Design, synthesis, and structure-activity-relationship of a novel series of CXCR4 antagonists

Article abstract of DOI:10.1016/j.ejmech.2018.02.042

The important roles of the CXCL12/CXCR4 axis in numerous pathogenic pathways involving HIV infection and cancer metastasis make the CXCR4 receptor an attractive target for the development of therapeutic agents. Through scaffold hybridization of a few known CXCR4 antagonists, a series of novel aminopyrimidine derivatives was developed. Compound 3 from this new scaffold demonstrates excellent binding affinity with CXCR4 receptor (IC₅₀ = 54 nM) and inhibits CXCL12 induced cytosolic calcium increase (IC₅₀ = 2.3 nM). Furthermore, compound 3 possesses good physicochemical properties (MW 353, clogP 2.0, PSA 48, pKa 6.7) and exhibits minimal hERG and CYP isozyme (e.g. 3A4, 2D6) inhibition. Collectively, these results strongly support further optimization of this novel scaffold to develop better CXCR4 antagonists.

Full text of DOI:10.1016/j.ejmech.2018.02.042

Accepted Manuscript
Design, synthesis, and structure-activity-relationship of a novel series of CXCR4
antagonists
Zhanhui Li, Yujie Wang, Chunyan Fu, Xu Wang, Jun Jun Wang, Yi Zhang, Dongping
Zhou, Yuan Zhao, Lusong Luo, Haikuo Ma, Wenfeng Lu, Jiyue Zheng, Xiaohu Zhang
PII:
S0223-5234(18)30172-7
10.1016/j.ejmech.2018.02.042
EJMECH 10219
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2017
Revised Date: 7 February 2018
Accepted Date: 12 February 2018
Please cite this article as: Z. Li, Y. Wang, C. Fu, X. Wang, J.J. Wang, Y. Zhang, D. Zhou, Y. Zhao,
L. Luo, H. Ma, W. Lu, J. Zheng, X. Zhang, Design, synthesis, and structure-activity-relationship of a
novel series of CXCR4 antagonists, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.042.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Design, Synthesis, and Structure-Activity-Relationship of a Novel Series of CXCR4 Antagonists
Zhanhui Li, Yujie Wang, Chunyan Fu, Xu Wang, Jun Jun Wang, Yi Zhang, Dongping Zhou, Yuan Zhao,
Lusong Luo, Haikuo Ma, Wenfeng Lu, Jiyue Zheng, and Xiaohu Zhang
A novel series of CXCR4 antagonists was developed by a scaffold hybridization strategy. The most
promising lead of this series, compound 3, binds potently with CXCR4 receptor (IC50 = 54 nM) and
inhibits CXCL12 induced cytosolic calcium increase (IC50 = 2.3 nM). Furthermore, compound 3
possesses good physicochemical properties (MW 353, clogP 2.0, PSA 48, pKa 6.7)，exhibits minimal
CYP isozyme and hERG inhibition.
1

ACCEPTED MANUSCRIPT
Design, Synthesis, and Structure-Activity-Relationship of a Novel Series of CXCR4 Antagonists
Zhanhui Li a,1, Yujie Wang a,1, Chunyan Fu b,1, Xu Wang a, Jun Jun Wang c, Yi Zhang a, Dongping Zhou
b, Yuan Zhao b, Lusong Luo b,*, Haikuo Ma a, Wenfeng Lu a, Jiyue Zheng a,*, 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 Department of Oncology, the Third Affiliated Hospital, Soochow University, P. R. China
1 these authors contributed equally to this paper
* Corresponding author. Tel.: +86 512 65880380; fax: +86 512 65880380; email:
xiaohuzhang@suda.edu.cn; jyzheng@suda.edu.cn, lusong.luo@beigene.com
Abstract: The important roles of the CXCL12/CXCR4 axis in numerous pathogenic pathways
involving HIV infection and cancer metastasis make the CXCR4 receptor an attractive target for the
development of therapeutic agents. Through scaffold hybridization of a few known CXCR4 antagonists,
a series of novel aminopyrimidine derivatives was developed. Compound 3 from this new scaffold
demonstrates excellent binding affinity with CXCR4 receptor (IC50 = 54 nM) and inhibits CXCL12
induced cytosolic calcium increase (IC50 = 2.3 nM). Furthermore, compound 3 possesses good
physicochemical properties (MW 353, clogP 2.0, PSA 48, pKa 6.7) and exhibits minimal hERG and
CYP isozyme (e.g. 3A4, 2D6) inhibition. Collectively, these results strongly support further
optimization of this novel scaffold to develop better CXCR4 antagonists.
Keywords: Chemokine, CXCR4, antagonist, GPCR, scaffold hybridization, therapy
Abbreviations: APC, allophycocyanin; BPO, benzoyl peroxide; CCD, charge coupled device; CYP,
cytochrome P450; DC, dendritic cells; DCE, 1,2-dichloroethane; DIPEA, N,N-diisopropylethylamine;
DMEM, Dulbecco's modified eagle medium; 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;
hERG, human ether-a-go-go-related gene; HI-FBS, heat inactivated FBS; HIV, human
immunodeficiency virus; HLM, human liver microsomes; ICCD, intensified CCD; MDSC, myeloid
derived suppressor cells; MLM, mouse liver microsomes;. mp, melting point; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBS, N-bromosuccinimide; NMP,
N-methyl-2-pyrrolidone; PFA, paraformaldehyde; SAR, structure-activity-relationship; S/B ratio,
signal-to-background ratio; SD, standard deviation; SDF-1, stromal cell-derived factor-1; SEM,
standard error measurement; TCCA, trichloroisocyanuric acid.
1

ACCEPTED MANUSCRIPT
1. Introduction
The chemokine receptor CXCR4 was first identified as a co-receptor (along with CCR5) for the
Human Immunodeficiency Virus (HIV) entrance to lymphocytes.[1] Recent studies demonstrated that
CXCR4 and its natural ligand CXCL12 (also called stromal cell-derived factor-1, or SDF-1) regulate a
broad range of physiological functions such as hematopoietic stem cell homing and retention.[2,3]
Dysfunction of the CXCL12/CXCR4 axis contributes to numerous human pathologies, including virus
infection, cancer and inflammatory diseases.[4-6] In addition, CXCR4 was reported to modulate
immune cell function via the trafficking of key immune cells including T-cells, dendritic cells (DC),
and myeloid derived suppressor cells (MDSC).[7] CXCL12/CXCR4 axis is therefore an important
player in immune evasion. Consequently, inhibition of CXCR4 activation is recognized as an attractive
strategy for the development of therapeutic agents.
A number of chemicals modifying CXCR4 activity have been evaluated in in vitro experimental
studies and pre-clinical animal models.[5,8,9] Among them, peptide analogues such as CTCE-9908 [10]
and POL6326 [11] had progressed into clinical testing. Small molecule CXCR4 antagonists were also
developed.[6,8,9] The most advanced small molecule CXCR4 antagonist, AMD3100 (plerixafor,
Figure 1), was approved by the FDA for the treatment of non-Hodgkin’s lymphoma and multiple
myeloma patients with hematopoietic stem cell transplantation.[12] Although many of the small
molecule CXCR4 antagonists were originally developed for anti-HIV treatment, the clinical usage of
AMD3100 in mobilizing stem cells in oncology indication and the emerging findings [13-15] of its
potential role in modulating immune cells at the tumor microenvironment prompted a significant
expansion of discovering the next generation of CXCR4 antagonists with better pharmaceutical
properties.
2. Design
The diverse role of the CXCL12/CXCR4 axis in the pathogenesis of HIV, cancer, and
inflammatory diseases has established CXCR4 as a promising therapeutic target.[5,6,8,9] Unfortunately,
only one CXCR4 antagonist, AMD3100 (plerixafor, Figure 1), has currently been approved by the
FDA for hematopoietic stem cell mobilization as an injectable agent for short-term treatments. Since
HIV infection and cancer are all chronic diseases, CXCR4 antagonists that are orally available and safe
for long-term use are highly desirable. Towards this end, we initiated our endeavor in pursuit of novel
CXCR4 antagonists. Numerous small molecule CXCR4 antagonists had been reported in the literature,
exemplified by AMD11070 [16,17], IT1t [18], MSX-122 [19] and KRH3955 [20] (Figure 1). Our goal
was to utilize a scaffold hybridization strategy to formulate a novel template based on the known
pharmacophore knowledge. In addition, we hope to reduce the high pKa frequently associated with
many of the CXCR4 antagonists. Highly basic functional groups not only hamper permeability (which
may lead to poor absorption), but may also lead to safety issues such as hERG potassium channel
inhibition, CYP enzyme inhibition and phospholipidosis.[21] We are particularly inspired by the
elegant work from AMD11070 [17], GSK812397 [22,23], and Emory-15 [24] (Figure 2). We therefore
hybridized the aforementioned templates to synthesize compounds 1 and 2. Compounds 1 and 2 were
highly potent in a cell based CXCR4 competitive binding assay. Compound 2, with the basic amine
protected by a Boc group, was equally potent as compound 1. Encouraged by this data and the SAR
2

ACCEPTED MANUSCRIPT
established by scientists from GSK [23,25], we truncated the aliphatic chain to obtain compound 3
(Figure 2). Compound 3 potently competed with APC-conjugate clone 12G5 for CXCR4 binding in
a cell based FACS assay (IC50 = 54 nM) and inhibited CXCL12 induced cytosolic calcium increase
(IC50 = 2.3 nM). Among the features that made compound 3 an attractive starting point for a medicinal
chemistry optimization program was its moderate basicity, relative low molecular weight and clogP,
and the simple chemistry required to prepare a diverse set of analogues within the pyrimidine
chemotype. Here we report the preliminary SAR utilizing compound 3 as a lead template for medicinal
chemistry optimization.
3. Chemistry
The general synthetic route used to prepare compounds 1 and 2 was outlined in Scheme 1.
Commercially available diethyl oxalacetate sodium salt was reacted with formamidine acetate in base
conditions to give pyrimidine 23. Chlorination of 23 with POCl3 and then treatment with EtOH
afforded ester 24, which was reduced with NaBH4 and chlorinated with SOCl2 to give key intermediate
26. Treatment of 5,6,7,8-tetrahydroquinoline with trichloroisocyanuric acid provided intermediate 28,
which was substituted with tert-butyl 4-aminobutylcarbamate to yield amine 29. Reaction of amine 29
with intermediate 24 in the presence of DIPEA and KI afforded intermediate 30. The synthesis of
compound 2 was achieved by reacting intermediate 30 with N-methyl piperazine in NMP. Removal of
BOC of compound 2 by HCl/EtOAc afforded hydrochloride salt of compound 1.
The synthesis of compounds 3, 9-21 was performed as shown in Scheme 2. Substitution of
intermediate 28 with methylamine gave amine 31, which was reacted with intermediate 26 to afford
intermediate 32. Hydrogenated reduction of 32 led to compound 9, or displacement of the chlorine of
32 with corresponding amines afforded compounds 10, 11, 13-21, respectively. Removal of BOC of
compound 11 yielded compound 12.
The general synthesis of compounds 4-8 is outlined in Scheme 3. Intermediates 35a-e were
synthesized by reductive amination of amine 31 with commercially available aromatic aldehydes 34b
and 34c, or by alkylation of amine 31 with intermediates 34a,d,e, which were obtained by bromination
of 33a,d,e with NBS and BPO in CCl4. Substitution of 35a-e with N-methyl piperazine in the presence
of DIPEA in NMP afforded final compounds 4-8.
The synthesis of compounds (S)-3 and (R)-3 was performed as shown in Scheme 4. According to the
established procedure [26], reductive amination of ketone 36 with (S)-1-(4-methoxyphenyl)ethanamine
afforded amine 37. After N-methylation and removal of the chiral auxiliary, amine 38 was obtained.
Treatment of amine 38 with intermediate 26 provided chloro derivative 39, which was reacted with
N-methyl piperazine to give the final product (S)-3. Similarly, according the same procedure,
compound (R)-3 was synthesized by using (R)-1-(4-methoxyphenyl)ethanamine in place of
(S)-1-(4-methoxyphenyl)ethanamine.
4. Results and discussion
4.1. Development and validation of a cell based binding assay for CXCR4
There are a number of assays in the literature characterizing the interaction of the CXCL12/CXCR4
axis. These assays include the CXCL12 competitive binding assay, CXCL12-induced calcium
mobilization, CXCR4 receptor internalization, CXCL12-guided chemotaxis and CXCR4-specific HIV
3

ACCEPTED MANUSCRIPT
entry and replication. Tom Van Loy’s group elegantly summarized most of the known CXCR4 ligands
(including peptides and small molecules) in a series of pharmacological and functional assays.[27]
They concluded that the CXCL12 competition binding assay to be highly reliable and indicative for the
pharmacological and functional profile of competitive CXCR4 antagonists. Encouraged by their results,
we decided to establish a cell base competitive binding assay as an initial screening assay for our
structural optimization campaign.
Four acute lymphoblastic leukemia cell lines (HPB-ALL, MOLT-4, CCRF-CEM and Jurkat) which
naturally express CXCR4 receptor were evaluated for the CXCL12 competitive binding assay.
APC-conjugate clone 12G5, an antibody competing for CXCR4 binding with CXCL12, was used as
the tool for readout. The cells with unoccupied, antagonist-free receptors were sorted with FACS and
all four cell lines yielded good signal readout (supplemental materials, Figure S1 and Figure S2).
HPB-ALL cell line was selected as the assay cell line based on its superior S/B ratio. The assay was
further optimized and validated to obtain the optimal signal and Z’ for routine compound screening
(supplemental materials, Figure S3, A and B).
4.2. Structure-Activity-Relationship of synthesized compounds
The CXCR4 inhibition of the synthesized compounds was tested using the cell based FACS 12G5
competitive binding assay as mention above. This is the primary assay we used to screen compounds.
The structure-activity-relationship was summarized in Tables 1 and 2.
We started structural modifications on the central aromatic ring to probe the key functional groups
associated with the blockade of CXCL12 and CXCR4 interaction. Numerous reports indicated that a
nitrogen atom is essential on the X position for CXCR4 antagonistic activity.[17,28] Indeed, compound
3 was active in the FACS based APC-conjugate clone 12G5 competitive binding assay at 54 nM. When
the Y position was changed from N to CH, the resulting pyridine analogue compound 4 was also active,
albeit four folds less potent than compound 3 at 200 nM. Compared with compound 3, the isomeric
pyrimidine compound 7 was also active at 180 nM. These results were in stark contrast to compounds 5,
6, and 8, of which the N atom on X position was replaced by a CH. Compounds 5, 6, and 8 were
completely inactive, regardless of the nature of atoms on position Y and Z. Our results were consistent
with previous SAR studies and validated that the N on position X is key for the activity of CXCR4
receptor antagonists.
Based on the above structure-activity relationship, we chose the 4,6-substituted pyrimidine as the
prototype for further optimization. Removal of the terminal methyl piperazine (compound 9)
completely abolished the binding affinity, indicating the critical interactions associated with this
functional group. Similarly, substitution of methyl piperazine with a simple dimethylamino group
resulted in an inactive compound (compound 10). The activity was partially recovered with a
piperazine group (compound 12, 2600 nM), but a Boc group on the terminal nitrogen abolished the
activity completely. This is in contrast to the case of compound 1 (18 nM) vs. compound 2 (18 nM)
where both compounds are very potent. We speculate a basic center is needed for good activity on the
pyrimidine ring, whereas polar interactions maybe involved around the upper long aliphatic arm.
Different substitutions on the terminal nitrogen of the piperazine were well tolerated, as demonstrated
by compounds 13, 14, 15 and 16 (320, 210, 1050, and 290 nM, respectively). Ring expansion was
tolerated as the methylhomopiperazine analogue compound 17 was also active (170 nM). Removal of
the basic nitrogen to the exocyclic locations was also tolerated as exemplified by compounds 18 and 19
4

ACCEPTED MANUSCRIPT
(630 and 150 nM, respectively). Finally, ring opening led to analogues with compromised activity
(compounds 20, 21; 1020 and 640 nM, respectively). Overall, the structure-activity relationship of this
pyrimidine scaffold largely follow the established SAR information and compound 3 remained to be
the most promising lead.
4.3. Functional evaluation of lead compound 3
CXCL12 binds to and activates CXCR4. CXCR4 activation results in transient increase of
cytosolic calcium concentration. The calcium mobilization assay performed by FLIPR Tetra can
evaluate the antagonistic potency of compounds inhibiting the CXCL12-induced calcium signal.[29]
Having established that compound 3 potently binds with CXCR4 in the CXCL12/ APC-conjugate clone
12G5 competitive binding assay, we next evaluated compound 3 in the FLIPR Tetra based calcium
mobilization assay to see if compound 3 was indeed a functional antagonist. Primary T-cells were
isolated from human whole blood and subsequently activated and expanded using a CD3/CD28
expansion kit (Life Technologies) and was used as the cell line to carry out the calcium mobilization
assay. FLIPR Tetra and homogeneous FLIPR Calcium 4 assay kit from Molecular Devices were used
as the detection system. The cells were pre-incubated with compounds and subsequently stimulated
with CXCL12. Compound 3 was a potent functional CXCR4 antagonist (IC50 = 2.3 nM) and the
standard AMD3100 was about 8-fold less potent (IC50 = 18 nM) when tested in this calcium
mobilization assay (Figure 3).
CXCL12 activates CXCR4, resulting in migration and invasion of the corresponding cells. Thus,
we used the Matrigel invasion assay to evaluate compound 3 for its inhibitory activity of
CXCL12/CXCR4 mediated chemotaxis and invasion. Compound 3 (100 nM) and cells were added to
the upper chamber, and the chemoattractant CXCL12 was added to the lower chamber. AMD3100 was
used as a positive control. The inhibition of cell invasion with compound 3 and AMD3100 was
calculated by comparing to the cell invasion without treatment. The results were summarized in Figure
4. In consistent with the 12G5 binding and the calcium mobilization data, compound 3 was more potent
in the Matrigel invasion assay than AMD3100 (Figure 4).
Compound 3 was also evaluated in cell viability (MTT) assay for its potential cytotoxicity.
Compound 3 did not inhibit the proliferation of MDA-MB-231, MCF-7 and HOSEpiC cells at
concentrations as high as 400 nM (Figure 5).
4.4. Preliminary in vitro safety evaluation of lead compounds
AMD11070 was a leading small molecule CXCR4 antagonist which could be administered orally.
However, it was reported that AMD11070 suffered from CYP isozyme (3A4, 2D6) inhibition, possibly
due to the benzimidazole functionality and/or its highly basic nitrogen (pKa = 10.4, Table 3) on the
aliphatic side chain. Highly basic functional groups not only hamper permeability (which may lead to
poor absorption), but may also lead to safety issues such as hERG potassium channel inhibition, CYP
enzyme inhibition and phospholipidosis.[21] Our initial goal was to utilize a scaffold hybridization
strategy to formulate a novel template based on the known pharmacophore knowledge, and in the
meantime, reduce the high pKa frequently associated with many of the CXCR4 antagonists. Compound
3 was demonstrated to bind potently to CXCR4 (IC50 = 54 nM) and inhibit CXCL12 induced cytosolic
calcium increase (IC50 = 2.3 nM). Furthermore, compound 3 possesses good physicochemical
properties (MW 353, clogP 2.0, PSA 48, pKa 6.7, Figure 2). Encouraged by the aforementioned results,
5

ACCEPTED MANUSCRIPT
we decided to test 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%, respectively). Since compound 3 has a chiral center,
we then synthesized the enantiomeric pure (S)-3 and (R)-3 according to the established procedure.[26]
Consistent with the literature reports [17,25,30], (S)-3 exhibited more potent binding and function than
(R)-3 (Table 4). Compound (S)-3 was further profiled by a standard patch clamp (express) experiment
for its inhibition of the human ether-a-go-go related gene (hERG) potassium channel. Compound (S)-3
exhibited very low inhibition (IC50 > 30 µM) of hERG, indicating minimal cardiotoxicity liability
associated with blockade of this key potassium channel. Compound (S)-3 was found to be highly
permeable (15× 10-6 cm/s, Caco-2), and not likely to be an efflux substrate (Table 4). Unfortunately,
compound (S)-3 was quickly metabolized in human and mouse liver microsomes (Table 4).
Improvement of metabolic stability must be considered in the future medicinal chemistry optimization.
5. Conclusion
We have utilized a scaffold hybridization strategy to design and synthesize a new series of CXCR4
antagonists. The most promising lead of this series, compound 3, binds potently with CXCR4 (IC50
=
54 nM) and inhibits CXCL12 induced cytosolic calcium increase (IC50 = 2.3 nM). Furthermore,
compound 3 possesses good physicochemical properties (MW 353, clogP 2.0, PSA 48, pKa 6.7) and
exhibits minimal hERG and CYP isozyme inhibition. Structure-activity-relationship study of other
parts of this novel scaffold is ongoing and results will be reported in due course.
6. Experimental protocols
6.1. Chemistry
General reaction progress was monitored by analytical thin layer chromatography performed on
silica gel HSGF254 pre-coated plates. Organic solutions were dried over anhydrous Na2SO4, and the
solvents were removed under reduced pressure. Final compounds were purified with silica gel 100-200
mesh for column chromatography. 1H NMR were obtained on 400 MHz (Varian) spectrometer, and 13
C
NMR were obtained on 600 MHz (Varian) spectrometer. Chemical shifts were given in ppm using
tetramethylsilane as internal standard. Mass spectra were obtained using an Agilent 1100 LC/MSD
Trap SL version Mass Spectrometer. HRMS analysis was recorded on an Agilent 6540 UHD
Accurate-Mass Q-TOF LC/MS. Melting points were determined by a SMP10 melting point apparatus.
Values of optical rotation were measured on a Rudolph Automatic Polarimeter A21101. IR spectra were
recorded on a Bruker Vertex 70 spectrophotometer with KBr pellets.
6.1.1. 6-Hydroxypyrimidine-4-carboxylic acid (23).
To a solution of formamidine acetate (10 g, 96 mmol) in 200 mL of water was added diethyl
oxalacetate sodium salt (21 g, 100 mmol) and sodium hydroxide (3.8 g, 96 mmol). The reaction
mixture was stirred overnight at room temperature, before 6 N HCl was added carefully to adjust PH =
1. The mixture was allowed to stand overnight at 0 °C, and the resultant precipitate was filtered and
dried in vacuum to give the desired product (2 g, 15%) as a red solid. 1H NMR (400 MHz, DMSO-d6) δ
12.90 (s, 1H), 8.25 (s, 1H), 6.83 (s, 1H).
6

ACCEPTED MANUSCRIPT
6.1.2. Ethyl 6-chloropyrimidine-4-carboxylate (24).
To a solution of 23 (1 g, 7.1 mmol) in POCl3 (16.6 g, 106 mmol) was added PCl5 (3 g, 14.2 mmol).
The mixture was stirred at reflux overnight, and then concentrated in vacuum. The residue was
dissolved in dichloromethane (30 mL) and EtOH (390 mg, 8.5 mmol) was added dropwise at 0 °C. The
mixture was stirred at 0 °C for 1 h, and then poured into ice-water. The organic layer was washed with
brine, 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 (500 mg, 38%) as a
white solid. 1H NMR (400 MHz, CDCl3) δ 9.16 (s, 1H), 8.05 (s, 1H), 4.50 (q, J = 7.0 Hz, 2H), 1.45 (t, J
= 7.2 Hz, 4H).
6.1.3. (6-Chloropyrimidin-4-yl)methanol (25).
To a solution of 24 (500 mg, 2.7 mmol) in methanol (10 mL) at 0 °C was added NaBH4 (120 mg, 3.2
mmol), and the mixture was stirred at room temperature for 2 h. The mixture was quenched with water
and extracted with dichloromethane (50 mL × 3). The combined organic layers was washed with brine,
dried over Na2SO4 and concentrated to give the desired product (280 mg, 72%) as a yellow solid. 1H
NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 7.48 (s, 1H), 4.78 (s, 2H).
6.1.4. 4-Chloro-6-(chloromethyl)pyrimidine (26).
To a solution of 25 (280 mg, 1.9 mmol) in dichloromethane (10 mL) was added SOCl2 (280 mg, 2.3
mmol). The reaction was stirred at room temperature overnight. The mixture was quenched with
saturated NaHCO3 aqueous solution (50 mL) and extracted with dichloromethane (20 mL × 3). The
combined organic layers was washed with brine, dried over Na2SO4 and concentrated to give the
desired product (180 mg, 58%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 7.61 (s, 1H),
4.61 (s, 2H).
6.1.5. 8-Chloro-5,6,7,8-tetrahydroquinoline (28).
To a solution of 5,6,7,8-tetrahydroquinoline (10 g, 60 mmol) in dichloromethane (200 mL) was
added TCCA (21 g, 90 mmol). The reaction mixture was stirred at reflux overnight. After cooling to
room temperature, the mixture was filtered. The filtrate was washed with saturated NaHCO3 aqueous
solution (100 mL), 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 (8.6 g, 86%)
as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 4.4 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.18
(dd, J = 7.2, 4.8 Hz, 1H), 5.43 (s, 1H), 2.92-2.83 (m, 1H), 2.80-2.76 (m, 1H), 2.42-2.39 (m, 1H),
2.26-2.18 (m, 2H), 1.90-1.87 (m, 1H).
6.1.6. tert-Butyl 4-(5,6,7,8-tetrahydroquinolin-8-ylamino)butylcarbamate (29).
To a solution of 28 (210 mg, 1.3 mmol) in EtOH (10 mL) were added tert-butyl
4-aminobutylcarbamate (250 mg, 1.4 mmol) and Et3N (390 mg, 3.9 mmol). The reaction mixture was
stirred at reflux overnight, and then quenched with saturated NaHCO3 aqueous solution (100 mL). The
aqueous layer was extracted with dichloromethane (50 mL × 3). The combined organic layers was
washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel column
chromatography (dichloromethane/methanol = 100/3) to give the desired product (200 mg, 48%) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 4.0 Hz, 1H), 7.36 (d, J = 7.2 Hz, 1H), 7.05 (dd,
J = 7.6, 4.4 Hz, 1H), 4.89 (s, 1H), 3.78-3.75 (m, 1H), 3.12 (s, 2H), 2.77-2.74 (m, 4H), 2.73 (s, 1H),
7

ACCEPTED MANUSCRIPT
2.15-2. 13 (m, 1H), 2.00-1.98 (m, 1H), 1.78-1.73 (m, 2H), 1.59 (s, 4H), 1.41 (s, 9H).
6.1.7.
tert-Butyl
4-(((6-chloropyrimidin-4-yl)methyl)(5,6,7,8-tetrahydroquinolin-8-yl)amino)butylcarbamate (30).
A solution of 29 (300 mg, 0.9 mmol), 26 (160 mg, 1 mmol), KI (16 mg, 0.09 mmol), DIPEA (350
mg, 2.3 mmol) in MeCN (10 mL) was stirred at 50 °C overnight. The reaction mixture was quenched
with saturated NaHCO3 aqueous solution (50 mL), and extracted with dichloromethane (40 mL × 3).
The combined organic layers was washed with brine, dried over Na2SO4 and concentrated. The residue
was purified by silica gel column chromatography (dichloromethane/methanol = 100/1 to 50/1) to give
1
the desired product (280 mg, 70%) as a colorless oil. H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 8.45
(d, J = 4.4 Hz, 1H), 7.99 (s, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.04 (dd, J = 7.6, 4.8 Hz, 1H), 4.63 (s, 1H),
4.08-4.07 (m, 1H), 3.94 (d, J = 17.6 Hz, 1H), 3.73 (d, J = 17.2 Hz, 1H), 3.05 (s, 2H), 2.77-2.71 (m, 4H),
2.17-2.14 (m, 1H), 2.01-1.99 (m, 1H), 1.84-1.75 (m, 1H), 1.71-1.68 (m, 1H), 1.46 (s, 4H), 1.42 (s, 9H).
6.1.8.
tert-Butyl
4-(((6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)(5,6,7,8-tetrahydroquinolin-8-yl)amino)butylcarb
amate (2).
To a solution of 30 (100 mg, 0.2 mmol) in EtOH (4 mL) was added Et3N (202 mg, 2 mmol) and
N-methyl piperazine (25 mg, 0.22 mmol). The reaction was stirred at 80 °C overnight, and then
quenched with saturated NaHCO3 aqueous solution (50 mL). The aqueous layer was extracted with
dichloromethane (20 mL × 3). The combined organic layers was washed with brine, dried over Na2SO4
and concentrated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol = 25/1) to give the desired product (100 mg, 98%) as a colorless oil. 1H
NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 8.43-8.42 (m, 1H), 7.46 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H),
7.04-7.01 (m, 1H), 4.69 (s, 1H), 4.06 (t, J = 8.0 Hz, 1H), 3.73 (s, 4H), 3.68 (s, 1H), 3.61-3.57 (m,
1H), 3.01 (s, 2H), 2.78 (s, 1H), 2.68-2.66 (m, 3H), 2.48 (t, 2J = 6.0 Hz, 4H), 2.34 (s, 3H), 2.11 (s,
1H), 1.99-1.97 (s, 1H), 1.88-1.80 (m, 1H), 1.44 (s, 4H), 1.41 (s, 9H). 13C NMR (150 MHz, CDCl3) δ
162.4, 157.4, 156.1, 147.1, 136.7, 134.4, 121.8, 101.4, 61.1, 56.5, 54.6, 52.0, 46.0, 45.9, 43.6, 40.4,
33.3, 31.4, 29.4, 28.5, 27.8, 25.9, 25.2, 21.5. IR v (cm-1): 3351, 2929, 1692, 1592, 1525, 1501, 1443,
1250, 1164, 975, 784. HRMS (ESI): calcd for C28H44N7O2 [M+H]+ 510.3551, found 510.3555.
6.1.9.
N1-((6-(4-Methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N1-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-
diamine (1).
To a solution of 2 (100 mg, 0.2 mmol) in ethyl acetate (3 mL) was added 3 M HCl/ ethyl acetate (4
mL) dropwise. The mixture was stirred at room temperature for 12 h. The reaction was concentrated to
1
give the title compound as a yellow solid (90 mg, 100%). mp: 120-123 °C. H NMR (400 MHz,
DMSO-d6) δ 11.94 (s, 1H), 8.84 (s, 1H), 8.77 (s, 1H), 8.35 (s, 1H), 8.18 (s, 1H), 7.89 (s, 3H), 7.72 (s,
1H), 4.86 (s, 2H), 4.47 (s, 1H), 4.00 (s, 2H), 3.54 (s, 4H), 3.23 (s, 2H), 2.93 (s, 2H), 2.79 (s, 3H), 2.66
(s, 3H), 2.39 (s, 1H), 2.26 (s, 1H), 2.03 (s, 1H), 1.75 (s, 3H), 1.54 (s, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 161.6, 156.0, 150.9, 146.1, 140.4, 138.7, 125.2, 102.7, 51.9, 51.4, 50.4, 41.8, 40.1, 38.3,
27.3, 24.6, 24.0, 20.0, 19.8. IR v (cm-1): 3383, 2937, 1644, 1611, 1545, 1454, 1287, 1021, 973, 793.
HRMS (ESI): calcd for C23H36N7 [M+H]+ 410.3027, found 410.3022.
8

ACCEPTED MANUSCRIPT
6.1.10. N-Methyl-5,6,7,8-tetrahydroquinolin-8-amine (31).
A solution of 28 (4.8 g, 29.3 mmol) in methylamine solution (30 wt. percent in absolute ethanol; 10
mL) was stirred at room temperature overnight. After completion of the reaction, the ethanol was
evaporated.
The
residue
was
purified
by
silica
gel
column
chromatography
(dichloromethane/methanol/NH4OH = 100/1/1) to give the desired product (3.4 g, 72%) as a brown oil.
1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 4.4 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.07 (dd, J = 7.6, 4.8
Hz, 1H), 3.66 (t, J = 6.0 Hz, 1H), 2.81-2.71 (m, 2H), 2.53 (s, 3H), 2.16-2.10 (m, 1H), 2.01-1.96 (m,
1H), 1.79-1.75 (m, 2H).
6.1.11. N-((6-Chloropyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (32)
A mixture of 31 (160 mg, 1 mmol), 28 (170 mg, 1.05 mmol), KI (16 mg, 0.1 mmol) and DIPEA (320
mg, 2.5 mmol) in MeCN (10 mL) was stirred at room temperature overnight. The reaction solution was
evaporated to remove most of MeCN, diluted with water (50 mL) and extracted with dichloromethane
(50 mL × 3). The combined organic layers was dried over Na2SO4, filtered and evaporated. The residue
was purified by silica gel column chromatography (dichloromethane/methanol= 100/1 to 50/1) to give
the desired product (200 mg, 69%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.50
(d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.08 (dd, J = 8.0, 4.0 Hz, 1H), 4.03-3.99
(m, 1H), 3.80 (s, 2H), 2.82-2.77 (m, 1H), 2.75-2.70 (m, 1H), 2.41 (s, 3H), 2.17-2.14 (m, 1H),
2.06-2.04 (m, 1H), 1.92-1.89 (m, 1H), 1.72-1.71 (m, 1H).
6.1.12. N-Methyl-N-(pyrimidin-4-ylmethyl)-5,6,7,8-tetrahydroquinolin-8-amine (9)
A mixture of 32 (70 mg, 0.24 mmol) and Pd/C (5%, 15 mg) in 5 mL of methanol was stirred at 50 °C
under N2 overnight. The reaction solution 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 (50 mg, 82%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.65 (d, J = 4.8 Hz,
1H), 8.50 (s, 1H), 7.80 (d, J = 4.4 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.08-7.05 (m, 1H), 4.04-4.01 (m,
1H), 3.82-3.72 (m, 2H), 2.82-2.78 (m, 1H), 2.73-2.69 (m, 1H), 2.39 (s, 3H), 2.13 (s, 1H), 2.03 (s, 2H),
1.97-1.88 (m, 1H), 1.72 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 170.0, 158.2, 157.2, 157.0, 147.4,
136.8, 134.2, 121.9, 120.4, 63.8, 59.3, 39.6, 29.2, 24.4, 21.1. IR v (cm-1): 2933, 2861, 2795, 1578, 1548,
1442, 1385, 1060, 971, 785. MS (ESI/APCI) m/z 254.9 [M + H]+.
6.1.13. General procedure for the synthesis of 3, 10, 11, 13-21
A mixture of 32 (0.12 mmol, 1.0 eq.), DIPEA (10 eq.) and corresponding amine (3.0 eq.) in 2 mL of
NMP was stirred at 120 °C overnight. The reaction was diluted with EtOAc (50 mL) and washed with
brine (30 mL × 5). The organic layer was dried over Na2SO4, filtered and evaporated. The residue was
purified by silica gel column chromatography (dichloromethane/methanol = 100/1 to 100/3) to give the
desired product.
6.1.13.1.
N-Methyl-N-((6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine (3)
Compound 3 was obtained as a colorless oil (yield 80%). 1H NMR (400 MHz, CDCl3) δ 8.45 (s,
2H), 7.33 (d, J = 6.8 Hz, 1H), 7.20 (s, 1H), 7.03 (s, 4.8 Hz, 1H), 3.99 (s, 1H), 3.68 (s, 4H), 3.62 (s,
2H), 2.77 (s, 1H), 2.68-2.64 (m, 1H), 2.44 (s, 4H), 2.35 (s, 3H), 2.30 (s, 3H), 1.97 (s, 1H),
1.91-1.88 (m, 1H), 1.85 (s, 1H), 1.67 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 167.5, 162.5, 157.7,
9

ACCEPTED MANUSCRIPT
157.4, 147.3, 136.8, 134.3, 121.8, 101.1, 62.6, 59.1, 54.8, 46.2, 43.8, 39.3, 29.3, 23.1, 21.3. IR v (cm-1):
2947, 2878, 1647, 1588, 1542, 1454, 1287, 973, 872, 795. HRMS (ESI): calcd for C20H29N6 [M+H]+
353.2448, found 353.2451.
6.1.13.2. N-((6-(Dimethylamino)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine
(10)
1
Compound 10 was obtained as a colorless oil (yield 48%). H NMR (400 MHz, CDCl3) δ 8.49 (s,
2H), 7.36 (s, 1H), 7.06 (s, 2H), 4.04 (s, 1H), 3.67 (s, 2H), 3.12 (s, 6H), 2.81 (s, 1H), 2.71-2.67 (m, 1H),
2.40 (s, 3H), 2.16-2.12 (m, 1H), 2.04-2.01 (m, 1H), 1.94-1.92 (m, 1H), 1.69 (s, 1H). 13C NMR (150
MHz, CDCl3) δ 166.6, 162.8, 157.5, 147.3, 136.7, 134.3, 121.7, 100.8, 62.7, 59.3, 39.4, 37.3, 29.3, 23.5,
21.3. IR v (cm-1): 2930, 2861, 1591, 1515, 1402, 1343, 1189, 1003, 971, 785. MS (ESI/APCI) m/z
297.9 [M + H]+.
6.1.13.3.
tert-Butyl
4-(6-((methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)pyrimidin-4-yl)piperazine-1-carboxylate
(11)
1
Compound 11 was obtained as a colorless oil (yield 94%). H NMR (400 MHz, CDCl3) δ 8.49 (s,
2H), 7.37 (d, J = 7.6 Hz, 1H), 7.28 (s, 1H), 7.07 (dd, J = 7.2, 4.8 Hz, 1H), 4.03-4.00 (m, 1H), 3.69-3.66
(m, 6H), 3.49 (s, 4H), 2.84-2.77 (m, 1H), 2.72-2.68 (m, 1H), 2.38 (s, 3H), 2.10 (s, 1H), 2.00 (s, 1H),
1.97-1.89 (m, 1H), 1.73-1.67 (m, 1H), 1.48 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 162.5, 157.7, 154.8,
147.4, 146.1, 136.8, 134.4, 121.9, 101.3, 80.3, 62.4, 59.1, 43.7, 39.3, 29.4, 28.5, 23.0, 21.3. IR v (cm-1):
2928, 2858, 1692, 1587, 1492, 1414, 1240, 1160, 1121, 992, 769. MS (ESI/APCI) m/z 438.8 [M + H]+.
6.1.13.4.
N-((6-(4-Ethylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (13)
Compound 13 was obtained as a colorless oil (yield 32%).1H NMR (400 MHz, CDCl3) δ 8.48 (s,
2H), 7.36 (d, J = 7.2 Hz, 1H), 7.22 (s, 1H), 7.06 (s, 1H), 4.02 (t, J = 6.8 Hz, 1H), 3.72 (s, 4H), 3.64 (s,
2H), 2.81-2.78 (m, 1H), 2.71-2.67 (m, 1H), 2.50 (s, 4H), 2.46-2.42 (m, 2H), 2.40 (s, 3H), 2.10 (s, 1H),
2.00 (s, 1H), 1.94-1.88 (m, 1H), 1.71 (s, 1H), 1.12 (t, J = 6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ
167.6, 162.5, 157.7, 157.5, 147.4, 136.8, 134.3, 121.8, 101.1, 62.6, 59.1, 52.6, 52.5, 43.8, 39.4, 29.4,
23.2, 21.3, 12.0. IR v (cm-1): 2931, 2856, 1587, 1494, 1441, 1338, 1124, 995, 784. MS (ESI/APCI) m/z
366.9 [M + H]+.
6.1.13.5.
N-((6-(4-Isopropylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine
(14)
1
Compound 14 was obtained as a colorless oil (yield 51%). H NMR (400 MHz, CDCl3) δ 8.49-8.47
(m, 2H), 7.36 (d, J = 7.2 Hz, 1H), 7.19 (s, 1H), 7.06 (s, 1H), 4.02 (s, 1H), 3.70 (s, 4H), 3.64 (s, 2H),
2.81-2.78 (m, 1H), 2.72 (s, 2H), 2.57 (s, 4H), 2.39 (s, 3H), 2.10 (s, 1H), 2.00 (s, 1H), 1.94-1.89 (m, 1H),
1.67 (s, 1H), 1.07 (d, J = 6.0 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 167.5, 162.4, 157.7, 157.5, 147.4,
136.7, 134.3, 121.7, 101.0, 62.6, 59.2, 54.7, 48.5, 44.2, 39.4, 29.4, 23.2, 21.3, 18.6, 18.5. IR v (cm-1):
2930, 2856, 1587, 1496, 1441, 1337, 1177, 1128, 995, 784. MS (ESI/APCI) m/z 380.9 [M + H]+.
6.1.13.6.
10

ACCEPTED MANUSCRIPT
N-((6-(4-(Cyclopropylmethyl)piperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinoli
n-8-amine (15)
1
Compound 15 was obtained as a colorless oil (yield 20%). H NMR (400 MHz, CDCl3) δ 8.49-8.47
(m, 2H), 7.36 (d, J = 7.6 Hz, 1H), 7.20 (s, 1H), 7.08-7.06 (m, 1H), 4.04-4.03 (m, 1H), 3.73 (s, 4H),
3.64 (s, 2H), 2.81-2.80 (m, 1H), 2.72-2.67 (m, 1H), 2.58 (s, 4H), 2.39 (s, 3H), 2.29 (d, J = 6.0 Hz, 2H),
2.10 (s, 1H), 2.01 (m, 1H), 1.97-1.92 (m, 1H), 1.69 (s, 1H), 0. 89 (s, 1H), 0.55-0.53 (m, 2H), 0.12-0.11
(m, 2H). 13C NMR (150 MHz, CDCl3) δ 167.5, 162.5, 157.7, 157.4, 147.4, 136.8, 134.3, 121.8, 101.1,
63.8, 62.6, 59.2, 52.9, 43.8, 39.4, 29.4, 23.2, 21.3, 8.3, 4.1. IR v (cm-1): 2930, 2851, 1587, 1496, 1441,
1335, 1133, 994, 785. MS (ESI/APCI) m/z 392.9 [M + H]+.
6.1.13.7.
2-(4-(6-((Methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)pyrimidin-4-yl)piperazin-1-yl)ethanol
(16)
1
Compound 16 was obtained as a colorless oil (yield 93%). H NMR (400 MHz, CDCl3) δ 8.48 (s,
2H), 7.37 (d, J = 7.2 Hz, 1H), 7.26 (s, 1H), 7.07 (s, 1H), 4.02 (s, 1H), 3.71-3.65 (m, 8H), 2.81-2.78 (m,
1H), 2.71-2.67 (m, 2H), 2.57 (s, 6H), 2.39 (s, 3H), 2.11 (s, 1H), 2.00 (s, 1H), 1.97-1.88 (m, 1H),
1.70-1.68 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 167.6, 162.5, 157.7, 147.4, 136.8, 134.4, 121.8,
101.1, 62.5, 59.5, 59.1, 57.9, 52.7, 43.9, 39.3, 29.4, 23.0, 21.3. IR v (cm-1): 3248, 2931, 2856, 1589,
1498, 1441, 1339, 1138, 994, 784. HRMS (ESI): calcd for C21H31N6O [M+H]+ 383.2554, found
383.2555.
6.1.13.8.
N-Methyl-N-((6-(4-methyl-1,4-diazepan-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amin
e (17)
1
Compound 17 was obtained as a colorless oil (yield 98%). H NMR (400 MHz, CDCl3) δ 8.47 (s,
2H), 7.35 (d, J = 7.2 Hz, 1H), 7.05 (s, 2H), 4.04 -4.01 (m, 1H), 3.84 (s, 1H), 3.69 (s, 1H), 3.64 (s, 3H),
2.81-2.77 (m, 1H), 2.71-2.67 (m, 3H), 2.55 (s, 2H), 2.39 (s, 3H), 2.35 (s, 3H), 2.09 (s, 1H), 1.99 (s, 1H),
1.94-1.89 (m, 4H), 1.72 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 167.1, 162.2, 157.6, 147.4, 136.7,
134.3, 121.7, 100.6, 62.6, 59.4, 57.5, 46.8, 39.4, 29.4, 23.4, 21.4. IR v (cm-1): 2933, 2857, 1588, 1497,
1442, 1349, 1124, 964, 784. MS (ESI/APCI) m/z 366.9 [M + H]+.
6.1.13.9.
N-((6-(4-(Dimethylamino)piperidin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8
-amine (18)
1
Compound 18 was obtained as a colorless oil (yield 38%). H NMR (400 MHz, CDCl3) δ 8.50-8.47
(m, 2H), 7.36 (d, J = 6.8 Hz, 1H), 7.23 (s, 1H), 7.06 (s, 1H), 4.53 (s, 2H), 4.02 (s, 1H), 3.72-3.71 (m,
1H), 3.63 (s, 2H), 2.90-2.84 (m, 3H), 2.71-2.67 (m, 1H), 2.38 (s, 4H), 2.29 (s, 6H), 2.10 (s, 1H), 2.01 (s,
1H), 1.91 (s, 3H), 1.68 (s, 1H), 1.43 (t, J = 11.0 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 167.5, 162.2,
157.8, 157.5, 147.4, 136.7, 134.3, 121.7, 101.0, 62.5, 62.4, 59.2, 43.5, 43.4, 41.7, 39.4, 29.4, 28.2, 28.1,
23.2, 21.3. IR v (cm-1): 2932, 2858, 2771, 1587, 1496, 1442, 1342, 1262, 1038, 978, 785. MS
(ESI/APCI) m/z 380.9 [M + H]+.
6.1.13.10.
N-((6-(3-(Dimethylamino)pyrrolidin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-
11

ACCEPTED MANUSCRIPT
8-amine (19)
1
Compound 19 was obtained as a colorless oil (yield 39%). H NMR (400 MHz, CDCl3) δ 8.48 (s,
2H), 7.35 (d, J = 7.2 Hz, 1H), 7.05 (s, 1H), 6.90 (s, 1H), 4.03 (s, 1H), 3.92-3.84 (m, 1H), 3.66 (s, 3H),
3.44 (s, 1H), 3.26 (s, 1H), 2.83-2.78 (m, 2H), 2.71-2.67 (m, 1H), 2.40 (s, 3H), 2.31 (s, 6H), 2.23 (s, 1H),
2.10 (s, 1H), 2.01 (s, 1H), 1.93-1.88 (m, 2H), 1.69 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 166.5, 160.5,
157.7, 147.3, 136.7, 134.3, 121.7, 101.5, 62.5, 59.2, 50.8, 45.6, 44.6, 44.5, 39.4, 29.4, 21.3. IR v (cm-1):
2932, 2863, 2773, 1591, 1502, 1445, 1323, 1156, 1037, 972, 784. MS (ESI/APCI) m/z 366.9 [M + H]+.
6.1.13.11.
N1,N1,N2-Trimethyl-N2-(6-((methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)pyrimidin-4-yl)ethan
e-1,2-diamine (20)
Compound 20 was obtained as a colorless oil (yield 97%). 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J
= 4.0 Hz, 1H), 8.47 (s, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.05 (dd, J = 7.6, 4.8 Hz, 1H), 6.98 (s, 1H),
4.04-4.01 (m, 1H), 3.69 (s, 2H), 3.65 (s, 2H), 3.09 (s, 3H), 2.81-2.80 (m, 1H), 2.78-2.71 (m, 1H), 2.47
(t, J = 7.2 Hz, 2H), 2.38 (s, 3H), 2.27 (s, 6H), 2.10-2.09 (m, 1H), 2.03-2.01 (m, 1H), 1.94-1.91 (m, 1H),
1.71-1.67 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 166.9, 162.3, 157.6, 157.5, 147.3, 136.7, 134.2,
121.7, 100.7, 62.9, 59.4, 56.4, 47.3, 45.7, 39.4, 36.1, 29.3, 23.8, 21.2. IR v (cm-1): 2934, 2860, 1590,
1509, 1442, 1412, 1349, 1119, 1007, 783.
M
S (ESI/APCI) m/z 354.9 [M + H]+.
6.1.13.12.
N-Methyl-N-((6-(2-morpholinoethylamino)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine
(21)
1
Compound 21 was obtained as a colorless oil (yield 75%). H NMR (400 MHz, CDCl3) δ 8.50 (s,
1H), 8.42 (s, 1H), 7.35 (s, 1H), 7.05 (s, 1H), 6.90 (s, 1H), 4.03 (s, 1H), 3.71 (s, 4H), 3.57 (s, 2H), 3.41
(s, 2H), 2.78 (s, 1H), 2.71-2.68 (m, 1H), 2.58 (s, 2H), 2.46 (s, 4H), 2.38 (s, 3H), 2.11 (s, 1H), 2.00 (m,
1H), 1.95-1.89 (m, 1H), 1.68 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 162.8, 158.0, 157.4, 147.4, 136.8,
134.3, 121.7, 67.0, 63.8, 63.2, 58.8, 56.8, 53.3, 39.4, 29.4, 23.3, 21.3. IR v (cm-1): 3256, 2933, 2855,
1599, 1510, 1443, 1331, 1114, 1035, 979, 785. HRMS (ESI): calcd for C21H31N6 [M+H]+ 383.2554,
found 383.2553.
6.1.14. N-Methyl-N-((6-(piperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine (12)
To a solution of 11 (270 mg, 0.6 mmol) in dichloromethane (5 mL) was added dropwise 3 M HCl/
ethyl acetate (5 mL). The mixture was stirred at room temperature for 12 h. Saturated aqueous NaHCO3
solution was added to adjust pH = 9, and the mixture was then extracted with dichloromethane (20 mL
× 3). The combined organic layers was dried over Na2SO4, filtered and evaporated. The residue was
purified by silica gel column chromatography (dichloromethane/methanol= 50/1 to 25/1) to give the
desired product (180 mg, 90%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.49 (s, 2H), 7.38 (d, J
= 7.2 Hz, 1H), 7.31 (s, 1H), 7.08 (s, 1H), 4.05-4.04 (m, 1H), 3.80-3.70 (m, 6H), 3.02-2.98 (m, 4H),
2.81-2.79 (m, 1H), 2.72-2.68 (m, 1H), 2.58 (s, 1H), 2.41 (s, 3H), 2.13 (s, 1H), 2.01 (m, 1H), 1.95-1.89
(m, 1H), 1.71-1.69 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 167.4, 162.5, 157.6, 157.3, 147.3, 136.7,
134.3, 121.7, 101.0, 62.4, 59.0, 45.7, 44.8, 39.2, 29.3, 22.9, 21.3. IR v (cm-1): 3372, 2929, 2853, 1587,
1495, 1441, 1339, 1148, 993, 785. MS (ESI/APCI) m/z 338.9 [M + H]+.
6.1.15. General procedure for the synthesis of 34a,d,e
12

ACCEPTED MANUSCRIPT
A mixture of corresponding aromatic heterocyclic compound (2.9 mmol, 1.0 eq.), NBS (566 mg, 3.2
mmol) and AIBN (50 mg, 0.3 mmol) in 10 mL of CCl4 was stirred at reflux overnight. After
concentration, the residue was diluted with water (50 mL) and extracted with dichloromethane (50 mL
× 3). The combined organic layers was dried over Na2SO4, filtered and evaporated. The residue was
purified by silica gel column chromatography (petroleum ether/ethyl acetate = 50/1) to give the desired
product.
6.1.15.1. 4-Bromo-2-(bromomethyl)pyridine (34a)
Compound 34a was obtained as a colorless oil (240 mg, 33%). 1H NMR (400 MHz, CDCl3) δ 8.39 (s,
1H), 7.65 (s, 1H), 7.39 (s, 1H), 4.64 (d, J = 3.8 Hz, 1H), 4.50 (s, 2H).
6.1.15.2. 2-(Bromomethyl)-4-chloropyrimidine (34d)
Compound 34d was obtained as a white solid (130 mg, 22%). 1H NMR (400 MHz, CDCl3) δ 8.62 (d,
J = 5.6 Hz, 1H), 7.27 (s, 1H), 4.54 (s, 2H).
6.1.15.3. 4-(Bromomethyl)-2-chloropyrimidine (34e)
Compound 34e was obtained as a white solid (150 mg, 25%). 1H NMR (400 MHz, CDCl3) δ 8.64 (s,
1H), 7.44 (s, 1H), 4.41 (s, 1H).
6.1.16. General procedure for the synthesis of 35a,d,e
A mixture of 34a/d/e (1.1 eq.), 31 (1.0 eq.), KI (0.1 eq.) and DIPEA (2.5 eq.) in MeCN (10 mL) was
stirred at 50 °C overnight. The reaction solution was evaporated to remove most of MeCN, diluted with
water (50 mL) and extracted with dichloromethane (50 mL × 3). The combined organic layers was
dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column
chromatography (dichloromethane/methanol = 100/1 to 100/3) to give the desired product.
6.1.16.1. N-((4-Bromopyridin-2-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (35a)
Compound 35a was obtained as a yellow oil (yield 56%). 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J =
4.0 Hz, 1H), 8.27 (d, J = 5.2 Hz, 1H), 7.91 (s, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.28 (d, J = 4.8 Hz, 1H),
7.06 (dd, J = 7.2, 4.8 Hz, 1H), 4.05-4.01 (m, 1H), 3.80 -3.70 (m, 2H), 2.83-2.78 (m, 1H), 2.72-2.68 (m,
1H), 2.38 (s, 3H), 2.16-2.13 (m, 1H), 2.06-2.03 (m, 1H), 1.97-1.89 (m, 1H), 1.73-1.70 (m, 1H).
6.1.16.2. N-((4-Chloropyrimidin-2-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (35d)
Compound 35d was obtained as an brown oil (yield 84%). 1H NMR (400 MHz, CDCl3) δ 8.62 (d, J
= 4.8 Hz, 1H), 8.46 (d, J = 2.8 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 4.8 Hz, 1H), 7.09-7.06 (m,
1H), 4.28-4.26 (m, 1H), 4.22 (s, 2H), 4.15-4.12 (m, 1H), 2.84-2.78 (m, 1H), 2.72-2.68 (m, 1H), 2.50 (s,
3H), 2.25-2.24 (m, 1H), 2.04-1.96 (m, 2H), 1.73-1.70 (m, 1H).
6.1.16.3. N-((2-Chloropyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (35e)
Compound 35e was obtained as a yellow oil (yield 30%). 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J =
4.8 Hz, 1H), 8.48 (d, J = 3.6 Hz, 1H), 7.83 (d, J = 4.8 Hz, 1H), 7.37 (d, J = 7.6 Hz, 1H), 7.07 (dd, J =
7.2, 4.8 Hz, 1H), 4.03-3.99 (m, 1H), 3.76 (s, 2H), 2.85-2.77 (m, 1H), 2.73-2.69 (m, 1H), 2.41 (s, 3H),
2.16-2.13 (m, 1H), 2.04-2.02 (m, 1H), 1.93-1.84 (m, 1H), 1.72-1.69 (m, 1H).
13

ACCEPTED MANUSCRIPT
6.1.17. General procedure for the synthesis of 35b,c
A mixture of corresponding aldehyde (1.1 eq.), 31 (1.0 eq.), AcOH (1.0 eq.) in 1,2-dichloroethane
(10 mL) was stirred at 0 °C for 10 min. NaBH(OAc)3 (1.5 eq.) was then added to the reaction solution
at 0 °C. The resulting suspension was stirred at room temperature overnight. The reaction solution was
diluted with water (10 mL) and extracted with dichloromethane (30 mL × 3). The combined organic
layers 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.
6.1.17.1. N-((2-Chloropyridin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (35b)
Compound 35b was obtained as a yellow oil (yield 91%). 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H),
8.25 (d, J = 4.0 Hz, 1H), 7.39-7.35 (m, 2H), 7.27 (s, 1H), 7.07 (s, 1H), 3.95 (s, 1H), 3.73-3.58 (m, 2H),
2.79-2.69 (m, 2H), 2.30 (s, 3H), 2.06 (s, 2H), 1.92-1.89 (m, 1H), 1.70 (s, 1H).
6.1.17.2. N-((6-Bromopyridin-2-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (35c)
Compound 35c was obtained as a yellow oil (yield 87%). 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H),
7.73 (d, J = 7.6 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H),
7.05 (t, J = 5.8 Hz, 1H), 4.03 (t, J = 7.0 Hz, 1H), 3.73 (s, 2H), 2.81-2.77 (m, 1H), 2.72-2.68 (m, 1H),
2.39 (s, 3H), 2.12 (s, 1H), 2.01(s, 1H), 1.95-1.87 (m, 1H), 1.71 (s, 1H).
6.1.18. General procedure for the synthesis of 4-8
A mixture of 35a-e (1.0 eq.), DIPEA (10.0 eq.) and 1-methylpiperazine (4.5 eq.) in NMP (2 mL) was
stirred at 200 °C under microwave for 2 h. The reaction was diluted with ethyl acetate (50 mL) and
washed with brine (30 mL × 5). The organic layer was dried over Na2SO4, filtered and evaporated. The
residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/1 to 100/3)
to give the desired product.
6.1.18.1.
N-Methyl-N-((4-(4-methylpiperazin-1-yl)pyridin-2-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine (4)
Compound 4 was obtained as a colorless oil (yield 43%). 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J =
4.0 Hz, 1H), 8.15 (d, J = 4.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.05 (dd, J = 8.0, 4.0 Hz,
1H), 6.51 (d, J = 4.0 Hz, 1H), 4.07-4.05 (m, 1H), 3.70 (s, 2H), 3.39 (t, J = 4.0 Hz, 4H), 2.81-2.79 (m,
1H), 2.77-2.71 (m, 1H), 2.52 (t, J = 6.0 Hz, 4H), 2.37 (s, 3H), 2.34 (s, 3H), 2.13-2.11 (m, 1H),
2.02-2.00 (m, 1H), 1.99-1.80 (m, 1H), 1.70-1.67 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 160.9, 157.8,
155.9, 149.1, 147.4, 136.7, 134.4, 121.7, 107.5, 106.9, 62.7, 59.8, 54.8, 46.3, 46.16, 39.3, 29.5, 22.9,
21.4. IR v (cm-1): 2932, 2840, 2794, 1594, 1543, 1443, 1378, 1293, 1257, 1141, 994, 786. MS
(ESI/APCI) m/z 351.9 [M + H]+.
6.1.18.2.
N-Methyl-N-((2-(4-methylpiperazin-1-yl)pyridin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine (5)
1
Compound 5 was obtained as a yellow oil (yield 31%). H NMR (400 MHz, CDCl3) δ 8.51 (d, J =
4.0 Hz, 1H), 8.07 (d, J = 5.2 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.06 (dd, J = 7.2, 4.8 Hz, 1H), 6.86 (s,
1H), 6.67 (d, J = 4.8 Hz, 1H), 3.99-3.96 (m, 1H), 3.67-3.64 (m, 1H), 3.56 (s, 4H), 3.52-3.48 (m, 1H),
2.84-2.78 (m, 1H), 2.71-2.67 (m, 1H), 2.51 (t, J = 4.6 Hz, 4H), 2.34 (s, 3H), 2.31 (s, 3H), 2.04-2.02 (m,
2H), 1.96-1.90 (m, 1H), 1.73-1.69 (m, 1H). 13C NMR (150 MHz, CDCl3) δ160.0, 157.7, 151.2, 147.6,
14

ACCEPTED MANUSCRIPT
147.2, 136.7, 134.2, 121.7, 114.1, 107.0, 62.9, 58.0, 55.1, 46.3, 45.4, 39.2, 29.2, 24.3, 21.0. IR v (cm-1):
2933, 2838, 2791, 1599, 1555, 1426, 1286, 1252, 1141, 995, 785. MS (ESI/APCI) m/z 351.9 [M + H]+.
6.1.18.3.
N-Methyl-N-((6-(4-methylpiperazin-1-yl)pyridin-2-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine (6)
Compound 6 was obtained as a colorless oil (yield 23%). 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J =
4.4 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 4.8 Hz, 1H), 7.00 (d, J = 7.2
Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 4.03 (s, 1H), 3.64 (q, J = 15.0 Hz, 2H), 3.53 (s, 4H), 2.82-2.78 (m,
1H), 2.71-2.66 (m, 1H), 2.50 (t, J = 4.4 Hz, 5H), 2.37 (s, 3H), 2.33 (s, 3H), 2.07 (s, 1H), 2.02-2.98 (m,
1H), 1.95-1.92 (m, 1H), 1.65 (s, 1H). 13C NMR (150 MHz, CDCl3) δ159.0, 147.3, 138.0, 136.6, 134.1,
121.6, 112.5, 104.8, 63.4, 60.1, 55.1, 46.3, 45.4, 39.5, 29.2, 23.9, 21.0. IR v (cm-1): 2932, 2838, 2792,
1587, 1570, 1443, 1250, 1142, 984, 781. HRMS (ESI): calcd for C21H30N5 [M+H]+ 352.2496, found
352.2514.
6.1.18.4.
N-Methyl-N-((4-(4-methylpiperazin-1-yl)pyrimidin-2-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine
(7)
1
Compound 7 was obtained as a yellow oil (yield 57%). H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H),
8.18 (d, J = 6.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.05-7.02 (m, 1H), 6.32 (d, J = 6.4 Hz, 1H),
4.15-4.12 (m, 1H), 3.81 (s, 2H), 3.65 (s, 4H), 2.87-2.77 (m, 1H), 2.72-2.69 (m, 1H), 2.65-2.44 (m, 7H),
2.32 (s, 3H), 2.11-1.98 (m, 3H), 1.73-1.64 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 168.1, 161.9, 157.7,
155.93, 147.0, 136.7, 134.2, 121.6, 100.8, 63.7, 61.5, 54.8, 46.3, 43.8, 39.2, 29.3, 24.9, 21.2. IR v
(cm-1): 2935, 2849, 2793, 1580, 1544, 1445, 1288, 1260, 1142, 980, 784. HRMS (ESI): calcd for
C20H29N6 [M+H]+ 353.2448, found 353.2448.
6.1.18.5.
N-Methyl-N-((2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amine
(8)
Compound 8 was obtained as a colorless oil (yield 81%). 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J =
4.0 Hz, 1H), 8.23 (d, J = 4.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.06-7.03 (m, 1H), 6.92 (d, J = 4.8 Hz,
1H), 4.02-3.99 (m, 1H), 3.81 (s, 4H), 3.58 (q, J = 14.4 Hz, 2H), 2.85-2.77 (m, 1H), 2.71-2.67 (m, 1H),
2.44 (t, J = 4.6 Hz, 4H), 2.37 (s, 3H), 2.32 (s, 3H), 2.07-2.02 (m, 2H), 1.97-1.89 (m, 1H), 1.77 (s, 1H).
13C NMR (150 MHz, CDCl3) δ170.4, 161.6, 157.7, 147.3, 136.7, 134.1, 121.7, 108.7, 63.6, 59.8, 55.1,
46.3, 43.7, 39.7, 29.2, 24.5, 21.0. IR v (cm-1): 2933, 2843, 2789, 1572, 1554, 1489, 1441, 1339, 1285,
1265, 1143, 995, 783. MS (ESI/APCI) m/z 352.9 [M + H]+.
6.1.19. (S)-N-((S)-1-(4-Methoxyphenyl)ethyl)-5,6,7,8-tetrahydroquinolin-8-amine (37)
A slurry of sodium triacetoxyborohydride (3.55 g, 16.7 mmol) in dichloromethane (60 mL) was
treated with 36 [31] (1.37 g, 9.3 mmol), followed by (S)-1-(4-methoxyphenyl)ethanamine (1.41 g, 9.3
mmol). The reaction was stirred vigorously at room temperature overnight. The reaction was quenched
with 1 N NaOH (20 mL) to adjust pH = 8. The organic layer was separated, dried over Na2SO4, and
concentrated. The resulting residue was purified by silica gel column chromatography
25
(dichloromethane/methanol = 50/1) to give the desired product (1.5 g, 58%) as a yellow oil. [α]D
=
+34 (c = 0.2, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 4.0 Hz, 1H), 7.47 (d, J = 7.2 Hz,
15

ACCEPTED MANUSCRIPT
1H), 7.31 (d, J = 8.4 Hz, 2H), 7.20-7.15 (m, 1H), 6.85 (d, J = 8.8 Hz, 2H), 4.04-3.99 (m, 1H), 3.72 (s,
3H), 3.65-3.62 (m, 1H), 2.75-2.69 (m, 2H), 1.83-1.73 (m, 1H), 1.69-1.59 (m, 1H), 1.57-1.48 (m, 1H),
1.45-1.38 (m, 1H), 1.26 (d, J = 7.2 Hz, 3H).
6.1.20. (S)-N-Methyl-5,6,7,8-tetrahydroquinolin-8-amine (38)
A slurry of sodium triacetoxyborohydride (1.62 g, 7.7 mmol) and 37 (1.45 g, 5.1 mmol) in
dichloromethane (30 mL) was cooled to 0 °C. Formaldehyde solution (37 wt.% in water, 1.2 g, 10.2
mmol) was added slowly to maintain the temperature < 25 °C. The solution was stirred at room
temperature for 5 h. Trifluoroacetic acid (10 mL) was added and the resulting mixture was stirred at
room temperature for another 10 h. The reaction mixture was concentrated and 1 N NaOH (20 mL) was
added. The aqueous layer was extracted with dichloromethane (30 mL × 3). The combined organic
layers was dried over Na2SO4 and concentrated. The residue was purified by silica gel column
chromatography (dichloromethane/methanol = 50/1) to give the desired product (520 mg, 43%) as a
1
yellow oil. [α]D25 = +112 (c = 0.2, MeOH). H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 3.6 Hz, 1H),
7.36 (d, J = 7.6 Hz, 1H), 7.07-7.03 (m, 1H), 3.68-3.59 (m, 1H), 2.84- 2.65 (m, 2H), 2.52 (s, 3H),
2.15-2.07 (m, 1H), 2.00-1.91 (m, 1H), 1.81-1.69 (m, 2H).
6.1.21. (S)-N-((6-chloropyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (39)
A mixture of 38 (162 mg, 1 mmol), 26 (162 mg, 1 mmol), KI (16 mg, 0.1 mmol) and DIPEA (320
mg, 2.5 mmol) in MeCN (10 mL) was stirred at room temperature overnight. The reaction solution was
evaporated to remove most of MeCN. The residue was diluted with water (30 mL) and extracted with
dichloromethane (30 mL × 3). The combined organic layers was dried over Na2SO4, filtered and
evaporated. The residue was purified by silica gel column chromatography (dichloromethane/methanol
= 100/1-50/1) to give the desired product (150 mg, 52%) as a yellow oil. [α]D25 = -58 (c = 0.2, MeOH).
1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.50 (d, J = 3.6 Hz, 1H), 7.89 (s, 1H), 7.37 (d, J = 7.6 Hz,
1H), 7.09-7.06 (m, 1H), 4.07-3.97 (m, 1H), 3.80 (s, 2H), 2.86-2.78 (m, 1H), 2.75-2.68 (m, 1H), 2.41 (s,
3H), 2.20-2.11 (m, 1H), 2.10-1.99 (m, 1H), 1.98-1.85 (m, 1H), 1.74-1.68 (m, 1H).
6.1.22.
(S)-N-Methyl-N-((6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amin
e ((S)-3)
To a solution of 39 (120 mg, 0.42 mmol) in EtOH (8 mL) was added Et3N (424 mg, 4.2 mmol) and
N-methyl piperazine (210 mg, 2.1 mmol). The reaction mixture was stirred at 80 °C overnight, and then
quenched with saturated NaHCO3 aqueous solution (50 mL). The aqueous layer was extracted with
dichloromethane (20 mL × 3). The combined organic layers was washed with brine, dried over Na2SO4
and concentrated. The residue was purified by silica gel column chromatography
25
(dichloromethane/methanol = 25/1) to give the desired product (70 mg, 48%) as a colorless oil. [α]D
=
1
-128 (c = 0.2, MeOH). H NMR (400 MHz, CDCl3) δ 8.48 (s, 2H), 7.35 (s, 1H), 7.22 (s, 1H), 7.06 (s,
1H), 4.08-3.95 (m, 1H), 3.71 (s, 4H), 3.64 (s, 2H), 2.87-2.75 (m, 1H), 2.75-2.64 (m, 1H), 2.46 (s, 4H),
2.39 (s, 3H), 2.33 (s, 3H), 2.19-2.05 (m, 1H), 2.05-1.95 (m, 1H), 1.95-1.87 (m, 1H), 1.75-1.62 (m, 1H).
13C NMR (150 MHz, CDCl3) δ 167.4 162.4, 157.6, 157.3, 147.2, 136.6, 134.2, 121.6, 100.9, 62.4, 59.0,
54.7, 46.1, 43.7, 39.2, 29.3, 23.1, 21.2. IR v (cm-1): 2933, 2847, 2791, 1587, 1523, 1441, 1338, 1141,
995, 784. HRMS (ESI): calcd for C20H29N6 [M+H]+ 353.2448, found 353.2515.
16

ACCEPTED MANUSCRIPT
6.1.23. (R)-N-((R)-1-(4-Methoxyphenyl)ethyl)-5,6,7,8-tetrahydroquinolin-8-amine (40)
A slurry of sodium triacetoxyborohydride (1.3 g, 6.1 mmol) in dichloromethane (25 mL) was treated
with 36 (500 mg, 3.4 mmol), followed by (R)-1-(4-methoxyphenyl)ethanamine (513 mg, 3.4 mmol).
The reaction was stirred vigorously at room temperature overnight. The reaction was quenched with 1
N NaOH (10 mL) to adjust pH = 8. The organic layer was separated, dried over Na2SO4, and
concentrated. The resulting residue was purified by silica gel column chromatography
(dichloromethane/methanol = 50/1) to give the title product (320 mg, 33%) as a yellow oil. [α]D25 = -28
(c = 0.2, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 1H), 7.50 (d, J = 6.8 Hz, 1H), 7.40 (s, 2H),
7.22 (s, 1H), 6.89 (d, J = 7.6 Hz, 2H), 4.28 (s, 1H), 3.84-3.65 (m, 4H), 2.69 (s, 2H), 1.81 (s, 2H), 1.57(s,
2H), 1.37 (s, 3H).
6.1.24. (R)-N-Methyl-5,6,7,8-tetrahydroquinolin-8-amine (41)
A slurry of sodium triacetoxyborohydride (382 mg, 2 mmol) and 40 (300 mg, 1.1 mmol) in
dichloromethane (10 mL) was cooled to 0 °C. Formaldehyde solution (37 wt.% in water, 431 mg, 5.3
mmol) was added slowly to maintain the temperature < 25 °C. The solution was stirred at room
temperature for 5 h. Trifluoroacetic acid (5 mL) was added and the resulting mixture was stirred at
room temperature for another 10 h. The reaction mixture was concentrated and 1 N NaOH (10 mL) was
added. The aqueous layer was extracted with dichloromethane (20 mL × 3). The combined organic
layers was dried over Na2SO4 and concentrated. The residue was purified by silica gel column
chromatography (dichloromethane/methanol = 50/1) to give the desired product (80 mg, 45%) as a
1
yellow oil. [α]D25 = -98 (c = 0.2, MeOH). H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.46 (d, J = 4.0
Hz, 1H), 7.20-7.11 (m, 1H), 3.91-3.53 (m, 1H), 2.95-2.77 (m, 2H), 2.73 (s, 3H), 2.27-2.05 (m, 2H),
1.91-1.67 (m, 2H).
6.1.25. (R)-N-((6-Chloropyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (42)
A mixture of 41 (70 mg, 0.43 mmol), 26 (69 mg, 0.43 mmol), KI (7 mg, 0.04 mmol) and DIPEA
(139 mg, 1.1 mmol) in MeCN (5 mL) was stirred at room temperature overnight. The reaction solution
was evaporated to remove most of MeCN. The resulting residue was diluted with water (20 mL) and
extracted with dichloromethane (20 mL × 3). The combined organic layers was dried over Na2SO4,
filtered and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol = 100/1-50/1) to give the desired product (60 mg, 49%) as a yellow oil.
1
[α]D25 = +56 (c = 0.2, MeOH). H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.51 (s, 1H), 7.89 (s, 1H),
7.38 (d, J = 7.6 Hz, 1H), 7.09-7.06 (m, 1H), 4.07-3.97 (m, 1H), 3.80 (s, 2H), 2.86-2.78 (m, 1H),
2.75-2.68 (m, 1H), 2.42 (s, 3H), 2.20-2.11 (m, 1H), 2.10-1.99 (m, 1H), 1.98-1.85 (m, 1H), 1.74-1.68 (m,
1H).
6.1.26.
(R)-N-Methyl-N-((6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-amin
e ((R)-3)
To a solution of 42 (50 mg, 0.2 mmol) in EtOH (5 mL) was added Et3N (200 mg, 2.0 mmol) and
N-methyl piperazine (100 mg, 1.0 mmol). The reaction was stirred at 80 °C overnight, and then
quenched with saturated NaHCO3 aqueous solution (30 mL). The aqueous layer was extracted with
dichloromethane (20 mL × 3). The combined organic layers was washed with brine, dried over Na2SO4
and concentrated. The residue was purified by silica gel column chromatography
17

ACCEPTED MANUSCRIPT
25
(dichloromethane/methanol = 25/1) to give the desired product (30 mg, 43%) as a colorless oil. [α]D
=
+138 (c = 0.2, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.48 (s, 2H), 7.37 (s, 1H), 7.22 (s, 1H), 7.07 (s,
1H), 4.08-3.95 (m, 1H), 3.73 (s, 4H), 3.67 (s, 2H), 2.87-2.75 (m, 1H), 2.75-2.64 (m, 1H), 2.46 (d, J =
4.0 Hz, 4H), 2.41 (s, 3H), 2.34 (s, 3H), 2.21-2.07 (m, 1H), 2.05-1.95 (m, 2H), 1.95-1.65 (m, 1H). 13C
NMR (150 MHz, CDCl3) δ 167.2 162.4, 157.5, 157.3, 147.2, 136.7, 134.2, 121.7, 101.1, 62.5, 59.0,
54.7, 46.1, 43.7, 39.2, 29.2, 23.0, 21.2. IR v (cm-1) 2934, 2845, 2791, 1587, 1523, 1441, 1338, 1141,
995, 784. HRMS (ESI): calcd for C20H29N6 [M+H]+ 353.2448, found 353.2515.
6.2. In vitro biological assays
6.2.1. 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 CXCR4. Then the compounds for testing 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 was then added into the wells
and with the addition of APC-conjugated anti-human CXCR4 clone 12G5 at its EC80 determined. The
mix 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.
6.2.2. 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 4 mM and a
bottom concentration of 0.01 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 10 µM to 0.035 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 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 and CXCL12 were added to the plates. 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
18

ACCEPTED MANUSCRIPT
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.
6.2.3. Matrigel invasion assay
The human breast cancer cell lines MDA-MB-231, MCF-7 and human ovarian epithelial cell line
HOSEpiC were purchased from ATCC (Manassas, VA). MDA-MB-231, MCF-7 cell lines were
cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml
of penicillin and 100 mg/ml of streptomycin. HOSEpiC cell line was cultured in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml of penicillin and 100 mg/ml
of streptomycin. Cells were cultured at 37 °C in a humidified atmosphere of 20% O2/5% CO2. All
cultures were monitored routinely and found to be free of contamination by mycoplasma or fungi,
discarded after three months, and new lines propagated from frozen stocks.
Matrigel invasion assays were carried out in modified Boyden chambers with filter inserts with 8-µm
pores in 24-well plates (Corning, NY, USA). The surfaces of the filters were coated with 50mg/L
ice-cold Matrigel (Matrigel basement membrane matrix, BD Bioscience, NJ, USA). The lower chamber
was filled with medium containing 10% serum. The target compounds (100 nM) and the human breast
cancer cell line MDA-MB-231 cells (1 × 105 cells/well) were added to in the upper chamber of a vessel
and CXCL12 was added in the lower chamber as a chemoattractant in serum free medium. After 24 h
incubation, the filters were gently removed from the chambers, and the cells on the upper surface were
removed by wiping with a cotton swab. Cells that had invaded to the lower surface areas were fixed
with ice cold methanol, stained with crystal violet, and counted in 10 randomly selected fields under a
microscope (100×). Results shown are representative of three independent experiments.
6.2.4. Cell viability (MTT) assay
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Briefly, cells were plated at a density of 5 × 103 cells/well on 96-well plates and subjected to compound
3 treatment. Following 48 h incubation at 37 ºC in a humidified atmosphere containing 5% CO2/95%
air,
the
samples
were
incubated
for
another
4
h
with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent. The formazan product was
dissolved in dimethyl sulfoxide and read at 570 nm on a Victor3 Multi Label plate reader (PerkinElmer,
Boston, MA, USA).
6.3. Preliminary in vitro safety test
CYP inhibitory potency and human/mouse liver microsomes metabolic stability were evaluated as
previously reported [32]. hERG inhibition and Caco-2 assays were reported before [33].
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No. 81773561,
81473090, 21502133), China Postdoctoral Science Foundation (2016M601884), the Priority Academic
Program Development of the Jiangsu Higher Education Institutes (PAPD) and the Jiangsu Key
Laboratory of Neuropsychiatric Diseases (BM2013003).
Appendix A. Supplementary data
19

ACCEPTED MANUSCRIPT
Supplementary data related to this article can be found at
References
[1] A.A. Haqqani, J.C. Tilton, Entry inhibitors and their use in the treatment of HIV-1 infection,
Antiviral Res. 98 (2013) 158-170.
[2] T. Lapidot, O. Kollet, The essential roles of the chemokine SDF-1 and its receptor CXCR4 in
human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and
NOD/SCID/B2m(null) mice, Leukemia 16 (2002) 1992-2003.
[3] O. Kollet, I. Petit, J. Kahn, S. Samira, A. Dar, A. Peled, V. Deutsch, M. Gunetti, W. Piacibello, A.
Nagler, T. Lapidot, Human CD34(+)CXCR4(-) sorted cells harbor intracellular CXCR4, which can be
functionally expressed and provide NOD/SCID repopulation, Blood 100 (2002) 2778-2786.
[4] T. Pozzobon, G. Goldoni, A. Viola, B. Molon, CXCR4 signaling in health and disease, Immunol.
Lett. 177 (2016) 6-15.
[5] W.T. Choi, S. Duggineni, Y. Xu, Z. Huang, J. An, Drug discovery research targeting the CXC
chemokine receptor 4 (CXCR4), J. Med. Chem. 55 (2012) 977-994.
[6] B. Debnath, S. Xu, F. Grande, A. Garofalo, N. Neamati, Small molecule inhibitors of CXCR4,
Theranostics 3 (2013) 47-75.
[7] N. Nagarsheth, M.S. Wicha, W. Zou, Chemokines in the cancer microenvironment and their
relevance in cancer immunotherapy, Nat. Rev. Immunol. 17 (2017) 559-572.
[8] F. Grande, G. Giancotti, G. Ioele, M.A. Occhiuzzi, A. Garofalo, An update on small molecules
targeting CXCR4 as starting points for the development of anti-cancer therapeutics, Eur. J. Med. Chem.
139 (2017) 519-530.
[9] H. Zhang, D. Kang, B. Huang, N. Liu, F. Zhao, P. Zhan, X. Liu, Discovery of non-peptide small
molecular CXCR4 antagonists as anti-HIV agents: Recent advances and future opportunities, Eur. J.
Med. Chem. 114 (2016) 65-78.
[10] S. Hotte, H. Hirte, A. Iacobucci, D. Wong, L. Cantin, W. Korz, W. Miller, Phase I/II study of
CTCE-9908, a novel anticancer agent that inhibits CXCR4, in patients with advanced solid cancers, in,
AACR, 2007.
[11] D. Karpova, S. Brauninger, E. Wiercinska, A. Kramer, B. Stock, J. Graff, H. Martin, A. Wach, C.
Escot, G. Douglas, B. Romagnoli, E. Chevalier, K. Dembowski, L. Hooftman, H. Bonig, Mobilization
of hematopoietic stem cells with the novel CXCR4 antagonist POL6326 (balixafortide) in healthy
volunteers-results of a dose escalation trial, J. Transl. Med. 15 (2017) 2.
[12] J.F. DiPersio, G.L. Uy, U. Yasothan, P. Kirkpatrick, Plerixafor, Nat. Rev. Drug Discov. 8 (2009)
105-106.
[13] K. Jung, T. Heishi, J. Incio, Y. Huang, E.Y. Beech, M. Pinter, W.W. Ho, K. Kawaguchi, N.N.
Rahbari, E. Chung, J.K. Kim, J.W. Clark, C.G. Willett, S.H. Yun, A.D. Luster, T.P. Padera, R.K. Jain, D.
Fukumura, Targeting CXCR4-dependent immunosuppressive Ly6C(low) monocytes improves
antiangiogenic therapy in colorectal cancer, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) 10455-10460.
[14] Y. Chen, R.R. Ramjiawan, T. Reiberger, M.R. Ng, T. Hato, Y. Huang, H. Ochiai, S. Kitahara, E.C.
Unan, T.P. Reddy, C. Fan, P. Huang, N. Bardeesy, A.X. Zhu, R.K. Jain, D.G. Duda, CXCR4 inhibition
in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in
sorafenib-treated hepatocellular carcinoma in mice, Hepatology 61 (2015) 1591-1602.
20

ACCEPTED MANUSCRIPT
[15] F. Grande, I. Barone, F. Aiello, A. Brancale, M. Cancellieri, M. Badolato, F. Chemi, C. Giordano,
V. Vircillo, D. Bonofiglio, A. Garofalo, S. Ando, S. Catalano, Identification of novel
2-(1H-indol-1-yl)-benzohydrazides CXCR4 ligands impairing breast cancer growth and motility,
Future Med. Chem. 8 (2016) 93-106.
[16] N.D. Stone, S.B. Dunaway, C. Flexner, C. Tierney, G.B. Calandra, S. Becker, Y.J. Cao, I.P.
Wiggins, J. Conley, R.T. MacFarland, J.G. Park, C. Lalama, S. Snyder, B. Kallungal, K.L. Klingman,
C.W. Hendrix, Multiple-dose escalation study of the safety, pharmacokinetics, and biologic activity of
oral AMD070, a selective CXCR4 receptor inhibitor, in human subjects, Antimicrob. Agents
Chemother. 51 (2007) 2351-2358.
[17] R.T. Skerlj, G.J. Bridger, A. Kaller, E.J. McEachern, J.B. Crawford, Y. Zhou, B. Atsma, J. Langille,
S. Nan, D. Veale, T. Wilson, C. Harwig, S. Hatse, K. Princen, E. De Clercq, D. Schols, Discovery of
novel small molecule orally bioavailable C-X-C chemokine receptor 4 antagonists that are potent
inhibitors of T-tropic (X4) HIV-1 replication, J. Med. Chem. 53 (2010) 3376-3388.
[18] G. Thoma, M.B. Streiff, J. Kovarik, F. Glickman, T. Wagner, C. Beerli, H.G. Zerwes, Orally
bioavailable isothioureas block function of the chemokine receptor CXCR4 in vitro and in vivo, J. Med.
Chem. 51 (2008) 7915-7920.
[19] Z. Liang, W. Zhan, A. Zhu, Y. Yoon, S. Lin, M. Sasaki, J.M. Klapproth, H. Yang, H.E.
Grossniklaus, J. Xu, M. Rojas, R.J. Voll, M.M. Goodman, R.F. Arrendale, J. Liu, C.C. Yun, J.P. Snyder,
D.C. Liotta, H. Shim, Development of a unique small molecule modulator of CXCR4, PLoS One 7
(2012) e34038.
[20] T. Murakami, S. Kumakura, T. Yamazaki, R. Tanaka, M. Hamatake, K. Okuma, W. Huang, J.
Toma, J. Komano, M. Yanaka, Y. Tanaka, N. Yamamoto, The novel CXCR4 antagonist KRH-3955 is an
orally bioavailable and extremely potent inhibitor of human immunodeficiency virus type 1 infection:
comparative studies with AMD3100, Antimicrob. Agents Chemother. 53 (2009) 2940-2948.
[21] P.S. Charifson, W.P. Walters, Acidic and basic drugs in medicinal chemistry: a perspective, J. Med.
Chem. 57 (2014) 9701-9717.
[22] S. Jenkinson, M. Thomson, D. McCoy, M. Edelstein, S. Danehower, W. Lawrence, P. Wheelan, A.
Spaltenstein, K. Gudmundsson, Blockade of X4-tropic HIV-1 cellular entry by GSK812397, a potent
noncompetitive CXCR4 receptor antagonist, Antimicrob. Agents Chemother. 54 (2010) 817-824.
[23] K.S. Gudmundsson, S.D. Boggs, J.G. Catalano, A. Svolto, A. Spaltenstein, M. Thomson, P.
Wheelan, S. Jenkinson, Imidazopyridine-5,6,7,8-tetrahydro-8-quinolinamine derivatives with potent
activity against HIV-1, Bioorg. Med. Chem. Lett. 19 (2009) 6399-6403.
[24] V.M. Truax, H. Zhao, B.M. Katzman, A.R. Prosser, A.A. Alcaraz, M.T. Saindane, R.B. Howard, D.
Culver, R.F. Arrendale, P.R. Gruddanti, T.J. Evers, M.G. Natchus, J.P. Snyder, D.C. Liotta, L.J. Wilson,
Discovery of tetrahydroisoquinoline-based CXCR4 antagonists, ACS Med. Chem. Lett. 4 (2013)
1025-1030.
[25] K.S. Gudmundsson, P.R. Sebahar, L.D. Richardson, J.F. Miller, E.M. Turner, J.G. Catalano, A.
Spaltenstein,
W.
Lawrence,
M.
Thomson,
S.
Jenkinson,
Amine
substituted
N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines as CXCR4 antagonists with
potent activity against HIV-1, Bioorg. Med. Chem. Lett. 19 (2009) 5048-5052.
[26] S. Boggs, V.I. Elitzin, K. Gudmundsson, M.T. Martin, M.J. Sharp, Kilogram-Scale Synthesis of
the CXCR4 Antagonist GSK812397, Organic Process Research & Development 13 (2009) 781-785.
[27] A. Van Hout, T. D'Huys, M. Oeyen, D. Schols, T. Van Loy, Comparison of cell-based assays for
the identification and evaluation of competitive CXCR4 inhibitors, PLoS One 12 (2017) e0176057.
21

ACCEPTED MANUSCRIPT
[28] J.F. Miller, K.S. Gudmundsson, L. D'Aurora Richardson, S. Jenkinson, A. Spaltenstein, M.
Thomson, P. Wheelan, Synthesis and SAR of novel isoquinoline CXCR4 antagonists with potent
anti-HIV activity, Bioorg. Med. Chem. Lett. 20 (2010) 3026-3030.
[29] K. Princen, S. Hatse, K. Vermeire, E. De Clercq, D. Schols, Evaluation of SDF-1/CXCR4-induced
Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry, Cytometry A 51
(2003) 35-45.
[30] J.F. Miller, E.M. Turner, K.S. Gudmundsson, S. Jenkinson, A. Spaltenstein, M. Thomson, P.
Wheelan, Novel N-substituted benzimidazole CXCR4 antagonists as potential anti-HIV agents, Bioorg.
Med. Chem. Lett. 20 (2010) 2125-2128.
[31] E.J. McEachern, W. Yang, G. Chen, R.T. Skerlj, G.J. Bridger, Convenient synthesis of
5,6,7,8-tetrahydroquinolin-8-ylamine and 6,7-dihydro-5H-quinolin-8-one, Synth. Commun. 33 (2003)
3497-3502.
[32] Y. Dong, K. Li, Z. Xu, H. Ma, J. Zheng, Z. Hu, S. He, Y. Wu, Z. Sun, L. Luo, J. Li, H. Zhang, X.
Zhang, Exploration of the linkage elements of porcupine antagonists led to potent Wnt signaling
pathway inhibitors, Bioorg. Med. Chem. 23 (2015) 6855-6868.
[33] W. Lu, Y. Liu, H. Ma, J. Zheng, S. Tian, Z. Sun, L. Luo, J. Li, H. Zhang, Z.J. Yang, X. Zhang,
Design, Synthesis, and Structure-Activity Relationship of Tetrahydropyrido[4,3-d]pyrimidine
Derivatives as Potent Smoothened Antagonists with in Vivo Activity, ACS Chem. Neurosci. 8 (2017)
1980-1994.
22

ACCEPTED MANUSCRIPT
Figure Captions
Fig. 1 Examples of small molecule CXCR4 antagonists reported in the literature
Fig. 2 Scaffold hybridization approach to a novel series of CXCR4 antagonists
Fig. 3 Dose response curves of compound 3 and AMD3100 in the FLIPR Tetra calcium mobilization
assay
Fig. 4 Effect of selected compounds on matrigel invasion of MDA-MB-231 cells. The selected
compounds (100 nM) and cells (1 × 105 cells/well) were added to in the upper chamber of a vessel and
CXCL12 was added in the lower chamber, and their ability to invade was determined as described in
Experimental protocols. P-values were determined by t-test. *P < 0.05, **P < 0.01 compared with
control
Fig. 5 Cytotoxicity evaluation of compound 3 in MDA-MB-231, MCF-7 and HOSEpiC cells.
MDA-MB-231, MCF-7 and human ovarian epithelial cells HOSEpiC were treated with compound 3 as
indicated for 24 h. Cell viability was measured by MTT assay. Bars are mean±SD of three
independent experiments
23

ACCEPTED MANUSCRIPT
Table 1. SAR of the central ring of designed CXCR4 compounds
12G5 binding
IC50 (nM) a
Compd
X
Y
Z
clogP c
pKa d
3
N
N
N
CH
N
CH
CH
CH
N
54 ± 8
2.0
2.5
2.4
2.5
1.6
2.5
6.7
10.2
8.4
4
200 ± 81
> 10000
> 10000
180 ± 30
> 10000
290 ± 53
5
CH
CH
N
6
CH
CH
N
7.8
7
N
7.0
8
CH
N
6.6
AMD3100
a
Inhibition of luminescence signaling in HPB-ALL CXCR4 competitive binding assay. Data are
expressed as geometric mean values of at least two runs ± the standard error measurement (SEM).
b AMD3100 was run as standard in each assay. Data are expressed as geometric mean values of nine
runs ± the standard error measurement (SEM).
c Calculated by Molinspiration.
d Calculated by ACD/Labs 6.0.
24

ACCEPTED MANUSCRIPT
Table 2. SAR of the pyrimidine substitution of designed CXCR4 compounds
12G5
12G5
binding
Compd
R1
binding
clogP c
2.0
pKa d
6.7
Compd
R1
clogP c
2.8
pKa d
6.7
IC50 (nM) a
IC50 (nM) a
3
54 ± 8
> 10000
15
1050 ± 390
290 ± 91
9
H
1.6
2.1
3.0
1.4
2.4
5.8
5.8
6.7
8.2
6.7
16
17
18
19
20
1.4
2.2
2.5
2.2
2.1
6.7
8.8
9.0
8.8
8.7
10
11
12
13
> 10000
170 ± 93
> 10000
630 ± 330
150 ± 45
2600 ± 900
320 ± 160
1020 ± 400
14
210 ± 100
290 ± 53
2.6
6.7
21
640 ± 370
1.7
7.1
AMD3100 b
a
Inhibition of luminescence signaling in HPB-ALL CXCR4 competitive binding assay. Data are
expressed as geometric mean values of at least two runs ± the standard error measurement (SEM).
b AMD3100 was run as standard in each assay. Data are expressed as geometric mean values of nine
runs ± the standard error measurement (SEM).
c Calculated by Molinspiration.
d Calculated by ACD/Labs 6.0.
25

ACCEPTED MANUSCRIPT
Table 3. Preliminary in vitro safety evaluation of lead compounds
CYP Inhibition (%) b
Compd
3 c
clogD a
pKa a
3A4
9
2D6
3
1.0
6.7
AMD11070 c
0.0
10.4
60
64
a
b
c
Calculated by ACD/Labs 6.0. All compounds were tested at 10 µM concentration. Measured as
HCl salts.
26

ACCEPTED MANUSCRIPT
Table 4.
1
Clint
(mL/min/kg)
HLM MLM
t1/2
(min)
HLM MLM
Caco-2
12G5 binding
IC50 (nM) a
Ca2+ flux
hERG
(µM)
Compd
IC50 (nM) b
0.73 ± 0.11
68 ± 32
Papp(A−B)
efflux ratio
(10−6 cm/s) (B−A)/(A−B)
c
d
c
d
(S)-3
88 ± 16
> 30
15
1.5
226
3864
7.7
1.4
(R)-3
1300 ± 340
a
Inhibition of luminescence signaling in HPB-ALL CXCR4 competitive binding assay. Data are
expressed as geometric mean values of at least two runs ± the standard error measurement (SEM).
b Inhibition of Ca2+ signal in the FLIPR assay. Data are expressed as geometric mean values of at least
two runs ± the standard error measurement (SEM).
c HLM = human liver microsomes. All compounds were tested at 1 µM concentration.
d MLM = mouse liver microsomes. All compounds were tested at 1µM concentration.
27

ACCEPTED MANUSCRIPT
Figure 1
28