Structural optimization of aminopyrimidine-based CXCR4 antagonists

Article abstract of DOI:10.1016/j.ejmech.2019.111914

Structural optimization of aminopyrimidine-based CXCR4 antagonists is reported. The optimization is guided by molecular docking studies based on available CXCR4-small molecule crystal complex. The optimization identifies a number of compounds with improved receptor binding affinity and functional activity exemplified by compound 23 (inhibition of APC-conjugate clone 12G5 for CXCR4 binding in a cell based assay: IC₅₀ = 8.8 nM; inhibition of CXCL12 induced cytosolic calcium increase: IC₅₀ = 0.02 nM). In addition, compound 23 potently inhibits CXCR4/CXLC12 mediated chemotaxis in a matrigel invasion assay. Furthermore, compound 23 exhibits good physicochemical properties (MW 367, clogP 2.1, PSA 48, pKa 7.2) and in vitro safety profiles (marginal/moderate inhibition of CYP isozymes and hERG). These results represent significant improvement over the initial hit from scaffold hybridization and suggest that compound 23 can be used as a starting point to support lead optimization.

Full text of DOI:10.1016/j.ejmech.2019.111914

Journal Pre-proof
Structural optimization of aminopyrimidine-based CXCR4 antagonists
Fang Zhu, Yujie Wang, Qian Du, Wenxiang Ge, Zhanhui Li, Xu Wang, Chunyan Fu,
Lusong Luo, Sheng Tian, Haikuo Ma, Jiyue Zheng, Yi Zhang, Xiaotian Sun, Sudan
He, Xiaohu Zhang
PII:
S0223-5234(19)31066-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111914
EJMECH 111914
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 September 2019
Revised Date: 13 November 2019
Accepted Date: 25 November 2019
Please cite this article as: F. Zhu, Y. Wang, Q. Du, W. Ge, Z. Li, X. Wang, C. Fu, L. Luo, S. Tian, H.
Ma, J. Zheng, Y. Zhang, X. Sun, S. He, X. Zhang, Structural optimization of aminopyrimidine-based
CXCR4 antagonists, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111914.
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Graphical Abstract
Structural Optimization of Aminopyrimidine-Based CXCR4 Antagonists
Fang Zhu, Yujie Wang, Qian Du, Wenxiang Ge, Zhanhui Li, Xu Wang, Chunyan Fu, Lusong Luo,
Sheng Tian, Haikuo Ma, Jiyue Zheng, Yi Zhang, Xiaotian Sun, Sudan He, and Xiaohu Zhang
Guided by molecular docking, the reported optimization identifies a number of compounds with
improved receptor binding affinity and functional activity exemplified by compound 23 (inhibition of
APC-conjugate clone 12G5 for CXCR4 binding in a cell based assay: IC50 = 8.8 nM; inhibition of
CXCL12 induced cytosolic calcium increase: IC50 = 0.02 nM). In addition, 23 potently inhibits
CXCR4/CXLC12 mediated chemotaxis in a matrigel invasion assay, exhibits good physicochemical
properties and in vitro safety profiles.
1

Structural Optimization of Aminopyrimidine-Based CXCR4 Antagonists
b,c,1
e
a,1
b,c
c,d
a
a
e
Fang Zhu
, Yujie Wang , Qian Du , Wenxiang Ge , Zhanhui Li , Xu Wang , Chunyan Fu ,
a
a,b
a,*
a
f
b,c,*
Lusong Luo , Sheng Tian , Haikuo Ma , Jiyue Zheng , Yi Zhang , Xiaotian Sun , Sudan He
,
a,*
Xiaohu Zhang
a
Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences,
Soochow University, Suzhou, Jiangsu 215123, P. R. China
b
Cyrus Tang Hematology Center, Jiangsu Institute of Hematology and Collaborative Innovation
Center of Hematology, Soochow University, Suzhou 215123, P. R. China
c
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,
2
15123 Jiangsu, P. R. China
d
School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, P. R. China
BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
02206, P. R. China
e
1
f
College of Chemistry and Chemical Engineering, and Henan Key Laboratory of Function-Oriented
Porous Materials, Luoyang Normal University, Luoyang 471934, 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; hesudan2018@163.com
Abstract: Structural optimization of aminopyrimidine-based CXCR4 antagonists is reported. The
optimization is guided by molecular docking studies based on available CXCR4-small molecule crystal
complex. The optimization identifies a number of compounds with improved receptor binding affinity
and functional activity exemplified by compound 23 (inhibition of APC-conjugate clone 12G5 for
CXCR4 binding in a cell based assay: IC = 8.8 nM; inhibition of CXCL12 induced cytosolic calcium
50
increase: IC50 = 0.02 nM). In addition, compound 23 potently inhibits CXCR4/CXLC12 mediated
chemotaxis in a matrigel invasion assay. Furthermore, compound 23 exhibits good physicochemical
properties (MW 367, clogP 2.1, PSA 48, pKa 7.2) and in vitro safety profiles (marginal/moderate
inhibition of CYP isozymes and hERG). These results represent significant improvement over the
initial hit from scaffold hybridization and suggest that compound 23 can be used as a starting point to
support lead optimization.
Keywords: Chemokine, CXCR4, antagonist, GPCR, structural optimization
1

Abbreviations: APC, allophycocyanin; CCD, charge coupled device; CuTC, copper(I)
thiophene-2-carboxylate; CYP, cytochrome P450; DCE, 1,2-dichloroethane; DIPEA,
N,N-diisopropylethylamine; DMEM, Dulbecco's modified eagle medium; 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; hERG, human
ether-a-go-go-related gene; HI-FBS, heat inactivated FBS; HIV, human immunodeficiency virus; HLM,
human liver microsomes; ICCD, intensified CCD; LDV, low dead volume microplate; LE, ligand
efficiency; LLE, ligand lipophilic efficiency; MEF, mouse embryonic fibroblasts; PFA,
paraformaldehyde; PK, pharmacokinetics; RLM, rat liver microsomes; rt, room temperature; SAR,
structure-activity-relationship; SDF-1, stromal cell-derived factor-1; SEM, standard error measurement;
SP, standard precision; THQ, tetrahydroquinoline; TIQ, tetrahydroisoquinoline; XP, extra precision.
2

1
.
Introduction
Chemokine CXCL12 (also known as stromal cell-derived factor-1, or SDF-1) and its receptor
CXCR4 regulate various cellular processes including chemotaxis, survival, and proliferation [1].
CXCR4 was first identified as a coreceptor for cellular entry by T cell-tropic (X4) HIV-1 virus [2]. As a
result, the majority of early research on CXCR4 antagonists focused on anti-HIV application. Small
molecule CXCR4 antagonists, exemplified by AMD3100 [3], AMD11070 [4, 5], IT1t [6], MSX-122
[
(
7], KRH3955 [8], and TIQ15 [9] (Figure 1), have been disclosed in the literature. AMD3100
plerixafor, Figure 1) was originally developed as an anti-HIV treatment, but its poor oral
bioavailability and dose-limiting toxicity prevented its further development for this chronic disease.
Surprisingly, AMD3100 was found to be a potent agent in mobilizing stem cells in human and was
subsequently approved by the FDA for the treatment of non-Hodgkin’s lymphoma and multiple
myeloma patients with hematopoietic stem cell transplantation [3]. More recently, CXCR4 was
reported to play important roles in modulating cancer microenvironment by trafficking of key immune
cells including T-cells, dendritic cells (DC), and myeloid derived suppressor cells (MDSC) into the
tumor tissue [10]. Since majority of the immune cells were immunosuppressive (e.g., Treg, MDSC),
CXCR4 was suggested to be an important culprit in evasion of immune surveillance. The clinical
application of CXCR4 antagonist AMD3100 in oncology, combined with the emerging evidence of
CXCR4 in modulating immune cells at the tumor microenvironment [11, 12], prompted a significant
resurgent effort to discover the next generation CXCR4 antagonists.
As the first and only small molecule CXCR4 antagonist in the market place, AMD3100 is an
injectable agent for short-term usage. Since autoimmune/inflammatory diseases, cancer, and HIV
infection are chronic diseases, CXCR4 antagonists which are intended to be used for the
aforementioned indications are expected to be administrated orally and safe for long-term use.
Following this theme, AMD11070 was developed with improved oral bioavailability and safety [4].
AMD11070 progressed to clinical testing despite showing moderate CYP450 (2D6, 3A4) inhibition
[
13]. Other small molecule CXCR4 antagonists are in preclinical or early clinical development [14-17].
Here we wish to report our efforts toward to the development of CXCR4 antagonists which are novel,
highly potent with improved in vitro safety profiles. Our ultimate goal is to obtain CXCR4 antagonists
with good pharmacokinetic properties to support oral dosing regimen for the treatment of chronic
diseases.
2
.
Design
Most CXCR4 antagonists contain highly basic centers. Since highly basic functional groups often
lead to poor permeability, hERG/CYP inhibition and phospholipidosis [18], we set our goal to reduce
basicity in our designed compounds. We previously utilized a scaffold hybridization strategy to obtain a
series of CXCR4 antagonists, exemplified by compound A (Figure 2c) [13]. Compound A possesses
good physicochemical properties (MW 353, clogP 2.0, pKa 6.7) and exhibits weak inhibition of
hERG/CYP (racemic A, hERG > 30 µM, CYP inhibition at 10 µM: 3A4, 9%; 2D6 3%). In addition,
compound A inhibits binding of APC-conjugate clone 12G5 to CXCR4 in a cell based assay (IC = 88
50
nM) and inhibits CXCL12 induced cytosolic calcium increase (IC50 = 0.73 nM) [13]. Here we report
the structural optimization based on the prototypical A.
Molecular docking study was performed to guide our optimization efforts. Specifically, A was
3

docked into the binding pocket of CXCR4 (PDB ID: 3ODU [19] from RCSB Protein Data Bank [20])
using standard precision (SP) and extra precision (XP) modes in Glide docking of Schrodinger 9.0
software [21]. The detailed Glide docking procedures were described in the previous studies [22-24].
The computational results demonstrated that A had good predicted binding affinity for CXCR4
(
docking score = -7.12 kcal/mol). Subsequently, the binding poses predicted by Glide docking and the
antagonist-residues interaction spectra were depicted and analyzed. As shown in Figures 2a and 2b, A
had tight interactions with three favorable residues including Glu32, Trp94 and His113. In addition, we
found that the pyrimidine functional group of A was in close proximity to residues Leu41 and Tyr45.
The residues within 5 Å of compound A in the binding pocket of CXCR4 were selected and labeled
(
Figure 2a). Besides residues Leu41 and Tyr45, other residues, including His281, Ser285 and Glu288,
were also in close range of A. We hypothesized that substitutions on the pyrimidine ring can be
optimized to form additional favorable interactions (hydrogen bond, electrostatic, or hydrophobic
interactions) with these nearby residues (Leu41, Tyr45, His281, Ser285 and Glu288) to improve
antagonistic activity of candidate compounds. Based on these observation and assessment, we
formulated chemical modification/optimization plan as outlined in Figure 2c.
3
.
Chemistry
The general synthetic route used to prepare compounds 1-18 was outlined in Scheme 1. Ester 39
[
25] was reacted with 2-methyl-2-thiopseudourea sulfate, followed by substitution with N-methyl
piperazine to give intermediate 41. Oxidation of 41 with Oxone and then substitution with
corresponding alcohols or amines afforded intermediates 44a-k. Direct coupling of 41 with
phenylboronic acid or 1-methyl-1H-pyrazol-4-ylboronic acid in the presence of Pd(PPh ) and CuTC
3
4
provided intermediates 44p and 44q, respectively. 44l-o and 44r were prepared from ester 39 in two
steps as described for intermediate 41. Compounds 1-18 were synthesized by deprotection of acetals
4
4a-r, followed by reductive amination with racemic N-methyl-5,6,7,8-tetrahydroquinolin-8-amine [13]
(
S-isomer was used in the case of compound 18).
The synthesis of compounds 19, 20, and 23-28 was performed as shown in Scheme 2. Cyclization
of ester 46 with acetamidine, followed by chlorination with POCl3 furnished intermediate 48.
Treatment of 49 [13] with ethylamine or cyclopropylmethylamine gave amines 50a and 50b, which
were then alkylated with intermediate 48 and subsequently substituted by N-methyl piperazine to yield
compounds 19 and 20, respectively. Reaction of (S)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (52)
[
26] with 48 produced intermediate 53. The synthesis of compounds 23-28 was achieved in 1-3 steps
by reacting 53 with diverse amines.
Compound 21 was prepared as described in Scheme 3. Claisen condensation of ester 54 afforded
ketoester 55. Cyclization of 55 with acetamidine, followed by substitution with N-methyl piperazine in
the presence of PyBOP gave intermediate 57. Treatment of 57 with methylamine under thermal
conditions [27] led to amine 58, which was reacted with chloride 49 in i-PrOH to yield compound 21.
The synthesis of compound 22 was performed as shown in Scheme 4. Cyclization of 59 with
acetamidine, followed by substitution with N-Boc-piperazine in the presence of PyBOP gave
intermediate 61. Treatment of 61 with trichloroisocyanuric acid afforded chloride 62, which was
substituted with (±)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine to provide intermediate 63. De-Boc
protection of 63, followed by reductive alkylation with formaldehyde led to compound 22.
4

4
4
.
Results and discussion
.1. Structure-Activity-Relationship of synthesized compounds
We used a cell based competitive binding assay to evaluate the binding affinity of compound to
CXCR4 receptor. The detailed description of assay development and validation had been reported [13].
Briefly, CXCR4 expressing HPB-ALL cells were incubated with APC-conjugate clone 12G5 antibody
and testing compound. The inhibition of APC-conjugate clone 12G5 antibody signal with different
concentrations of compound provided inhibition curve and readout of IC . At the early stage of
50
structural optimization, we did not synthesize the enantiomerically pure compounds associated with the
chiral center on the tetrahydroquinoline (THQ) ring. We reasoned that once more potent compounds
were identified, we can synthesize the chirally pure (S)-stereoisomer. As shown in Table 1, when the
hydrogen was substituted by a methoxy group on R , the binding affinity was improved (1, 25 nM).
1
Increasing the size of the substitution was detrimental for binding affinity (2 and 3, 78 and 151 nM,
respectively). Substitution of methoxy to amino maintained binding affinity (4, 23 nM). Consistently,
larger substitutions were detrimental for binding, especially for the secondary amines (compounds
5
-11). When methyl group was installed on the R position, more than four folds of binding
1
improvement was achieved compared to compound A (12 and A, 12 and 54 nM, respectively). Again,
decrease of binding affinity was observed when the size of the substitution increased (compounds
1
3-17). Lastly, a strong electronwithdrawing trifluoromethyl led to decrease of binding affinity.
In order to examine the observed differences in binding activity, the two most potent CXCR4
antagonists, compounds 4 and 12 were docked into the binding site of CXCR4 (PDB ID: 3ODU) using
the XP mode in Glide docking. The computational results demonstrated that compounds 4 (docking
score = -8.26 kcal/mol) and 12 (docking score = -8.91 kcal/mol) can form additional interactions with
the five nearby residues in the binding site of CXCR4 as expected (Figure 3) and reserved the tight
interactions with the Glu32, Trp94 and His113 residues as identified in compound A. The amine group
in the R position of compound 4 can form hydrogen bonds with residues Ser285 and Glu288 (Figure
1
3
b) and the methyl group in the R position of compound 12 can interact with residue Leu41 (alkyl
1
hydrophobic interaction, Figure 3c). Based on these findings, we found that chemical modifications
after rational drug design using molecular docking in the R position of the pyrimidine on compound A
1
are reasonably reliable.
Having established methyl as the best tentative substitution on R , we decided to explore
1
modifications on R and R positions. Moderate increase of size on R was well tolerated, but provided
2
3
2
no significant improvement on binding affinity (19 and 20, 17 and 51 nM, respectively). Similarly,
small substitutions on R led to slight decrease in binding affinity (21 and 22, 12 and 33 nM,
3
respectively). Overall, the structure-activity data was consistent with the molecular docking study, as
all positions modified thus far were in the helical cavity which may not tolerate big structural change.
We next focused on exploration of R . Since R is solvent-exposed according to our docking study,
4
4
we expect more tolerance to structure diversity. At this stage, we had identified the preferred
substitutions on R , R , and R , and the best compounds showed binding affinity at low nM range. We
1
2
3
therefore decided to synthesize the chirally pure (S)-stereoisomers. In agreement with literature reports
and our own data [4, 13], the (S)-stereoisomer 23 (8.8 nM) was more potent than the racemic 12 (12
nM). As shown in Table 2, increasing the size of capping group on the terminal nitrogen of the
piperazine led to decrease of binding affinity to CXCR4 (compounds 24-26). Addition of methyl group
5

to the piperazine was well tolerated, resulting in compounds with equal or slightly decreased affinity
compared with compound 23 (compounds 27-31). Ring closure to form a bicyclic system did not help
to improve binding affinity (32 and 33). Extension of the nitrogen out of the piperazine ring and
addition of an extra nitrogen both led to decrease of binding affinity (34 and 35). Open chain analogues
with different linker length were well tolerated (36 and 37) but removal of the basic nitrogen
completely abolished binding (38, > 10,000 nM). In summary, R could tolerate diverse functional
4
groups with different size, rigidity, stereoisomeric configuration. However, the most important
pharmacological element remained to be the basic nitrogen.
Collectively, optimization on R , R , R , and R led to numerous compounds with improved
1
2
3
4
binding affinity (e.g., compounds 23, 29, 31, and 33, 8.8, 8.3, 8.3, and 18 nM, respectively). We
carefully controlled key physicochemical properties in a favorable range (e.g., clogP 2-3; pKa 6-8)
throughout the structural modification. Compounds with moderate clogP and pKa are less likely to
encounter safety issues (CYP/hERG inhibition) and may have better chance to be progressed further
[
18, 28]. Since compounds 23, 29, 31, and 33 were similar in structure and showed comparable binding
affinity, we decided to use LE (ligand efficiency) and LLE (ligand lipophilic efficiency) as stratification
criterion to choose a compound for further proof of concept evaluation. Compound 23 had the lowest
molecular weight/lipophilicity and was therefore selected as the prototypical compound for functional
testing.
4
.2. Functional evaluation of lead compound 23
One of the hallmarks of CXCR4 activation by CXCL12 is the transient increase of cytosolic
calcium concentration. Inhibition of the CXCL12-induced calcium signal can be used to evaluate
functional activity of CXCR4 antagonists. The calcium mobilization assay can be efficiently performed
by FLIPR Tetra system [29]. Since compound 23 demonstrated high binding affinity to CXCR4 in the
1
2G5 competitive binding assay, we tested compound 23 for its functional activity in the FLIPR Tetra
based calcium mobilization assay. The detailed experimental procedure was reported previously [13].
Compound 23 proved to be a potent functional CXCR4 antagonist (IC50 = 0.02 nM) while the
positive control AMD3100 was much less potent (IC50 = 6 nM) under the same assay conditions
(
Figure 4a). We further assessed the preliminary functional activities/specificities of compound 23 by
testing it in CXCR1 (within the CXCR sub-family) and CCR6 (outside of the CXCR sub-family)
functional assays. Compound 23 proved to be inactive up to 10,000 nM (Figure 4b, c). Compound 23
binds with CXCR4 with high affinity (IC50 = 8.8 nM in the 12G5 competitive binding assay) and
inhibits CXCR4 function with excellent potency (IC50 = 0.02 nM in the FLIPR Tetra based calcium
mobilization assay). The divergence of the binding and functional data might reflect the interaction
mechanism of compound 23 with CXCR4 receptor. It is possible that compound 23 abolishes CXCL12
induced calcium flux (D97, E288 mediated) more efficiently than it blocks engagement of 12G5 with
CXCR4 (ECL-2 mediated) [30]. However, crystal/mutational data are needed to draw conclusion.
We next investigated compound 23 in the matrigel invasion assay to evaluate its inhibition of
CXCL12/CXCR4 mediated chemotaxis. MDA-MB-231 cells which naturally express CXCR4 and
compound 23 (100 nM) were added to the upper chamber. Human CXCL12 (200 ng/mL) was added to
the lower chamber. The inhibition of cell invasion with compound 23 and AMD3100 was calculated by
comparing to the cell invasion without treatment (Figure 5a, b). In agreement with the 12G5 binding
and the calcium mobilization data, compound 23 exhibited superior potency in the matrigel invasion
6

assay than AMD3100. Since compounds with general cellular toxicity can impede cell mobility, we
further investigated the potential cytotoxicity effect of compound 23 using the cell viability assay
(
ATP-based Cell Titer-Glo Luminescent Cell Viability Assay). As shown in Figure 5c, compound 23
did not inhibit the proliferation of multiple human and murine cells, including MDA-MB-231 and
mouse embryonic fibroblasts (MEF), at concentrations up to 1000 nM.
4
.3. Preliminary in vitro safety evaluation of lead compound
Based on its excellent in vitro pharmacological properties, we decided to evaluate compound 23
for its in vitro safety profiles. As shown in Table 3, compound 23 displayed moderate inhibition of
CYP3A4 and 2D6 at 10 µM concentration (48% and 57%, respectively), similar to these of AMD11070
(
60% and 64%, respectively) [13]. In order to assess its potential cardiotoxicity, compound 23 was
tested in a standard patch clamp (express) experiment for its inhibition of the human ether-a-go-go
related gene (hERG) potassium channel. Again, compound 23 displayed moderate inhibition (IC50 = 8
µM) of hERG (positive control: cisapride, 0.03 µM). Although the in vitro safety window seemed to be
reasonable for hERG (1000 folds based on binding affinity, over 100,000 folds based on calcium
mobilization), the inhibition potency of 8 µM remained a safety concern. On the other hand, most of
the published CXCR4 antagonists displayed poor to moderate pharmacokinetic profiles [6, 8, 9, 31],
including low Cmax, which suggested better tolerance for hERG inhibition. Nevertheless, future
optimization must consider a balanced CXCR4 potency and safety/PK profiles. Compound 23 was
-6
found to be highly permeable (27 × 10 cm/s) with low efflux ratio when measured in a Caco-2
permeability assay (Table 3). In addition, compound 23 displayed moderate plasma protein binding in
mouse and rat (89% and 82%, respectively) but high plasma protein binding in human (99%). Lastly,
compound 23 was quickly metabolized in rat liver microsomes (Table 3).
5
.
Conclusion
Guided by molecular docking studies based on available CXCR4-small molecule crystal complex,
the current optimization identifies a number of compounds with improved receptor binding affinity and
functional activity exemplified by compound 23 (inhibition of APC-conjugate clone 12G5 for CXCR4
binding in a cell based assay: IC = 8.8 nM; inhibition of CXCL12 induced cytosolic calcium increase:
5
0
IC = 0.02 nM). In addition, compound 23 potently inhibits CXCR4/CXLC12 mediated chemotaxis in
50
a matrigel invasion assay. Furthermore, compound 23 exhibits good physicochemical properties (MW
67, clogP 2.1, PSA 48, pKa 7.2) and in vitro safety profiles (marginal/moderate inhibition of CYP
3
isozymes and hERG). However, the metabolic stability in liver microsomes is suboptimal and needs to
be optimized. While this work was in progress, we encountered a report from Emory University in
which they presented metabolic data on their tetrahydroisoquinoline (TIQ) series of compounds [32].
They showed hydroxylation on the THQ moiety in mouse liver microsomes was the culprit for high
clearance. Blockade of the metabolic soft spot on the THQ moiety shifted the metabolic liability to the
TIQ moiety. Encouraged by their results, we are currently working on structural modification on the
THQ moiety on our scaffold and the results will be reported in due course.
6
6
.
Experimental protocols
.1. Chemistry
7

All reagents and solvents were used without any further purification. Reactions were monitored by
1
thin-layer chromatography or by Agilent 1100 LC/MSD Trap SL version Mass Spectrometer. H and
1
3
C 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.
6
.1.1. 6-(Dimethoxymethyl)-2-(methylthio)pyrimidin-4-ol (40)
A mixture of 39 (3.0 g, 17 mmol), 2-methyl-2-thiopseudourea sulfate (9.5 g, 34 mmol) in100 mL
of H O was added K CO (10 g, 76 mmol). The mixture was stirred at room temperature overnight and
2
2
3
acidified pH to 5 by AcOH. The solution was extracted with dichloromethane (30 mL x 3). The
combined organic layer was dried over Na SO and concentrated. The residue was purified by silica gel
2
4
column chromatography (dichloromethane/methanol = 100/1) to give the desired product (3.4 g, 92%)
1
as a white solid. H NMR (400 MHz, CDCl ) δ 12.79 (s, 1H), 6.46 (s, 1H), 5.06 (s, 1H), 3.39 (s, 6H),
3
+
2
.60 (s, 3H). MS (ESI/APCI) m/z 216.9 [M+H] .
6
.1.2. 4-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidine (41)
To a solution of 40 (3.4 g, 16 mmol), Et N (16 g, 160 mmol) and N-methyl piperazine (4.0 g, 40
3
mmol) in 100 mL of MeCN was added PyBOP (9.0 g, 17 mmol). The mixture was stirred at reflux
overnight. The reaction mixture was quenched with saturated NaHCO aqueous solution (200 mL) and
3
extracted with dichloromethane (200 mL). The organic layer was dried over Na SO and concentrated.
2
4
The residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/3) to
1
give the product (2.8 g, 59%) as a yellow oil. H NMR (400 MHz, CDCl ) δ 6.44 (s, 1H), 5.06 (s, 1H),
3
3
2
6
.69 (s, 4H), 3.40 (s, 6H), 2.50 (s, 3H), 2.46 (t, J = 5.0 Hz, 4H), 2.34 (s, 3H). MS (ESI/APCI) m/z
+
98.8 [M+H] .
.1.3. 4-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)-2-(methylsulfonyl)pyrimidine (42)
To a solution of 41 (2.8 g, 9.4 mmol) in THF (90 mL) and H O (4.5 mL) was added Oxone (6.7 g,
2
11 mmol) at room temperature. The mixture was stirred at room temperature for 4 h. The reaction
mixture was quenched with saturated NaHCO aqueous solution (200 mL) and extracted with ethyl
3
acetate (200 mL). The organic layer was dried over Na SO and concentrated to give the desired
2
4
product (2.4 g, crude) as a yellow oil, which was used directly in the next step without further
1
purification. H NMR (400 MHz, CDCl ) δ 6.85 (s, 1H), 5.17 (s, 1H), 4.00-3.60 (m, 4H), 3.42 (s, 6H),
3
+
3
.29 (s, 3H), 2.53-2.47 (m, 4H), 2.35 (s, 3H). MS (ESI/APCI) m/z 330.8 [M+H] .
6
.1.4. General procedure for the synthesis of 43a-e
To a mixture of 39 (1.7 mmol, 1.0 eq.), corresponding amidine (2.0 eq.) in 10 mL of H O was
2
added K CO (4.5 eq.). The mixture was stirred at room temperature overnight and acidified pH to 5 by
2
3
AcOH. The solution was extracted with dichloromethane (30 mL * 3). The combined organic layer was
dried over Na SO and concentrated. The residue was purified by silica gel column chromatography
2
4
(
dichloromethane/methanol = 50/1) to give the desired product.
6
-(Dimethoxymethyl)-2-methylpyrimidin-4-ol (43a)
1
Compound 43a was obtained as a white solid (yield 35%). H NMR (400 MHz, CDCl ) δ 13.12 (s,
3
+
1
H), 6.56 (s, 1H), 5.09 (s, 1H), 3.38 (s, 6H), 2.51 (s, 3H). MS (ESI/APCI) m/z 185.0 [M+H] .
6
-(Dimethoxymethyl)-2-ethylpyrimidin-4-ol (43b)
1
Compound 43b was obtained as a colorless oil (yield 90%). H NMR (400 MHz, CDCl ) δ 6.57 (s,
3
1
1
6
H), 5.10 (s, 1H), 3.39 (s, 6H), 2.75 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). MS (ESI/APCI) m/z
+
98.9 [M+H] .
-(Dimethoxymethyl)-2-isopropylpyrimidin-4-ol (43c)
8

1
Compound 43c was obtained as a white solid (yield 40%). H NMR (400 MHz, CDCl ) δ 12.28 (s,
3
1
H), 6.57 (s, 1H), 5.10 (s, 1H), 3.39 (s, 6H), 3.03-2.93 (m, 1H), 1.34 (d, J = 7.2 Hz, 6H).
2
-Cyclopropyl-6-(dimethoxymethyl)pyrimidin-4-ol (43d)
1
Compound 43d was obtained as a white solid (yield 48%). H NMR (400 MHz, CDCl ) δ 13.00 (s,
3
1
H), 6.49 (s, 1H), 5.00 (s, 1H), 3.37 (s, 6H), 1.98-1.87 (m, 1H), 1.28-1.22 (m, 2H), 1.17-1.03 (m, 2H).
+
MS (ESI/APCI) m/z 210.9 [M+H] .
6
-(Dimethoxymethyl)-2-(trifluoromethyl)pyrimidin-4-ol (43e)
1
Compound 43e was obtained as a yellow solid (yield 36%). H NMR (400 MHz, CDCl ) δ 6.99 (s,
3
+
1
H), 5.22 (s, 1H), 3.42 (s, 6H). MS (ESI/APCI) m/z 238.8 [M+H] .
6
.1.5. General procedure for the synthesis of 44a and 44b
To a solution of 42 (0.61 mmol, 1.0 eq.) in 5 mL of MeOH or EtOH was added MeONa or EtONa
(
5.0 eq.). The mixture was stirred at reflux overnight. The reaction mixture was quenched with
saturated NaHCO aqueous solution and extracted with dichloromethane. The organic layer was dried
3
over Na SO and concentrated. The residue was purified by silica gel column chromatography
2
4
(
dichloromethane/methanol = 100/1 to 100/3) to give the desired product.
4
-(Dimethoxymethyl)-2-methoxy-6-(4-methylpiperazin-1-yl)pyrimidine (44a)
1
Compound 44a was obtained as a yellow oil (yield 88%). H NMR (400 MHz, CDCl ) δ 11.71 (s,
3
1
H), 6.38 (s, 1H), 5.02 (s, 1H), 4.02 (s, 3H), 3.40 (s, 6H).
4
-(Dimethoxymethyl)-2-ethoxy-6-(4-methylpiperazin-1-yl)pyrimidine (44b)
1
Compound 44b was obtained as a yellow oil (yield 89%). H NMR (400 MHz, CDCl ) δ 6.42 (s,
3
1
3
6
H), 5.04 (s, 1H), 4.35 (q, J = 7.2 Hz, 2H), 3.68 (s, 4H), 3.40 (s, 6H), 2.45 (t, J = 5.0 Hz, 4H), 2.32 (s,
+
H), 1.38 (t, J = 7.0 Hz, 3H). MS (ESI/APCI) m/z 296.9 [M+H] .
.1.6. 4-(Dimethoxymethyl)-2-isopropoxy-6-(4-methylpiperazin-1-yl)pyrimidine (44c)
To a solution of 42 (240 mg, 0.73 mmol) in THF (5 mL) and i-PrOH (2 mL) was added NaH (60%
dispersion in mineral oil, 35 mg, 0.87 mmol). The mixture was stirred at 80°C for 2 h. The reaction
mixture was quenched with saturated NaHCO aqueous solution and extracted with dichloromethane.
3
The organic layer was dried over Na SO and concentrated to give the product (220 mg, 97%) as a
2
4
1
yellow oil. H NMR (400 MHz, CDCl ) δ 6.39 (s, 1H), 5.28-5.18 (m, 1H), 5.03 (s, 1H), 3.68 (s, 4H),
3
3
.41 (s, 6H), 2.45 (t, J = 5.0 Hz, 4H), 2.33 (s, 3H), 1.35 (d, J = 6.2 Hz, 6H). MS (ESI/APCI) m/z 310.9
+
[
M+H] .
6
.1.7. General procedure for the synthesis of 44d-k
A mixture of 42 (1.4 mmol, 1.0 eq.) in 5 mL of corresponding amine/THF (2 M) was stirred at
reflux overnight in a sealed tube. The reaction mixture was quenched with saturated NaHCO aqueous
3
solution and extracted with dichloromethane. The organic layer was dried over Na SO4 and
2
concentrated. The residue was purified by silica gel column chromatography
(
dichloromethane/methanol = 100/1 to 100/3) to give the desired product.
4
-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine (44d)
1
Compound 44d was obtained as a yellow oil (yield 62%). H NMR (400 MHz, CDCl ) δ 6.20 (s,
3
1
H), 5.02 (s, 1H), 4.77 (s, 2H), 3.63 (s, 4H), 3.38 (s, 6H), 2.43 (t, J = 5.0 Hz, 4H), 2.32 (s, 3H). MS
+
(
ESI/APCI) m/z 267.9 [M+H] .
4
-(Dimethoxymethyl)-N-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine (44e)
1
Compound 44e was obtained as a yellow oil (yield 97%). H NMR (400 MHz, CDCl ) δ 6.12 (s,
3
1
4
H), 5.01 (s, 1H), 3.67 (s, 4H), 3.39 (s, 6H), 2.94 (d, J = 5.2 Hz, 3H), 2.45 (t, J = 5.2 Hz, 4H), 2.33 (s,
H).
9

4
-(Dimethoxymethyl)-N-ethyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine (44f)
1
Compound 44f was obtained as a yellow oil (yield 80%). H NMR (400 MHz, CDCl ) δ 6.11 (s,
3
1
1
4
H), 5.00 (s, 1H), 4.87 (s, 1H), 3.65 (s, 4H), 3.43-3.35 (m, 8H), 2.44 (t, J = 5.0 Hz, 4H), 2.33 (s, 3H),
.18 (t, J = 7.2 Hz, 3H).
-(Dimethoxymethyl)-N-isopropyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine (44g)
Compound 44g was obtained as a yellow oil (340 mg, crude). This material was used directly in
1
the next step without further purification. H NMR (400 MHz, CDCl ) δ 6.08 (s, 1H), 5.75 (br s, 1H),
3
5
.03 (s, 1H), 4.12–3.98 (m, 1H), 3.67 (s, 4H), 3.38 (s, 6H), 2.46 (s, 4H), 2.32 (s, 3H), 1.19 (d, J = 6.0
+
Hz, 6H). MS (ESI/APCI) m/z 309.9 [M+H] .
4
-(Dimethoxymethyl)-N,N-dimethyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-amine (44h)
1
Compound 44h was obtained as a yellow oil (yield 88%). H NMR (400 MHz, CDCl ) δ 6.07 (s,
3
1
H), 4.99 (s, 1H), 3.63 (s, 4H), 3.40 (s, 6H), 3.13 (s, 6H), 2.44 (s, 4H), 2.32 (s, 3H).
2
-((4-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)pyrimidin-2-yl)(methyl)amino)ethan-1-ol (44i)
1
Compound 44i was obtained as a yellow oil (yield 92%). H NMR (400 MHz, CDCl ) δ 6.11 (s,
3
1
3
4
H), 5.00 (s, 1H), 3.84 (t, J = 4.6 Hz, 2H), 3.71 (t, J = 4.6 Hz, 2H), 3.68-3.60 (m, 4H), 3.38 (s, 6H),
+
.17 (s, 3H), 2.44 (t, J = 5.2 Hz, 4H), 2.32 (s, 3H). MS (ESI/APCI) m/z 325.9 [M+H] .
-(4-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)pyrimidin-2-yl)morpholine (44j)
1
Compound 44j was obtained as a yellow oil (yield 89%). H NMR (400 MHz, CDCl ) δ 6.14 (s,
3
1
H), 4.98 (s, 1H), 3.74 (s, 8H), 3.63 (s, 4H), 3.40 (s, 6H), 2.44 (t, J = 5.0 Hz, 4H), 2.32 (s, 3H).
4
-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)-2-(pyrrolidin-1-yl)pyrimidine (44k)
1
Compound 44k was obtained as a yellow oil (yield 96%). H NMR (400 MHz, CDCl ) δ 6.09 (s,
3
1
3
H), 5.01 (s, 1H), 3.69-3.61 (m, 4H), 3.58-3.50 (m, 4H), 3.41 (s, 6H), 2.44 (t, J = 5.0 Hz, 4H), 2.32 (s,
H), 1.94-1.88 (m, 4H).
6
.1.8. General procedure for the synthesis of 44p and 44q
A mixture of 41 (1.0 mmol, 1.0 eq.), phenylboronic acid or 1-methyl-1H-pyrazole-4-boronicacid
(
2.0 eq.), copper(I) thiophene-2-carboxylate (2.6 eq.), and Pd(PPh ) (0.1 eq.) in 20 mL of THF was
3 4
stirred at 80°C for 12 h under N . The resulting solution was evaporated to remove most of THF. The
2
residue was purified by silica gel column chromatography (dichloromethane/methanol = 100/1.5) to
give the desired product.
4
-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)-2-phenylpyrimidine (44p)
1
Compound 44p was obtained as a colorless oil (yield 73%). H NMR (400 MHz, CDCl ) δ
3
8
.49-8.28 (m, 2H), 7.44-7.43 (m, 3H), 6.70 (s, 1H), 5.24 (s, 1H), 3.81 (s, 4H), 3.46 (s, 6H), 2.59-2.42
+
(
m, 4H), 2.36 (s, 3H). MS (ESI/APCI) m/z 328.9[M+H] .
4
-(Dimethoxymethyl)-2-(1-methyl-1H-pyrazol-4-yl)-6-(4-methylpiperazin-1-yl)pyrimidine (44q)
1
Compound 44q was obtained as a colorless oil (yield 73%). H NMR (400 MHz, CDCl ) δ 8.10 (s,
3
1
4
H), 8.04 (s, 1H), 6.57 (s, 1H), 5.15 (s, 1H), 3,93 (s, 3H), 3.67-3.77 (m, 4H), 3.42 (s, 6H), 2.53-2.43 (m,
+
H), 2.34 (s, 3H). MS (ESI/APCI) m/z 332.9 [M+H] .
6
.1.9. General procedure for the synthesis of 44l-o and 44r
To a solution of 43a/b/c/d/e (0.6 mmol, 1.0 eq.), Et N (6.0 eq.) and N-methyl piperazine (1.5 eq.)
3
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 and extracted with
dichloromethane. The organic layer was dried over Na SO and concentrated to give the desired
2
4
product, which was used directly in the next step without further purification.
4
-(Dimethoxymethyl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine (44l)
1
Compound 44l was obtained as a yellow oil (140 mg, crude). H NMR (400 MHz, CDCl ) δ 6.58
3
1
0

(
s, 1H), 5.13 (s, 1H), 3.71 (s, 4H), 3.39(s, 6H), 2.52 (s, 3H), 2.49 (s, 4H), 2.35 (s, 3H). MS (ESI/APCI)
+
m/z 266.9 [M+H] .
4
-(Dimethoxymethyl)-2-ethyl-6-(4-methylpiperazin-1-yl)pyrimidine (44m)
1
Compound 44m was obtained as a yellow oil (460 mg, crude). H NMR (400 MHz, CDCl ) δ 6.59
3
(
s, 1H), 5.14 (s, 1H), 3.73 (s, 4H), 3.40 (s, 6H), 2.78 (q, J = 7.6 Hz, 2H), 2.58-2.44 (m, 4H), 2.36 (s,
+
3
H), 1.29 (t, J = 7.6 Hz, 3H). MS (ESI/APCI) m/z 280.9 [M+H] .
4
-(Dimethoxymethyl)-2-isopropyl-6-(4-methylpiperazin-1-yl)pyrimidine (44n)
1
Compound 44n was obtained as a brown oil (270 mg, crude). H NMR (400 MHz, CDCl ) δ 6.57
3
(
s, 1H), 5.13 (s, 1H), 3.71 (s, 4H), 3.40 (s, 6H), 3.08-2.97 (m, 1H), 2.54-2.42 (m, 4H), 2.34 (s, 3H),
+
1
.26 (d, J = 6.8 Hz, 6H). MS (ESI/APCI) m/z 294.9 [M+H] .
2
-Cyclopropyl-4-(dimethoxymethyl)-6-(4-methylpiperazin-1-yl)pyrimidine (44o)
1
Compound 44o was obtained as a yellow oil (380 mg, crude). H NMR (400 MHz, CDCl ) δ 6.54
3
(
s, 1H), 5.13 (s, 1H), 3.68 (s, 4H), 3.40 (s, 6H), 2.50 (s, 4H), 2.36 (s, 3H), 2.10 (s, 1H), 1.06 (s, 2H),
+
0
.98-0.92 (m, 2H). MS (ESI/APCI) m/z 292.9 [M+H] .
4
-(Dimethoxymethyl)-6-(4-methylpiperazin-1-yl)-2-(trifluoromethyl)pyrimidine (44r)
1
Compound 44r was obtained as a yellow oil (800 mg, crude). H NMR (400 MHz, CDCl ) δ 6.84
3
(
[
s, 1H), 5.18 (s, 1H), 3.76 (s, 4H), 3.42 (s, 6H), 2.50 (s, 4H), 2.35 (s, 3H). MS (ESI/APCI) m/z 320.8
+
M+H] .
6
.1.10. General procedure for the synthesis of 45l-o and 45r
A mixture of 44l/m/n/o/r (1.0 eq.) in 5 mL of 20% H SO was stirred at reflux overnight. The
2
4
reaction mixture was alkalified pH to 9 by saturated NaHCO aqueous solution and extracted with
3
dichloromethane (30 mL). The organic layer was dried over Na SO and concentrated to give the
2
4
desired product.
2
-Methyl-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45l)
1
Compound 45l was obtained as a yellow oil (yield 83%). H NMR (400 MHz, CDCl ) δ 9.88 (s,
3
1
H), 6.88 (s, 1H), 3.74 (s, 4H), 2.59 (s, 3H), 2.48 (t, J = 5.2 Hz, 4H), 2.34 (s, 3H).
2
-Ethyl-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45m)
1
Compound 45m was obtained as a brown oil (yield 26%). H NMR (400 MHz, CDCl ) δ 9.89 (s,
3
1
7
2
H), 6.88 (s, 1H), 3.75 (s, 4H), 2.84 (q, J = 7.6 Hz, 2H), 2.53-2.43 (m, 4H), 2.34 (s, 3H), 1.33 (t, J =
.4 Hz, 3H).
-Isopropyl-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45n)
1
Compound 45n was obtained as a yellow solid (yield 37%). H NMR (400 MHz, CDCl ) δ 9.89 (s,
3
1
6
2
H), 6.87 (s, 1H), 3.75 (m, 4H), 3.16-2.98 (m, 1H), 2.53-2.44 (m, 4H), 2.34 (s, 3H), 1.30 (d, J = 7.2 Hz,
H).
-Cyclopropyl-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45o)
1
Compound 45o was obtained as a yellow solid (yield 91%). H NMR (400 MHz, CDCl ) δ 9.86 (s,
3
1
1
6
H), 6.83 (s, 1H), 3.69 (s, 4H), 2.50-2.41 (m, 4H), 2.33 (s, 3H), 2.17-2.08 (m, 1H), 1.12-1.07 (m, 2H),
.03-0.95 (m, 2H).
-(4-Methylpiperazin-1-yl)-2-(trifluoromethyl)pyrimidine-4-carbaldehyde (45r)
1
Compound 45r was obtained as a yellow solid (yield 80%). H NMR (400 MHz, CDCl ) δ 9.94 (s,
3
1
H), 7.13 (s, 1H), 3.75 (s, 4H), 2.52 (s, 4H), 2.36 (s, 3H).
6
.1.11. General procedure for the synthesis of 45a-k, 45p, and 45q
A mixture of 44a/b/c/d/e/f/g/h/i/j/k/p/q (1.3 mmol, 1.0 eq.) in 5 mL of 20% H SO was stirred at
2
4
reflux overnight. The reaction mixture was alkalified pH to 9 by saturated NaHCO aqueous solution
3
11

and extracted with dichloromethane (30 mL). The organic layer was dried over Na SO4 and
2
concentrated to give the desired product.
2
-Methoxy-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45a)
1
Compound 45a was obtained as a yellow oil (yield 59%). H NMR (400 MHz, CDCl ) δ 9.80 (s,
3
1
H), 6.74 (s, 1H), 3.98 (s, 4H), 3.72 (s, 3H), 2.46 (s, 4H), 2.32 (s, 3H).
2
-Ethoxy-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45b)
1
Compound 45b was obtained as a yellow solid (yield 80%). H NMR (400 MHz, CDCl ) δ 9.79 (s,
3
1
H), 6.73 (s, 1H), 4.40 (q, J = 7.2 Hz, 2H), 3.92-3.44 (m, 4H), 2.54-2.40 (m, 4H), 2.33 (s, 3H), 1.41 (t,
+
J = 7.2 Hz, 3H). MS (ESI/APCI) m/z 282.9 [M+MeOH+H] .
2
-Isopropoxy-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45c)
1
Compound 45c was obtained as a yellow solid (yield 64%). H NMR (400 MHz, CDCl ) δ 9.79 (s,
3
1
6
2
H), 6.71 (s, 1H), 5.40-5.18 (m, 1H), 3.75 (s, 4H), 2.56-2.48 (m, 4H), 2.36 (s, 3H), 1.38 (d, J = 6.0 Hz,
+
H). MS (ESI/APCI) m/z 296.9 [M+MeOH+H] .
-Amino-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45d)
1
Compound 45d was obtained as a yellow solid (yield 79%). H NMR (400 MHz, CDCl ) δ 9.73 (s,
3
1
H), 6.52 (s, 1H), 5.35 (s, 2H), 3.69 (s, 4H), 2.47 (s, 4H), 2.34 (s, 3H). MS (ESI/APCI) m/z 221.9
+
[
M+H] .
2
-(Methylamino)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45e)
1
Compound 45e was obtained as a yellow solid (yield 82%). H NMR (400 MHz, CDCl ) δ 9.74 (s,
3
1
H), 6.45 (s, 1H), 3.69 (s, 4H), 2.98 (d, J = 4.8 Hz, 3H), 2.47-2.43 (m, 4H), 2.33 (s, 3H).
2
-(Ethylamino)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45f)
1
Compound 45f was obtained as a yellow solid (yield 91%). H NMR (400 MHz, CDCl ) δ 9.73 (s,
3
1
H), 6.44 (s, 1H), 3.69 (s, 4H), 3.52-3.35 (m, 2H), 2.46 (t, J = 5.2 Hz, 4H), 2.34 (s, 3H), 1.23 (t, J = 7.2
Hz, 4H).
2
-(Isopropylamino)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde(45g)
1
Compound 45g was obtained as a yellow solid (yield 83%). H NMR (400 MHz, CDCl ) δ 9.71 (s,
3
1
5
2
H), 6.42 (s, 1H), 4.93 (s, 1H), 4.22–3.96 (m, 1H), 3.72 (s, 4H), 2.53 (s, 4H), 2.38 (s, 3H), 1.23 (d, J =
+
.6 Hz, 6H). MS (ESI/APCI) m/z 263.9 [M+H] .
-(Dimethylamino)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45h)
1
Compound 45h was obtained as a yellow solid (yield 65%). H NMR (400 MHz, CDCl ) δ 9.76 (s,
3
1
H), 6.39 (s, 1H), 3.68 (s, 4H), 3.18 (s, 6H), 2.45 (s, 4H), 2.33 (s, 3H).
2
-((2-Hydroxyethyl)(methyl)amino)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45i)
1
Compound 45i was obtained as a yellow solid (yield 85%). H NMR (400 MHz, CDCl ) δ 9.73 (s,
3
1
H), 6.42 (s, 1H), 3.92–3.84 (m, 2H), 3.84–3.76 (m, 2H), 3.70 (s, 4H), 3.22 (s, 3H), 2.61 (s, 1H), 2.49
+
(
s, 4H), 2.35 (s, 3H). MS (ESI/APCI) m/z 311.9 [M+MeOH+H] .
6
-(4-Methylpiperazin-1-yl)-2-morpholinopyrimidine-4-carbaldehyde (45j)
1
Compound 45j was obtained as a yellow solid (yield 75%). H NMR (400 MHz, CDCl ) δ 9.74 (s,
3
1
2
6
H), 6.44 (s, 1H), 3.83-3.78 (m, 4H), 3.76-3.74 (m, 4H), 3.67-3.65 (m, 4H), 2.45 (t, J = 5.0 Hz, 4H),
.33 (s, 3H).
-(4-Methylpiperazin-1-yl)-2-(pyrrolidin-1-yl)pyrimidine-4-carbaldehyde (45k)
1
Compound 45k was obtained as a yellow solid (yield 99%). H NMR (400 MHz, CDCl ) δ 9.77 (s,
3
1
H), 6.40 (s, 1H), 3.67 (s, 4H), 3.58 (s, 4H), 2.44 (s, 4H), 2.33 (s, 3H), 1.96 (s, 4H).
6
-(4-Methylpiperazin-1-yl)-2-phenylpyrimidine-4-carbaldehyde (45p)
1
Compound 45p was obtained as a colorless oil (yield 82%). H NMR (400 MHz, CDCl ) δ 10.01
3
1
2

(
(
s, 1H), 8.53-8.41 (m, 2H), 7.52-7.45 (m, 3H), 6.98 (s, 1H), 3.85 (s, 4H), 2.53 (d, J = 4.6 Hz, 4H), 2.37
s, 3H).
2
-(1-Methyl-1H-pyrazol-4-yl)-6-(4-methylpiperazin-1-yl)pyrimidine-4-carbaldehyde (45q)
1
Compound 45q was obtained as a colorless oil (yield 76%). H NMR (400 MHz, CDCl ) δ 9.92 (s,
3
1
3
H), 8.14 (s, 1H), 8.08 (s, 1H), 6.86 (s, 1H), 3.97 (s, 3H), 3.94-3.74 (m, 4H), 2.68-2.49 (m, 4H), 2.41 (s,
H).
6
.1.12. General procedure for the synthesis of 1-18
To a solution of 45a-r (1.1 eq.) and N-methyl-5,6,7,8-tetrahydroquinolin-8-amine or 52 (0.21
mmol, 1.0 eq.) in 5 mL of DCE was added AcOH (1.0 eq.). After stirred at room temperature for 10
min, NaBH(OAc) (1.5 eq.) was added. The mixture was stirred at room temperature overnight and
3
then quenched with saturated NaHCO aqueous solution (10 mL). The aqueous layer was extracted
3
with dichloromethane (10 mL). The organic layer was dried over Na SO and concentrated. The residue
2
4
was purified by silica gel column chromatography (dichloromethane/methanol = 100/1 to 50/1) to give
the desired product.
N-((2-Methoxy-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-
8
-amine (1)
1
Compound 1 was obtained as a colorless oil (yield 28%). H NMR (400 MHz, CDCl ) δ 8.48 (d, J
3
=
4.4 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.09-7.02 (m, 1H), 6.87 (s, 1H), 4.03 (t, J = 7.4 Hz, 1H), 3.88
(
s, 3H), 3.76-3.66 (m, 4H), 3.58 (s, 2H), 2.86-2.75 (m, 1H), 2.74-2.65 (m, 1H), 2.44 (t, J = 5.2 Hz, 4H),
2
.37 (s, 3H), 2.32 (s, 3H), 2.15-2.06 (m, 1H), 2.05-1.97 (m, 1H), 1.97-1.87 (m, 1H), 1.72-1.66 (m, 1H).
1
3
C NMR (150 MHz, CDCl ) δ 170.1, 165.2, 164.5, 157.5, 147.4, 136.8, 134.3, 121.8, 95.0, 62.6, 59.2,
3
+
5
4.9, 54.1, 46.3, 44.1, 39.4, 29.4, 23.0, 21.0. HRMS (ESI): calcd for C H N O [M+H] 383.2559,
21
31
6
found 383.2553. Purity: 98.4%
N-((2-Ethoxy-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-
amine (2)
1
Compound 2 was obtained as a colorless oil (yield 50%). H NMR (400 MHz, CDCl ) δ 8.49 (d, J
3
=
4.0 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.13-7.00 (m, 1H), 6.87 (s, 1H), 4.31 (q, J = 7.2 Hz, 2H),
4
2
1
1
.08-3.96 (m, 1H), 3.69 (s, 4H), 3.57 (s, 2H), 2.88-2.76 (m, 1H), 2.74-2.65 (m, 1H), 2.49-2.42 (m, 4H),
.39 (s, 3H), 2.32 (s, 3H), 2.14-2.06 (m, 1H), 2.05-1.98 (m, 1H), 1.98-1.87 (m, 1H), 1.75-1.70 (m, 1H),
1
3
.37 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl ) δ 170.2, 164.8, 164.5, 157.5, 147.4, 136.8, 134.3,
3
21.8, 94.8, 62.5, 62.5, 59.2, 55.0, 46.3, 44.0, 39.4, 29.4, 22.9, 21.4, 14.8. MS (ESI/APCI) m/z 396.9
+
[
M+H] . Purity: 97.9%
N-((2-Isopropoxy-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinoli
n-8-amine (3)
1
Compound 3 was obtained as a colorless oil (yield 26%). H NMR (400 MHz, CDCl ) δ 8.48 (d, J
3
=
4.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.08-7.02 (m, 1H), 6.86 (s, 1H), 5.25-5.12 (m, 1H), 4.10-3.99
(
m, 1H), 3.75-3.64 (m, 4H), 3.56 (s, 2H), 2.85-2.76 (m, 1H), 2.74-2.65 (m, 1H), 2.44 (t, J = 5.0 Hz,
4
1
1
4
H), 2.38 (s, 3H), 2.32 (s, 3H), 2.13-2.06 (m, 1H), 2.05-1.98 (m, 1H), 1.98-1.87 (m, 1H), 1.69-1.64 (m,
1
3
H), 1.33 (d, J = 6.0 Hz, 6H). C NMR (150 MHz, CDCl ) δ 170.3, 164.5, 164.4, 157.6, 147.4, 136.7,
3
34.3, 121.7, 94.6, 69.0, 62.6, 59.3, 54.9, 46.3, 44.1, 39.4, 29.4, 22.9, 22.2, 21.3. MS (ESI/APCI) m/z
+
10.9 [M+H] . Purity: 96.3%
N-((2-Amino-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-
amine (4)
1
Compound 4 was obtained as a white solid (yield 52%). H NMR (400 MHz, CDCl ) δ 8.48 (d, J
3
1
3

=
4.4 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.09-6.98 (m, 1H), 6.57 (s, 1H), 4.75 (s, 2H), 4.08-3.99 (m,
1
3
H), 3.68-3.60 (m, 4H), 3.51 (s, 2H), 2.86-2.74 (m, 1H), 2.74-2.63 (m, 1H), 2.47-2.40 (m, 4H), 2.34 (s,
13
H), 2.31 (s, 3H), 2.10-2.06 (m, 1H), 2.04-1.98 (m, 1H), 1.96-1.87 (m, 1H), 1.74-1.64 (m, 1H).
C
NMR (150 MHz, CDCl ) δ 168.6, 163.8, 162.4, 157.6, 147.3, 136.7, 134.3, 121.7, 92.7, 62.7, 59.4,
3
+
5
3
5.0, 46.3, 43.9, 39.1, 29.4, 23.2, 21.4. HRMS (ESI): calcd for C H N [M+H] 368.2563, found
20
30
7
68.2553. Purity: 96.2%
N-Methyl-N-((2-(methylamino)-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroqui
nolin-8-amine (5)
1
Compound 5 was obtained as a colorless oil (yield 24%). H NMR (400 MHz, CDCl ) δ 8.48 (d, J
3
=
4.4 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.10-7.00 (m, 1H), 6.47 (s, 1H), 4.87 (s, 1H), 4.13-3.98 (m,
H), 3.67 (m, 4H), 3.50 (s, 2H), 2.92 (d, J = 4.4 Hz, 3H), 2.86-2.74 (m, 1H), 2.73-2.64 (m, 1H), 2.44 (t,
J = 5.0 Hz, 4H), 2.32 (s, 6H), 2.13-2.05 (m, 1H), 2.03-1.97 (m, 1H), 1.96-1.87 (m, 1H), 1.69-1.61 (m,
1
1
3
1
5
H). C NMR (150 MHz, CDCl ) δ 168.3, 163.7, 162.5, 157.7, 147.4, 136.7, 134.3, 121.7, 91.3, 62.7,
3
+
9.4, 55.0, 46.4, 43.9, 39.3, 29.5, 28.5, 23.3, 21.4. MS (ESI/APCI) m/z 381.9 [M+H] . Purity: 98.5%
N-((2-(Ethylamino)-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquin
olin-8-amine (6)
1
Compound 6 was obtained as a colorless oil (yield 34%). H NMR (400 MHz, CDCl ) δ 8.48 (d, J
3
=
4.4 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.08-7.00 (m, 1H), 6.48 (s, 1H), 4.76 (s, 1H), 4.04 (t, J = 7.6
Hz, 1H), 3.65 (s, 4H), 3.55-3.43 (m, 2H), 3.43-3.31 (m, 2H), 2.85-2.75 (m, 1H), 2.74-2.62 (m, 1H),
2
1
1
.43 (s, 4H), 2.35 (s, 3H), 2.32 (s, 3H), 2.14-2.04 (m, 1H), 2.03-1.96 (m, 1H), 1.96-1.86 (m, 1H),
13
.74-1.62 (s, 1H), 1.17 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl ) δ 168.4, 163.7, 161.9, 157.7,
3
47.4, 136.7, 134.3, 121.7, 91.3, 62.7, 59.4, 55.0, 46.4, 43.9, 39.3, 36.3, 29.5, 23.3, 21.4, 15.2. MS
+
(
ESI/APCI) m/z395.9 [M+H] . Purity: 98.1%
N-((2-(Isopropylamino)-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydro
quinolin-8-amine (7)
1
Compound 7 was obtained as a colorless oil (yield 95%). H NMR (400 MHz, CDCl ) δ 8.49 (d, J
3
=
4.4 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.08-7.02 (m, 1H), 6.48 (s, 1H), 4.60 (s, 1H), 4.13-4.00 (m,
2
2
1
1
4
H), 3.72-3.60 (m, 4H), 3.55-3.38 (m, 2H), 2.84-2.75 (m, 1H), 2.73-2.64 (m, 1H), 2.47-2.40 (m, 4H),
.36 (s, 3H), 2.32 (s, 3H), 2.14-2.04 (s, 1H), 2.03-1.96 (m, 1H), 1.92-1.86 (m, 1H), 1.68-1.64 (m, 1H),
1
3
.18 (d, J = 6.4 Hz, 6H). C NMR (150 MHz, CDCl ) δ 168.0, 163.7, 161.0, 157.7, 147.4, 136.7, 134.3,
3
21.7, 91.1, 62.7, 59.4, 55.0, 46.4, 44.0, 42.8, 39.3, 29.8, 29.5, 23.3, 23.1, 21.4. MS (ESI/APCI) m/z
+
10.0 [M+H] . Purity: 95.9%
N-((2-(Dimethylamino)-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydro
quinolin-8-amine (8)
1
Compound 8 was obtained as a colorless oil (yield 18%). H NMR (400 MHz, CDCl ) δ 8.49 (d, J
3
=
4.4 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.08-7.00 (m, 1H), 6.48 (s, 1H), 4.10-4.00 (m, 1H), 3.65 (s,
4
3
H), 3.55-3.49 (m, 2H), 3.10 (s, 6H), 2.86-2.73 (m, 1H), 2.74-2.62 (m, 1H), 2.46-2.39 (m, 4H), 2.37 (s,
13
H), 2.31 (s, 3H), 2.14-2.06 (m, 1H), 2.05-1.96 (m, 1H), 1.97-1.92 (m, 1H), 1.74-1.62 (m, 1H).
C
NMR (150 MHz, CDCl ) δ 168.4, 163.5, 162.3, 157.7, 147.3, 136.7, 134.2, 121.6, 90.2, 62.6, 59.7,
3
+
5
3
2
5.0, 46.3, 43.9, 39.3, 37.0, 29.4, 23.1, 21.3. HRMS (ESI): calcd for C H N [M+H] 396.2876, found
22
34
7
96.2882. Purity: 99.4%
-(Methyl(4-((methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-6-(4-methylpiperazin-1-yl)pyrimi
din-2-yl)amino)ethan-1-ol (9)
Compound 9 was obtained as a colorless oil (yield 52%). H NMR (400 MHz, CDCl ) δ 8.48 (d, J
1
3
1
4

=
4.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.10-6.98 (m, 1H), 6.49 (s, 1H), 4.10-3.98 (m, 1H), 3.83 (t, J =
4
1
1
1
4
.6 Hz, 2H), 3.69-3.63 (m, 6H), 3.50 (d, J = 5.6 Hz, 2H), 3.15 (s, 3H), 2.86-2.74 (m, 1H), 2.72-2.63 (m,
H), 2.46-2.40 (m, 4H), 2.34 (s, 3H), 2.32 (s, 3H), 2.13-2.05 (m, 1H), 2.04-1.97 (m, 1H), 1.94-1.86 (m,
1
3
H), 1.68-1.60 (m, 1H). C NMR (150 MHz, CDCl ) δ 167.9, 163.2, 162.5, 157.7, 147.3, 136.7, 134.3,
3
21.7, 91.0, 63.7, 62.7, 59.3, 54.9, 53.4, 46.3, 44.0, 39.3, 36.9, 29.4, 23.3, 21.3. MS (ESI/APCI) m/z
+
25.9 [M+H] . Purity: 99.8%
N-Methyl-N-((6-(4-methylpiperazin-1-yl)-2-morpholinopyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquino
lin-8-amine (10)
1
Compound 10 was obtained as a colorless oil (yield 29%). H NMR (400 MHz, CDCl ) δ 8.48 (d,
3
J = 4.8 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.13-7.00 (m, 1H), 6.57 (s, 1H), 4.04 (t, J = 7.2 Hz, 1H), 3.72
(
s, 8H), 3.68-3.60 (m, 4H), 3.55-3.47 (m, 2H), 2.87-2.76 (m, 1H), 2.74-2.63 (m, 1H), 2.43 (t, J = 5.2
Hz, 4H), 2.37 (s, 3H), 2.32 (s, 3H), 2.14-2.06 (m, 1H), 2.05-1.98 (m, 1H), 1.98-1.87 (m, 1H), 1.75-1.62
1
3
(
m, 1H). C NMR (150 MHz, CDCl ) δ 168.8, 163.5, 161.8, 157.8, 147.4, 136.7, 134.2, 121.7, 91.6,
3
+
6
7.2, 62.7, 59.6, 55.0, 46.4, 44.6, 44.0, 39.5, 29.4, 23.2, 21.3. MS (ESI/APCI) m/z 437.9 [M+H] .
Purity: 97.4%
N-Methyl-N-((6-(4-methylpiperazin-1-yl)-2-(pyrrolidin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroq
uinolin-8-amine (11)
1
Compound 11 was obtained as a colorless oil (yield 79%). H NMR (400 MHz, CDCl ) δ 8.48 (d,
3
J = 4.4 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.10-6.98 (m, 1H), 6.51 (s, 1H), 4.07 (t, J = 7.4 Hz, 1H),
3
.70-3.63 (m, 4H), 3.57-3.48 (m, 6H), 2.87-2.74 (m, 1H), 2.73-2.62 (m, 1H), 2.43 (t, J = 5.0 Hz, 4H),
2
.37 (s, 3H), 2.32 (s, 3H), 2.15-2.07 (m, 1H), 2.04-1.98 (m, 1H), 1.96-1.87 (m, 5H), 1.68-1.62 (m, 1H).
1
3
C NMR (150 MHz, CDCl ) δ 168.5, 163.5, 160.5, 157.8, 147.4, 136.6, 134.2, 121.6, 90.2, 62.6, 59.7,
3
+
5
5.1, 46.6, 46.4, 43.9, 39.3, 29.5, 25.7, 23.1, 21.4. MS (ESI/APCI) m/z 421.9 [M+H] . Purity: 98.0%
N-Methyl-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-
amine (12)
1
Compound 12 was obtained as a colorless oil (yield 39%). H NMR (400 MHz, CDCl ) δ 8.48 (d,
3
J = 3.6 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.08-7.01 (m, 2H), 4.04 (t, J = 7.4 Hz, 1H), 3.76-3.67 (m,
4
2
H), 3.60 (s, 2H), 2.86-2.74 (m, 1H), 2.74-2.65 (m, 1H), 2.49-2.43 (m, 7H), 2.38 (s, 3H), 2.33 (s, 3H),
13
.15-2.07 (m, 1H), 2.06-1.98 (m, 1H), 1.98-1.85 (m, 1H), 1.74-1.62 (m, 1H). C NMR (150 MHz,
CDCl ) δ 167.8, 166.6, 162.9, 157.5, 147.4, 136.7, 134.3, 121.8, 97.9, 62.7, 59.1, 54.9, 46.3, 43.8, 39.4,
3
+
2
9
9.4, 26.1, 23.1, 21.4. HRMS (ESI): calcd for C H N [M+H] 367.2610, found 367.2604. Purity:
21
31
6
9.1%
N-((2-Ethyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-a
mine (13)
1
Compound 13 was obtained as a yellow oil (yield 44%). H NMR (400 MHz, CDCl ) δ 8.49 (d, J
3
=
4.4 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.08-7.01 (m, 2H), 4.04 (t, J = 7.4 Hz, 1H), 3.72 (s, 4H), 3.61
(
s, 2H), 2.85-2.76 (m, 1H), 2.73-2.68 (m, 3H), 2.46 (t, J = 5.0 Hz, 4H), 2.39 (s, 3H), 2.33 (s, 3H),
13
2
.18-2.06 (m, 1H), 2.05-1.98 (m, 1H), 1.97-1.90 (m, 1H), 1.75-1.70 (m, 1H), 1.28-1.25 (m, 3H).
C
NMR (150 MHz, CDCl ) δ 170.7, 167.8, 163.0, 157.6, 147.4, 136.7, 134.3, 121.7, 98.0, 62.7, 59.1,
3
+
5
5.0, 46.4, 43.8, 39.4, 32.7, 29.5, 23.1, 21.4, 12.8. MS (ESI/APCI) m/z 380.9 [M+H] . Purity: 95.0%
N-((2-Isopropyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin
8-amine (14)
Compound 14 was obtained as a colorless oil (yield 63%). H NMR (400 MHz, CDCl ) δ 8.49 (d,
-
1
3
1
5

J = 4.4Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.07-7.00 (m, 2H), 4.04 (t, J = 7.4 Hz, 1H), 3.78-3.68 (m, 4H),
3
2
6
5
9
.61 (s, 2H), 2.98-2.87(m, 1H), 2.85-2.75 (m, 1H), 2.72-2.65 (m, 1H), 2.52-2.43 (m, 4H), 2.40 (s, 3H),
.33 (s, 3H), 2.18-2.08 (m, 1H), 2.06-1.98 (m, 1H), 1.97-1.86 (m, 1H), 1.72-1.65 (m, 1H), 1.28-1.21 (m,
1
3
H). C NMR (150 MHz, CDCl ) δ 173.9, 167.8, 163.0, 157.6, 147.4, 136.7, 134.3, 121.7, 98.1, 62.8,
3
+
9.1, 55.0, 46.4, 43.8, 39.5, 37.6, 29.5, 23.1, 22.0, 21.4. MS (ESI/APCI) m/z 394.9 [M+H] . Purity:
5.1%
N-((2-Cyclopropyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquino
lin-8-amine (15)
1
Compound 15 was obtained as a colorless oil (yield 26%). H NMR (400 MHz, CDCl ) δ 8.48 (d,
3
J = 4.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.08-6.98 (m, 2H), 4.03 (t, J = 7.4 Hz, 1H), 3.70-3.64 (m,
4
2
0
1
H), 3.60 (s, 2H), 2.86-2.75 (m, 1H), 2.74-2.64 (m, 1H), 2.46-2.40 (m, 4H), 2.39 (s, 3H), 2.32 (s, 3H),
.15-2.06 (m, 2H), 2.03-1.99 (m, 1H), 1.95-1.86 (m, 1H), 1.74-1.64 (m, 1H), 1.04-1.00 (m, 2H),
1
3
.91-0.84 (m, 2H). C NMR (150 MHz, CDCl ) δ 170.3, 167.4, 162.8, 157.6, 147.4, 136.7, 134.3,
3
21.7, 97.9, 62.6, 59.1, 54.9, 46.3, 43.7, 39.3, 29.4, 23.0, 21.4, 18.0, 9.7. HRMS (ESI): calcd for
+
C H N [M+H] 393.2767, found 393.2760. Purity: 97.5%
2
3
33
6
N-Methyl-N-((6-(4-methylpiperazin-1-yl)-2-phenylpyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-
amine (16)
1
Compound 16 was obtained as a colorless oil (yield 20%). H NMR (400 MHz, CDCl ) δ 8.51 (s,
3
1
4
2
H), 8.39-8.29 (m, 2H), 7.46-7.38 (m, 3H), 7.36 (d, J = 7.6 Hz, 1H), 7.18 (s, 1H), 7.12-7.01 (m, 1H),
.17-4.02 (m, 1H), 3.82 (s, 4H), 3.74 (s, 2H), 2.90-2.76 (m, 1H), 2.75-2.64 (m, 1H), 2.55-2.47 (m, 4H),
.45 (s, 3H), 2.35 (s, 3H), 2.22-2.10 (m, 1H), 2.09-2.01 (m, 1H), 2.01-1.92 (m, 1H), 1.78-1.64 (m, 1H).
1
3
C NMR (150 MHz, CDCl ) δ 168.4, 163.3, 163.1, 157.6, 147.5, 139.0, 136.8, 134.3, 130.0, 128.3,
3
1
28.2, 121.8, 99.1, 62.7, 59.4, 55.0, 46.4, 44.0, 39.5, 29.5, 23.1, 21.4. HRMS (ESI): calcd for
+
C H N [M+H] 429.2767, found 429.2760. Purity: 96.1%
2
6
33
6
N-Methyl-N-((2-(1-methyl-1H-pyrazol-4-yl)-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-t
etrahydroquinolin-8-amine (17)
1
Compound 17 was obtained as a colorless oil (yield 35%). H NMR (400 MHz, CDCl ) δ 8.50 (d,
3
J = 4.8 Hz, 1H), 8.07 (s, 1H), 8.00 (s, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.09-7.02 (m, 2H), 4.12-4.02 (m,
1
4
1
1
H), 3.92 (s, 3H), 3.78-3.72 (m, 4H), 3.66 (s, 2H), 2.86-2.76 (m, 1H), 2.75-2.65 (m, 1H), 2.52-2.45 (m,
H), 2.41 (s, 3H), 2.34 (s, 3H), 2.15-2.08 (m, 1H), 2.09-2.03 (m, 1H), 1.98-1.88 (m, 1H), 1.75-1.65 (m,
1
3
H)). C NMR (150 MHz, CDCl ) δ 168.2, 162.9, 159.8, 157.6, 147.4, 139.8, 136.8, 134.3, 131.0,
3
24.2, 121.8, 98.2, 62.7, 59.3, 55.0, 46.4, 43.9, 39.4, 39.2, 29.5, 23.1, 21.4. HRMS (ESI): calcd for
+
C H N [M+H] 433.2828, found 433.2819. Purity: 99.5%
2
4
33
8
(S)-N-Methyl-N-((6-(4-methylpiperazin-1-yl)-2-(trifluoromethyl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahy
droquinolin-8-amine (18)
1
Compound 18 was obtained as a colorless oil (yield 15%). H NMR (400 MHz, CDCl ) δ 8.47 (d,
3
J = 3.6 Hz, 1H), 7.46 (s, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.08-7.04 (m, 1H), 4.06-3.96 (m, 1H), 3.84-3.72
(
m, 4H), 3.72-3.60 (m, 2H), 2.87-2.75 (m, 1H), 2.74-2.61 (m, 1H), 2.53-2.42 (m, 4H), 2.42 (s, 3H),
13
2
.34 (s, 3H), 2.16-2.08 (m, 1H), 2.07-1.96 (m, 1H), 1.96-1.86 (m, 1H), 1.76-1.64 (m, 1H). C NMR
(
150 MHz, CDCl ) δ 168.8, 162.6, 157.1, 155.5 (q, J = 34.5 Hz), 147.3, 136.8, 134.3, 121.8, 119.7 (q, J
3
=
283.5 Hz), 102.0, 62.5, 58.5, 54.6, 46.0, 43.8, 39.3, 29.3, 22.9, 21.2. HRMS (ESI): calcd for
+
25
C H F N [M+H] 421.2328, found 421.2312. [α] = -30 (c = 0.2, CHCl ). Purity: 98.1%
2
1
28
3
6
D
3
6
.1.13. 6-(Chloromethyl)-2-methylpyrimidin-4-ol (47)
To a mixture of 46 (22 g, 136 mmol) and acetamidine hydrochloride (17 g, 150 mmol) in 180 mL
1
6

of EtOH was added DBU (41 g, 272 mmol) at 0°C. The mixture was stirred at room temperature
overnight and then concentrated. 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 Na SO and concentrated.
2
4
The crude material was purified by trituration with ethyl acetate to give the desired compound (8 g,
1
3
2
6
7%) as a yellow solid. H NMR (400 MHz, DMSO-d ) δ 12.49 (br s, 1H), 6.30 (s, 1H), 4.44 (s, 2H),
6
+
.28 (s, 3H). MS (ESI/APCI) m/z 158.9 [M+H] .
.1.14. 4-Chloro-6-(chloromethyl)-2-methylpyrimidine (48)
A mixture of compound 47 (8.0 g, 50 mmol) and POCl (40 mL) was stirred at reflux for 2 h. The
3
resulting solution was evaporated and ethyl acetate (200 mL) was added. The mixture was poured
into iced water, and the organic layer was separated. The organic layer was dried over Na SO and
2
4
concentrated. The residue was purified by silica gel column chromatography (petroleum ether /ethyl
1
acetate = 50/1) to give the desired product (5.2 g, 59%) as a slight yellow solid. H NMR (400 MHz,
+
CDCl ) δ 7.41 (s, 1H), 4.55 (s, 2H), 2.71 (s, 3H). MS (ESI/APCI) m/z 176.9 [M+H] .
3
6
.1.15. General procedure for the synthesis of 50a and 50b
A solution of 49 (3.0 mmol, 1.0 eq.) and corresponding amine (10.0 eq.) in 10 mL of THF
was stirred at 80°C overnight in a sealed tube. The reaction mixture was quenched with saturated
NaHCO aqueous solution (10 mL) and extracted with dichloromethane (10 mL x 3). The combined
3
organic layer was dried over Na SO and concentrated to give the desired product, which was used
2
4
directly in the next step without further purification.
N-Ethyl-5,6,7,8-tetrahydroquinolin-8-amine (50a)
1
Compound 50a was obtained as a brown oil (960 mg, crude). H NMR (400 MHz, CDCl ) δ 8.39
3
(
s, 1H), 7.37 (d, J =7.2 Hz, 1H), 7.11-7.01 (m, 1H), 3.86-3.74 (m, 1H), 2.88-2.70 (m, 4H), 2.19-2.14 (m,
+
1
H), 2.05-1.95 (m, 2H), 1.78-1.71 (m, 2H), 1.21-1.18 (m, 3H). MS (ESI/APCI) m/z 177.0 [M+H] .
N-(Cyclopropylmethyl)-5,6,7,8-tetrahydroquinolin-8-amine (50b)
1
Compound 50b was obtained as a brown oil (240 mg, crude). H NMR (400 MHz, CDCl ) δ 8.39
3
(
s, 1H), 7.37 (d, J =7.2 Hz, 1H), 7.11-7.01 (m, 1H), 3.86-3.74 (m, 1H), 2.88-2.70 (m, 4H), 2.19-2.14 (m,
+
1
H), 2.05-1.95 (m, 2H), 1.78-1.71 (m, 2H), 1.21-1.18 (m, 3H). MS (ESI/APCI) m/z 203.0 [M+H] .
6
.1.16. General procedure for the synthesis of 51a and 51b
A mixture of 50a or 50b (0.28 mmol, 1.0 eq.), 48 (1.1 eq.), KI (0.01 eq.) and DIPEA (2.5 eq.) in
1
.5 mL of MeCN was stirred at room temperature overnight. The reaction solution was evaporated to
remove most of MeCN, diluted with saturated aqueous NaHCO solution (10 mL) and extracted with
3
dichloromethane (10 mL x 3). The combined organic layer was dried over Na SO , filtered and
2
4
evaporated. The residue was purified by silica gel column chromatography (dichloromethane/methanol
200/1) to give the desired product.
=
N-((6-Chloro-2-methylpyrimidin-4-yl)methyl)-N-ethyl-5,6,7,8-tetrahydroquinolin-8-amine (51a)
1
Compound 51a was obtained as a slight yellow oil (yield 41%). H NMR (400 MHz, CDCl ) δ
3
8
3
2
.45 (d, J = 4.4 Hz, 1H), 7.80 (s, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.06-6.98 (m, 1H), 4.18-4.07 (m, 1H),
.81 (d, J = 17.6 Hz, 1H), 3.65 (d, J = 17.6 Hz, 1H), 2.93-2.70 (m, 4H), 2.62 (s, 3H), 2.17-2.13 (m, 1H),
.05-1.99 (m, 1H), 1.82-1.76 (m, 1H), 1.72-1.66 (m, 1H), 1.06 (t, J = 7.2 Hz, 3H).
N-((6-Chloro-2-methylpyrimidin-4-yl)methyl)-N-(cyclopropylmethyl)-5,6,7,8-tetrahydroquinolin-8-ami
ne (51b)
1
Compound 51b was obtained as a yellow oil (yield 83%). H NMR (400 MHz, CDCl ) δ 8.44 (d, J
3
=
4.4 Hz, 1H), 7.91 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.06-6.98 (m, 1H), 4.32-4.18 (m, 1H), 3.95-3.80
(
m, 1H), 3.80-3.60 (m, 1H), 2.75-2.66 (m, 4H), 2.63 (s, 3H), 2.25-2.13 (m, 1H), 2.05-1.96 (m, 1H),
1
7

1
.87-1.75 (m, 2H), 0.88-0.76 (m, 1H), 0.42-0.30 (m, 2H), 0.12-0.06 (m, 1H).
6
.1.17. General procedure for the synthesis of 19 and 20
To a solution of 51a or 51b (0.12 mmol, 1.0 eq.) in EtOH (1 mL) was added Et N (2.0 eq.) and
3
N-methyl piperazine (1.2 eq.). The reaction was stirred at 80°C overnight, and then quenched with
saturated NaHCO aqueous solution (20 mL). The aqueous layer was extracted with dichloromethane
3
(
20 mL). The organic layer was dried over Na SO and concentrated. The residue was purified by silica
2 4
gel column chromatography (dichloromethane/methanol = 200/1) to give the desired product.
N-Ethyl-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinolin-8-a
mine (19)
1
Compound 19 was obtained as a colorless oil (yield 58%). H NMR (400 MHz, CDCl ) δ 8.43 (s,
3
1
2
1
1
H), 7.38-7.29 (m, 1H), 7.21 (s, 1H), 7.03 (s, 1H), 4.13 (s, 1H), 3.73 (s, 4H), 3.64-3.45 (m, 2H),
.90-2.60 (m, 4H), 2.55-2.40 (m, 7H), 2.34 (s, 3H), 2.20-2.06 (m, 1H), 2.06-1.90 (m, 1H), 1.90-1.78 (m,
13
H), 1.78-1.70 (m, 1H), 1.10-0.98 (m, 3H). C NMR (150 MHz, CDCl ) δ 169.8, 166.4, 163.0, 158.3,
3
47.3, 136.5, 134.4, 121.6, 98.0, 61.1, 56.7, 55.0, 47.4, 46.4, 43.9, 29.5, 26.1, 25.9, 21.6, 14.6. MS
+
(
ESI/APCI) m/z 380.9 [M+H] . Purity: 99.4%
N-(Cyclopropylmethyl)-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahy
droquinolin-8-amine (20)
1
Compound 20 was obtained as a yellow oil (yield 52%). H NMR (400 MHz, CDCl ) δ 8.42 (s,
3
1
2
1
1
H), 7.32 (d, J = 7.2 Hz, 2H), 7.02 (s, 1H), 4.30-4.18 (m, 1H), 3.80-3.55 (m, 6H), 2.85-2.52 (m, 4H),
.52-2.40 (m, 7H), 2.35 (s, 3H), 2.20-2.10 (m, 1H), 2.02-1.90 (m, 1H), 1.88-1.76 (m, 2H), 0.90-0.78 (m,
13
H), 0.45-0.30 (m, 2H), 0.12-0.03 (m, 2H). C NMR (150 MHz, CDCl ) δ 169.8, 166.3, 163.0, 158.3,
3
47.2, 136.5, 134.3, 121.5, 98.0, 61.5, 58.1, 56.7, 54.9, 46.3, 43.8, 29.5, 26.1, 26.1, 21.7, 10.8, 4.5, 3.8.
+
HRMS (ESI): calcd for C H N [M+H] 407.2923, found 407.2912. Purity: 98.0%
2
4
35
6
6
.1.18. (S)-N-((6-Chloro-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine
(53)
A mixture of 52 (90 mg, 0.56 mmol), 48 (108 mg, 0.61 mmol), KI (10 mg, 0.06 mmol) and
DIPEA (180 mg, 1.4 mmol) in 10 mL of MeCN was stirred at room temperature overnight. The
reaction solution was evaporated to remove most of MeCN, diluted with saturated aqueous NaHCO3
solution and extracted with dichloromethane. The organic layer was dried over Na SO , filtered and
2
4
evaporated. The residue was purified by silica gel column chromatography (dichloromethane/methanol
1
=
100/1 to 50/1) to give the desired product (110 mg, 65%) as a slight yellow oil. H NMR (400 MHz,
CDCl ) δ 8.49 (s, 1H), 7.74-7.66 (m, 1H), 7.40-7.32 (m, 1H), 7.11-7.02 (m, 1H), 4.05-3.95 (m, 1H),
3
3
1
.72 (s, 2H), 2.86-2.68 (m, 2H), 2.63 (s, 3H), 2.40 (s, 3H), 2.18-2.09 (m, 1H), 2.07-1.98 (m, 1H),
25
+
.91-1.82 (m, 1H), 1.76-1.64 (m, 1H). [α] = -29 (c = 0.2, CHCl ). MS (ESI/APCI) m/z 302.8 [M+H] .
D
3
6
.1.19. General procedure for the synthesis of 23-26, 29-30, 32-38
To a solution of 53 (0.1 mmol, 1.0 eq.) in EtOH (2 mL) was added Et N (10.0 eq.) and
3
corresponding amine (5.0 eq.). The reaction was stirred at 85 °C overnight, and then quenched with
saturated NaHCO aqueous solution. The aqueous layer was extracted with dichloromethane. The
3
organic layer was dried over Na SO and concentrated. The residue was purified by silica gel column
2
4
chromatography (dichloromethane/methanol = 100/1) to give the desired product.
S)-N-Methyl-N-((2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroquinoli
n-8-amine (23)
(
1
Compound 23 was obtained as a yellow oil (yield 98%). H NMR (400 MHz, CDCl ) δ 8.49 (d, J
3
=
4.4 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.09-7.01 (m, 2H), 4.04 (t, J = 7.6 Hz, 1H), 3.71 (s, 4H), 3.60
1
8

(
(
s, 2H), 2.88-2.75 (m, 1H), 2.74-2.65 (m, 1H), 2.48-2.44 (m, 7H), 2.39 (s, 3H), 2.33 (s, 3H), 2.16-2.06
13
m, 1H), 2.04-1.98(m, 1H), 1.95-1.84 (m, 1H), 1.77-1.68 (m, 1H). C NMR (150 MHz, CDCl ) δ
3
1
2
0
67.7, 166.6, 162.9, 157.5, 147.4, 136.8, 134.4, 121.8, 98.0, 62.7, 59.0, 54.9, 46.3, 43.8, 39.3, 29.5,
+
25
6.1, 22.9, 21.4. HRMS (ESI): calcd for C H N [M+H] 367.2610, found 367.2605. [α] = -29 (c =
21
31
6
D
.2, CHCl ). Purity: 99.3%
3
(
S)-N-((6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-
-amine (24)
Compound 24 was obtained as a yellow oil (yield 80%). H NMR (400 MHz, CDCl ) δ 8.54-8.46
8
1
3
(
m, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.10-7.02 (m, 2H), 4.10-4.00 (m, 1H), 3.72 (s, 4H), 3.60 (s, 2H),
2
1
1
2
.87-2.76 (m, 1H), 2.75-2.64 (m, 1H), 2.53-2.42 (m, 9H), 2.40 (s, 3H), 2.16-2.07 (m, 1H), 2.05-1.98 (m,
13
H), 1.96-1.89 (m, 1H), 1.76-1.66 (m, 1H), 1.12 (t, J = 7.4 Hz, 3H). C NMR (150 MHz, CDCl ) δ
3
67.7, 166.6, 162.9, 157.5, 147.4, 136.8, 134.4, 121.8, 97.9, 62.7, 59.1, 52.7, 52.5, 43.9, 39.4, 29.5,
+
25
6.1, 23.1, 21.4, 12.1. HRMS (ESI): calcd for C H N [M+H] 381.2767, found 381.2766. [α] = -24
22
33
6
D
(
c = 0.2, CHCl ). Purity: 99.4%
3
(S)-N-((6-(4-Cyclopropylpiperazin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroq
uinolin-8-amine (25)
1
Compound 25 was obtained as a yellow oil (yield 99%). H NMR (400 MHz, CDCl ) δ 8.49 (d, J
3
=
3.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.07-7.03 (m, 2H), 4.08-4.01 (m, 1H), 3.70-3.63 (m, 4H), 3.61
(
s, 2H), 2.83-2.74 (m, 1H), 2.73-2.69 (m, 1H), 2.68-2.63 (m, 4H), 2.47 (s, 3H), 2.40 (s, 3H), 2.16-2.07
m, 1H), 2.05-1.96 (m, 1H), 1.95-1.86 (m, 1H), 1.69-1.66 (m, 1H), 1.65-1.59 (m, 1H), 0.51-0.43 (m,
(
1
3
4
5
H). C NMR (150 MHz, CDCl ) δ 167.5, 166.5, 162.9, 157.5, 147.4, 136.8, 134.4, 121.8, 98.0, 62.6,
3
+
25
9.0, 53.2, 43.9, 39.3, 38.6, 29.5, 26.1, 22.9, 21.4, 6.0. MS (ESI/APCI) m/z 392.9 [M+H] . [α] = -11 (c
D
=
0.2, CHCl ). Purity: 95.1%
3
(S)-N-Methyl-N-((2-methyl-6-(4-(oxetan-3-yl)piperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydro
quinolin-8-amine (26)
1
Compound 26 was obtained as a yellow solid (yield 26%). H NMR (400 MHz, CDCl ) δ 8.49 (d,
3
J = 4.0 Hz, 1H), 7.36 (d, J = 7.2 Hz, 1H), 7.10-7.01 (m, 2H), 4.71-4.60 (m, 4H), 4.10-3.95 (m, 1H),
3
.74 (s, 4H), 3.61 (s, 2H), 3.55-3.46 (m, 1H), 2.86-2.75 (m, 1H), 2.71-2.67 (m, 1H), 2.47 (s, 3H), 2.38
13
(
s, 7H), 2.20-2.07 (m, 1H), 2.06-1.96 (m, 1H), 1.93-1.88 (m, 1H), 1.78-1.65 (m, 1H). C NMR (150
MHz, CDCl ) δ 167.7, 166.6, 162.8, 157.4, 147.4, 136.8, 134.4, 121.8, 98.0, 75.5, 62.6, 59.3, 58.9, 49.5,
3
+
25
4
3.6, 39.2, 29.4, 26.1, 22.8, 21.4. MS (ESI/APCI) m/z 408.9 [M+H] . [α] = -21 (c = 0.2, CHCl ).
D
3
Purity: 98.7%
(S)-N-((6-((R)-3,4-Dimethylpiperazin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydr
oquinolin-8-amine (29)
1
Compound 29 was obtained as a slight yellow solid (yield 40%). H NMR (400 MHz, CDCl ) δ
3
8
7
2
1
1
.52-8.47 (m, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.09-7.00 (m, 2H), 4.35 (s, 1H), 4.25 (s, 1H), 4.04 (t, J =
.6 Hz, 1H), 3.68-3.55 (m, 2H), 3.14-3.03 (m, 1H), 2.88-2.82 (m, 1H), 2.82-2.76 (m, 1H), 2.74-2.63 (m,
H), 2.46 (s, 3H), 2.37 (s, 3H), 2.31 (s, 3H), 2.29-2.18 (m, 1H), 2.14-2.06 (m, 2H), 2.04-1.97 (m, 1H),
13
.93-1.87 (m, 1H), 1.73-1.64 (m, 1H), 1.17-1.09 (m, 3H). C NMR (150 MHz, CDCl ) δ 167.7, 166.6,
3
62.7, 157.6, 147.5, 136.8, 134.4, 121.8, 97.9 (J = 3.0 Hz), 62.5, 59.1, 57.7 (J = 6.0 Hz), 55.3 (J = 7.5
+
Hz), 50.3, 44.0, 42.7, 39.3, 29.5, 26.1, 22.9, 21.5, 17.0. HRMS (ESI): calcd for C H N [M+H]
22
33
6
2
5
3
81.2767, found 381.2763. [α] = -6 (c = 0.2, CHCl ). Purity: 90.1%
D
3
(S)-N-((6-((S)-3,4-Dimethylpiperazin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydr
1
9

oquinolin-8-amine (30)
1
Compound 30 was obtained as a colorless oil (yield 44%). H NMR (400 MHz, CDCl ) δ 8.50 (d,
3
J = 4.4 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.09-7.01 (m, 2H), 4.35 (s, 1H), 4.25 (s, 1H), 4.05 (t, J = 7.6
Hz, 1H), 3.68-3.55 (m, 2H), 3.14-3.02 (m, 1H), 2.89-2.82 (m, 1H), 2.80-2.74 (m, 1H), 2.74-2.63 (m,
2
1
1
H), 2.46 (s, 3H), 2.37 (s, 3H), 2.31 (s, 3H), 2.28-2.19 (m, 1H), 2.14-2.06 (m, 2H), 2.04-1.98 (m, 1H),
13
.97-1.87 (m, 1H), 1.73-1.64 (m, 1H), 1.16-1.10 (m, 3H). C NMR (150 MHz, CDCl ) δ 167.6, 166.6,
3
62.8, 157.5, 147.4, 136.8, 134.2, 121.8, 97.9 (J = 4.5 Hz), 62.5, 59.0, 57.7 (J = 4.5 Hz), 55.3 (J = 6.0
+
25
Hz), 50.3, 44.0, 42.7, 39.3, 29.5, 26.1, 22.8, 21.4, 17.0. MS (ESI/APCI) m/z 380.9 [M+H] . [α] = -22
D
(
c = 0.2, CHCl ). Purity: 96.6%
3
(S)-N-((6-((R)-Hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,
6
,7,8-tetrahydroquinolin-8-amine (32)
Compound 32 was obtained as a yellow oil (yield 51%). H NMR (400 MHz, CDCl ) δ 8.55-8.48
1
3
(
m, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.14-7.02 (m, 2H), 4.75-4.35 (m, 2H), 4.11-3.97 (m, 1H), 3.61 (s,
2
3
1
5
H), 3.15-3.08 (m, 2H), 3.05-2.96 (m, 1H), 2.86-2.76 (m, 1H), 2.74-2.57 (m, 2H), 2.46 (s, 3H), 2.38 (s,
13
H), 2.23-1.83 (m, 9H), 1.74-1.63 (m, 1H), 1.55-1.42 (m, 1H). C NMR (150 MHz, CDCl ) δ 167.5,
3
66.5, 163.0, 157.5, 147.5 (J = 3.0 Hz), 136.8, 134.4, 121.8, 98.2, 62.6 (J = 4.5 Hz), 62.4 (J = 6.0 Hz),
9.0, 53.7, 51.6 (J = 7.5 Hz), 48.7, 43.4, 39.3, 29.5, 27.5, 26.1, 22.9, 21.4 (J = 9.0 Hz). MS (ESI/APCI)
+
25
m/z 392.9 [M+H] . [α] = -9 (c = 0.2, CHCl ). Purity: 99.0%
D
3
(S)-N-((6-((S)-Hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,
6
,7,8-tetrahydroquinolin-8-amine (33)
1
Compound 33 was obtained as a yellow oil (yield 56%). H NMR (400 MHz, CDCl ) δ 8.49 (s,
3
1
2
3
H), 7.36 (d, J = 7.6 Hz, 1H), 7.11-7.03 (m, 2H), 4.68-4.37 (m, 2H), 4.09-3.99 (m, 1H), 3.68-3.55 (m,
H), 3.17-3.07 (m, 2H), 3.06-2.97 (m, 1H), 2.84-2.74 (m, 1H), 2.71-2.60 (m, 2H), 2.47 (s, 3H), 2.38 (s,
13
H), 2.28-2.06 (m, 3H), 2.04-1.82 (m, 6H), 1.71-1.59 (m, 1H), 1.54-1.43 (m, 1H). C NMR (150 MHz,
CDCl ) δ 167.3, 166.4, 163.0, 157.5, 147.5 (J = 3.0 Hz), 136.8, 134.4, 121.8, 98.2, 62.6 (J = 4.5 Hz),
3
6
9
2.5 (J = 6.0 Hz), 59.0, 53.7, 51.6 (J = 7.5 Hz), 48.7, 43.4, 39.3, 29.8, 29.5, 27.6, 26.1, 22.9, 21.4 (J =
+
25
.0 Hz). MS (ESI/APCI) m/z 392.9 [M+H] . [α] = -5 (c = 0.2, CHCl ). Purity: 97.3%
D
3
(S)-N-Methyl-N-((2-methyl-6-(4-morpholinopiperidin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroqu
inolin-8-amine (34)
1
Compound 34 was obtained as a yellow oil (yield 46%). H NMR (400 MHz, CDCl ) δ 8.51-8.47
3
(
m, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.11-7.02 (m, 2H), 4.58 (d, J = 12.0 Hz, 2H), 4.03 (t, J = 7.2 Hz, 1H),
3
2
.72 (s, 4H), 3.61 (s, 2H), 2.88-2.76 (m, 3H), 2.74-2.65 (m, 1H), 2.56 (s, 4H), 2.46 (s, 4H), 2.38 (s, 3H),
13
.15-1.96 (m, 2H), 1.95-1.85 (m, 3H), 1.76-1.65 (m, 1H), 1.55-1.42 (m, 2H). C NMR (150 MHz,
CDCl ) δ 167.5, 166.6, 162.7, 157.5, 147.4, 136.8, 134.4, 121.8, 97.9, 67.4, 62.6, 62.4, 59.0, 49.9, 43.5,
3
+
25
3
9.4, 29.5, 28.1 (J = 6.0 Hz), 26.1, 23.0, 21.4. MS (ESI/APCI) m/z 436.9 [M+H] . [α] = -11 (c = 0.2,
D
CHCl ). Purity: 96.5%
3
(S)-N-Methyl-N-((2-methyl-6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-
Tetrahydroquinolin-8-amine (35)
1
Compound 35 was obtained as a yellow oil (yield 63%). H NMR (400 MHz, CDCl ) δ 8.48 (s,
3
1
2
2
1
H), 7.41-7.31 (m, 1H), 7.13-6.92 (m, 2H), 4.63-4.50 (m, 2H), 4.08-3.92 (m, 1H), 3.59 (s, 2H),
.87-2.75 (m, 3H), 2.74-2.55 (m, 4H), 2.54-2.40 (m, 6H), 2.37 (s, 3H), 2.28 (s, 3H), 2.16-2.07 (m, 4H),
13
.02-1.85 (m, 4H), 1.73-1.62 (m, 1H), 1.50-1.37 (m, 2H). C NMR (150 MHz, CDCl ) δ 167.7, 166.7,
3
62.7, 157.6, 147.5, 136.7, 134.3, 121.7, 97.9, 62.6, 62.2, 59.1, 55.5, 49.1, 46.1, 43.6, 39.4, 29.5, 28.2
+
25
(
J = 6.0 Hz), 26.2, 23.1, 21.4. MS (ESI/APCI) m/z 449.9 [M+H] . [α] = -29 (c = 0.2, CHCl ). Purity:
D
3
2
0

9
5.9%
S)-N-Methyl-N-((2-methyl-6-((2-morpholinoethyl)amino)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroqui
nolin-8-amine (36)
(
1
Compound 36 was obtained as a yellow oil (yield 76%). H NMR (400 MHz, CDCl ) δ 8.53-8.48
3
(
m, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.10-7.02 (m, 1H), 6.75 (s, 1H), 5.52 (br s, 1H), 4.08-4.02 (m, 1H),
3
.72 (s, 4H), 3.56 (s, 2H), 3.42 (s, 2H), 2.84-2.77 (m, 1H), 2.74-2.65 (m, 1H), 2.62-2.56 (m, 2H), 2.46
13
(
s, 7H), 2.38 (s, 3H), 2.15-2.08 (m, 1H), 2.06-1.98 (m, 1H), 1.96-1.85 (m, 1H), 1.80-1.75 (m, 1H).
C
NMR (150 MHz, CDCl ) δ167.0, 166.4, 163.0, 157.2, 147.2, 136.6, 134.2, 121.6, 98.5, 86.9, 83.1, 58.5,
3
+
25
5
7.0, 53.2, 39.0, 37.3, 29.3, 25.7, 23.0, 21.2. MS (ESI/APCI) m/z 396.9 [M+H] . [α] = -26 (c = 0.2,
D
CHCl ). Purity: 96.3%
3
(S)-N-Methyl-N-((2-methyl-6-((3-morpholinopropyl)amino)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydroq
uinolin-8-amine (37)
1
Compound 37 was obtained as a yellow oil (yield 49%). H NMR (400 MHz, CDCl ) δ 8.50 (s,
3
1
4
2
H), 7.37 (d, J = 6.8 Hz, 1H), 7.12-7.02 (m, 1H), 6.73 (s, 1H), 5.50 (s, 1H), 4.13-3.95 (m, 1H), 3.72 (s,
H), 3.59 (s, 2H), 3.44 (s, 2H), 2.88-2.76 (m, 1H), 2.75-2.63 (m, 1H), 2.50-2.40 (m, 9H), 2.38 (s, 3H),
13
.18-2.08 (m, 1H), 2.06-1.96 (m, 1H), 1.96-1.88 (m, 2H), 1.76-1.73 (m, 2H). C NMR (150 MHz,
CDCl ) δ167.3, 166.6, 163.2, 157.3, 147.2, 136.5, 134.1, 121.5, 97.9, 66.9, 62.9, 58.9, 56.9, 53.7, 40.2,
3
+
25
3
9.1, 29.2, 25.8, 25.6, 23.3, 21.2. MS (ESI/APCI) m/z 410.9 [M+H] . [α] = -13 (c = 0.2, CHCl ).
D
3
Purity: 96.2%
(S)-2-(Methyl(2-methyl-6-((methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)pyrimidin-4-yl)amino
)ethan-1-ol (38)
1
Compound 38 was obtained as a yellow oil (yield 88%). H NMR (400 MHz, CDCl ) δ 8.50-8.45
3
(
m, 1H), 7.41-7.32 (m, 1H), 7.10-7.00 (m, 1H), 7.00 (s, 1H), 4.10-3.96 (m, 1H), 3.88-3.80 (m, 2H),
3
.78-3.68 (m, 2H), 3.62 (s, 2H), 3.14 (s, 3H), 2.86-2.74 (m, 1H), 2.75-2.63 (m, 1H), 2.46 (s, 3H), 2.38
13
(
s, 3H), 2.16-2.06 (m, 1H), 2.06-1.98 (m, 1H), 1.96-1.85 (m, 2H). C NMR (150 MHz, CDCl ) δ 167.5,
3
1
65.9, 163.5, 157.4, 147.4, 136.9, 134.4, 121.8, 98.3, 62.7, 59.0, 53.6, 39.3, 37.3, 29.4, 25.8, 23.1, 21.4.
+
25
MS (ESI/APCI) m/z 341.9 [M+H] . [α] = -44 (c = 0.2, CHCl ). Purity: 97.8%
D
3
6
.1.20. General procedure for the synthesis of 27 and 28
To a solution of 53 (0.1 mmol, 1.0 eq.), Et N (10.0 eq.) and corresponding amine (5.0 eq.) in 2 mL
3
of EtOH was stirred at 80°C overnight. The reaction mixture was quenched with saturated NaHCO3
aqueous solution (20 mL) and extracted with dichloromethane (20 mL). The organic layer was dried
over Na SO and concentrated. The residue was diluted with ethyl acetate (2 mL) and added HCl/ ethyl
2
4
acetate (3 M, 3 mL). The mixture was stirred at room temperature overnight and quenched with
saturated NaHCO aqueous solution (50 mL). The aqueous layer was extracted with ethyl acetate (30
3
mL x 3). The organic layers were dried over Na SO and concentrated. The resulting residue was
2
4
diluted with DCE (4 mL) and HCHO/H O (37 percent, 5.0 eq.) was added, followed by NaBH(OAc)3
2
(
5.0 eq.). The mixture was stirred at room temperature overnight and then quenched with saturated
NaHCO aqueous solution. The aqueous layer was extracted with dichloromethane (30 mL x 3). The
3
combined organic layer was dried over Na SO and concentrated. The residue was purified by silica gel
2
4
column chromatography (dichloromethane/methanol = 100/1) to give the desired product.
S)-N-((6-((R)-2,4-Dimethylpiperazin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydr
oquinolin-8-amine (27)
(
1
Compound 27 was obtained as a colorless oil (yield 25%). H NMR (400 MHz, CDCl ) δ 8.48 (s,
3
1
H), 7.35 (d, J = 7.6 Hz, 1H), 7.09-6.92 (m, 2H), 4.62 (s, 1H), 4.35-4.28 (m, 1H), 4.09-3.98 (m, 1H),
2
1

3
3
1
.74-3.49 (m, 2H), 3.23-3.08 (m, 1H), 2.91-2.83 (m, 1H), 2.82-2.75 (m, 1H), 2.74-2.65 (m, 2H), 2.46 (s,
H), 2.42-2.36 (m, 3H), 2.28 (s, 3H), 2.22-2.17 (m, 1H), 2.14-2.07 (m, 1H), 2.05-1.91 (m, 3H),
1
3
.74-1.63 (m, 1H), 1.29-1.23 (m, 3H). C NMR (150 MHz, CDCl ) δ 166.9 (d, J = 16.5 Hz), 166.2 (d,
3
J = 9.0 Hz), 162.2 (d, J = 10.5 Hz), 157.2, 147.1, 136.7, 134.2 (d, J = 7.5 Hz), 121.6 (d, J = 3.0 Hz),
7.9 (d, J = 7.5 Hz), 62.6 (d, J = 90.0 Hz), 60.0 (d, J = 4.5 Hz), 58.7 (d, J = 16.5 Hz), 55.2 (d, J = 6.0
Hz), 46.6, 46.5, 38.9 (d, J = 46.5 Hz), 38.7, 29.2, 25.8, 22.7 (d, J = 51.0 Hz), 21.2 (d, J = 9.0 Hz), 14.7
9
+
25
(
d, J = 6.0 Hz). MS (ESI/APCI) m/z 380.9 [M+H] . [α] = -77 (c = 0.2, CHCl ). Purity: 96.4%
D
3
(S)-N-((6-((S)-2,4-Dimethylpiperazin-1-yl)-2-methylpyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydr
oquinolin-8-amine (28)
1
Compound 28 was obtained as a colorless oil (yield 72%). H NMR (400 MHz, CDCl ) δ
3
8
.51-8.44 (m, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.09-7.02 (m, 1H), 7.02-6.89 (m, 1H), 4.61 (s, 1H), 4.31 (d,
J = 13.2 Hz, 1H), 4.05 (q, J = 7.8 Hz, 1H), 3.63 (d, J = 8.0 Hz, 1H), 3.56 (s, 1H), 3.21-3.10 (m, 1H),
2
3
1
1
3
.89-2.83 (m, 1H), 2.81-2.74 (m, 1H), 2.74-2.67 (m, 2H), 2.46 (s, 3H), 2.37 (d, J = 6.0 Hz, 3H), 2.23 (s,
H), 2.21-2.15 (m, 1H), 2.15-2.05 (m, 1H), 2.04-1.95 (m, 2H), 1.95-1.92 (m, 1H), 1.74-1.63 (m, 1H),
13
.30-1.25 (m, 2H), 1.18 (d, J = 6.8 Hz, 1H). C NMR (150 MHz, CDCl ) δ 167.3 (d, J = 19.5 Hz),
3
66.4 (d, J = 9.0 Hz), 162.2 (d, J = 10.5 Hz), 157.3, 147.2, 136.6, 134.1 (d, J = 7.5 Hz), 121.6 (d, J =
.0 Hz), 97.9 (d, J = 7.5 Hz), 62.6 (d, J = 96 Hz), 60.0 (d, J = 6.0 Hz), 58.9 (d, J = 16.5 Hz), 55.2 (d, J
=
7.5 Hz), 46.6 (d, J = 3.0 Hz), 46.5, 39.1 (d, J = 49.5 Hz), 38.7, 29.3, 26.0, 22.8 (d, J = 55.5 Hz) ,21.2
+
25
(
d, J = 9.0 Hz), 14.6 (d, J = 7.5 Hz). MS (ESI/APCI) m/z 380.9 [M+H] . [α] = +45 (c = 0.2, CHCl ).
D
3
Purity: 95.2%
6
.1.21.
(S)-N-Methyl-N-((2-methyl-6-((3S,5R)-3,4,5-trimethylpiperazin-1-yl)pyrimidin-4-yl)methyl)-5,6,7,8-tetr
ahydroquinolin-8-amine (31)
To a solution of 53 (30 mg, 0.1 mmol) in EtOH (2 mL) was added Et N (101 mg, 1.0 mmol) and
3
cis-2,6-dimethylpiperazine (57 mg, 0.5 mmol). The reaction mixture was stirred at 80°C overnight, and
then quenched with saturated NaHCO aqueous solution (20 mL). The aqueous layer was extracted
3
with dichloromethane (20 mL). The organic layer was dried over Na SO and concentrated. The
2
4
resulting residue was diluted with DCE (4 mL) and HCHO/H O (37 percent, 0.5 mL) was added,
2
followed by NaBH(OAc) (34 mg, 0.16 mmol). The mixture was stirred at room temperature overnight
3
and then quenched with saturated NaHCO aqueous solution (50 mL). The aqueous layer was extracted
3
with dichloromethane (30 mL x 3). The combined organic layer was dried over Na SO4 and
2
concentrated. The residue was purified by silica gel column chromatography
1
(
dichloromethane/methanol = 100/1) to give the desired product (30 mg, 76%) as a colorless oil. H
NMR (400 MHz, CDCl ) δ 8.52 (s, 1H), 7.36 (d, J = 7.2 Hz, 1H), 7.06 (s, 2H), 4.32 (s, 2H), 4.05 (t, J =
3
7
2
.6 Hz, 1H), 3.63 (s, 2H), 2.85-2.76 (m, 1H), 2.75-2.62 (m, 3H), 2.47 (s, 3H), 2.39 (s, 3H), 2.29 (s, 3H),
.24-2.16 (m, 2H), 2.13-2.07 (m, 1H), 2.05-1.96 (m, 1H), 1.95-1.87 (m, 1H), 1.71-1.65 (m, 1H), 1.18 (t,
1
3
J = 6.0 Hz, 6H). C NMR (150 MHz, CDCl ) δ 167.3, 166.6, 162.4, 157.5, 147.5, 136.8, 134.4, 121.8,
3
9
7.8, 62.3, 59.0, 57.8 (J = 4.5 Hz), 50.4, 39.1, 37.8, 29.5, 26.1, 22.5, 21.5, 18.0 (J = 4.5 Hz). MS
+
25
(
ESI/APCI) m/z 394.9 [M+H] . [α] = -20 (c = 0.2, CHCl ). Purity: 99.5%
D
3
6
.1.22. Ethyl 2,4-difluoro-3-oxobutanoate (55)
Compound 55 was synthesized with slight modification of the procedure reported in the literature.
33] To a suspension of NaH (60% dispersion in mineral oil, 936 mg, 23 mmol) in 50 mL of ether was
added ethyl 2-fluoroacetate (5.0 g, 47 mmol) at room temperature. The mixture was stirred at 40°C for
h. The reaction mixture was poured into H SO (2 M, 15 mL) and extracted with ether (50 mL x 3).
[
4
2
4
2
2

The organic layers were dried over Na SO and concentrated to obtain the desired product (2 g, crude)
2
4
as a yellow oil, which was used directly in the next step without further purification.
6
.1.23. 5-Fluoro-6-(fluoromethyl)-2-methylpyrimidin-4-ol (56)
A mixture of 55 (1.9 g, 11 mmol), acetamidine hydrochloride (2.2 g, 22 mmol) and EtONa (2.3 g,
4 mmol) in 40 mL of EtOH was stirred at 80°C overnight. 6 N HCl (2 mL) was added and the reaction
3
mixture was concentrated. The residue was purified by silica gel column chromatography (petroleum
1
ether/ethyl acetate = 3/1 to 1/1) to give the desired product (800 mg, 43%) as a yellow solid. H NMR
(
400 MHz, CDCl ) δ 13.07 (br s, 1H), 5.35 (d, J = 46.8 Hz, 2H), 2.53 (s, 3H).
3
6
.1.24. 5-Fluoro-4-(fluoromethyl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine (57)
To a solution of 56 (800 mg, 5.0 mmol), Et N (1.5 g, 15 mmol) and N-methyl piperazine (750 mg,
3
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 Na SO and concentrated. The residue was purified by silica gel column
2
4
1
chromatography (ethyl acetate) to give the product (1 g, 83%) as a yellow oil. H NMR (400 MHz,
CDCl ) δ 5.36 (d, J = 47.2 Hz, 2H), 3.87-3.73 (m, 4H), 2.57-2.50 (m, 4H), 2.49 (s, 3H), 2.33 (s, 3H).
3
+
MS (ESI/APCI) m/z 242.9 [M+H] .
6
.1.25. 1-(5-Fluoro-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)-N-methylmethanamine (58)
A mixture of 57 (1.5 g, 6.2 mmol) and 2 N methylamine/methanol (6 mL) in i-PrOH (15 mL) and
H O (15 mL) was stirred at 80°C overnight in a sealed tube. The reaction mixture was concentrated,
2
diluted with H O (20 mL) and extracted with dichloromethane (30 mL x 3). The combined organic
2
layer was dried over Na SO and concentrated. The residue was purified by silica gel column
2
4
chromatography (dichloromethane/methanol = 40/1 to 20/1) to give the product (800 mg, 51%) as a
1
yellow oil. H NMR (400 MHz, CDCl ) δ 3.80-3.74 (m, 4H), 3.73 (s, 2H), 2.48-2.46 (m, 4H), 2.44 (s,
3
+
6
H), 2.31 (s, 3H). MS (ESI/APCI) m/z 254.0 [M+H] .
6
.1.26.
N-((5-Fluoro-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroq
uinolin-8-amine(21)
To a solution of 58 (100 mg, 0.4 mmol), 49 (81 mg, 0.48 mmol) and DIPEA (103 mg, 0.8 mmol)
in 5 mL of i-PrOH was stirred at 90°C for 3 days. 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). The organic layer was dried over Na SO and concentrated. The residue was
2
4
purified by silica gel column chromatography (dichloromethane/methanol = 20/1) to give the product
1
(
73 mg, 48%) as a yellow oil. H NMR (400 MHz, CDCl ) δ 8.48 (d, J = 4.0 Hz, 1H), 7.34 (d, J = 7.6
3
Hz, 1H), 7.06-7.02 (m, 1H), 4.11-4.00 (m, 1H), 3.81-3.60 (m, 6H), 2.89-2.75 (m, 1H), 2.75-2.62 (m,
1
1
H), 2.50-2.45 (m, 4H), 2.46-2.42 (m, 3H), 2.40 (s, 3H), 2.32 (s, 3H), 2.16-1.96 (m, 3H), 1.76-1.68 (m,
1
3
H). C NMR (150 MHz, CDCl ) δ 161.4 (d, J = 9.0 Hz), 157.4, 152.2 (d, J = 13.5 Hz), 147.2, 144.5
3
(
d, J = 256.5 Hz), 136.6, 134.2, 121.7, 64.5, 55.2, 53.5, 46.3, 46.1, 46.1, 39.2, 29.3, 25.5, 24.7, 21.3.
+
HRMS (ESI): calcd for C H FN [M+H] 385.2516, found 385.2514. Purity: 98.6%
2
1
30
6
6
.1.27. 4-Hydroxy-2,6-dimethylpyrimidine-5-carbonitrile (60)
A mixture of 59 (5.0 g, 27 mmol), acetamidine hydrochloride (3.9 g, 41 mmol) and K CO (11.3 g,
2
3
8
2 mmol) in 80 mL of EtOH was stirred at room temperature overnight. The reaction mixture was
acidified pH to 5 by 3 N HCl and then extracted with butyl alcohol (50 mL x 6). The combined organic
layer was dried over Na SO and concentrated to give the desired product (3.5 g, 87%) as a slight
2
4
2
3

1
yellow solid. H NMR (400 MHz, DMSO-d ) δ 13.30 (s, 1H), 2.39 (s, 3H), 2.35 (s, 3H). MS
6
+
(
ESI/APCI) m/z 150.0 [M+H] .
6
.1.28. tert-Butyl 4-(5-cyano-2,6-dimethylpyrimidin-4-yl)piperazine-1-carboxylate (61)
A mixture of 60 (2.5 g, 16 mmol), 1-Boc-piperazine (4.7 g, 25 mmol), PyBOP (9.6 g, 18 mmol)
and Et N (5.1 g, 50 mmol) in 60 mL of MeCN was stirred at 80°C overnight. The reaction solution was
3
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
1
a yellow solid. H NMR (400 MHz, CDCl ) δ 3.94 (s, 4H), 3.55 (s, 4H), 2.57 (s, 3H), 2.50 (s, 3H), 1.47
3
+
(
s, 9H). MS (ESI/APCI) m/z 317.9 [M+H] .
6
.1.29. tert-Butyl 4-(6-(chloromethyl)-5-cyano-2-methylpyrimidin-4-yl)piperazine-1-carboxylate (62)
To a solution of 61 (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 Na S O aqueous solution (20 mL). The aqueous layer was extracted with
2
2
3
dichloromethane (50 mL x 3). The combined organic layer was dried over Na SO and concentrated.
2
4
The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10/1 to
1
5
4
6
4
/1) to give the desired product (2.4 g, 54%) as a slight yellow solid. H NMR (400 MHz, CDCl ) δ
3
.58 (s, 2H), 3.99 (s, 4H), 3.57 (s, 4H), 2.56 (s, 3H), 1.48 (s, 9H).
.1.30.
tert-Butyl
-(5-Cyano-2-methyl-6-((methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)pyrimidin-4-yl)piperazi
ne-1-carboxylate (63)
A mixture of N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (421 mg, 2.6 mmol), 62 (1.0 g, 2.8 mmol),
KI (45 mg, 0.3 mmol) and DIPEA (671 mg, 5.2 mmol) in 10 mL of MeCN was stirred at room
temperature 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 = 1/1 to 0/1)
1
to give the desired product (800 mg, 64%) as a yellow oil. H NMR (400 MHz, CDCl ) δ 8.44 (d, J =
3
3
2
3
6
2
.6 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.04 (dd, J = 7.2, 4.8 Hz, 1H), 4.15-4.08 (m, 1H), 4.08-3.97 (m,
H), 3.97-3.88 (m, 4H), 3.02-2.93 (m, 4H), 2.86-2.76 (m, 1H), 2.73-2.63 (m, 1H), 2.53 (s, 3H), 2.28 (s,
H), 2.22-2.12 (m, 1H), 2.10-1.92 (m, 2H), 1.76 (s, 10H).
.1.31.
-Methyl-4-((methyl(5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-6-(4-methylpiperazin-1-yl)pyrimidi
ne-5-carbonitrile (22)
To a solution of 63 (600 mg, 1.3 mmol) in 2 mL of dichloromethane was added CF COOH (1 mL).
3
The mixture was stirred at room temperature for 2 h and then evaporated to give the product (700 mg,
crude) as a red oil. To a solution of the crude product in 1 mL of methanol was added HCHO/H O (37
2
percent, 1 mL) and NaBH CN (125 mg, 2.0 mmol). The mixture was stirred at room temperature for 2
3
h and then quenched with saturated NaHCO aqueous solution (10 mL). The aqueous layer was
3
extracted with dichloromethane (10 mL x 3). The combined organic layer was dried over Na SO and
2
4
concentrated. The residue was purified by Al O column chromatography (petroleum ether/ethyl
2
3
1
acetate = 5/1 to 0/1) to give the desired product (200 mg, 39%) as a slight yellow solid. H NMR (400
MHz, CDCl ) δ 8.44 (d, J = 4.0 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.04 (dd, J = 8.0, 4.8 Hz, 1H),
3
4
3
1
2
.15-4.08 (m, 1H), 4.08-3.93 (m, 6H), 2.87-2.75 (m, 1H), 2.75-2.62 (m, 1H), 2.55-2.45 (m, 7H), 2.33 (s,
13
H), 2.23 (s, 3H), 2.08-1.90 (m, 3H), 1.77-1.64 (m, 1H). C NMR (100 MHz, CDCl ) δ 174.7, 168.5,
3
62.4, 157.5, 147.0, 136.7, 134.3, 121.7, 117.4, 87.6, 64.8, 61.0, 55.0, 46.5, 46.1, 38.1, 29.3, 26.6, 26.4,
+
1.4. HRMS (ESI): calcd for C H N [M+H] 392.2563, found 392.2559. Purity: 98.2%
2
2
30
7
2
4

6
.2. In vitro biological assays
6
.2.1. HPB-ALL CXCR4 competitive binding assay [13]
HPB-ALL cells were maintained in RPMI-1640 (Gibico) supplemented with 10% FBS (Hyclone).
APC-conjugated anti-human CXCR4 was from Sungene. EC80 (0.83 µg/mL) was first determined for
2G5 binding to CXCR4. Then the compounds for testing were added into 96-well plates serially
1
diluted at a ratio of 1:3. Cells were washed once with ice-cold assay buffer (DPBS+2% HI-FBS) and
6
then re-suspended in the same buffer at a final concentration of 1 × 10 /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 [13]
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
4
+
range of compound was 1 µM to 0.0035 nM in 0.5% DMSO. Human CD 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
1
X HBSS/20 mM HEPES/0.005% P-104 assay buffer, adjusted the volume of the suspension to
6
achieve a cell concentration of 2.5 × 10 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 were added. After 20 min, CXCL12 (EC , 5 nM) was added
80
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 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 (IC ) was determined with a four-parameter logistic dose response equation.
50
6
.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
2
5

5
counted using Countess (Invitrogen). PBS was added to adjust cell density to 2.7 x 10 /ml.
IL-8 was serial diluted in LDV (low dead volume microplate) with 100 µM top dose, 4-fold
dilution, 10 points, and dispensed 100 nL/well into assay plate. Cells (7.5 µL, 2000/well) were added,
and the mixture was incubated at room temperature for 15 min. Forskolin (4.4 µM, 2.5 µL) was added
to the assay plate, and the mixture was incubated at room temperature for another 15 min. cAMP-d2 (5
µL) and Eu-Anti-cAMP working solutions (5 µL) were added, and the mixture was incubated at room
temperature for 60 min. The raw data were read on Envision (665 nm/615 nm).
Compound 23 and Navarixin were serial diluted in LDV with 10 mM top dose, 4-fold dilution, 10
points, and dispensed 100 nL/well into assay plate. Cells (7.5 µL, 2000/well) were added, and the
mixture was incubated at room temperature for 15 min. A solution of Forskolin and IL-8 in assay buffer
(
2.5 µL, 4.4 µM for Forskolin and 100 nM for IL-8) was added to the assay plate, and the mixture was
incubated at room temperature for another 15 min. cAMP-d2 (5 µL) and Eu-Anti-cAMP working
solutions (5 µL) were added, and the mixture was incubated at room temperature for 60 min. The raw
data were read on Envision (665 nm/615 nm).
6
.2.4. CCR6 functional assay
The CCR6 functional 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
4+
compound was 10 µM to 0.035 nM in 0.5% DMSO. Human CD , CCR6 enriched T-Cells were
previously activated, expanded and subsequently 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
6
suspension to achieve a cell concentration of 2.5 × 10 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 CCL20 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 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 (IC ) was determined with a four-parameter logistic dose response equation.
5
0
6
.2.5. Matrigel invasion assay
The human breast cancer cell line MDA-MB-231 was purchased from ATCC (Manassas, VA).
MDA-MB-231cell line was cultured in DMEM medium supplemented with 10% heat-inactivated fetal
2
6

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% O /5% CO . All cultures were monitored routinely and found to be
2
2
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
4
cancer cell line MDA-MB-231 cells (4 × 10 cells/well) were added to 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
.2.6. Cell viability assay
The cell viability was analyzed by using the CellTiter-Glo Luminescent Cell Viability Assay kit
following the manufacturer’s instructions (Promega). Luminescence was measured with SpectraMax
i3x (Molecular Devices).
6
.3. Preliminary in vitro safety and DMPK test
6
.3.1. Evaluation of CYP inhibitory potency [34]
Inhibitory potency of test compounds against human P450 enzymes was investigated in human
liver microsomes. The five major CYP isoforms and their respective probe substrates were as follows:
CYP3A4 (midazolam, 1µM), CYP2D6 (dextromethorphan, 5 µM), CYP1A2 (phenacitine, 20 µM),
CYP2C9 (tolbutamide, 100 µM), and CYP2C19 (S-mephenytoin, 40 µM). All probe substrates were
used at concentrations near or below their K s (Michaelis constant). Triplicate incubations were
m
conducted in the 200 µL incubation mixtures at 37 °C. The mixture contained HLM (0.2 mg/mL),
phosphate buffer (100 mM, pH 7.4), NADPH (1 mM), tested compounds (10 µM), and individual CYP
probe substrate. The mixtures were pre-incubated for 10 min to allow inhibitor/enzyme interactions
before the initiation of the reaction with NADPH. After 10 min, reactions were terminated by the
addition of 100 µL of cold acetonitrile containing an appropriate internal standard. Samples were then
centrifuged and injected into the LCMS/MS apparatus to quantify the concentrations of specific
metabolites formed by individual CYP enzymes.
6
.3.2. hERG assays [35,36]
Compound 23 was evaluated for block of the hERG potassium channel using CHO cells stably
expressing the hERG gene and the QPatch platform (Sophion, Ballerup, Denmark). K tail currents were
measured at −50 mV following a 500 ms depolarization to +20 mV from a holding voltage of −80 mV.
+
2+
2+
The external solution contained 4 mM K , 1 mM Mg , and 2 mM Ca . Compound effects were
quantified 4 min after application to the cells. Pulses were elicited every 20 s.
2
7

6
.3.3. Metabolic stability test [34]
To test the metabolic stability of compound 23 in vitro, each compound was incubated with rat
liver microsomes in the presence of NADPH. The reaction mixtures include NADPH (1 mM),
microsomes (0.2 mg/mL), phosphate buffer (100 mM, pH 7.4), and test compounds (1 µM). The
reaction was initiated by the addition of NADPH after a 10 min pre-incubation. The incubations were
taken at three time points (0, 30, 60 min) and placed into 100 µL of cold acetonitrile with internal
standard. The samples were then centrifuged at 13,000 rpm for 10 min and the supernatants were
subjected to LC-MS/MS analysis. The in vitro kinetic parameters were calculated using the following
equations:
0
.693
k
t
1/2
=
(1)
Where k is the slope from linear regression analysis of the natural Ln percent remaining of test
compound as a function of incubation time.
a
k
mgof protein
gof liver
Clint,in vitro
=
×
mgof proteinper mlincubation
(
2)
b
gof liver
×
kgof body weight
Where a is 20 mg/g for mouse and human, b is 87.5 g/kg for mouse and 25.7 g/kg for human.
Q
h
×Clint, in vitro
+ Clint, in vitro
Clhepatic
=
(3)
Q
h
Where Q is the hepatic blood flow (90 mL/min for mouse and 20.7 mL/min for human).
h
6
.3.4. Caco-2 permeability study [35,37]
The Caco-2 permeability study was performed by ChemPartner. Caco-2 cells were obtained
from American Tissue Culture Collection (Rockville, MD). The cells were maintained in Modified
Eagle’s medium (MEM), containing 10% heat-inactivated fetal bovine serum, and 1%
non-essential amino acids, in CO at 37°C. Cells were seeded on polycarbonate filter inserts
2
(Millipore, CAT#PSHT 010 R5).
The cells were cultured for 21–28 days prior to the transport experiments. The transepithelial electric
resistance and Lucifer Yellow permeability were checked before and after the assay. Compounds were
dissolved at 10 mM in 100% dimethyl sulfoxide (DMSO) and diluted for studies in Hanks Balanced
Salt Solution (HBSS, Invitrogen, Cat# 14025-092) with 25 mM HEPES, pH 7.4. Compounds were
tested at 10 µM, and in both the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) directions,
and were conducted at 37°C for 90 min. At the end of incubation, donor samples were diluted 10-fold
by assay buffer, then 60 µL of receiver and diluted-donor samples were mixed with 60 µL of
acetonitrile, and analyzed by LC-MS/MS. The concentrations of the compounds were quantified by
standard curve.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No. 81773561,
2
8