Design, synthesis, and evaluation of novel CXCR4 antagonists based on an aminoquinoline template

Article abstract of DOI:10.1016/j.bioorg.2020.103824

The chemokine receptor CXCR4 has been explored as a drug target due to its involvement in pathological conditions such as HIV infection and cancer metastasis. Here we report the structure–activity relationship study of novel CXCR4 antagonists based on an aminoquinoline template. This template is devoid of the chiral center in the classical tetrahydroquinoline (THQ) ring moiety and therefore can be easily synthesized. A number of potent CXCR4 antagonists were identified, exemplified by compound 3, which demonstrated excellent binding affinity with CXCR4 receptor (IC₅₀ = 57 nM) and inhibited CXCL12 induced cytosolic calcium increase (IC₅₀ = 0.24 nM). Furthermore, compound 3 potently inhibited CXLC12/CXCR4 mediated cell migration in a transwell invasion assay. The simplified synthetic approach combined with good physicochemical properties (e.g. MW 362, clogP 2.1, PSA 48, pKa 7.0 for compound 3) demonstrate the potential of this aminoquinoline template as a novel scaffold to develop CXCR4 antagonists.

Full text of DOI:10.1016/j.bioorg.2020.103824

Journal Pre-proofs
Design, Synthesis, and Evaluation of Novel CXCR4 Antagonists Based on an
Aminoquinoline Template
Yu Lin, Zhanhui Li, Chen Xu, Kaijiang Xia, Shuwei Wu, Yongjin Hao, Qing
Yang, Haikuo Ma, Jiyue Zheng, Lusong Luo, Fang Zhu, Sudan He, Xiaohu
Zhang
PII:
S0045-2068(20)30139-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103824
YBIOO 103824
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
19 January 2020
23 February 2020
5 April 2020
Please cite this article as: Y. Lin, Z. Li, C. Xu, K. Xia, S. Wu, Y. Hao, Q. Yang, H. Ma, J. Zheng, L. Luo, F. Zhu,
S. He, X. Zhang, Design, Synthesis, and Evaluation of Novel CXCR4 Antagonists Based on an Aminoquinoline
Template, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103824
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Design, Synthesis, and Evaluation of Novel CXCR4 Antagonists Based on an Aminoquinoline
Template
Yu Lin,a,1 Zhanhui Li,a,1 Chen Xu,a Kaijiang Xia,a Shuwei Wu,a Yongjin Hao,a Qing Yang,a Haikuo
Ma,a Jiyue Zheng,a,* Lusong Luo,b Fang Zhu,c,d Sudan He,c,d,* and Xiaohu Zhang a,*
a Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences,
Soochow University, Suzhou, Jiangsu 215123, P. R. China
b BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, P. R. China
c Cyrus Tang Hematology Center, Jiangsu Institute of Hematology and Collaborative Innovation Center
of Hematology, Soochow University, Suzhou 215123, P.R. China.
d Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences & Peking Union Medial College, Beijing; Suzhou Institute of Systems Medicine, Suzhou,
215123 Jiangsu, P. R. China
1 These two authors contribute equally to this study.
* Corresponding author. Tel.: +86 512 65880380; fax: +86 512 65880380; email:
xiaohuzhang@suda.edu.cn; jyzheng@suda.edu.cn; hesudan2018@163.com
Abstract: The chemokine receptor CXCR4 has been explored as a drug target due to its involvement in
pathological conditions such as HIV infection and cancer metastasis. Here we report the structure-activity
relationship study of novel CXCR4 antagonists based on an aminoquinoline template. This template is
devoid of the chiral center in the classical tetrahydroquinoline (THQ) ring moiety and therefore can be
easily synthesized. A number of potent CXCR4 antagonists were identified, exemplified by compound
3, which demonstrated excellent binding affinity with CXCR4 receptor (IC50 = 57 nM) and inhibited
CXCL12 induced cytosolic calcium increase (IC50 = 0.24 nM). Furthermore, compound 3 potently
inhibited CXLC12/CXCR4 mediated cell migration in a transwell invasion assay. The simplified
synthetic approach combined with good physicochemical properties (e.g. MW 362, clogP 2.1, PSA 48,
pKa 7.0 for compound 3) demonstrate the potential of this aminoquinoline template as a novel scaffold
to develop CXCR4 antagonists.
Keywords: aminoquinoline, antagonist, Chemokine, CXCL12, CXCR4, GPCR
Abbreviations: APC, allophycocyanin; BINAP, 1.1'-Binaphthyl-2.2'-diphemyl phosphine; Boc, tert-
butyloxycarbonyl; CCD, charge coupled device; CYP, cytochrome P450; DBU, 1,8-
Diazabicyclo[5.4.0]undec-7-ene; DCE, 1,2-dichloroethane; DIPEA, N,N-diisopropylethylamine; DMF,
N,N-dimethyl formamide; DMPK, drug metabolism and pharmacokinetics; DMSO, dimethyl sulfoxide;
DPBS, Dulbecco's phosphate buffered saline; EMCCD, electron-multiplying CCD; FACS, fluorescence
activated cell sorter; FBS, fetal bovine serum; FLIPR, fluorescent imaging plate reader; GPCR, G
protein-coupled receptor; HBSS, Hank's balanced salt solution; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; HI-FBS, heat inactivated FBS; HIV, human immunodeficiency virus;
ICCD, intensified CCD; MEF, mouse embryonic fibroblasts; Oxone, Potassiumperoxomonosulfate; PK,
pharmackinetices;
PPB,
Plasma
protein
binding;
PyBOP,
Benzotriazol-1-yl-
1

oxytripyrrolidinophosphonium hexafluorophosphate; RLM, rat liver microsomes; rt, room temperature;
SAR, structure-activity-relationship; SDF-1, stromal cell-derived factor-1; selectfluor, 1-Chloromethyl-
4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate); SEM, standard error measurement;
TBS, tert-butyldimethylsilyl; THQ, tetrahydroquinoline; TIQ, tetrahydroisoquinoline; TLC, thin-
layer chromatography.
1. Introduction
Chemokines are a family of small proteins with chemotactic functions. Chemokines bind to specific
G-protein coupled receptors (chemokine receptors) to mediate their biological activities. One of the most
extensively studied chemokine receptors is CXCR4 receptor. CXCR4 was first identified as a co-receptor
utilized by the Human Immunodeficiency Virus (HIV) to gain cellular entry [1]. In addition to this
important function, CXCR4 and its endogenous ligand CXCL12 (also known as stromal cell-derived
factor-1, or SDF-1) regulate a broad range of physiological functions (e.g., chemotaxis,
immunomodulation, and hematopoietic stem cell homing and retention) [2,3]. CXCL12/CXCR4 axis is
also involved in human pathological conditions, including virus infection, inflammatory/autoimmune
diseases and cancer [4-6]. For example, CXCR4 is overexpressed in a plethora of hematopoietic and
solid tumors and has been proposed as a biomarker for clinical prognosis [7,8]. Furthermore,
CXCL12/CXCR4 axis contributes significantly to tumor metastasis [9]. Consequently, inhibition of the
pathological functions of CXCL12/CXCR4 axis has been proposed as an important strategy for the
development of drug candidates to treat HIV infection, inflammatory diseases and cancer.
Historically, the majority of the small molecule CXCR4 antagonists were intended for anti-HIV
treatment. For example, AMD3100 (plerixafor, Figure 1) initially entered clinical trial as an anti-HIV
agent [10]. However, poor oral bioavailability and dose-limiting adverse events made it unsuitable for
the treatment of HIV infection [11]. Unexpected, AMD3100 exhibited strong mobilization effect on
hematopoietic cells during clinical testing. Through further investigation, AMD3100 was subsequently
approved by the FDA to treat patients with multiple myeloma and non-Hodgkin’s lymphoma who have
undergone stem cell transplantation [10]. This serendipitous discovery, which resulted in unprecedented
therapeutic application, combined with the recent discovery of the multiple functions of
CXCL12/CXCR4 axis in inflammatory disease and cancer, prompted significant expansion of efforts to
pursue the next generation CXCR4 antagonists. The main challenge is to combine high potency with low
toxicity and acceptable oral bioavailability. Towards this end, numerous small molecule CXCR4
antagonists had been synthesized, exemplified by AMD11070 [12-14], GSK812397 [15], TIQ15 [16],
MSX-122 [17,18], KRH-3955 [19] and IT1t [20] (Figure 1). The majority of these molecules were
synthesized in an effort to reduce the relatively high molecular weight and overall charge displayed by
AMD3100. AMD11070 is the first oral CXCR4 antagonist that has been advanced to clinical
investigation. Other compounds are in preclinical or early clinical testing.
2

N
N
N
N
N
NH HN
HN
N
N
N
NH
N
NH2
NH2
N
HN
NH
OH
NH HN
N
N
N
AMD3100 (plerixafor)
AMD11070
GSK812397
TIQ15
HN
N
N
N
N
N
N
S
N
N
S
H
H
N
NH
N
N
N
N
N
N
MSX-122
KRH-3955
IT1t
Fig. 1 Examples of small molecule CXCR4 antagonists reported in the literature.
We recently published a series of CXCR4 antagonists based on an aminopyrimidine scaffold as
exemplified by compound 1 in Figure 2 [21,22]. In the process of structural optimization, we designed
compound 2A by introducing di-fluoro to mitigate metabolic liability of THQ ring (Scheme 1). In the
process of the final substitution reaction, instead of 2A, we accidentally synthesized compound 2, the
elimination product (Figure 2 and Scheme 1). Unexpectedly, compound 2 exhibited excellent binding
affinity (inhibition of 12G5 competitive binding, IC50 = 15 nM) and functional activity (inhibition of
CXCL12 induced calcium release, IC50 = 1.3 nM). These results were intriguing given that it had been
well characterized that the chirality of the tetrahydroquinoline (THQ) ring was important for CXCR4
activity. Ordinarily, the S-enantiomer is at least 10 times more active than the R-enantiomer, and the
configurational preference at this chiral center suggests that the orientation of the substituents is
important for small molecule to interact with the CXCR4 receptor [13,14,21]. In contrast to the
established structure-activity relationship, compound 2 demonstrated that replacement of the chiral
center by an achiral double bond had no significant impact on activity. Following through on this
observation, we synthesized the quinoline analogue 3 (Figure 2), which was also active (inhibition of
12G5 competitive binding, IC50 = 57 nM; inhibition of CXCL12 induced calcium release, IC50 = 0.24
nM). Elimination of the chiral center greatly simplifies the synthetic effort, which enables efficient SAR
study. In addition, the moderate basicity combined with relative low molecular weight and clogP made
compound 3 an attractive starting point for medicinal chemistry optimization. Here we report the SAR
investigation based on compound 3 as a prototypical template.
N
N
N
N
F
N
N
N
N
Remove of the
chiral center
N
N
N
Aromatization
SAR
N
R3
R4
N
N
N
N
N
N
R2
N
R1
N
N
N
1
2
3
binding IC50 = 8.8 1.0 nM
binding IC50 = 15 3.5 nM
binding assay IC50 = 57 8.0 nM
±
±
±
Ca2+ Flux IC50 = 0.021 0.004 nM
Ca2+ Flux IC50 = 1.3 0.4 nM
Ca2+ Flux IC50 = 0.24 0.01 nM
±
±
±
clogP = 2.06
pKa = 7.2
clogP = 2.07
pKa = 6.9
clogP = 2.13
pKa = 7.0
Fig. 2 Proposed SAR study plan.
3

2. Results and Discussion
2.1. Chemistry
The synthesis of compounds 2, 3 and 11-15 was performed as shown in Scheme 1. Fluorination of
ketone 4 with selectfluor, followed by reductive amination with CH3NH2 yielded methylamine 6.
Cyclization of ester 7 with acetamidine hydrochloride, followed by chlorination with POCl3 furnished
intermediate 9. Amine 6 was alkylated with intermediate 9, subsequently substituted by N-methyl
piperazine, and competitive elimination of hydrogen fluoride yielded compound 2 rather than the desired
compound 2A. Substitution of intermediate 9 with N-methylquinolin-8-amine gave intermediate 10. The
synthesis of compounds 3 and 11-15 was achieved by reacting 10 with diverse amines.
a
b
F
F
N
N
N
F
F
F
N
N
HN
O
O
F
N
4
5
6
intended
product
X
not obtained
Cl
Cl
N
N
O
O
N
c
e, f
d
2A
Cl
N
N
EtO
N
OH
N
Cl
N
N
F
actual
product
N
7
8
9
N
N
N
Cl
2
N
N
N
g
f
N
N
3
(R1 = 4-methylpiperazin-1-yl)
N
11
12
13
(R1 = 4-cyclopropylpiperazin-1-yl)
(R1 = 4-(2-hydroxyethyl)piperazin-1-yl)
(R1 = 4-methyl-1,4-diazepan-1-yl)
N
N
Cl
N
R1
N
Cl
14 (R1 = 4-(dimethylamino)piperidin-1-yl)
15
(R1 = 3-(dimethylamino)pyrrolidin-1-yl)
9
10
Scheme 1 Reagents and conditions: (a) NaH, THF, 0oC, 10 min, then selectfluor, rt, 1 h; (b)
CH3NH2/EtOH, DCE, AcOH, DCE, NaBH3CN, rt, overnight; (c) acetamidine hydrochloride, DBU,
o
EtOH, 0 C - rt, 12 h; (d) POCl3, reflux, 1 h; (e) 6, KI, DIPEA, MeCN, rt, overnight; (f) N-methyl
piperazine, Et3N, EtOH, reflux, overnight; (g) N-methylquinolin-8-amine, K2CO3, KI, MeCN, reflux,
overnight; (h) corresponding amine, Et3N, EtOH, reflux, overnight.
The general synthetic route used to prepare compounds 20-22 and 32-34 was outlined in Scheme
2. Chlorides 18a-c were prepared from the corresponding amidines 16a-c in two steps as the same route
as intermediate 9. Compounds 20-22 were synthesized from 18a-c by substitution by N-methylquinolin-
8-amine and N-methyl piperazine in sequence. Ester 23 [23] was reacted with 2-methyl-2-thiopseudourea
sulfate or 2-hydroxyacetimidamide hydrochloride to provide pyrimidine 24 and 28, followed by
condensation with N-methyl piperazine in the presence of PyBOP to give intermediates 25 and 29.
Oxidation of 25 with Oxone followed by substitution with corresponding MeONa or dimethylamine
afforded intermediates 27a and 27b. Aldehyde compounds 30a-c were synthesized by deprotection of
acetals 27a, 27b and 29. Compounds 32-34 were obtained from aldehydes 30a-c by reductive amination
with quinolin-8-amine and subsequently with formaldehyde.
4

Cl
Cl
N
N
N
N
N
NH
NH2.HCl
N
N
N
a
b
d
c
R2
R2
N
OH
R2
N
Cl
R2
N
N
R2
N
Cl
N
16a
16b
16c
17a
17b
17c
18a
18b
18c
19a
19b
19c
20
21
22
(R2 = Et)
(R2 = c-Pr)
(R2 = MeS)
(R2 = Et)
(R2 = c-Pr)
(R2 = MeS)
(R2 = Et)
(R2 = c-Pr)
(R2 = MeS)
(R2 = Et)
(R2 = c-Pr)
(R2 = MeS)
(R2 = Et)
(R2 = c-Pr)
(R2 = MeS)
O
O
O
O
O
O
O
N
O
g
h
f
N
N
N
O
S
S
N
N
N
N
R2
N
N
S
N
OH
O
N
N
N
e
24
25
26
27a
27b
(R2 = MeO)
(R2 = Me2N)
i
O
O
O
O
O
N
O
O
O
O
O
N
N
N
j
NH
N
23
i
f
k
l
N
N
N
R2
N
N
N
N
N
OH
N
OH
N
OH
R2
N
N
R2
N
N
N
N
28
29
30a
30b
30c
31a
31b
31c
32
33
34
(R2 = MeO)
(R2 = Me2N)
(R2 = CH2OH)
(R2 = MeO)
(R2 = Me2N)
(R2 = CH2OH)
(R2 = MeO)
(R2 = Me2N)
(R2 = CH2OH)
o
Scheme 2 Reagents and conditions: (a) 7, DBU, EtOH, 0 C - rt, 12 h; (b) POCl3, reflux, 1 h; (c) N-
methylquinolin-8-amine, K2CO3, KI, MeCN, reflux, overnight; (d) N-methyl piperazine, Et3N, EtOH,
reflux, overnight; (e) 2-methyl-2-thiopseudourea sulfate; K2CO3, H2O, rt, overnight; (f) N-methyl
piperazine, PyBOP, Et3N, MeCN, reflux, overnight; (g) Oxone, THF, H2O, rt, 4 h; (h) for 27a: MeONa,
MeOH, reflux, overnight; for 27b: dimethylamine, THF, reflux, overnight; (i) 20% H2SO4, reflux,
overnight; (j) 2-hydroxyacetimidamide hydrochloride, K2CO3, H2O, rt, overnight; (k) quinolin-8-amine,
NaBH3CN, AcOH, MeOH, rt, overnight; (l) HCHO/H2O, NaBH3CN, AcOH, MeOH, rt, overnight.
Compounds 37 and 41 were prepared as described in Scheme 3. Substitution of chloride 9 with
quinolin-8-amine and N-methyl piperazine in sequence gave intermediate 36. Reductive amination of 36
with acetaldehyde yielded compound 37. Compound 39 was formed from 38 by Buchwald coupling
reaction with cyclopropylamine in the presence of Pd2(dba)3 and BINAP. Substitution of 9 with
intermediate 39 gave intermediate 40, which was reacted with N-methyl piperazine to afford compound
41.
N
N
N
N
Cl
N
N
NH
N
c
b
a
NH
N
N
Cl
N
N
N
N
N
Cl
N
N
9
35
36
37
N
N
N
N
d
e
b
N
N
N
HN
Br
N
N
N
N
Cl
N
38
39
40
41
Scheme 3 Reagents and conditions: (a) quinolin-8-amine, K2CO3, KI, MeCN, reflux, overnight; (b) N-
5

methyl piperazine, Et3N, EtOH, reflux, overnight; (c) acetaldehyde, NaBH3CN, AcOH, MeOH, rt,
overnight; (d) cyclopropanamine, Pd2(dba)3, BINAP, Cs2CO3, PhMe, 100 oC, overnight; (e) 9, K2CO3,
KI, MeCN, reflux, overnight.
The synthetic routes of compounds 47, 52, 57, 63 and 73 are shown in Scheme 4. Compound 47
was obtained from 42 [23] by using the same route as compound 34. The Claisen condensation reaction
of ester 48 was used to get the intermediate 50. Cyclization of 50 with acetimidamide hydrochloride gave
intermediate 51, which was substituted by N-methyl piperazine to provide compound 52. Compound 57
can be prepared from 53 [24] by using the same method as compound 3. Cyclization of 58 with
acetimidamide hydrochloride, followed by condensation with N-Boc-piperazine in the presence of
PyBOP provided intermediate 60. Trichloroisocyanuric acid was used to convert 60 to chloride 61, which
was substituted with N-methylquinolin-8-amine to generate intermediate 62. Removal of the Boc group
of 62 to get amine, followed by reductive amination with formaldehyde furnished compound 63.
Compound 73 could be synthesized in nine steps from 2-methylpyrimidine-4,6-diol 64. The Vilsmeier-
Haack reaction of 64 afforded aldehyde 65, followed by reduction to alcohol with NaBH4. Protection of
the alcohol group with TBS followed by Suzuki–Miyaura reaction with potassium vinyltrifluoroborate
furnished 68. Oxidation of the double bond to aldehyde 69 with ozone, followed by the reductive
amination with quinolin-8-amine led to intermediate 70. Desilylation of 70 and reductive amination with
formaldehyde gave intermediate 72. Finally, treatment of 72 with N-methyl piperazine afforded
compound 73.
All final compounds were characterized by LC-MS, 1H NMR, 13C NMR and HRMS with at least
95% purity. The results of spectral analyses can be found in Supplementary Information.
6

O
N
O
O
N
N
N
N
O
N
O
O
NH
N
b
d
e
N
a
c
O
O
N
N
N
N
O
O
N
OH
N
N
N
N
N
N
N
N
42
43
44
45
46
47
F
N
N
F
O
O
N
F
O
g
f
b
h
N
F
F
F
EtO
N
F
OEt
N
N
F
N
OH
N
N
N
N
48
49
50
51
52
N
Cl
Cl
N
O
O
N
j
k
a
i
N
Cl
Cl
Cl
Cl
OEt
N
N
Cl
N
N
Cl
Cl
Cl
N
N
OH
N
N
N
N
53
54
55
56
57
Cl
N
N
N
N
CN
O
OEt
N
N
CN
N
N
CN
OH
j
N
l
a
m
n, e
N
EtO
CN
N
CN
N
N
N
N
CN
N
N
N
Boc
N
N
Boc
N
N
Boc
58
59
60
61
62
63
O
OH
Cl
Cl
N
Cl
q
r
p
o
s
N
OTBS
N
N
N
OH
N
OTBS
O
N
OTBS
N
Cl
69
N
OH
N
Cl
Cl
N
Cl
67
N
Cl
64
65
66
68
N
N
N
N
N
N
N
k
d
NH
t
NH
N
e
N
OH
OTBS
OH
N
OH
N
N
N
Cl
70
N
Cl
N
Cl
N
71
72
73
Scheme 4 Reagents and conditions: (a) acetamidine hydrochloride, K2CO3, H2O, rt, overnight; (b) N-
methyl piperazine, PyBOP, Et3N, MeCN, reflux, overnight; (c) 20% H2SO4, reflux, overnight; (d)
quinolin-8-amine, NaBH3CN, AcOH, MeOH, rt, overnight; (e) HCHO/H2O, NaBH3CN, AcOH, MeOH,
rt, overnight; (f) NaH, Et2O, reflux, 4 h; (g) acetamidine hydrochloride, EtONa, EtOH, reflux, overnight;
(h) N-methylquinolin-8-amine, 2-ethyl-2-(hydroxymethyl)propane-1,3-diol, KI, H2O, reflux, overnight;
(i) POCl3, reflux, 1 h; (j) N-methylquinolin-8-amine, K2CO3, KI, MeCN, reflux, overnight; (k) N-methyl
piperazine, Et3N, EtOH, reflux, overnight; (l) N-Boc-piperazine, PyBOP, Et3N, MeCN, reflux, overnight;
(m) trichloroisocyanuric acid, CH2Cl2, rt, 6 h; (n) TFA, CH2Cl2, rt, 2 h; (o) POCl3, DMF, 0 oC, 1h; reflux,
o
overnight; (p) NaBH4, THF, H2O, 0 C, 30 min; (q) TBSCl, imidazole, CH2Cl2, rt, overnight; (r)
potassium vinyltrifluoroborate, Pd(PPh3)4, Cs2CO3, dioxane, H2O, 100 oC, overnight; (s) ozone, MeOH,
CH2Cl2, -65 oC, 1 h; Me2S, -65 oC, 10 min; (t) pyridine hydrofluoride, THF, rt, 4 h.
2.2. Structure-activity relationship based on calcium mobilization assay
Numerous assays have been used to characterize the CXCL12/CXCR4 axis. The reported assays
include competitive binding assay, CXCL12-induced calcium mobilization, CXCR4 receptor
7

internalization, CXCL12-guided chemotaxis and CXCR4-specific HIV entry and replication. In our
previous publications, we used a cell based FACS 12G5 competitive binding assay as the primary
screening assay followed by a FLIPR Tetra based cytosolic calcium flux assay as the functional assay
[21]. In this report, we used FLIPR Tetra based cytosolic calcium flux assay [21,25] as our primary
screening assay followed by the 12G5 competitive binding assay [21,26] as the on-target assay.
Competitive ligand binding assays are good for characterization of target engagement but convey no
functional information. On the other hand, calcium flux assay provides information regarding receptor
activation, inhibition, or potentiation. In addition, competitive ligand binding assays tend to miss
allosteric binders which do not compete for the same binding pocket. More importantly, based on our
experience, the results from the 12G5 competitive binding assay correlate very well with the results from
the functional calcium flux assay. Furthermore, the expense of the calcium flux assay was lower and the
throughput was higher compared with the 12G5 competitive binding assay. Based on these
considerations, we decided to use the FLIPR Tetra based calcium flux functional assay as the screening
assay and the 12G5 competitive binding assay as the confirmation assay. The structure-activity-
relationship is summarized in Tables 1 and 2.
We started structural modifications on the R1 position, which is solvent exposed according to our
previous docking study. We used a small set of basic amines to test if the SAR on R1 would follow the
established trend. Replacing the methyl group on the terminal nitrogen of the piperazine by a larger
cyclopropyl group had a negative effect, leading to a 7-fold decrease of potency (1.5 nM vs. 0.24 nM for
compounds 11 and 3, respectively). Similarly, introducing a polar hydroxyl group resulted in a 10-fold
decrease of potency (compound 12, 2.1 nM). Ring expansion (compound 13, 0.81 nM), extension of the
nitrogen out of the piperazine ring (compound 14, 2.3 nM), and introduction of amino pyrrolidine
(compound 15, 0.71 nM) to increase pKa (8.8-9.0, Table 1) all led to decreased potency. The SAR on
the basic R1 position largely followed our previous SAR results [21,22]. With piperazine fixed on R1, we
next explored R2 with different substituents. Increase size on R2 led to decreased potency (compounds
20 and 21; 0.73 and 2.9 nM, respectively). Introduction of heteroatoms have distinct impact: while
methylthio and methoxy were tolerated (compound 22 and 23, 0.57 and 3.0 nM, respectively), the
dimethylamino led to significant decrease of potency (compound 33, 1926 nM). Reduction of clogP by
incorporation of a hydroxymethyl group resulted in 30-fold decrease of potency (compound 34, 7.3 nM).
Table 1. SAR for R1 and R2
N
N
N
R2
N
R1
Ca2+
Flux
Ca2+
Flux
Cmpd
R1
R2
clogPb pKac Cmpd
R1
R2
Et
clogPb pKac
IC50
IC50
(nM) a
0.24 ±
0.01
(nM) a
0.73 ±
0.48
N
N
N
3
Me
Me
2.13
2.50
7.0
7.0
20
21
2.71
2.50
7.1
7.2
N
N
N
N
1.5 ±
0.04
2.9 ±
0.03
N
11
8

2.1 ±
0.005
0.81 ±
0.47
0.57 ±
0.06
3.0 ±
0.6
N
N
N
N
N
OH
12
13
14
15
Me
Me
Me
Me
1.50
2.40
2.66
2.39
-1.04
7.0
8.8
22
32
33
34
MeS
MeO
Me2N
3.52
2.42
3.00
1.49
6.3
6.3
8.8
5.9
N
N
N
N
N
N
N
N
2.3 ±
0.5
1926
± 470
7.3 ±
0.7
9.0
N
0.76 ±
0.08
HOCH
N
N
8.8
2
AMD310
0
23 ±
2.3
10.6
a Inhibition of luminescence signaling in FLIPR Tetra calcium mobilization assay. Data are expressed as
b
geometric mean values of at least two runs ± the standard error measurement (SEM). Calculated by
Molinspiration. c Calculated by ACD/Labs 6.0.
Having established piperazine and methyl as the preferred substituents on R1 and R2 positions
respectively, we set to focus on the SAR of R3 and R4 positions. Replacing the methyl with an ethyl on
R3 led to similar activity (compound 37, 0.16 nM) to compound 3. Further increase on size led to
decreased potency (compound 41, 2.5 nM). R4 position had been used to introduce polar functional
groups as a way to fine tune physicochemical properties [27,28]. The synthesis of R4 analogues was
challenging because R4 was difficult to be modified at the final steps. As a result, each of the five
analogues was synthesized by a different synthetic route, albeit in a similar fashion. Replacing the
hydrogen with a methyl or a fluorine led to compounds with similar activity (compounds 47 and 52, 0.49
and 0.86 nM, respectively) to compound 3. Chlorine, cyano and hydroxymethyl group were all tolerated
but showed decrease of potency to various extents (compounds 57, 63, and 73; 1.2, 3.5, 0.95 nM,
respectively). Overall, the SAR of the aforementioned locations generally followed the established SAR
on the tetrahydroquinoline series. The most intriguing result was the lack of impact on potency with the
elimination of the chiral center on the tetrahydroquinoline scaffold.
Table 2. SAR for R3 and R4
N
N
R3
R4
N
N
N
N
Cmpd
3
R3
Me
Et
R4
H
Ca2+ flux IC50 (nM) a clogPb pKac
0.24 ± 0.01
0.16 ± 0.01
2.5 ± 0.9
2.13
2.51
2.50
2.51
2.22
2.74
1.81
1.40
7.0
8.1
7.2
7.4
5.9
5.6
5.2
6.7
37
H
41
H
47
Me
Me
Me
Me
Me
Me
F
0.49 ± 0.08
0.86 ± 0.18
1.2 ± 0.3
52
57
Cl
63
CN
CH2OH
3.5 ± 0.4
73
0.95 ± 0.04
9

AMD3100
23 ± 2.3
-1.04
10.6
a Inhibition of luminescence signaling in FLIPR Tetra calcium mobilization assay. Data are expressed as
b
geometric mean values of at least two runs ± the standard error measurement (SEM). Calculated by
Molinspiration. c Calculated by ACD/Labs 6.0.
2.3. Binding assay and preliminary selectivity assays
Compounds 3 and 37 emerged as the leads of this aminoquinoline series of analogues based on their
excellent functional activity and overall properties (e.g., compound 3, MW 362, clogP 2.1, PSA 48, pKa
7.0). We next tested compounds 3 and 37 in the 12G5 competitive binding assay to see if they bind with
the CXCR4 receptor. In this assay, HPB-ALL cells which naturally express CXCR4 were incubated with
APC-conjugate clone 12G5 antibody and testing compound. The inhibition of the 12G5 antibody signal
with different concentrations of compound provided inhibition curve and readout of IC50. The detailed
description of assay development and validation had been reported [21,26]. Compounds 3 and 37
potently replaced 12G5 antibody at IC50 = 57 ± 8.0 nM and 144 ± 7.3 nM, respectively; while the positive
control AMD3100 replaced the 12G5 antibody at IC50 = 561 ± 27 nM. Representative single assay curves
were shown in Figure 3. These data established compounds 3 and 37 as competitive CXCR4 antagonists.
Fig. 3 Dose response curves of compound 3 and AMD3100 in the 12G5 HPB-ALL CXCR4 competitive
binding assay.
Having demonstrated that compound 3 potently binds to CXCR4 (IC50 = 57 nM) and inhibits
CXCL12 induced cytosolic calcium flux (IC50 = 0.24 nM), we further assessed the preliminary functional
specificities of compound 3. We chose CXCR1 as a representative within the CXCR sub-family and
CCR6 as a representative outside of the CXCR sub-family and tested compound 3 in the corresponding
functional assays. Compound 3 was shown to be inactive up to 10,000 nM (Figure 4b, c).
10

Fig. 4 Compound 3 is a potent CXCR4 antagonist, but is inactive to CXCR1 and CCR6. (a) CXCR4 dose
response curve for SDF-1 (CXCL12) stimulation and inhibition curves of compound 3/AMD3100
(positive control for CXCR4) based on a calcium flux assay. (b) CXCR1 dose response curve for IL-8
stimulation and inhibition curves of compound 3/Navarixin (positive control for CXCR1) based on a
functional cAMP assay. (c) CCR6 dose response curve for CCL20 stimulation and inhibition curves of
compound 3/PF-9654-00 (positive control for CCR6) based on a calcium flux assay.
2.4. Chemotaxi assay based on transwell and cell toxicity assays
CXCR4 is a chemokine receptor. One of the hallmarks of CXCR4 activation by CXCL12 is the
migration and invasion of the corresponding cells. We investigated compound 3 in the matrigel invasion
assay to evaluate its inhibition of CXCL12/CXCR4 mediated chemotaxis and invasion. To the upper
chamber were added compound 3 or AMD3100 (100 nM) and CXCR4 expressing MDA-MB-231 cells.
To the lower chamber was added human CXCL12 (200 ng/mL). The inhibition of cell invasion with
compound 3 or AMD3100 was calculated by comparison of the cell invasion without compound
treatment (Figure 5a, b). Compound 3 inhibited cell migration more potently in the matrigel invasion
assay than AMD3100, consistent with the calcium flux and the 12G5 binding assays. Cell mobility can
be affected by compounds with general cellular toxicity. To rule out this possibility, we used cell viability
assay (ATP-based Cell Titer-Glo Luminescent Cell Viability Assay) to investigate the potential
cytotoxicity effect of compound 3. As shown in Figure 5c, compound 3 did not inhibit cell proliferation
11

of multiple human and murine cells at concentrations up to 1000 nM.
Fig. 5 Effect of compound 3 on matrigel invasion of MDA-MB-231 cells and cytotoxicity assessment of
3 in the cultured human and murine cells. (a) Photo images of matrigel 22 h after invasion experiment.
(b) Quantification of transwell analysis of cell invasion experiment. Data represent mean value ± standard
deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Magnification: 4x. (c) Effect of
compound 3 on proliferation of human and murine cells.
2.5. Preliminary in vitro safety and DMPK evaluation
AMD11070 was derived from AMD3100 to reduce molecular weight and over charges. As a result,
AMD11070 can be administered orally and has been progressed into clinical testing. However, CYP
isozyme (3A4, 2D6) inhibition was reported and the possible culprit had been attributed to the
benzimidazole functionality [14,21]. In our previous work, we used aminopyrimidine to replace the
benzimidazole as a way to mitigate CYP inhibition. In order to understand the CYP inhibition profile of
this new aminoquinoline template, we tested compound 3 in CYP isozyme inhibition assays. As shown
in Table 3, compound 3 exhibited minimal inhibition of CYP3A4 and 2D6 at 10 μM concentration, while
AMD11070 demonstrated moderate inhibition (60% and 64%; 3A4 and 2D6, respectively) [21].
Compound 3 was found to be highly permeable (45× 10-6 cm/s, Caco-2), and not likely to be an efflux
substrate (Table 3). Moreover, compound 3 exhibited high plasma protein binding in human (99%) but
more moderate binding in mouse and rat (94% and 90%, respectively). However, despite the marginally
improved metabolic stability in human liver microsomes, compound 3 was quickly metabolized in rat
liver microsomes (Table 3). Improvement of metabolic stability cross species must be considered in the
future medicinal chemistry optimization.
Table 3. Preliminary in vitro safety and DMPK evaluation of compound 3
CYP
Cmpd
Caco-2a
PPB (%)b
RLMc
HLMd
Inhibition
12

(%)a
Papp(A−B)
(10−6 cm/s)
45
efflux ratio
(B−A)/(A−B)
0.52
Clint
(mL/min/kg) (min) (mL/min/kg) (min)
656 3.8 122 14.2
t1/2
Clint
t1/2
3A4 2D6
mouse rat human
94 90 99
3
13
15
a The compound was tested at 10 μM concentration. b The compound was tested at 1 µM concentration.
c RLM = Rat liver microsomes. d HLM = Human liver microsomes.
3. Conclusion
An aminoquinoline template was used to develop a novel series of CXCR4 antagonists. This
template is devoid of the chiral center in the classical tetrahydroquinoline (THQ) ring moiety and
therefore can be easily synthesized. A number of potent CXCR4 antagonists were identified, exemplified
by compound 3, which demonstrated excellent binding affinity with CXCR4 receptor (IC50 = 57 nM)
and inhibited CXCL12 induced cytosolic calcium increase (IC50 = 0.24 nM). Furthermore, compound 3
potently inhibited CXLC12/CXCR4 mediated cell migration in a transwell invasion assay. The
simplified synthetic approach combined with good physicochemical properties (e.g. MW 362, clogP 2.1,
PSA 48, pKa 7.0 for compound 3) demonstrate the potential of this aminoquinoline template as a novel
scaffold to develop CXCR4 antagonists. Structural modification on the aminoquinoline is ongoing and
the results will be reported in due course.
4. Experimental protocols
4.1. Chemistry
All reagents and solvents were used without further purification. Reactions were monitored by thin-
1
layer chromatography or by Agilent 1100 LC/MSD Trap SL version Mass Spectrometer. H and 13C
NMR spectra were performed on Varian 400 MHz or 600 MHz spectrometers with tetramethylsilane as
an internal reference. HRMS analysis was recorded on an Agilent 6540 UHD Accurate-Mass Q-TOF
LC/MS. HPLC method: Waters Acquity UPLC, BEH C18 2.1 mm × 50 mm, 1.7 μm particles. Mobile
phase A: 5 mM aqueous ammonium acetate. Mobile phase B: MeOH. Temperature: 24 °C. Gradient:
5−40% B over 1 min, 40-70% B over 1 min, 70-95% B over 4 min, then a 1 min hold at 95% B. Flow:
1.2 mL/min. Detection: UV at 214 and 254 nm. The detailed procedures of chemical experiment can be
found in the supporting information.
4.1.1. 7,7-Difluoro-6,7-dihydroquinolin-8(5H)-one (5)
To a solution of NaH (60 wt percent moistened with oil, 511 mg, 12.8 mmol) in THF (20 mL) was
added dropwise the THF solution (5 mL) containing compound 4 (588 mg, 4 mmol) at 0°C. The reaction
was stirred at 0°C for 10 min followed by adding selectfluro (3.0 g, 8.4 mmol) in portions. The reaction
mixture was stirred at room temperature for 1h. Water (20 mL) was added to quench the reaction. The
water phase was extracted with diethyl ether (20 mL x 3). The combined organic layer was dried over
Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography
1
(petroleum ether/ethyl acetate = 10/1) to give the desired product (380 mg, 52%) as a white solid. H
NMR (400 MHz, CDCl3) δ 8.79 (d, J = 4.4 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.55-7.45 (m, 1H), 3.20 (t,
J = 6.4 Hz, 1H), 2.72-2.56 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 183.9 (t, J = 26.3 Hz), 150.4, 145.9,
139.8, 137.8, 128.2, 113.8 (t, J = 247.8 Hz), 32.0 (t, J = 23.0 Hz), 24.4 (t, J = 5.9 Hz). MS (ESI/APCI)
13

m/z 183.9 [M+H]+.
4.1.2. 7,7-Difluoro-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (6)
To a solution of 5 (18.3 mg, 0.1 mmol), MeNH2 solution (30 wt percent in ethanol, 1 mL) and HOAc
(5 mg, 0.083 mmol) in 1,2-dichloroethane (2 mL) was added NaBH3CN (13 mg, 0.2 mmol). The resulting
suspension was stirred at room temperature overnight. The reaction mixture was filtered and the filtrate
was evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol = 150/1) to give the desired product (15 mg, 75%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) δ 8.48 (d, J = 4.4 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.21-7.14 (m, 1H), 3.89 (t, J =
10.0 Hz, 1H), 3.01-2.92 (m, 2H), 2.72 (s, 3H), 2.57-2.44 (m, 1H), 2.28-2.18 (m, 1H). MS (ESI/APCI)
m/z 199.0 [M+H]+.
4.1.3. 6-(Chloromethyl)-2-methylpyrimidin-4-ol (8)
To a solution of ethyl 4-chloro-3-oxobutanoate 7 (22 g, 136 mmol) and acetimidamide
hydrochloride (17 g, 150 mmol) in ethanol (180 mL) was slowly added DBU (41 g, 272 mmol) at 0 °C.
The reaction was stirred at room temperature for 12 h. The reaction solution was evaporated. The residue
was acidified with 2 N HCl to pH = 4 and extracted with ethyl acetate (200 mL x 6). The combined
organic layer was dried over Na2SO4 and concentrated. The crude material was purified by silica gel
column chromatography (petroleum ether/ethyl acetate = 2.5/1) to give the desired product (8 g, 37%)
as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.54 (br s, 1H), 6.29 (s, 1H), 4.44 (s, 2H), 2.28 (s,
3H). 13C NMR (150 MHz, DMSO-d6) δ 162.4, 161.8, 159.8, 110.4, 45.3, 21.1. MS (ESI/APCI) m/z 156.9
[M-H]-.
4.1.4. 4-Chloro-6-(chloromethyl)-2-methylpyrimidine (9)
The solution of 8 (8.0 mmol, 50 mmol) in POCl3 (40 mL) was stirred at reflux for 1 h. The reaction
mixture was concentrated and dissolved in ethyl acetate (200 mL), then added saturated NaHCO3 solution
to adjust pH to 7, extracted with ethyl acetate (100 mL * 3). The combined organic layer was dried over
Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 10/1) to give the desired product (5.2 g, 59%) as a yellow solid. 1H NMR (400 MHz,
CDCl3) δ 7.40 (s, 1H), 4.55 (s, 2H), 2.70 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 169.1, 166.7, 162.2,
116.7, 44.7, 25.8. MS (ESI/APCI) m/z 176.9 [M+H]+.
4.1.5.
7-Fluoro-N-methyl-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6-
dihydroquinolin-8-amine (2)
A mixture of 6 (55 mg, 0.28 mmol), 9 (54 mg, 0.31 mmol), KI (5 mg, 0.028 mmol) and DIPEA (90
mg, 0.7 mmol) in CH3CN (10 mL) was stirred at room temperature overnight. The reaction solution was
evaporated to remove most of CH3CN, diluted with dichloromethane (15 mL) and washed with saturated
brine solution (10 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The residue
was dissolved in ethanol (10 mL). TEA (252 mg, 2.5 mmol) and N-methylpiperazine (125 mg, 1.25 mmol)
were added and stirred at reflux overnight. The reaction mixture was cooled to room temperature and
concentrated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/ammonium hydroxide = 50/1/1) to give the desired product (15 mg, 15%)
as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 4.0 Hz, 1H), 7.33(d, J = 7.2 Hz, 1H), 7.04-
6.90 (m, 2H), 4.26 (s, 2H), 3.68-3.58 (m, 4H), 2.95-2.80 (m, 5H), 2.61-2.53 (m, 2H), 2.47 (s, 3H), 2.45-
2.39 (s, 4H), 2.32 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 167.1, 166.4, 162.8, 158.8, 157.0, 152.8 (d, J
= 9.6 Hz), 147.3, 134.5, 129.7, 127.0 (d, J = 7.1 Hz), 121.0, 97.4, 59.5, 54.7, 46.1, 43.7, 41.9 (d, J = 4.2
Hz), 27.0 (d, J = 8.0 Hz), 25.8, 25.1 (d, J = 23.4 Hz). HRMS (ESI): calcd for C21H28FN6 [M+H]+ 383.2354,
found 383.2365. Purity: 98.8%.
14

4.1.6. N-((6-Chloro-2-methylpyrimidin-4-yl)methyl)-N-methylquinolin-8-amine (10)
A mixture of 9 (890 mg, 5.0 mmol), N-methylquinolin-8-amine (530 mg, 3.4 mmol), KI (56 mg,
0.34 mmol) and K2CO3 (925 mg, 6.7 mmol) in CH3CN (20 mL) was stirred at reflux overnight. The
reaction solution was evaporated to remove most of CH3CN. The residue was purified by silica gel
column chromatography (dichloromethane/methanol = 100/1) to give the desired product (1.04 g, 98%)
as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.75 (d, J = 2.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.67 (s,
1H), 7.48-7.34 (m, 3H), 7.14 (d, J = 7.6 Hz, 1H), 4.83 (s, 2H), 3.04 (s, 3H), 2.72 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 171.9, 168.4, 161.4, 148.1, 147.7, 142.0, 136.9, 129.9, 126.9, 121.2, 120.8, 117.1, 115.7,
61.7, 41.1, 25.9. MS (ESI/APCI) m/z 298.8 [M+H]+.
4.1.7. General procedure for the synthesis of 3 and 11-15
A mixture of 10 (0.17 mmol, 1.0 eq.), TEA (10.0 eq.) and corresponding amine (5.0 eq.) in ethanol
(5 mL) was stirred at reflux overnight. The reaction solution was evaporated. The residue was purified
by silica gel column chromatography (dichloromethane/methanol/ammonium hydroxide = 50/1/1) to
give the desired product.
4.1.7.1. N-Methyl-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine (3)
Compound 3 was obtained as a yellow oil (yield 69%). 1H NMR (400 MHz, CDCl3) δ 8.82 (d, J =
2.4 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.44-7.35 (m, 3H), 7.12 (d, J = 7.2 Hz, 1H), 6.67 (s, 1H), 4.68 (s,
2H), 3.56 (s, 4H), 3.05 (s, 3H), 2.51 (s, 3H), 2.42-2.36 (m, 4H), 2.29 (s, 3H).13C NMR (150 MHz, CDCl3)
δ 167.1, 166.8, 162.8, 148.9, 147.8, 142.7, 136.5, 129.9, 126.8, 121.0, 120.6, 116.2, 97.9, 61.8, 54.8,
46.3, 43.8, 41.5, 26.2. HRMS (ESI): calcd for C21H27N6 [M+H]+ 363.2292, found 363.2296. Purity:
96.9%.
4.1.7.2.
N-((6-(4-Cyclopropylpiperazin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methylquinolin-8-
amine (11)
Compound 11 was obtained as a yellow oil (yield 50%). 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H),
8.09 (d, J = 8.4 Hz, 1H), 7.46-7.30 (m, 3H), 7.11 (d, J = 7.2 Hz, 1H), 6.67 (s, 1H), 4.69 (s, 2H), 3.59-
3.40 (m, 4H), 3.05 (s, 3H), 2.63-2.54 (m, 4H), 2.51 (s, 3H), 1.67-1.51 (m, 1H), 0.53-0.33 (m, 4H). 13
C
NMR (150 MHz, CDCl3) δ 166.7, 166.6, 162.6, 148.9, 147.7, 142.6, 136.5, 129.8, 126.7, 121.0, 120.6,
116.2, 97.9, 61.6, 53.0, 43.8, 41.5, 38.5, 26.1, 5.9. HRMS (ESI): calcd for C23H29N6 [M+H]+ 389.2448,
found 389.2454. Purity: 98.0%.
4.1.7.3. 2-(4-(2-Methyl-6-((methyl(quinolin-8-yl)amino)methyl)pyrimidin-4-yl)piperazin-1-yl)ethan-1-
ol (12)
Compound 12 was obtained as a yellow oil (yield 50%). 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J =
4.4 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.43-7.29 (m, 3H), 7.11 (d, J = 7.2 Hz, 1H), 6.66 (s, 1H), 4.65(s,
2H), 3.66-3.59 (m, 2H), 3.58-3.48 (m, 4H), 3.03 (s, 3H), 2.64-2.41 (m, 10H). 13C NMR (150 MHz, CDCl3)
δ 167.1, 166.7, 162.6, 148.8, 147.7, 142.6, 136.5, 129.8, 126.7, 121.0, 120.6, 116.1, 97.8, 61.6, 59.6,
57.9, 52.6, 43.8, 41.5, 26.1. HRMS (ESI): calcd for C22H29N6O [M+H]+ 393.2397, found 393.2399.
Purity: 95.7%.
4.1.7.4. N-Methyl-N-((2-methyl-6-(4-methyl-1,4-diazepan-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine
(13)
Compound 13 was obtained as a yellow oil (yield 31%). 1H NMR (400 MHz, CDCl3) δ 8.82 (d, J =
2.4 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.45-7.32 (m, 3H), 7.11 (d, J = 7.2 Hz, 1H), 6.44 (s, 1H), 4.72 (s,
2H), 4.05 -3.16 (m, 4H), 3.08 (s, 3H), 2.72-2.38(m, 7H), 2.30 (s, 3H), 1.78 (s, 2H). 13C NMR (150 MHz,
CDCl3) δ 166.5, 166.3, 162.1, 148.8, 147.7, 142.6, 136.4, 129.8, 126.7, 121.0, 120.5, 116.2, 97.4, 61.5,
58.0, 57.3, 46.7, 46.0, 45.5, 41.7, 27.2, 26.3. HRMS (ESI): calcd for C22H29N6 [M+H]+ 377.2448, found
15

377.2440. Purity: 95.5%.
4.1.7.5. N-((6-(4-(Dimethylamino)piperidin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methylquinolin-8-
amine (14)
Compound 14 was obtained as a yellow oil (yield 31%). 1H NMR (400 MHz, CDCl3) δ 8.88-8.76
(m, 1H), 8.11 (dd, J = 8.4, 2.4 Hz, 1H), 7.46-7.32 (m, 3H), 7.12 (dd, J = 7.2, 2.0 Hz, 1H), 6.66 (s, 1H),
4.68 (s, 2H), 4.37(d, J = 13.2 Hz, 2H), 3.05 (s, 3H), 2.75 (t, J = 7.2 Hz,2H), 2.52-2.46 (m, 4H), 2.32 (s,
6H), 1.86 (d, J = 12.8 Hz, 2H), 1.43-1.33 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 167.0, 166.8, 162.4,
148.9, 147.8, 142.7, 136.5, 129.9, 126.8, 121.1, 120.7, 116.3, 97.9, 62.6, 61.7, 43.3, 41.6, 41.4, 27.7,
26.2. HRMS (ESI): calcd for C23H31N6 [M+H]+ 391.2605, found 391.2613. Purity: 97.8%.
4.1.7.6. N-((6-(3-(Dimethylamino)pyrrolidin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methylquinolin-8-
amine (15)
Compound 15 was obtained as a yellow oil (yield 31%). 1H NMR (400 MHz, CDCl3) δ 8.82 (d, J =
4.4 Hz,1H), 8.09 (d, J = 8.0 Hz,1H), 7.46-7.30 (m, 3H), 7.12 (d, J = 7.2 Hz, 1H), 6.34 (s, 1H), 4.74 (s,
2H), 4.15-3.14 (m, 4H), 3.08 (s, 3H), 2.68 (s, 1H), 2.53 (s, 3H), 2.25 (s, 6H), 2.18-2.05 (m, 1H), 1.80 (s,
1H). 13C NMR (150 MHz, CDCl3) δ 166.5, 165.6, 160.5, 148.7, 147.5, 142.4, 136.3, 129.7, 126.6, 120.8,
120.4, 116.0, 98.1, 65.1, 61.3, 50.5, 45.4, 44.3, 41.4, 30.0, 26.0. HRMS (ESI): calcd for C22H29N6 [M+H]+
377.2448, found 377.2456. Purity: 99.0%.
4.1.8. General procedure for the synthesis of 17a-c
To a solution of 16a/b/c (9 mmol, 1.0 eq.) and 7 (1.0 eq.) in ethanol (15 mL) was slowly added
DBU (2.0 eq.) at 0 °C. The reaction was stirred at room temperature for 12 h. The reaction solution was
evaporated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate
= 2.5/1) to give the desired product.
4.1.8.1. 6-(Chloromethyl)-2-ethylpyrimidin-4-ol (17a)
Compound 17a was obtained as a yellow oil (yield 48%). 1H NMR (400 MHz, CDCl3) δ 12.97 (s,
1H), 6.54 (s, 1H), 4.39 (s, 2H), 2.73 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ 166.0, 164.3, 163.7, 110.2, 45.3, 28.8, 11.7. MS (ESI/APCI) m/z 170.8 [M-H]-.
4.1.8.2. 6-(Chloromethyl)-2-cyclopropylpyrimidin-4-ol (17b)
Compound 17b was obtained as a yellow oil (yield 36%). 1H NMR (400 MHz, CDCl3) δ 13.52 (s,
1H), 6.46 (s, 1H), 4.30 (s, 2H), 2.00-1.81 (m, 1H), 1.26-1.17 (m, 2H), 1.15-1.06 (m, 2H). 13C NMR (150
MHz, CDCl3) δ 166.0, 164.7, 164.6, 108.9, 45.4, 14.5, 11.0. MS (ESI/APCI) m/z 184.9 [M+H]+.
4.1.8.3. 4-Chloro-6-(chloromethyl)-2-(methylthio)pyrimidine (17c)
Compound 17c was obtained as a yellow oil (yield 73%). 1H NMR (400 MHz, CDCl3) δ 12.71 (s,
1H), 6.41 (s, 1H), 4.35 (s, 2H), 2.59 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 165.5, 163.4, 162.4, 108.3,
45.2, 13.4. MS (ESI/APCI) m/z 191.1 [M+H]+.
4.1.9. General procedure for the synthesis of 18a-c
The solution of 17a/b/c (4.1 mmol, 1.0 eq.) in POCl3 (2.5 mL) was heated to 90 °C, and stirred for
1 h. Then reaction mixture was concentrated and dissolved in ethyl acetate (20 mL), then added saturated
NaHCO3 solution to adjust pH to 7, extracted with ethyl acetate (10 mL * 3). The combined organic layer
was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography
(petroleum ether/ethyl acetate = 10/1) to give the desired product.
4.1.9.1. 4-Chloro-6-(chloromethyl)-2-ethylpyrimidine (18a)
Compound 18a was obtained as a yellow oil (yield 75%). 1H NMR (400 MHz, CDCl3) δ 7.42 (s,
1H), 4.57 (s, 2H), 2.96 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 173.3,
166.7, 162.3, 116.8, 44.8, 32.5, 12.6. MS (ESI/APCI) m/z 190.9 [M+H]+.
16

4.1.9.2. 4-Chloro-6-(chloromethyl)-2-cyclopropylpyrimidine (18b)
Compound 18b was obtained as a white solid (yield 95%). 1H NMR (400 MHz, CDCl3) δ 7.31 (s,
1H), 4.51 (s, 2H), 2.22 (s, 1H), 1.20-1.05 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 173.5, 166.3, 162.1,
116.1, 44.9, 18.3, 11.8. MS (ESI/APCI) m/z 202.9 [M+H]+.
4.1.9.3. 4-Chloro-6-(chloromethyl)-2-(methylthio)pyrimidine (18c)
Compound 18c was obtained as a white solid (yield 80%). 1H NMR (400 MHz, CDCl3) δ 7.22 (s,
1H), 4.51 (s, 2H), 2.57 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 173.7, 166.6, 162.1, 114.4, 44.6, 14.4.
MS (ESI/APCI) m/z 208.8 [M+H]+.
4.1.10. General procedure for the synthesis of 19a-c
A mixture of N-methylquinolin-8-amine (3 mmol, 1.0 eq.), 18a/b/c (1.2 eq.), KI (0.1 eq.) and K2CO3
(2.0 eq.) in CH3CN (10 mL) was stirred at reflux overnight. The reaction solution was evaporated to
remove most of CH3CN. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/ammonium hydroxide = 100/1/1) to give the desired product.
4.1.10.1. N-((6-Chloro-2-ethylpyrimidin-4-yl)methyl)-N-methylquinolin-8-amine (19a)
Compound 19a was obtained as a yellow oil (yield 67%). 1H NMR (400 MHz, CDCl3) δ 8.74 (d, J
= 2.8 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.46-7.34 (m, 3H), 7.11 (d, J = 7.2 Hz, 1H), 4.86
(s, 2H), 3.05 (s, 3H), 2.95 (q, J = 7.6 Hz, 2H), 1.36 (q, J = 7.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ
172.7, 171.9, 161.4, 148.3, 147.7, 142.3, 136.6, 129.9, 126.8, 121.1, 120.6, 117.2, 115.4, 61.7, 41.0, 32.6,
12.8. MS (ESI/APCI) m/z 312.8 [M+H]+.
4.1.10.2. N-((6-Chloro-2-cyclopropylpyrimidin-4-yl)methyl)-N-methylquinolin-8-amine (19b)
Compound 19b was obtained as a yellow oil (yield 33%). 1H NMR (400 MHz, CDCl3) δ 8.76 (d, J
= 2.4 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.50-7.31 (m, 4H), 7.09 (d, J = 7.2 Hz, 1H), 4.85 (s, 2H), 3.04
(s, 3H), 2.25-2.13 (m, 1H), 1.16-1.09 (m, 2H), 1.08-1.01 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 172.7,
171.2, 161.1, 148.2, 147.6, 142.2, 136.5, 129.8, 126.7, 121.0, 120.5, 116.4, 115.5, 61.4, 40.9, 18.2, 11.2.
MS (ESI/APCI) m/z 324.8 [M+H]+.
4.1.10.3. N-((6-Chloro-2-(methylthio)pyrimidin-4-yl)methyl)-N-methylquinolin-8-amine (19c)
1
Compound 19c was obtained as a yellow oil (yield 25%). H NMR (400 MHz, CDCl3) δ 8.76 (s,
1H), 8.11 (d, J = 8.4 Hz, 1H), 7.48-7.32 (m, 4H), 7.10 (d, J = 7.2 Hz, 1H), 4.85 (s, 2H), 3.04 (s, 3H),
2.51 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 172.7, 171.8, 161.2, 148.1, 147.6, 142.2, 136.5, 129.8, 126.7,
121.1, 120.5, 115.4, 114.9, 61.4, 40.9, 14.3. MS (ESI/APCI) m/z 330.8 [M+H]+.
4.1.11. General procedure for the synthesis of 20-22
A mixture of 19a/b/c (2 mmol, 1.0 eq.), TEA (2.0 eq.) and N-methylpiperazine (1.2 eq.) in ethanol
(10 mL) was stirred at reflux overnight. The reaction solution was evaporated. The residue was purified
by silica gel column chromatography (dichloromethane/methanol/ammonium hydroxide = 100/2/1) to
give the desired product.
4.1.11.1. N-((2-Ethyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methylquinolin-8-amine (20)
Compound 20 was obtained as a yellow oil (yield 13%). 1H NMR (400 MHz, CDCl3) δ 8.82 (d, J =
2.4 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.46-7.29 (m, 3H), 7.12 (d, J = 6.8 Hz, 1H), 6.65 (s, 1H), 4.71 (s,
2H), 3.57 (s, 4H), 3.06 (s, 3H), 2.75 (q, J = 7.6 Hz, 2H), 2.41-2.35 (m, 4H), 2.29 (s, 3H), 1.30 (t, J = 7.6
Hz, 3H).13C NMR (150 MHz, CDCl3) δ 170.8, 167.0, 162.8, 148.9, 147.7, 142.6, 136.5, 129.8, 126.8,
121.0, 120.6, 116.2, 98.0, 61.8, 54.8, 46.3, 43.8, 41.5, 32.7, 12.8. HRMS (ESI): calcd for C22H29N6
[M+H]+ 377.2448, found 377.2443. Purity: 99.5%.
4.1.11.2.
N-((2-Cyclopropyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methylquinolin-8-
amine (21)
17

Compound 21 was obtained as a yellow oil (yield 21%). 1H NMR (400 MHz, CDCl3) δ8.78 (d, J =
4.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.44-7.30 (m, 3H), 7.09 (d, J = 7.2 Hz, 1H), 6.57 (s, 1H), 4.70 (s,
2H), 3.60-3.40 (m, 4H), 3.03 (s, 3H), 2.38-2.30 (s, 4H), 2.26 (s, 3H), 2.09-1.96 (m, 1H), 1.06-0.97 (m,
2H), 0.92-0.82 (m, 2H).13C NMR (150 MHz, CDCl3) δ 170.2, 166.3, 162.4, 148.8, 147.5, 142.5, 136.4,
129.7, 126.7, 120.9, 120.4, 116.1, 97.8, 61.4, 54.5, 46.1, 43.6, 41.4, 17.8, 9.7. HRMS (ESI): calcd for
C23H29N6 [M+H]+ 389.2448, found 389.2448. Purity: 95.6%.
4.1.11.3.
N-Methyl-N-((6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)methyl)quinolin-8-
amine (22)
Compound 22 was obtained as a yellow oil (yield 13%). 1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H),
8.10 (d, J = 8.0 Hz, 1H), 7.47-7.30 (m, 3H), 7.10 (d, J = 6.8 Hz, 1H), 6.46 (s, 1H), 4.70 (s, 2H), 3.56 (s,
4H), 3.05 (s, 3H), 2.54 (s, 3H), 2.42-2.35 (m, 4H), 2.29 (s, 3H).13C NMR (150 MHz, CDCl3) δ 170.5,
167.4, 162.1, 148.8, 147.7, 142.6, 136.5, 129.8, 126.8, 121.0, 120.5, 116.3, 96.6, 61.5, 54.7, 46.2, 43.8,
41.4, 14.2. HRMS (ESI): calcd for C21H27N6S [M+H]+ 395.2012, found 395.1998. Purity: 99.0%.
4.1.12. General procedure for the synthesis of 24 and 28
To a mixture of methyl 4,4-dimethoxy-3-oxobutanoate 23 (2 mmol, 1.0 eq.), corresponding amidine
(for 24: 2-methyl-2-thiopseudourea sulfate; for 28: 2-hydroxyacetimidamide hydrochloride) (1.2 eq.) in
10 mL of H2O was added K2CO3 (3 eq.). The mixture was stirred at room temperature overnight and
adjusted pH to acid by AcOH. The solution was extracted with dichloromethane (30 mL * 3). The
combined organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel
column chromatography (dichloromethane/methanol = 50/1) to give the desired product.
4.1.12.1. 6-(Dimethoxymethyl)-2-(methylthio)pyrimidin-4-ol (24)
Compound 24 was obtained as a white solid (yield 92%). 1H NMR (400 MHz, CDCl3) δ 12.79 (s,
1H), 6.46 (s, 1H), 5.06 (s, 1H), 3.39 (s, 6H), 2.60 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 165.6, 163.1,
162.4, 108.0, 102.1, 53.7, 13.4. MS (ESI/APCI) m/z 216.9 [M+H]+.
4.1.12.2. 6-(Dimethoxymethyl)-2-(hydroxymethyl)pyrimidin-4-ol (28)
Compound 28 was obtained as a yellow solid (yield 31%). 1H NMR (400 MHz, DMSO-d6) δ 12.17
(s, 1H), 6.21 (s, 1H), 5.63 (t, J = 5.6 Hz, 1H), 5.02 (s, 2H), 3.27 (s, 6H). 13C NMR (150 MHz, CDCl3) δ
162.3, 162.2, 161.6, 110.1, 101.7, 61.3, 53.3. MS (ESI/APCI) m/z 200.9 [M+H]+.
4.1.13. General procedure for the synthesis of 25 and 29
To a solution of 24 or 28 (16 mmol, 1.0 eq.), Et3N (6.0 eq.) and N-methyl piperazine (1.5 eq.) in 10
mL of MeCN was added PyBOP (1.1 eq.). The mixture was stirred at reflux overnight. The reaction
mixture was quenched with saturated NaHCO3 aqueous solution (200 mL) and extracted with
dichloromethane (200 mL). The organic layer was dried over Na2SO4 and concentrated. The residue was
purified by silica gel column chromatography (dichloromethane/methanol = 100/3) to give the product.
4.1.13.1. 4-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidine (25)
Compound 25 was obtained as a yellow oil (yield 59%). 1H NMR (400 MHz, CDCl3) δ 6.44 (s, 1H),
5.06 (s, 1H), 3.69 (s, 4H), 3.40 (s, 6H), 2.50 (s, 3H), 2.46 (t, J = 5.2 Hz, 4H), 2.34 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 171.3, 164.1, 162.0, 103.4, 95.0, 54.7, 54.1, 46.2, 43.8, 14.2. MS (ESI/APCI) m/z 298.8
[M+H]+.
4.1.13.2. (4-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)pyrimidin-2-yl)methanol (29)
Compound 29 was obtained as a yellow oil (yield 93%). 1H NMR (400 MHz, CDCl3) δ 6.65 (s, 1H),
5.15 (s, 1H), 4.61 (s, 2H), 3.90-3.61 (m, 4H), 3.40 (s, 6H), 2.51-2.45 (s, 4H), 2.34 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 167.6, 164.0, 162.3, 103.2, 98.0, 64.4, 54.7, 54.0, 46.2, 43.9. MS (ESI/APCI) m/z 282.9
[M+H]+.
18

4.1.14. 4-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)-2-(methylsulfonyl)pyrimidine (26)
To a solution of 25 (2.8 g, 9.4 mmol) in THF (90 mL) and H2O (4.5 mL) was added Oxone (6.7 g,
11 mmol) at room temperature. The mixture was stirred at room temperature for 4 h. The reaction mixture
was quenched with saturated NaHCO3 aqueous solution (200 mL) and extracted with ethyl acetate (200
mL). The organic layer was dried over Na2SO4 and concentrated to give the desired product (2.4 g, crude)
as a yellow oil, which was used directly in the next step without further purification. 1H NMR (400 MHz,
CDCl3) δ 6.84 (s, 1H), 5.16 (s, 1H), 4.00-3.60 (m, 4H), 3.41 (s, 6H), 3.28 (s, 3H), 2.52-2.44 (m, 4H),
2.33 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 165.5, 165.1, 162.7, 102.9, 101.6, 54.5, 54.4, 46.1, 44.3,
38.9. MS (ESI/APCI) m/z 330.8 [M+H]+.
4.1.15. 4-(Dimethoxymethyl)-2-methoxy-6-(4-methylpiperazin-1-yl)pyrimidine (27a)
To a solution of 26 (202 mg, 0.61 mmol) in 5 mL of MeOH was added MeONa (5.0 eq.). The
mixture was stirred at reflux overnight. The reaction mixture was quenched with saturated NaHCO3
aqueous solution and extracted with dichloromethane. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by silica gel column chromatography (dichloromethane/methanol
= 100/1 to 100/3) to give the desired product as a yellow oil (151 mg, yield 88%). 1H NMR (400 MHz,
CDCl3) δ6.43 (s, 1H), 5.05 (s, 1H), 3.92 (s, 3H), 3.70 (s, 4H), 3.41 (s, 6H), 2.49-2.44 (m, 4H), 2.33 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 166.2, 165.6, 164.4, 103.6, 93.6, 54.7, 54.4, 54.2, 46.2, 44.1. MS
(ESI/APCI) m/z 282.9 [M+H]+.
4.1.16. 4-(Dimethoxymethyl)-N,N-dimethyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine (27b)
To a solution of 26 (340 mg, 1 mmol) in THF (5 mL) was added dimethylamine (5 mL) and stirred
at reflux overnight in a sealed tube. Then the reaction solution was cooled to room temperature, added
saturated NaHCO3 aqueous solution (100 mL) and extracted with dichloromethane (100 mL). The
organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel
column chromatography (dichloromethane/methanol/ammonium hydroxide = 100/1/1 to 100/3/1) to give
the desired product (260 mg, 88%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 6.07 (s, 1H), 4.99 (s,
1H), 3.63 (s, 4H), 3.40 (s, 6H), 3.13 (s, 6H), 2.44 (s, 4H), 2.32 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
164.8, 163.3, 162.4, 104.3, 88.4, 54.8, 54.0, 46.2, 43.8, 37.0. MS (ESI/APCI) m/z 295.9 [M+H]+.
4.1.17. General procedure for the synthesis of 30a-c
A mixture of 27a/27b/29 (1.3 mmol, 1.0 eq.) in 5 mL of 20% H2SO4 was stirred at reflux overnight.
The reaction mixture was adjusted pH to 9 by saturated NaHCO3 aqueous solution and extracted with
dichloromethane (30 mL). The organic layer was dried over Na2SO4 and concentrated to give the desired
product.
4.1.17.1. 2-Methoxy-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (30a)
1
Compound 30a was obtained as a yellow oil (yield 59%). H NMR (400 MHz, CDCl3) δ 9.77 (s,
1H), 6.71 (s, 1H), 3.95 (s, 3H), 3.71 (s, 4H), 2.45 (s, 4H), 2.31 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
193.7, 166.4, 164.3, 159.5, 94.2, 54.7, 54.6, 46.1, 44.2. MS (ESI/APCI) m/z 268.9 [M+MeOH+H]+.
4.1.17.2. 2-(Dimethylamino)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (30b)
Compound 30b was obtained as a yellow solid (yield 65%). 1H NMR (400 MHz, CDCl3) δ 9.76 (s,
1H), 6.39 (s, 1H), 3.68 (s, 4H), 3.18 (s, 6H), 2.51-2.41 (m, 4H), 2.33 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 195.3, 163.3, 163.0, 159.1, 89.1, 54.8, 46.2, 43.9, 37.0. MS (ESI/APCI) m/z 250.0 [M+H]+.
4.1.17.3. 2-(Hydroxymethyl)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (30c)
1
Compound 30c was obtained as a yellow oil (yield 67%). H NMR (400 MHz, CDCl3) δ 9.90 (s,
1H), 6.95 (s, 1H), 4.68 (s, 2H), 3.88-3.59 (m, 5H), 2.59-2.45 (m, 4H), 2.35 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 193.6, 169.1, 162.4, 157.2, 98.3, 64.4, 54.6, 46.2, 44.2. MS (ESI/APCI) m/z 269.0
19

[M+MeOH+H]+.
4.1.18. General procedure for the synthesis of 31a-c
A mixture of 30a/b/c (0.66 mmol, 1.0 eq.), quinolin-8-amine (1.5 eq.), and AcOH (1 drop) in
methanol (5 mL) was stirred for 30 min. NaBH3CN (2.0 eq.) was then added to the reaction solution. The
resulting suspension was stirred at room temperature for 6h. The reaction solution was evaporated. The
residue was purified by silica gel column chromatography (dichloromethane/methanol/ammonium
hydroxide = 200/2/1) to give the desired product.
4.1.18.1. N-((2-Methoxy-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine (31a)
Compound 31a was obtained as a yellow oil (yield 65 %). 1H NMR (400 MHz, CDCl3) δ 8.74 (dd,
J = 8.4 Hz, 1.6 Hz, 1H), 8.06 (dd, J = 8.4, 1.6 Hz, 1H), 7.40 (dd, J = 8.4, 4.0 Hz, 1H), 7.34-7.29 (m, 1H),
7.07 (dd, J = 8.4, 1.2 Hz, 1H), 6.84-6.75 (m, 1H), 6.58 (dd, J = 7.6, 1.2 Hz, 1H), 6.31 (s, 1H), 4.43 (d, J
= 5.6 Hz, 2H), 3.95 (s, 3H), 3.67-3.46 (m, 4H), 2.36 (t, J = 5.2 Hz, 4H), 2.26 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 168.9, 165.5, 164.4, 147.1, 144.5, 138.4, 136.1, 128.6, 127.8, 121.5, 114.7, 105.7, 93.1,
54.6, 54.3, 49.3, 46.1, 44.0. MS (ESI/APCI) m/z 364.9 [M+H]+.
4.1.18.2.
N-((2-(Dimethylamino)-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine
(31b)
Compound 31b was obtained as a yellow oil (yield 42 %). 1H NMR (400 MHz, CDCl3) δ 8.75 (d, J
= 3.2 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.41-7.31 (m, 2H), 7.06 (d, J = 8.4 Hz, 1H), 6.96-6.89 (m, 1H),
6.63 (d, J = 7.6 Hz, 1H), 5.98 (s, 1H), 4.36 (d, J = 5.2 Hz, 2H), 3.56 (s, 4H), 3.19 (s, 6H), 2.40 (s, 4H),
2.29 (s, 3H).13C NMR (150 MHz, CDCl3) δ 166.9, 163.4, 162.3, 147.1, 145.0, 138.5, 136.1, 128.7, 127.9,
121.5, 114.2, 105.7, 88.4, 54.9, 49.4, 46.3, 43.9, 37.0. MS (ESI/APCI) m/z 377.9 [M+H]+.
4.1.18.3. (4-(4-Methylpiperazin-1-yl)-6-((quinolin-8-ylamino)methyl)pyrimidin-2-yl)methanol (31c)
Compound 31c was obtained as a yellow oil (yield 90 %). 1H NMR (400 MHz, CDCl3) δ 8.76 (d, J
= 3.2 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.44-7.37 (m, 1H), 7.36-7.30 (m, 1H), 7.10 (d, J = 8.0 Hz, 1H),
6.83-6.75 (m, 1H), 6.55 (d, J = 7.6 Hz, 1H), 6.52 (s, 1H), 4.62 (s, 2H), 4.50 (d, J = 6.0 Hz, 2H), 3.59 (s,
4H), 2.44-2.35 (m, 4H), 2.28 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 167.4, 166.7, 162.4, 147.3, 144.4,
138.4, 136.2, 128.7, 127.8, 121.6, 115.0, 105.8, 97.3, 64.3, 54.6, 49.3, 46.2, 43.9. MS (ESI/APCI) m/z
364.9 [M+H]+.
4.1.19. General procedure for the synthesis of 32-34
A mixture of 31a/b/c (0.28 mmol, 1.0 eq.) and formaldehyde (30 wt percent in water, 5.0 eq.) in
methanol (5 mL) was stirred for 2h. Then NaBH3CN (2.0 eq.) was added. The resulting suspension was
stirred at room temperature overnight. The reaction solution was evaporated. The residue was purified
by silica gel column chromatography (dichloromethane/methanol/ammonium hydroxide = 200/1/1) to
give the desired product.
4.1.19.1. N-((2-Methoxy-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methylquinolin-8-amine
(32)
Compound 32 was obtained as a yellow oil (yield 13%). 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J =
2.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.41-7.28 (m, 3H), 7.09 (d, J =7.2 Hz, 1H), 6.37 (s, 1H), 4.67 (s,
2H), 3.83 (s, 3H), 3.52 (s, 4H), 3.04 (s, 3H), 2.39-2.31 (m, 4H), 2.25 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 169.1, 165.1, 164.1, 148.7, 147.5, 142.4, 136.4, 129.7, 126.6, 120.8, 120.4, 116.2, 94.8, 61.4, 54.5,
54.0, 46.1, 43.9, 41.4. HRMS (ESI): calcd for C21H27N6O [M+H]+ 379.2241, found 379.2252. Purity:
96.5%.
4.1.19.2. N-((2-(Dimethylamino)-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methylquinolin-
8-amine (33)
20

Compound 33 was obtained as a yellow oil (yield 27%). 1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H),
8.09 (d, J = 8.4 Hz, 1H), 7.45-7.28 (m, 3H), 7.12 (d, J = 7.2 Hz, 1H), 6.08 (s, 1H), 4.69 (s, 2H), 3.58 (s,
4H), 3.15-2.95 (m, 9H), 2.45 (s, 4H), 2.33 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 165.7, 162.8, 160.7,
148.9, 147.5, 142.4, 136.7,129.8, 126.8, 120.9, 120.4, 116.6, 90.8, 61.0, 54.5, 45.9, 43.7, 41.2, 37.1.
HRMS (ESI): calcd for C22H30N7 [M+H]+ 392.2557, found 392.2557. Purity: 98.7%.
4.1.19.3. (4-((Methyl(quinolin-8-yl)amino)methyl)-6-(4-methylpiperazin-1-yl)pyrimidin-2-yl)methanol
(34)
Compound 34 was obtained as a yellow oil (yield 35%). 1H NMR (400 MHz, CDCl3) δ 8.77 (d, J =
2.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.43-7.28 (m, 3H), 7.08 (d, J = 7.2 Hz, 1H), 6.67 (s, 1H), 4.72 (s,
2H), 4.53 (s, 2H), 3.82 (br s, 1H), 3.55 (m, 4H), 3.01 (s, 3H), 2.40-2.30 (m, 4H), 2.25 (s, 3H). 13C NMR
(150 MHz, CDCl3) δ 166.9, 166.7, 162.1, 148.6, 147.5, 142.3, 136.4, 129.7, 126.6, 120.9, 120.5, 116.0,
98.9, 64.1, 61.4, 54.4, 46.0, 43.7, 41.1. HRMS (ESI): calcd for C21H27N6O [M+H]+ 379.2241, found
379.2249. Purity: 97.5%.
4.1.20. N-((6-Chloro-2-methylpyrimidin-4-yl)methyl)quinolin-8-amine (35)
A mixture of 9 (273 mg, 1.5 mmol), quinolin-8-amine (200 mg, 1.4 mmol), KI (23 mg, 0.14 mmol)
and K2CO3 (386 mg, 2.8 mmol) in CH3CN (5 mL) was stirred at reflux overnight. The reaction solution
was evaporated to remove most of CH3CN, diluted with saturated NaHCO3 (50 mL) and extracted with
dichloromethane (20 mL x 3). The combined organic layer was dried over Na2SO4, filtered and
evaporated.
The
residue
was
purified
by
silica
gel
column
chromatography
(dichloromethane/methanol/ammonium hydroxide = 100/2/1) to give the desired product (230 mg, 58%)
as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.84-8.74 (m, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.43 (dd, J =
8.4, 4.0 Hz, 1H), 7.37-7.30 (m, 1H), 7.28 (s, 1H), 7.14 (d, J = 8.4 Hz, 1H), 6.96-6.83 (m, 1H), 6.47 (d, J
= 7.6 Hz, 1H), 4.65(d, J = 6.4 Hz, 1H), 2.76 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 170.8, 169.1, 162.0,
147.5, 143.7, 138.3,136.3, 128.8, 127.7, 121.8, 115.5, 115.4, 105.4, 48.8, 25.9. MS (ESI/APCI) m/z 284.8
[M+H]+.
4.1.21. N-((2-Methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine (36)
A mixture of 35 (100 mg, 0.35 mmol), TEA (354 mg, 3.5 mmol) and N-methylpiperazine (42 mg,
0.42 mmol) in ethanol (4 mL) was stirred at reflux overnight. The reaction mixture was concentrated and
added saturated NaHCO3 aqueous solution (20 mL), then extracted with dichloromethane (10 mL x 3).
The combined organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified
by silica gel column chromatography (dichloromethane/methanol/ammonium hydroxide = 100/1/1) to
give the desired product (75 mg, 56 %) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.77 (d, J = 2.8
Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 8.0, 4.0 Hz, 1H), 7.36-7.31 (m, 1H), 7.10 (d, J = 8.0 Hz,
1H), 6.80-6.74 (m, 1H), 6.57 (d, J = 7.6 Hz, 1H), 6.48 (s, 1H), 4.49 (d, J = 6.0 Hz, 2H), 3.58 (s, 4H),
2.55 (s, 3H), 2.42-2.35 (m, 4H), 2.28 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 167.3, 166.7, 162.9, 147.2,
144.6, 138.4, 136.2, 128.7, 127.9, 121.6, 114.8, 105.8, 96.0, 54.7, 49.5, 46.2, 43.7, 26.2. MS (ESI/APCI)
m/z 348.9 [M+H] +.
4.1.22. N-Ethyl-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine (37)
A mixture of 36 (80 mg, 0.23 mmol) and acetaldehyde (37 wt percent in water, 137 mg, 1.2 mmol)
in methanol (10 mL) was added NaBH3CN (36 mg, 0.58 mmol). The resulting suspension was stirred at
room temperature overnight. Water (10 mL) was added to quench the reaction. The water phase was
extracted with dichloromethane (10 mL x 3). The combined organic layer was dried over Na2SO4, filtered
and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/ammonium hydroxide = 100/1/1) to give the desired product (20 mg, 22%)
21

as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.85 (d, J = 2.4 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.42-
7.29 (m, 3H), 7.17-7.10 (m, 1H), 6.58 (s, 1H), 4.61 (s, 2H), 3.65 (q, J = 7.2 Hz, 2H), 3.45 (s, 4H), 2.49
(s, 3H), 2.34-2.28 (m, 4H), 2.25 (s, 3H), 1.12 (t, J = 6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 167.1,
166.6, 162.6, 147.9, 146.8, 143.4, 136.6,130.0, 126.6, 121.0, 120.9, 118.7, 97.9, 58.3, 54.7, 48.6, 46.2,
43.7, 26.2, 11.9. HRMS (ESI): calcd for C22H29N6 [M+H]+ 377.2448, found 377.2451. Purity: 95.9%.
4.1.23. N-Cyclopropylquinolin-8-amine (39)
The mixture of 38 (104 mg, 0.5 mmol), cyclopropanamine (300 mg, 2.5 mmol), Cs2CO3 (326 mg,
1 mmol), Pd2(dba)3 (22 mg, 0.025 mol) and BINAP (31 mg, 0.05 mmol) in toluene (10 mL) was stirred
at 100°C overnight under N2 atmosphere. The reaction mixture was cooled to room temperature and
filtered. The filtrate was concentrated. The residue was purified by silica gel column chromatography
(petroleum ether/ethyl acetate = 50/1) to give the desired product (50 mg, 54%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) δ 8.70 (dd, J = 4.0, 1.6 Hz,1H), 8.07 (dd, J = 8.4, 1.6 Hz,1H), 7.48-7.40 (m, 1H),
7.36 (dd, J = 8.0, 4.0 Hz,1H), 7.11 (s, 1H), 7.09 (s, 1H), 6.42 (s, 1H), 2.62-2.54 (m, 1H), 0.87-0.80 (m,
2H), 0.70-0.64 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 147.0, 145.4, 136.5, 136.2, 128.7, 127.9, 121.4,
114.6, 106.5, 24.8, 7.3. MS (ESI/APCI) m/z 184.9 [M+H] +.
4.1.24. N-((6-Chloro-2-methylpyrimidin-4-yl)methyl)-N-cyclopropylquinolin-8-amine (40)
A mixture of 39 (50 mg, 0.27 mmol), 9 (62 mg, 0.81 mmol), KI (5 mg, 0.03 mmol) and K2CO3 (75
mg, 0.54 mmol) in CH3CN (10 mL) was stirred at reflux overnight. The reaction solution was evaporated
to remove most of CH3CN, diluted with saturated NaHCO3 (50 mL) and extracted with dichloromethane
(20 mL x 3). The combined organic layer was dried over Na2SO4, filtered and evaporated. The residue
was purified by silica gel column chromatography (dichloromethane/methanol = 40/1) to give the desired
product (70 mg, 79%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 2.4 Hz, 1H), 8.10 (d, J
= 8.0 Hz, 1H), 7.54-7.45 (m, 2H), 7.44-7.38 (m, 1H), 7.37-7.31 (m, 2H), 5.19 (s, 2H), 2.83-2.75 (m, 1H),
2.67 (s, 3H), 0.85-0.78 (m, 2H), 0.68-0.55 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 172.8, 168.3, 160.6,
147.7, 147.4, 141.8, 136.8, 129.7, 126.8, 121.0, 120.7, 117.5, 117.4, 60.8, 34.6, 25.9, 9.3. MS (ESI/APCI)
m/z 324.8 [M+H] +.
4.1.25. N-Cyclopropyl-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine
(41)
A mixture of 40 (70 mg, 0.25 mmol), TEA (250 mg, 2.5 mmol) and N-methylpiperazine (75 mg,
0.75 mmol) in ethanol (4 mL) was stirred at reflux overnight. The reaction mixture was concentrated and
added saturated NaHCO3 aqueous solution (20 mL), then extracted with dichloromethane (10 mL x 3).
The combined organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified
by silica gel column chromatography (dichloromethane/methanol/ammonium hydroxide = 100/1/1) to
give the desired product (40 mg, 41%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 2.4
Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.50-7.40 (m, 2H), 7.39-7.29 (m, 2H), 6.21 (s, 1H), 5.14 (s, 2H), 3.50-
3.35 (m, 4H), 2.84-3.75 (m, 1H), 2.45 (s, 3H), 2.38-2.31 (m, 4H), 2.27 (s, 3H), 0.84-0.74 (m, 2H), 0.70-
0.61 (m, 2H).13C NMR (150 MHz, CDCl3) δ 167.8, 166.7, 162.3, 148.3, 147.4, 142.1, 136.5,129.7, 126.5,
120.8, 120.3, 117.9, 98.2, 60.3, 54.7, 46.2, 43.7, 34.1, 26.2, 9.5. HRMS (ESI): calcd for C23H29N6 [M+H]+
389.2448, found 389.2448. Purity: 99.4%.
4.1.26. 6-(Dimethoxymethyl)-2,5-dimethylpyrimidin-4-ol (43)
To a mixture of methyl 4,4-dimethoxy-2-methyl-3-oxobutanoate 42 (670 mg, 3.5 mmol),
acetamidine hydrochloride (994 mg, 10.6 mmol) in 10 mL of H2O was added K2CO3 (966 mg, 7 mmol).
The mixture was stirred at room temperature overnight and adjusted pH to acid by AcOH. The solution
was extracted with dichloromethane (30 mL * 3). The combined organic layer was dried over Na2SO4
22

and concentrated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol = 100/1) to give the desired product (290 mg, 42%) as a brown oil. 1H NMR
(400 MHz, CDCl3) δ 13.06 (s, 1H), 5.27 (s, 1H), 3.43 (s, 6H), 2.48 (s, 3H), 2.13 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 166.2, 157.8, 155.8, 119.8, 103.1, 54.8, 21.7, 9.7. MS (ESI/APCI) m/z 198.9 [M+H]+.
4.1.27. 4-(Dimethoxymethyl)-2,5-dimethyl-6-(4-methylpiperazin-1-yl)pyrimidine (44)
To a solution of 43 (290 mg, 1.5 mmol), Et3N (1.5 g, 14.6 mmol) and N-methyl piperazine (293 mg,
2.9 mmol) in 10 mL of MeCN was added PyBOP (838 mg, 1.6 mmol). The mixture was stirred at reflux
overnight. The reaction mixture was quenched with saturated NaHCO3 aqueous solution (200 mL) and
extracted with dichloromethane (200 mL). The organic layer was dried over Na2SO4 and concentrated.
The residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/3) to
give the product (240 mg, 59%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 5.27 (s, 1H), 3.43 (s, 6H),
3.38-3.32 (m, 4H), 2.57-2.47 (m, 7H), 2.34 (s, 3H), 2.22 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 167.1,
163.9, 161.7, 113.8, 105.4, 55.1, 54.9, 48.5, 46.3, 25.9, 13.6. MS (ESI/APCI) m/z 280.9 [M+H]+.
4.1.28. 2,5-Dimethyl-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45)
A mixture of 44 (100 mg, 0.36 mmol) in 5 mL of 20% H2SO4 was stirred at reflux overnight. The
reaction mixture was adjusted pH to 9 by saturated NaHCO3 aqueous solution and extracted with
dichloromethane (30 mL). The organic layer was dried over Na2SO4 and concentrated to give the desired
product (50 mg, 60%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 10.05 (s, 1H), 3.49-3.40 (m, 4H),
2.62 (s, 3H), 2.56-2.52 (m, 4H), 2.39 (s, 3H), 2.35 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 195.8, 167.7,
164.9, 155.1, 116.2, 55.0, 48.5, 46.2, 25.6, 14.6. MS (ESI/APCI) m/z 266.9 [M+MeOH+H]+.
4.1.29. N-((2,5-Dimethyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)quinolin-8-amine (46)
A mixture of 45 (150 mg, 0.63 mmol), quinolin-8-amine (144 mg, 1.0 mmol), and AcOH (1 drop)
in methanol (5 mL) was stirred for 30 min. NaBH3CN (144 mg, 2.0 mmol) was then added to the reaction
solution. The resulting suspension was stirred at room temperature for 6h. The reaction solution was
evaporated.
The
residue
was
purified
by
silica
gel
column
chromatography
(dichloromethane/methanol/ammonium hydroxide = 200/2/1) to give the desired product (52 mg, 22%)
as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 2.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.44-
7.36 (m, 2H), 7.09 (d, J =8.0 Hz, 1H), 6.75 (d, J =7.2 Hz, 1H), 4.45 (s, 2H), 3.63 (s, 4H), 2.93 (s, 4H),
2.65 (s, 3H), 2.61 (s, 3H), 2.23 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 166.1, 164.1, 163.1, 147.3, 144.7,
138.7, 136.0, 128.8, 127.8, 121.5, 114.3, 112.8, 105.1, 55.2, 48.6, 46.6, 46.3, 26.0, 13.8. MS (ESI/APCI)
m/z 362.9 [M+H]+.
4.1.30. N-((2,5-Dimethyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methylquinolin-8-amine
(47)
A mixture of 46 (36 mg, 0.10 mmol) and formaldehyde (30 wt percent in water, 50 mg, 0.5 mmol)
in methanol (5 mL) was stirred for 0.5 h. Then NaBH3CN (62 mg, 1 mmol) was added. The resulting
suspension was stirred at room temperature overnight. The reaction solution was evaporated. The residue
was purified by silica gel column chromatography (dichloromethane/methanol/ammonium hydroxide =
200/1/1) to give the desired product (16 mg, 42%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.88
(d, J = 2.4 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.45-7.32 (m, 3H), 7.11 (d, J = 4.4 Hz, 1H), 4.85 (s, 2H),
3.24 (s, 4H), 3.05 (s, 3H), 2.53 (s, 3H), 2.47 (s, 4H), 2.31 (s, 3H), 1.86 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 166.5, 164.3, 163.4, 149.4, 147.7, 143.0, 136.6, 129.7, 126.7, 120.9, 120.8, 117.5, 114.6, 59.5,
55.1, 48.5, 46.3, 41.6, 25.8, 14.2. HRMS (ESI): calcd for C22H29N6 [M+H]+ 377.2448, found 377.2445.
Purity: 99.6%.
4.1.31. Ethyl 2,4-difluoro-3-oxobutanoate (49)
23

To a suspension of NaH (60% dispersion in mineral oil, 936 mg, 24 mmol) in 50 mL of ether was
added 48 (5.0 g, 47 mmol) at room temperature. The mixture was stirred at reflux for 4 h. The reaction
mixture was poured into H2SO4 (2 M, 15 mL) and extracted with ether (50 mL x 3). The organic layers
were dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography
(petroleum ether/ethyl acetate = 10/1) to give the desired product (2 g, 25%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) δ 5.45 (d, J = 48.0 Hz, 0.86H), 5.19 (d, J = 46.8 Hz, 1.68H), 4.89 (d, J = 47.2 Hz,
0.42H), 4.31 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 195.4 (dd, J =
22.4, 17.4 Hz), 163.3 (d, J = 23.3 Hz), 89.7 (d, J = 194.7 Hz), 83.3 (dd, J = 183.3, 3.3 Hz), 63.3, 13.9.
MS (ESI/APCI) m/z 164.9 [M-H]-.
4.1.32. 5-Fluoro-6-(fluoromethyl)-2-methylpyrimidin-4-ol (50)
A mixture of 49 (1.9 g, 11 mmol), acetamidine hydrochloride (2.2 g, 22 mmol) and EtONa (2.3 g,
34 mmol) in 40 mL of EtOH was stirred at reflux overnight. Then 6 N HCl (2 mL) was added and the
reaction mixture was concentrated. The residue was purified by silica gel column chromatography
(petroleum ether/ethyl acetate = 3/1 to 1/1) to give the desired product (800 mg, 43%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 13.22 (s, 1H), 5.35 (d, J = 46.8 Hz, 2H), 2.52 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 158.4 (d, J = 24.2 Hz), 154.6 (d, J = 6.9 Hz), 146.4 (dd, J = 256.8, 3.0 Hz), 144.5 (dd, J
= 15.8, 10.4 Hz), 78.0 (d, J = 171.2 Hz), 21.5. MS (ESI/APCI) m/z 158.9 [M-H]-.
4.1.33. 5-Fluoro-4-(fluoromethyl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine (51)
To a solution of 50 (800 mg, 5.0 mmol), Et3N (1.5 g, 15 mmol) and N-methyl piperazine (750 mg,
7.5 mmol) in 40 mL of MeCN was added PyBOP (2.9 g, 5.5 mmol). The mixture was stirred at reflux
overnight. The resulting solution was evaporated to remove MeCN. The residue was diluted with
dichloromethane (100 mL) and washed with saturated NaCl aqueous solution (50 mL x 3). The organic
layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column
chromatography (ethyl acetate) to give the product (1 g, 83%) as a yellow oil. 1H NMR (400 MHz, CDCl3)
δ 5.30 (dd, J = 47.2 Hz, 2.4 Hz, 2H), 3.79-3.73 (m, 4H), 2.46-2.39 (m, 4H), 2.26 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 162.3 (d, J = 8.6 Hz), 151.4 (d, J = 4.1 Hz), 147.3 (t, J = 15.3 Hz), 143.7 (dd, J = 258.8,
3.8 Hz), 79.1 (d, J = 168.9 Hz), 54.9, 46.0 (d, J = 12.8 Hz), 45.9, 25.3. MS (ESI/APCI) m/z 242.9 [M+H]+.
4.1.34.
N-((5-Fluoro-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-
methylquinolin-8-amine (52)
A mixture of 51 (48mg, 0.2 mmol), N-methylquinolin-8-amine (63 mg, 0.4 mmol), KI (6 mg, 0.036
mmol) and 2-ethyl-2-(hydroxymethyl)propane-1,3-diol (295 mg, 2.2 mmol) in water (1 mL) was stirred
at reflux overnight. The reaction mixture was diluted with saturated NaHCO3 (10 mL) and extracted with
dichloromethane (20 mL x 3). The combined organic layer was dried over Na2SO4, filtered and
evaporated. The residue was purified by silica gel column chromatography (dichloromethane/methanol
= 50/1) to give the product (20 mg, 13%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.88 (d, J = 4.0
Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.44-7.29 (m, 3H), 7.01 (s, 1H), 4.95 (s, 2H), 3.67-3.48 (m, 4H), 3.06
(s, 3H), 2.47-2.32 (m,7H), 2.26 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 161.3 (d, J = 8.6 Hz), 151.4 (d,
J = 5.1 Hz), 150.7 (d, J = 15.6 Hz), 148.8, 147.7, 143.9 (d, J = 255.2 Hz), 142.9, 136.4, 129.5, 126.4,
120.9, 120.8, 117.3, 55.0, 46.2, 46.0, 45.9, 40.9, 25.5 (d, J = 1.8 Hz). HRMS (ESI): calcd for C21H26FN6
[M+H]+ 381.2197, found 381.2182. Purity: 95.3%.
4.1.35. 5-Chloro-6-(chloromethyl)-2-methylpyrimidin-4-ol (54)
To a solution of ethyl 53 (500mg, 2.5 mmol) and acetamidine hydrochloride (262mg, 2.8 mmol) in
water (20 mL) was added K2CO3 (524 mg, 3.8 mmol). The reaction was stirred at room temperature
overnight. The reaction was quenched with 1M HCl aqueous solution to adjust pH to 5 and then extracted
24

with ethyl acetate (15 mL x 4). The combined organic layer was dried over Na2SO4, concentrated and
was purified by silica gel column chromatography (dichloromethane/methanol = 100/1) to give the
desired product (140 mg, 29%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 12.88 (s, 1H), 4.56 (s,
2H), 2.55 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 161.4, 159.1, 157.1, 119.5, 43.0, 21.6. MS (ESI/APCI)
m/z 192.8 [M+H] +.
4.1.36. 4,5-Dichloro-6-(chloromethyl)-2-methylpyrimidine (55)
The solution of 54 (110 mg, 0.57 mmol) in POCl3 (2 mL) was stirred at reflux for 1 h. The reaction
mixture was concentrated, added water (20 mL) and extracted with ethyl acetate (2 mL x 2). The
combined organic layer was concentrated and the residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 10/1) to give the desired product (53 mg, 42%) as a
1
yellow oil. H NMR (400 MHz, CDCl3) δ 4.68 (s, 2H), 2.71 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
166.3, 162.8, 159.5, 126.2, 43.3, 25.3. MS (ESI/APCI) m/z 210.8 [M+H] +.
4.1.37. N-((5,6-Dichloro-2-methylpyrimidin-4-yl)methyl)-N-methylquinolin-8-amine (56)
A mixture of 55 (53 mg, 0.25 mmol), N-methylquinolin-8-amine (40 mg, 0.25 mmol), KI (5 mg,
0.03 mmol) and K2CO3 (70 mg, 0.50 mmol) in CH3CN (10 mL) was stirred at reflux for 2h. The reaction
mixture was filtered and the filtrate was evaporated. The residue was purified by silica gel column
chromatography (dichloromethane/methanol = 100/1) to give the desired product (60 mg, 72%) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.75 (d, J = 2.4 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.46-7.39
(m, 1H), 7.39-7.32 (m, 2H), 7.16 (d, J = 7.2 Hz, 1H), 5.24 (s, 2H), 3.25 (s, 3H), 2.60 (s, 3H). 13C NMR
(150 MHz, CDCl3) δ 167.1, 165.3, 158.0, 148.1, 147.4, 142.1, 136.7, 129.7, 126.7, 125.0, 120.9, 120.4,
116.7, 58.4, 41.6, 25.4. MS (ESI/APCI) m/z 332.7 [M+H] +.
4.1.38. N-((5-Chloro-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methylquinolin-8-
amine (57)
A mixture of 56 (57 mg, 0.17 mmol), TEA (172 mg, 1.7 mmol) and N-methylpiperazine (86 mg,
0.86 mmol) in ethanol (5 mL) was stirred at reflux overnight. The reaction mixture was concentrated.
The residue was purified by silica gel column chromatography (dichloromethane/methanol/ammonium
hydroxide = 100/2/1) to give the desired product (40 mg, 40%) as a colorless oil. 1H NMR (400 MHz,
CDCl3) δ 8.84 (d, J = 2.4 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.42-7.33 (m, 3H), 7.11 (d, J = 6.8 Hz, 1H),
5.10 (s, 2H), 3.53 (s, 4H), 3.16 (s, 3H), 2.52-2.46 (m, 4H), 2.44 (s, 3H), 2.32 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 163.8, 163.7, 161.4, 149.1, 147.5, 142.8, 136.5, 129.6, 126.5, 120.8, 120.4, 117.3, 114.2,
58.0, 54.9, 47.8, 46.1, 41.4, 25.5. HRMS (ESI): calcd for C21H26ClN6 [M+H]+ 397.1902, found 397.1911.
Purity: 96.2%.
4.1.39. 4-Hydroxy-2,6-dimethylpyrimidine-5-carbonitrile (59)
A mixture of 58 (5.0 g, 27 mmol), acetamidine hydrochloride (3.9 g, 41 mmol) and K2CO3 (11.3 g,
82 mmol) in 80 mL of EtOH was stirred at room temperature overnight. The reaction mixture was
acidified to pH 5 by 3 N HCl and then extracted with butyl alcohol (50 mL x 6). The combined organic
layer was dried over Na2SO4 and concentrated to give the desired product (3.5 g, 87%) as a slight yellow
solid. 1H NMR (400 MHz, DMSO-d6) δ 13.30 (s, 1H), 2.39 (s, 3H), 2.35 (s, 3H). MS (ESI/APCI) m/z
150.0 [M+H]+.
4.1.40. tert-Butyl 4-(5-cyano-2,6-dimethylpyrimidin-4-yl)piperazine-1-carboxylate (60)
A mixture of 59 (2.5 g, 16 mmol), N-Boc-piperazine (4.7 g, 25 mmol), PyBOP (9.6 g, 18 mmol)
and Et3N (5.1 g, 50 mmol) in 60 mL of MeCN was stirred at 80°C overnight. The reaction solution was
evaporated to remove most of MeCN. The resulting residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 5/1 to 3/1) to give the desired product (4.3 g, 81%) as
25

a yellow solid. 1H NMR (400 MHz, CDCl3) δ 4.01-3.87 (m, 4H), 3.62-3.48 (m, 4H), 2.57 (s, 3H), 2.51
(s, 3H), 1.48 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 172.8, 168.8, 161.9, 154.7, 117.6, 87.2, 80.6, 46.3,
43.9, 28.5, 26.5, 23.9. MS (ESI/APCI) m/z 317.9 [M+H]+.
4.1.41. tert-Butyl 4-(6-(chloromethyl)-5-cyano-2-methylpyrimidin-4-yl)piperazine-1-carboxylate (61)
To a solution of 60 (4.0 g, 12 mmol) in 100 mL of dichloromethane was added trichloroisocyanuric
acid (2.9 g, 12 mmol) in portions at 0°C. The mixture was stirred at room temperature for 6 h and then
quenched with saturated Na2S2O3 aqueous solution (20 mL). The aqueous layer was extracted with
dichloromethane (50 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated. The
residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10/1 to 5/1)
to give the desired product (2.4 g, 54%) as a slight yellow solid. 1H NMR (400 MHz, CDCl3) δ 4.57 (s,
2H), 3.98 (d, J = 5.2 Hz, 4H), 3.57 (d, J = 5.2 Hz, 4H), 2.55 (s, 3H), 1.48 (s, 9H). 13C NMR (150 MHz,
CDCl3) δ 170.0, 169.8, 161.8, 154.6, 116.2, 86.6, 80.7, 46.4, 44.2, 42.9, 28.5, 26.5. MS (ESI/APCI) m/z
351.8 [M+H]+.
4.1.42.
tert-Butyl
4-(5-cyano-2-methyl-6-((methyl(quinolin-8-yl)amino)methyl)pyrimidin-4-
yl)piperazine-1-carboxylate (62)
A mixture of 61 (85mg, 0.24 mmol), N-methylquinolin-8-amine (38 mg, 0.24 mmol), KI (5 mg,
0.03 mmol) and K2CO3 (70 mg, 0.5 mmol) in 10 mL of MeCN was stirred at reflux for 2h. The reaction
solution was cold to room temperature and filtered. The filtrate was concentrated. The resulting residue
was purified by silica gel column chromatography (dichloromethane/methanol = 100/1) to give the
desired product (20 mg, 24%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.10 (d, J =
7.2 Hz, 1H), 7.51-7.34 (m, 3H), 7.09 (s, 1H), 5.12 (s, 2H), 3.82 (s, 4H), 3.67-3.40 (m, 4H), 3.18 (s, 3H),
2.47 (s, 3H), 1.47 (s, 9H). MS (ESI/APCI) m/z 473.9 [M+H]+.
4.1.43.
carbonitrile (63)
To a solution of 62 (300 mg, 0.63 mmol) in 2 mL of dichloromethane was added CF3CO2H (1 mL).
2-Methyl-4-((methyl(quinolin-8-yl)amino)methyl)-6-(4-methylpiperazin-1-yl)pyrimidine-5-
The mixture was stirred at room temperature for 2 h and then evaporated to give the residue as a red oil.
To a solution of this crude product in 1 mL of methanol was added formaldehyde (30 wt percent in water,
1 mL) and NaBH3CN (65 mg, 1.0 mmol). The mixture was stirred at room temperature overnight and
then quenched with saturated NaHCO3 aqueous solution (10 mL). The aqueous layer was extracted with
dichloromethane (10 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated. The
residue was purified by Al2O3 column chromatography (petroleum ether/ethyl acetate = 5/1 to 1/10) to
give the desired product (80 mg, 33%) as a slight yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.81 (d, J =
2.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.42-7.30 (m, 3H), 7.08 (d, J = 5.6 Hz, 1H), 5.09 (s, 2H), 3.90-
3.82 (m, 4H), 3.16 (s, 3H), 2.48-2.38 (m, 7H), 2.30 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 173.0, 168.5,
162.3, 148.3, 147.6, 142.6, 136.4, 129.7, 126.4, 120.9, 120.7, 116.9, 116.8, 86.3, 59.6, 54.8, 46.5, 46.0,
41.6, 26.6. HRMS (ESI): calcd for C22H26N7 [M+H]+ 388.2244, found 388.2248. Purity: 98.9%.
4.1.44. 4,6-Dichloro-2-methylpyrimidine-5-carbaldehyde (65)
To POCl3 (60.6 g, 397 mmol) was added dropwise DMF (9.8 g, 135 mmol) at 0°C. The resulting
suspension was stirred at the same temperature for 1h. Then 2-methylpyrimidine-4,6-diol 64 (10 g, 79
mmol) was added in portions and stirred at room temperature for 1h, followed by stirring at reflux
overnight. The reaction solution was concentrated and diluted with cold ethyl acetate (100mL). This
solution was added dropwise into ice-water and filtered. The filtrate was extracted with ethyl acetate
(200 mL). The organic layer was dried over Na2SO4, filtered and evaporated to give the desired product
(9 g, 59%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 2.75 (s, 3H). 13C NMR (150
26

MHz, CDCl3) δ 185.7, 171.6, 162.6, 121.9, 26.1. MS (ESI/APCI) m/z 222.8 [M+MeOH+H]+.
4.1.45. (4,6-Dichloro-2-methylpyrimidin-5-yl)methanol (66)
To the solution of 65 (9 g, 47.1 mmol) in THF (50 mL) and water (10 mL) was added NaBH4 (3.6
g, 94.2 mmol) in portions at 0°C. The resulting suspension was stirred at the same temperature for 30min.
Water (50 mL) was added, and extracted with ethyl acetate (50 mL). The organic layer was dried over
Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography
(petroleum ether/ethyl acetate = 10/1) to give the desired product (4.2 g, 46%) as a yellow solid. 1H NMR
(400 MHz, CDCl3) δ 4.89 (s, 2H), 2.67 (s, 3H), 2.51 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 168.4, 162.2,
127.1, 59.0, 25.4. MS (ESI/APCI) m/z 192.8 [M+H]+.
4.1.46. 5-((tert-Butyldimethylsilyloxy)methyl)-4,6-dichloro-2-methylpyrimidine (67)
To the solution of 66 (2.1 g, 10.9 mmol) and imidazole (814 mg, 12.0 mmol) in dichloromethane
(20mL) was added TBSCl (1.78 g, 12.0 mmol) in portions. The resulting suspension was stirred at room
temperature overnight. Water (20 mL) was added, and extracted with dichloromethane (20 mL). The
combined organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 100/1) to give the desired product
(2.6 g, 78 %) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 4.85 (s, 2H), 2.68 (s, 3H), 0.91 (s, 9 H),
0.14 (s, 6H).13C NMR (150 MHz, CDCl3) δ 168.0, 162.4, 127.4, 59.7, 25.9, 25.5, 18.6, -5.3. MS
(ESI/APCI) m/z 192.9 [M-TBS+2H]+.
4.1.47. 5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-chloro-2-methyl-6-vinylpyrimidine (68)
To the solution of 67 (2.1 g, 6.84 mmol), potassium vinyltrifluoroborate (917 mg, 6.84 mmol), and
Cs2CO3 (4.46 g, 13.68 mmol) in 1,4-dioxane (60 mL) and water (12 mL) was added Pd(PPh3)4 (790 mg,
0.68 mmol) under N2 atmosphere. The resulting suspension was stirred at reflux overnight. The reaction
mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 100/1) to give the desired product (1.3 g, 64 %) as a
yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.11 (dd, J = 16.8, 10.8 Hz, 1H), 6.67 (d, J = 16.8 Hz, 1H),
5.73 (d, J = 10.8 Hz, 1H), 4.85 (s, 2H), 2.68 (s, 3H), 0.89 (s, 9 H), 0.11 (s, 6H). 13C NMR (150 MHz,
CDCl3) δ 167.2, 163.5, 161.4, 131.7, 125.3, 124.9, 58.5, 25.9, 25.8, 18.4, -5.1. MS (ESI/APCI) m/z 184.9
[M-TBS+2H]+.
4.1.48. 5-(((tert-Butyldimethylsilyl)oxy)methyl)-6-chloro-2-methylpyrimidine-4-carbaldehyde (69)
The intermediate 68 (1.3 g, 4.35 mmol) was dissolved in methanol (120 mL) and dichloromethane
(30 mL). Then ozone was introduced into this reaction solution at -65°C and stirred at this temperature
for 4h before dimethyl sulfide (3 mL) was added to quench the reaction. The resulting solution was
evaporated to remove the solvent. The residue was added water (20 mL) and extracted with
dichloromethane (50 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The residue
was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 20/1) to give the
desired product (600 mg, 46 %) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1H), 5.09 (s,
2H), 2.79 (s, 3H), 0.89 (s, 9 H), 0.13 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 191.8, 168.6, 163.4, 157.6,
128.5, 57.0, 25.9, 25.6, 18.5, -5.3. MS (ESI/APCI) m/z 300.9 [M+H]+.
4.1.49. N-((5-(((tert-Butyldimethylsilyl)oxy)methyl)-6-chloro-2-methylpyrimidin-4-yl)methyl)quinolin-
8-amine (70)
A mixture of 69 (270 mg, 0.9 mmol), quinolin-8-amine (194 mg, 1.35 mmol), and AcOH (1 drop)
in methanol (15 mL) was stirred at room temperature for 1h. Then NaBH3CN (170 mg, 2.0 mmol) was
added to the reaction solution. The resulting suspension was stirred at room temperature overnight. The
reaction solution was evaporated. The residue was added saturated NaHCO3 aqueous solution (10 mL)
27

and extracted with dichloromethane (50 mL). The organic layer was dried over Na2SO4, filtered and
evaporated. The residue was purified by silica gel column chromatography (petroleum
ether/dichloromethane = 1/1) to give the desired product (200 mg, 52%) as a yellow oil. 1H NMR (400
MHz, CDCl3) δ 8.79 (d, J = 2.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.48-7.34 (m, 3H), 7.10 (d, J =8.0 Hz,
1H), 6.72 (d, J = 7.6 Hz, 1H), 4.95 (s, 2H), 4.76 (s, 2H), 2.77 (s, 3H), 0.94 (s, 9 H), 0.17 (s, 6H). 13C
NMR (150 MHz, CDCl3) δ 167.8, 167.5, 160.0, 147.4, 144.3, 138.7, 136.0, 128.8, 127.8, 126.0, 121.6,
114.7, 105.2, 58.8, 46.3, 26.0, 25.9, 18.5, -5.2. MS (ESI/APCI) m/z 314.7 [M-TBS+2H]+.
4.1.50. (4-Chloro-2-methyl-6-((quinolin-8-ylamino)methyl)pyrimidin-5-yl)methanol (71)
To the solution of 70 (190 mg, 0.44 mmol) in THF (5 mL) was added pyridine hydrofluoride (60
wt percent in pyridine, 3 mL). The resulting suspension was stirred at room temperature for 4h. The
reaction solution was evaporated. The residue was added water (10 mL) and extracted with ethyl acetate
(10 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified by
silica gel column chromatography (dichloromethane/methanol/ammonium hydroxide = 100/2/1) to give
the desired product (100 mg, 72%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1H), 8.11
(d, J = 7.6 Hz, 1H), 7.46-7.37 (m, 2H), 7.16 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 6.8 Hz, 1H), 4.93 (s, 2H),
4.75 (s, 2H), 2.78 (s, 3H). MS (ESI/APCI) m/z 314.8 [M+H]+.
4.1.51. (4-Chloro-2-methyl-6-((methyl(quinolin-8-yl)amino)methyl)pyrimidin-5-yl)methanol (72)
A mixture of 71 (40 mg, 0.13 mmol), formaldehyde (37 wt percent in water, 0.5 mL) and AcOH (1
drop) in methanol (10 mL) was stirred for 30min. NaBH3CN (62 mg, 1 mmol) was then added to the
reaction solution. The resulting suspension was stirred at room temperature overnight. The reaction
mixture was evaporated. The residue was added saturated NaHCO3 aqueous solution (10 mL) and
extracted with dichloromethane (50 mL). The organic layer was dried over Na2SO4, filtered and
evaporated. The residue was purified by silica gel column chromatography (dichloromethane/methanol
= 100/1) to give the desired product (31 mg, 74%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.86
(d, J = 4.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.65-7.36 (m, 4H), 4.81 (s, 2H), 4.62 (s, 2H), 2.79 (s, 3H),
2.72 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 167.7, 166.9, 162.8, 148.7, 142.3, 137.6, 129.8, 129.4, 127.1,
123.5, 121.8, 119.3, 99.6, 61.2, 57.8, 43.5, 25.7. MS (ESI/APCI) m/z 328.8 [M+H]+.
4.1.52.
yl)methanol (73)
A mixture of 72 (30 mg, 0.09 mmol), TEA (91 mg, 0.9 mmol) and N-methylpiperazine (45 mg, 0.45
(2-Methyl-4-((methyl(quinolin-8-yl)amino)methyl)-6-(4-methylpiperazin-1-yl)pyrimidin-5-
mmol) in ethanol (5 mL) was stirred at reflux overnight. The reaction mixture was concentrated to
remove the solvent. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/ammonium hydroxide = 100/2/1) to give the desired product (28 mg, 80%)
as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.90 (dd, J = 4.0, 1.6 Hz, 1H), 8.17 (dd, J = 4.0, 1.6 Hz,
1H), 7.58-7.48 (m, 2H), 7.45-7.38 (m, 2H), 6.68 (t, J = 7.2 Hz, 1H), 4.51-4.44 (m, 4H), 3.85-3.71 (m,
4H), 2.85 (s, 3H), 2.59-2.52 (m, 7H), 2.34 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 166.3, 166.2, 165.2,
149.7, 148.8, 142.9, 136.9, 129.7, 127.0, 123.2, 121.5, 118.6, 117.3, 61.6, 58.0, 55.5, 49.1, 46.3, 43.5,
26.0. HRMS (ESI): calcd for C22H29N6O [M+H]+ 393.2397, found 393.2394. Purity: 99.5%.
4.2. In vitro biological assays
4.2.1. HPB-ALL CXCR4 competitive binding assay
HPB-ALL cells were maintained in RPMI-1640 (Gibico) supplemented with 10% FBS (Hyclone).
APC-conjugated anti-human CXCR4 was from Sungene. EC80 was first determined for 12G5 binding to
28

CXCR4. Then the compounds for testing (5 µL) were added into 96-well plates serially diluted at a ratio
of 1:3. Cells were washed once with ice-cold assay buffer (DPBS+2% HI-FBS) and then re-suspended
in the same buffer at a final concentration of 1 × 106/mL. Cell suspension (95 µL) was then added into
the wells and with the addition of APC-conjugated anti-human CXCR4 clone 12G5 at its EC80
determined. The mix (100 µL, final) of cell, compounds and APC-conjugated anti-human CXCR4 were
incubated at 4 ºC for 3 h before addition of 100 µL of 4% PFA. Cells were then washed once and
resuspended in assay buffer and examined by FACS. Compounds 3 and 37 were tested by at least two
independent repeat experiments. Each independent experiment contained two technical replicates.
4.2.2. FLIPR Tetra calcium mobilization assay
The FLIPR Tetra calcium mobilization assay was performed by HD Bioscience. Briefly, The
Molecular Devices, Fluorescent Imaging Plate Reader (FLIPR) Tetra was used in this assay. Excitation
was achieved through unique placement of LED’s within the instrument and emission captured by a CCD
camera (EMCCD camera for FI and ICCD camera for luminescence). The homogeneous FLIPR Calcium
4 assay kit from Molecular Devices was used as the fluorescence reagent. Compounds were solubilized
in 100% dimethyl sulfoxide (DMSO) to a concentration of 30 mM. A 10-point, 4-fold, intermediate
dilution series was created in 100% DMSO with a top concentration of 400 µM and a bottom
concentration of 0.001 µM. A near assay ready, direct dilution plate (ddNARP) was prepared from this
compound dilution plate by transferring 1 µL of each dilution of compound in 100% DMSO to a
Greiner#781201 plate. In addition, each ddNARP plate also contained positive and negative control wells
to define the upper and lower limits for the assay signal. The final assay concentration range of compound
was 1 µM to 0.0035 nM in 0.5% DMSO. Human CD4+ T-Cells were isolated from human whole blood
and subsequently activated and expanded using a CD3/CD28 expansion kit (Life Technologies). The
cells were frozen in ThermoFisher-formulated Recovery Cell Culture Freezing Medium containing 10%
Dimethyl sulfoxide (DMSO) and 10% Fetal Bovine Serum (FBS) (ThermoFisher Catalog No. 10100147).
When used, cells were resuspended using room temperature 1X HBSS/20 mM HEPES/0.005% P-104
assay buffer, adjusted the volume of the suspension (20 µL/well) to achieve a cell concentration of 2.5 ×
106 cells/mL. 2X Calcium 4 dye (20 µL/well) were added and the mixture were centrifuged briefly (~10
s) and stopped when it reached 1000 rpm. The plates were allowed to equilibrate before compounds (10
µL/well) and CXCL12 (10 µL/well) were added to the plates. The final volume of the mixture was 60
µL/well. The raw data were analyzed using Abase. The percent (%) effect at each concentration of
compound was calculated by Abase and was based on and relative to the amount of calcium produced in
the positive and negative control wells contained within each assay plate. The concentrations and %
effect values for tested compounds were plotted by Abase and the concentration of compound required
for 50% effect (IC50) was determined with a four-parameter logistic dose response equation. Each
compound was tested by at least two independent repeat experiments. Each independent experiment
contained two technical replicates.
4.2.3. CXCR1 functional assay
Recombinant HEK293 Cells with CXCR1 over-expression were dissociated using Trypsin-EDTA
at 37°C for 3 mins, before fresh culture medium was added onto cells to stop Trypsin-EDTA. The cells
were centrifuged at 1200 rpm for 3 mins, and then resuspended using assay buffer. Cell number was
counted using Countess (Invitrogen). PBS was added to adjust cell density to 2.7 x 105/ml.
IL-8 was serial diluted in LDV with 100 µM top dose, 4-fold dilution, 10 points, and dispensed 100
29