77 (m, 2H), 7.17 (s, 2H), 6.63 (s, 2H), 3.76-3.67 (m, 4H), 2.95 (t, J = 4.8 Hz,
H).
.1.4.14.5-chloro-4-(3-nitrophenoxy)-N-(4-(piperidin-1-yl)phenyl)pyrimidin-2-amine
(7n)
To a solution of 3n and 6a in 2-BuOH were added TFA. Orange solid, 62.7%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.53 (s, 1H), 8.44 (s, 1H), 8.29-8.22 (m, 2H),
6
7
4
4
2
.85-7.79 (m, 2H), 7.13 (s, 2H), 6.61 (s, 2H), 2.96 (d, J = 5.4 Hz, 4H), 1.64-1.57 (m,
H), 1.53-1.47 (m, 2H).
.1.4.15.5-chloro-N-(4-(4-methylpiperidin-1-yl)phenyl)-4-(3-nitrophenoxy)pyrimidin-
-amine (7o)
To a solution of 3o and 6a in 2-BuOH were added TFA. Orange solid, 59.0%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.53 (s, 1H), 8.44 (s, 1H), 8.28-8.20 (m, 2H),
6
7
.85-7.78 (m, 2H), 7.12 (s, 2H), 6.60 (s, 2H), 3.46 (d, J = 12.0 Hz, 2H), 2.50-2.45 (m,
H), 1.69-1.62 (m, 2H), 1.50-1.40 (m, 1H), 1.19 (qd, J = 12.2, 3.9 Hz, 2H), 0.92 (d, J
2
=
6.5 Hz, 3H).
.1.4.16.5-chloro-N-(4-(4-methoxypiperidin-1-yl)phenyl)-4-(3-nitrophenoxy)pyrimidin
2-amine (7p)
To a solution of 3p and 6a in 2-BuOH were added TFA. Orange solid, 71.8%
4
-
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.54 (s, 1H), 8.44 (s, 1H), 8.30-8.20 (m, 2H),
6
7
2
.85-7.78 (m, 2H), 7.11 (s, 2H), 6.61 (s, 2H), 3.32-3.26 (m, 3H), 3.26 (s, 3H),
.77-2.68 (m, 2H), 1.92-1.87 (m, 2H), 1.48 (dtd, J = 12.9, 9.2, 3.7 Hz, 2H).
27

4
2
.1.4.17.5-chloro-N-(4-(4-methylpiperazin-1-yl)phenyl)-4-(3-nitrophenoxy)pyrimidin-
-amine (7q)
To a solution of 3q and 6a in 2-BuOH were added TFA. Orange solid, 71.5%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.52 (s, 1H), 8.44 (s, 1H), 8.25-8.20 (m, 2H),
6
7
.84-7.77 (m, 2H), 7.16 (s, 2H), 6.62 (s, 2H), 3.00 (t, J = 5.0 Hz, 4H), 2.50 (t, J = 5.0
Hz, 4H), 2.27 (s, 3H).
.1.4.18.N-(4-(4-methylpiperazin-1-yl)phenyl)-4-(3-nitrophenoxy)pyrimidin-2-amine
7r)
4
(
To a solution of 3q and 6b in 2-BuOH were added TFA. Orange solid, 66.3%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.35 (s, 1H), 8.35 (d, J = 5.5 Hz, 1H), 8.19
6
(
dt, J = 7.5, 2.1 Hz, 1H), 8.14 (d, J = 2.3 Hz, 1H), 7.82-7.72 (m, 2H), 7.25 (s, 2H),
.65 (d, J = 8.3 Hz, 2H), 6.47 (d, J = 5.5 Hz, 1H), 2.98 (t, J = 5.0 Hz, 4H), 2.47-4.39
m, 4H), 2.21 (s, 3H).
.1.4.19.5-chloro-N-(2-chloro-4-(4-methylpiperazin-1-yl)phenyl)-4-(3-nitrophenoxy)p
6
(
4
yrimidin-2-amine (7s)
To a solution of 3r and 6a in 2-BuOH were added TFA. Orange solid, 75.4%
1
yield; H NMR (500 MHz, DMSO-d ) δ 9.01 (s, 1H), 8.38 (s, 1H), 8.19-8.08 (m, 2H),
6
7
.80-7.67 (m, 2H), 7.10 (d, J = 8.8 Hz, 1H), 6.83 (s, 1H), 6.66 (s, 1H), 3.06 (t, J = 5.0
Hz, 4H), 2.43-2.40 (m, 4H), 2.22 (s, 3H).
4
.1.5 General procedures for the preparation of compound 8a-s
Compound 7a-s (1.0 mmol, 1 eq) was dissolved in a mixture of THF (6 mL) and
MeOH (4 mL) and this solution was cooled to 0 ºC. Subsequently, zinc powder (5.0
mmol, 5 eq), followed by ammonium chloride (5.0 mmol, 5 eq) were added and the
reaction mixture was stirred at room temperature for 5 h. After completion of the
reaction, the resulting mixture was filtered through Celite and the filtrate was
concentrated in vacuo. The crude was purified by flash chromatography with 5:1 (v/v)
dichloromethane - methanol.
1
4
4
4
.1.5.1.N -(4-(3-aminophenoxy)-5-chloropyrimidin-2-yl)-2-methoxy-N ,N -dimethylbe
nzene-1,4-diamine (8a)
2
8