Design, synthesis, and evaluation of pyrrolidine based CXCR4 antagonists with in vivo anti-tumor metastatic activity

Article abstract of DOI:10.1016/j.ejmech.2020.112537

The chemokine receptor CXCR4 has been proposed as a drug target based on its important functions in HIV infection, inflammation/autoimmune diseases and cancer metastasis. Herein we report the design, synthesis and evaluation of novel CXCR4 antagonists based on a pyrrolidine scaffold. The structural exploration/optimization identified numerous potent CXCR4 antagonists, represented by compound 46, which displayed potent binding affinity to CXCR4 receptor (IC₅₀ = 79 nM competitively displacing fluorescent 12G5 antibody) and inhibited CXCL12 induced cytosolic calcium flux (IC₅₀ = 0.25 nM). Moreover, in a transwell invasion assay, compound 46 significantly mitigated CXCL12/CXCR4 mediated cell migration. Compound 46 exhibited good physicochemical properties (MW 367, logD_7.4 1.12, pKa 8.2) and excellent in vitro safety profiles (e.g., hERG patch clamp IC₅₀ > 30 μM and minimal CYP isozyme inhibition). Importantly, 46 displayed much improved metabolic stability in human and rat liver microsomes. Lastly, 46 demonstrated marked efficacy in a cancer metastasis model in mice. These results strongly support 46 as a prototypical lead for the development of promising CXCR4 antagonists as clinical candidates.

Full text of DOI:10.1016/j.ejmech.2020.112537

Journal Pre-proof
Design, synthesis, and evaluation of pyrrolidine based CXCR4 antagonists with in
vivo anti-tumor metastatic activity
Zhanhui Li, Xu Wang, Yu Lin, Yujie Wang, Shuwei Wu, Kaijiang Xia, Chen Xu, Haikuo
Ma, Jiyue Zheng, Lusong Luo, Fang Zhu, Sudan He, Xiaohu Zhang
PII:
S0223-5234(20)30509-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112537
EJMECH 112537
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 April 2020
Revised Date: 20 May 2020
Accepted Date: 3 June 2020
Please cite this article as: Z. Li, X. Wang, Y. Lin, Y. Wang, S. Wu, K. Xia, C. Xu, H. Ma, J. Zheng, L. Luo,
F. Zhu, S. He, X. Zhang, Design, synthesis, and evaluation of pyrrolidine based CXCR4 antagonists with
in vivo anti-tumor metastatic activity, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112537.
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Design, synthesis, and evaluation of pyrrolidine based CXCR4 antagonists with in vivo
anti-tumor metastatic activity
Zhanhui Li a,1, Xu Wang a,1, Yu Lin a, Yujie Wang a, Shuwei Wu a, Kaijiang Xia a, Chen Xu a, Haikuo Ma
a, Jiyue Zheng a, Lusong Luo b,*, Fang Zhu c, d, Sudan He c, d,*, and Xiaohu Zhang a,*
a Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences,
Soochow University, Suzhou, Jiangsu 215123, P. R. China
b BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, P. R. China
c Cyrus Tang Hematology Center, Jiangsu Institute of Hematology and Collaborative Innovation Center
of Hematology, Soochow University, Suzhou 215123, P.R. China.
d Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences & Peking Union Medial College, Beijing; Suzhou Institute of Systems Medicine, Suzhou,
215123 Jiangsu, P. R. China
1 these authors contributed equally to this paper
* Corresponding author. Tel.: +86 512 65880380; fax: +86 512 65880380; email:
xiaohuzhang@suda.edu.cn; hesudan2018@163.com; lusong.luo@beigene.com
Abstract: The chemokine receptor CXCR4 has been proposed as a drug target based on its important
functions in HIV infection, inflammation/autoimmune diseases and cancer metastasis. Herein we report
the design, synthesis and evaluation of novel CXCR4 antagonists based on a pyrrolidine scaffold. The
structural exploration/optimization identified numerous potent CXCR4 antagonists, represented by
compound 46, which displayed potent binding affinity to CXCR4 receptor (IC50 = 79 nM competitively
displacing fluorescent 12G5 antibody) and inhibited CXCL12 induced cytosolic calcium flux (IC50
=
0.25 nM). Moreover, in a transwell invasion assay, compound 46 significantly mitigated
CXLC12/CXCR4 mediated cell migration. Compound 46 exhibited good physicochemical properties
(MW 367, logD7.4 1.12, pKa 8.2) and excellent in vitro safety profiles (e.g., hERG patch clamp IC50
>
30 ꢀM and minimal CYP isozyme inhibition). Importantly, 46 displayed much improved metabolic
stability in human and rat liver microsomes. Lastly, 46 demonstrated marked efficacy in a cancer
metastasis model in mice. These results strongly support 46 as a prototypical lead for the development
of promising CXCR4 antagonists as clinical candidates.
Keywords: antagonist, chemokine, CXCL12, CXCR4, GPCR
Abbreviations: APC, allophycocyanin; CCD, charge coupled device; CYP, cytochrome P450; D
CM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco's modified eagle m
edium; DMSO, dimethyl sulfoxide; DPBS, Dulbecco's phosphate buffered saline; EMCCD, elect
ron-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 s
alt solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; hERG, human ether-a-g
o-go-related gene; HI-FBS, heat inactivated FBS; HIV, human immunodeficiency virus; HLM, h
1

uman liver microsomes; HPLC, high performance liquid chromatography; ICCD, intensified CC
D; IPA, isopropanol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PFA, pa
raformaldehyde; RFP, red fluorescence protein; RLM, rat liver microsomes; RPMI, roswell park
memorial institute; rt, room temperature; SAR, structure-activity-relationship; SDF-1, stromal cel
l-derived factor-1; SEM, standard error measurement; TEA, triethylamine; TFA, trifluoroacetic a
cid; THF, tetrahydrofuran; THQ, tetrahydroquinoline; TIQ, tetrahydroisoquinoline; TsOH, p-tolue
nesulfonic acid.
1. Introduction
CXCR4 is a chemokine receptor which belongs to the G protein-coupled receptor super family.
CXCR4 and its natural ligand CXCL12 (also known as stromal cell-derived factor-1, or SDF-1)
regulate an array of important physiological functions such as chemotaxis, hematopoietic stem cell
homing and retention [1, 2]. Dysregulation of CXCR4/CXCL12 axis has been linked to human
pathological conditions (e.g., HIV virus infection, inflammatory/autoimmune diseases and cancer
metastasis) [3, 4]. Consequently, development of CXCR4 antagonists to modulate the pathological
functions of CXCL12/CXCR4 axis has been proposed as an important strategy for the treatment of
HIV infection, inflammatory diseases and cancer [5-9].
CXCR4 was first identified as a coreceptor used by T cell-tropic (X4) HIV-1 virus to gain cellular
entry [10]. As a result, the majority of early research of CXCR4 antagonists focused on anti-HIV
application. This effort culminated in the identification and development of AMD3100 (plerixafor,
Figure 1) [11, 12], the most advanced small molecule CXCR4 antagonist. Unfortunately, dose-limiting
toxicity and poor oral bioavailability impeded its application as an orally administered daily drug for
chronic HIV treatment. During clinical trials, however, AMD3100 surprisingly displayed strong effect
on mobilizing hematopoietic stem cell. Consequently, AMD3100 was approved by the FDA for the
treatment of non-Hodgkin’s lymphoma and multiple myeloma patients with autologous stem cell
transplants. This unexpected clinical utilization of AMD3100 significantly expanded the scope of
CXCR4 research [13]. More recent discoveries of CXCR4/CXCL12 function include cancer cell
proliferation, angiogenesis, and metastasis. In addition, CXCR4 was reported to modulate cancer
immunity by trafficking of key immune cells into tumor microenvironment. Since many of the immune
cells were immunosuppressive (e.g., Treg, myeloid derived suppressor cells) rather than
immunoreactive (e.g., CD8+ effector T lymphocytes), CXCL12/CXCR4 axis plays an important role in
evasion of immune surveillance [4]. The dose-limiting toxicity and poor oral bioavailability of
AMD3100, combined with the emerging evidence of CXCR4/CXCL12 in modulating cancer cell
proliferation, metastasis and immunity at the tumor microenvironment, sparked significant renewed
interests to discover new CXCR4 antagonists with high potency, low toxicity, and acceptable oral
bioavailability.
In order to address the poor oral bioavailability associated with AMD3100, systemic effort aimed
to reduce molecular weight and cationic charges was carried out [14-24]. These exercises led to the
identification of AMD11070 [17, 18, 25]. AMD11070 displayed improved bioavailability and was
progressed into clinical investigation. However, AMD11070 moderately inhibited CYP450 isozymes
(e.g., CYP3A4, 2D6), likely due to the benzimidazole moiety presented in the molecule. Further
2

structural modification to replace this functional group resulted in the identification of GSK812397 [24,
26] and TIQ15 [23, 27] (Figure 1).
Fig. 1. Representatives of small molecule CXCR4 antagonists reported in the literature.
Taking a similar approach, we have developed and reported a series of CXCR4 antagonists based
on aminopyrimidine central core structure (exemplified by compound 1 in Figure 2) [28, 29]. These
compounds competitively displace 12G5 antibody, inhibit CXCL12 induced cytosolic calcium flux,
and mitigate CXCL12 induced cell migration in a transwell invasion assay. Moreover, they generally
show good physicochemical properties and safety profiles (moderate to low hERG and CYP isozyme
inhibition). However, an outstanding issue related to these compounds is the fast clearance in liver
microsomes, which could result in poor oral bioavailability and short half-life. In order to address this
problem, we did an extensive structure-metabolic relationship study (Table 1). For example, the
2-position (R1) of the central pyrimidine ring was explored by substitutions with increased size and
lipophilicity (compounds 2 and 3) or with increased polarity and hydrophilicity (compounds 4 and 5).
Unfortunately, the metabolic stability of these compounds was unanimously poor in rat liver
microsomes. Similar results were obtained for R2, R3, and R4 variants. Since all these changes at R1-R4
had no impact to alter the metabolic stability, we posited that the tetrahydroquinoline (THQ) was likely
the culprit for high clearance (the only unchanged structural element in this exercise). This hypothesis
was supported by the metabolic stability data of AMD11070 and GSK812397 (Table 1), both of which
contain a THQ tethered to a distinctive heterocyclic group. Direct evidence concerning the metabolic
liability of the THQ functionality was presented by scientists from Emory University based on data of
their tetrahydroisoquinoline (TIQ) series of compounds [27]. They showed that hydroxylation on the
THQ moiety in mouse liver microsomes was responsible for high clearance. Blockade of the metabolic
soft spot on the THQ moiety shifted the metabolic liability to the TIQ moiety and ring opening of the
THQ moiety had helped to improve metabolic stability. Encouraged by these results, we formulated a
structural modification plan as described in Figure 2. Herein we report the design, synthesis and
evaluation of novel CXCR4 antagonists based on a pyrrolidine scaffold that showed improved
metabolic stability in human and rat liver microsomes. Furthermore, compound 46 demonstrated
marked efficacy in a mouse cancer metastasis model.
Table 1. Metabolic stability of compounds 1-13, AMD11070, and GSK812397
N
N
R2
R3
N
R1
N
R4
Cmpd
Structure
RLMa
Cmpd
Structure
RLMa
3

Clint
t1/2
Clint
t1/2
(mL/min/kg)
(min)
(mL/min/kg)
(min)
1
2
3
4
5
6
7
8
1567
2092
2439
1129
767
1.59
1.19
1.02
2.20
3.24
1.95
2.31
1.51
9
2179
1549
>497
>497
>497
1776
651
1.14
1.60
<1
10
11
12
13
<1
<1
AMD
11070
1275
1077
1644
1.40
3.82
GSK
812397
4

a All the compounds were tested at 1 µM concentration. RLM = Rat liver microsomes.
Fig. 2. Proposed SAR study plan.
2. Chemistry
The synthesis of compounds 26-28 was performed as shown in Scheme 1. Intermediate 15 was
synthesized by reduction of commercially available 14 with tin powder followed by Pd/C. Intermediate
17 was synthesized by cyclization of commercially available 16 [30]. Reduction of 17 with NaBH4
yielded intermediate 18. Commercially available 19 and diphenyl ketone were reacted in toluene to
give imine intermediate, which was then reacted with ethyl (E)-but-2-enoate to afford 20 under alkaline
conditions. Cyclization of 20 in the presence of concentrated HCl afforded 21, which was then reduced
by LiAlH4 in THF to give intermediate 22. Compounds 15, 18, and 22 were reacted with
4-chloro-6-(chloromethyl)-2-methylpyrimidine [29] in the presence of DIPEA and KI to give
intermediates 23-25. The syntheses of compounds 26-28 were achieved by reacting intermediates
23-25 with N-methylpiperazine in EtOH.
The synthesis of compounds 35 and 39 was performed as shown in Scheme 2. Commercially
available 2-(2-bromoethyl)-1,3-dioxolane was reacted with Mg in THF to give Grignard reagent, which
was reacted with commercially available 29 to give 30. Deprotection of 30 with 3 N HCl in acetone
afforded 31, which was reacted with (R)-1-(4-methoxyphenyl)ethan-1-amine in the presence of
NaBH(OAc)3 to give 32 [31]. Debenzylation of 32 with TFA provided 33, which was reacted with
4-chloro-6-(chloromethyl)-2-methylpyrimidine [29] in the presence of DIPEA and KI to give 34.
Compound 35 was achieved by reacting intermediate 33 with N-methylpiperazine in EtOH. Similarly,
compound 39 was synthesized by using (S)-1-(4-methoxyphenyl)ethan-1-amine in place of
(R)-1-(4-methoxyphenyl)ethan-1-amine.
The synthesis of compounds 46-49, 50, and 52-55 was performed as shown in Scheme 3.
Commercially available 2-(2-bromoethyl)-1,3-dioxolane was reacted with Mg in THF to give Grignard
reagent, which was reacted with commercially available 40a-d to give 41a-d. Deprotection of 41a-d
with
3
N
HCl
in
acetone
afforded
42a-d,
which
were
reacted
with
(R)-1-(4-methoxyphenyl)ethan-1-amine in the presence of NaBH(OAc)3 to give 43a-d. Debenzylation
of 43a-d with TFA provided 44a-d, which were reacted respectively with
4-chloro-6-(chloromethyl)-2-methylpyrimidine [29] in the presence of DIPEA and KI to give 45a-d.
Compounds 46-49 were achieved by reacting intermediates 45a-d with N-methylpiperazine in EtOH.
Compounds 53-55 were achieved by reacting intermediate 45a with N-methyl-1,4-diazepane,
5

N,N-dimethylpiperidin-4-amine, and 2-morpholinoethan-1-amine, respectively. Compound 50 was
achieved by reacting intermediate 44a with
5-fluoro-4-(fluoromethyl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine [29] in the presence of
2-ethyl-2-(hydroxymethyl)propane-1,3-diol and KI [32]. Intermediate 44a was reacted with tert-butyl
4-(6-(chloromethyl)-5-cyano-2-methylpyrimidin-4-yl)piperazine-1-carboxylate [29] in the presence of
DIPEA and KI to give intermediate 51. Deprotection of BOC group followed by reaction with
HCHO/NaBH3CN yielded compound 52.
Scheme 1. Reagents and conditions: (a) 1) Sn, 6 N HCl (aq.), EtOH, 100 oC, 12 h; 2) Pd/C, H2, MeOH,
o
rt, 16 h; (b) 1) 1-vinyl pyrrolidin-2-one, NaH, THF, 60 C, 2 h; 2) 4 N HCl (aq.), rt, overnight; (c)
o
o
NaBH4, acetic acid, MeOH, -20 C, 3 h; (d) 1) diphenyl ketone, TsOH, toluene, 120 C, overnight; 2)
ethyl (E)-but-2-enoate, benzyltriethylammonium chloride, 50% NaOH (aq.), MeCN, rt, overnight; (e)
conc.
HCl,
MeCN,
rt,
2
h;
(f)
LiAlH4,
THF,
50
oC,
overnight;
(g)
4-chloro-6-(chloromethyl)-2-methylpyrimidine, DIPEA, KI, MeCN, rt, overnight; (h)
N-methylpiperazine, TEA, EtOH, 80 oC,3 h.
Scheme 2. Reagents and conditions: (a) 1) 2-(2-bromoethyl)-1,3-dioxolane, Mg, iodine, THF, 30 oC, 1
6

h; 2) 29, THF,
0
oC;
3
h; (b)
3
N
HCl (aq.), acetone, rt, overnight; (c)
o
o
(R)-1-(4-methoxyphenyl)ethan-1-amine, NaBH(OAc)3, DCM, -70 C to rt, overnight; (d) TFA, 50 C,
12 h; (e) 4-chloro-6-(chloromethyl)-2-methylpyrimidine, DIPEA, KI, MeCN, rt, overnight; (f)
N-methylpiperazine, TEA, EtOH, 80 oC, 3 h; (g) (S)-1-(4-methoxyphenyl)ethan-1-amine, NaBH(OAc)3,
DCM, -70 oC to rt, overnight.
Scheme 3. Reagents and conditions: (a) 1) 2-(2-bromoethyl)-1,3-dioxolane, Mg, iodine, THF, 30 oC, 1
h; 2) 40a-d, THF,
0
oC;
3
h; (b)
3
N
HCl (aq.), acetone, rt, overnight; (c)
o
o
(R)-1-(4-methoxyphenyl)ethan-1-amine, NaBH(OAc)3, DCM, -70 C to rt, overnight; (d) TFA, 50 C,
12 h; (e) 4-chloro-6-(chloromethyl)-2-methylpyrimidine, DIPEA, KI, MeCN, rt, overnight; (f)
corresponding
amine,
TEA,
EtOH,
80
oC,
3
h;
(g)
for
50:
5-fluoro-4-(fluoromethyl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine,
2-ethyl-2-(hydroxymethyl)propane-1,3-diol, H2O, KI, 120 oC, overnight; for 51: tert-butyl
4-(6-(chloromethyl)-5-cyano-2-methylpyrimidin-4-yl)piperazine-1-carboxylate, DIPEA, KI, MeCN, rt,
overnight; (h) 1) TFA, DCM, rt, 2 h; 2) HCHO/H2O, NaBH3CN, MeOH, rt, 2 h.
3. Results and discussion
3.1. Structure-activity-relationship of synthesized compounds
In our previous publications [28, 29], we used the cell based FACS 12G5 competitive binding assay
as the primary screening assay. Once an active compound was identified, the compound was subjected
to a FLIPR Tetra based cytosolic calcium flux assay. In this report, we used both assays to characterize
our compounds and we are in a transition to switch the primary screening assay from the 12G5
competitively binding assay to the FLIPR Tetra based calcium flux functional assay. This change is
based on numerous considerations. For example, competitive ligand binding assays convey no
7

functional information while calcium flux assay provides information regarding receptor activation,
potentiation, and inhibition. Competitive ligand binding assays may also miss allosteric binders which
do not compete for the same binding pocket [33]. More importantly, based on our experience, the
results from the 12G5 competitive binding assay correlate very well with those from the functional
calcium flux assay. Moreover, the expense of the calcium flux assay was lower and the throughput was
higher compared with the 12G5 competitive binding assay. Based on these assessments, we decided to
use the FLIPR Tetra based calcium flux functional assay as the screening assay and the 12G5
competitively binding assay as the confirmation assay. The structure-activity-relationship is
summarized in Tables 2 and 3.
Controlling key physicochemical properties in a desirable range (e.g., pKa 6-8, clogP 0-3) has been
one of our strategies in optimization of CXCR4 antagonists. Here we report the pKa and clogP
alongside of the biological activity of synthesized analogues. Compounds 26 and 27 are respectively
formed from the ring opening/ring closure of compound 1 (Figure 2) to form piperidine or pyrrolidine
as the scaffold structures. Compound 27 (319 nM) was twice as potent as 26 (620 nM) in the 12G5
binding assay. In addition, compound 27 has a lower clogP than 26. Therefore, we chose to conduct
further exploration based on compound 27. Addition of a methyl group on the pyrrolidine ring
(compound 28) led to five folds binding affinity decrease compared with 27. Since compound 27
contains a chiral center, we synthesized chirally pure (S)-stereoisomer 35 (220 nM) and
(R)-stereoisomer 39 (> 10000 nM). As expected, compound 35 showed more potent binding compared
with the racemate 27, while its enantiomer 39 was virtually inactive. These results are consistent with
our previous research and literature reports [28, 29]. Therefore, all of our subsequent compounds are
synthesized to be (S)-stereoisomers. In order to explore substitution effects on the pyridine ring, we
introduced methyl group at the 3, 4, 5, and 6 positions of pyridine to obtain compounds 46-49. Among
them, the 3-methyl-substituted compound 46 (79 nM) displayed significantly higher binding affinity
than the other substituted analogues. In addition, by comparing the performance of compounds 46 and
35 in binding assay, we found that the 3-methyl substitution of the pyridine ring can increase binding
affinity by about three folds. We also tested the functional potencies in the FLIPR Tetra based calcium
mobilization assay of some compounds (35, 46, 48), and the results were largely consistent with the
binding affinity assay. AMD3100 was used as the positive control.
Using compound 46 as the prototype, we continued to explore R2 and R3 substituents. The results are
shown in Table 3. Highly basic functional group may lead to safety issues such as hERG potassium
channel inhibition, CYP enzyme inhibition and phospholipidosis [34], we therefore replace the
hydrogen at the R2 position with fluorine (compound 50) and cyano (compound 52) as a way to
mitigate overall basicity. Compared to compound 46 (pKa: 7.3), the pKa of the two compounds (50
pKa: 6.3, 52 pKa: 5.6) were significantly reduced. However, the functional potency of compounds 50
and 52 was decreased by 4 and 7 folds compared to that of compound 46, respectively. A basic center
on the R3 position was reported to be essential for CXCR4 antagonist activity [28, 29]. We therefore
designed and synthesized compounds 53-55 with varied pKa (7.0-9.0). In addition, this small set of
amines offered structural diversity such as ring expansion (compound 53), exocyclic basic center
(compound 54), and basic center with less restriction (compound 55). Unfortunately, compounds 54
(13 nM) and 55 (2.7 nM) displayed significantly decreased functional potency compared with
compound 46. Compound 53 (0.40 nM) showed comparable functional potency to compound 46, but
with significantly increased basicity (pKa: 8.9). Overall, compound 46 remained to be the most
attractive lead with balanced potency and physicochemical properties. We carefully controlled clogP
8

and pKa of our compounds throughout the optimization process. Although calculated data provide
efficiency and guidelines, they do not always reflect the actual values. We therefore experimentally
determined the logD7.4 (shake flask) and pKa (titration) of compound 46 (Table 3). The actual pKa (8.2)
of compound 46 was almost a unit higher compared with the calculated pKa (7.3). This discrepancy
was accurately reflected on actual logD7.4 (1.12) compared with the calculated clogP (2.08). The
relatively large variations between the actual and calculated data demonstrated the necessity to
experimentally determine the physicochemical properties of key compounds.
Table 2. SAR for ring A and R1
Ca2+
flux
Ca2+
flux
Binding
IC50
(nM)a
Binding
IC50
(nM)a
Ring
A
Cmpd Ring A R1
clogPc pKad Cmpd
R1
clogPc pKad
IC50
(nM)b
IC50
(nM)b
0.25
±
620 ±
191
H
NDe
ND
ND
1.80
1.29
1.77
7.8
7.3
7.3
3-Me 79 ± 11
2.08
2.08
1.75
7.3
7.3
7.3
26
46
47
48
0.08
319 ±
153
216 ±
4-Me
11
H
H
ND
27
28
1546 ±
342
278 ±
5-Me
1.3
4.9 ±
1.0
220 ±
76
2.7 ±
0.006
2391 ±
6-Me
H
H
1.29
1.29
7.3
7.3
ND
1.35
7.3
35
39
49
108
561 ±
27
23 ±
2.3
AMD
3100
>10000
ND
-1.04
10.6
a
Inhibition of luminescence signaling in HPB-ALL CXCR4 competitive binding assay. Data are
b
expressed as geometric mean values of at least two runs ± the standard error measurement (SEM).
Inhibition of fluorescence signaling in calcium flux assay based on FLIPR Tetra. Data are expressed as
c
geometric mean values of at least two runs ± the standard error measurement (SEM). Calculated by
Molinspiration. d Calculated by ACD/Labs 6.0. e ND = not determined.
Table 3. SAR for R2 and R3
9

Ca2+ flux
IC50 (nM)a
Cmpd
46
R2
H
R3
clogPb pKac
2.08b
1.12d
7.3c
8.2e
0.25 ± 0.08
1.1 ± 0.06
1.8 ± 0.5
F
2.17
1.76
2.35
2.61
6.3
5.6
8.9
9.0
50
CN
H
52
0.40 ± 0.18
13 ± 6.6
53
H
54
H
2.7 ± 0.2
23 ± 2.3
1.82
7.0
55
-1.04
10.6
AMD3100
a
Inhibition of fluorescence signaling in calcium flux assay based on FLIPR Tetra. Data are expressed
as geometric mean values of at least two runs ± the standard error measurement (SEM). b Calculated by
c
e
Molinspiration. Calculated by ACD/Labs 6.0. d logD7.4 was determined by shake flask technique.
pKa was determined by titration using the Sirius T3 system
.
3.2. Preliminary selectivity assessment and functional evaluation of lead compound 46
Having established compound 46 as a promising CXCR4 antagonist (displacement of 12G5, IC50
= 74 nM; inhibition of CXCL12 mediated cytosolic calcium flux, IC50 = 0.25 nM), we next assessed
the preliminary functional specificities of compound 46. CXCR1 was selected as a representative
within the CXCR sub-family and CCR6 was selected as a representative outside of the CXCR
sub-family. Compound 46 was tested in the corresponding functional assays. Compound 46 was shown
to be inactive up to 10,000 nM (Figure 3b, c).
CXCR4 is a chemokine receptor. One of its hallmark functions is migration and invasion of the
CXCR4 expressing cells toward CXCL12 gradient. Thus, we used the Matrigel invasion assay to
evaluate the effect of compound 46 on CXCL12/CXCR4 mediated cell migration. In this experiment,
CXCR4 expressing MDA-MB-231 cells and compound 46 (100 nM) were added to the upper chamber.
Chemoattractant CXCL12 was added to the lower chamber. AMD3100 was used as a positive control.
The inhibition of cell invasion with compound 46 and AMD3100 was calculated by comparing to the
cell invasion without treatment. In consistent with the 12G5 binding and the calcium mobilization data,
compound 46 was more potent in the Matrigel invasion assay than AMD3100 (Figure 4). General
cellular toxicity can impede cell mobility, resulting in false positive effect in cell migration assay. To
exclude this possibility, Cell viability assay (ATP-based Cell Titer-Glo Luminescent Cell Viability
Assay) was performed to evaluate the cytotoxicity effect of compound 46. As shown in Figure 4c,
compound 46 (up to 1000 nM) did not inhibit proliferation of multiple murine and human cells.
10

Fig. 3. Compound 46 is a potent CXCR4 antagonist, but is inactive to CXCR1 and CCR6. (a) CXCR4
dose response curve for SDF-1 (CXCL12) stimulation and inhibition curves of compound
46/AMD3100 (positive control for CXCR4) based on a calcium flux assay. (b) CXCR1 dose response
curve for IL-8 stimulation and inhibition curves of compound 46/Navarixin (positive control for
CXCR1) based on a functional cAMP assay. (c) CCR6 dose response curve for CCL20 stimulation and
inhibition curves of compound 46/PF-9654-00 (positive control for CCR6) based on a calcium flux
assay.
11

Fig. 4. Effect of compound 46 on matrigel invasion of MDA-MB-231 cells and cytotoxicity assessment
of 46 in the cultured human and murine cells. (a) Photo images of matrigel 22 h after invasion
experiment. (b) Quantification of transwell analysis of cell invasion experiment. Data represent mean
value ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Magnification: 100x.
(c) Effect of compound 46 on proliferation of human and murine cells.
4.3. Preliminary in vitro safety and metabolic stability evaluation of lead compound 46
AMD11070 was derived from AMD3100 with reduce molecular weight and cationic charges. As a
result, AMD11070 can be dosed orally and has been progressed into clinical testing. However, it was
reported that AMD11070 suffered from CYP isozyme inhibition (3A4, 2D6; 60% and 64%,
respectively, at 10 ꢀM), possibly due to the benzimidazole functionality [28]. Compound 46 possesses
good physicochemical properties (MW 367, logD7.4 1.12, pKa 8.2) and was evaluated by in vitro safety
tests. As shown in Table 4, compound 46 exhibited minimal inhibition of CYP 1A2, 2C9, 2C19, 2D6,
and 3A4 at 10 ꢀM concentration. Moreover, compound 46 exhibited very low inhibition (IC50 > 30 ꢀM)
of hERG (standard patch clamp), indicating minimal cardiotoxicity liability associated with blockade
of this key potassium channel. In addition, as shown in Table 5, compound 46 displayed low plasma
protein binding in mouse and rat (33% and 44%, respectively) but high plasma protein binding in
human (95%). Although it is common for compounds to have higher plasma protein binding in human
than in preclinical species, the variation of plasma protein binding in rodents and in human is large in
this case.[35] Since compound 46 is lipophilic (logD7.4 1.12) and basic (pKa 8.2), it likely binds to
albumin and more so, to α1-acid glycoprotein.[36] The observed species difference of the plasma
protein binding might reflect the different interactions between compound 46 and α1-acid
glycoprotein/albumin in rodents and human. More importantly, the higher unbound fractions in rodents
may lead to higher clearance compare with human.[37] Similarly, the higher unbound fractions in
12

rodents may also impose advantage on efficacy in rodent disease models. These factors must be
considered for further progression of compound 46. Lastly, compound 46 demonstrated moderate
intrinsic clearance in rat (123 mL/min/kg) and human (66 mL/min/kg) liver microsomes. This is in
contrast to the poor metabolic stability exhibited by our former results (Table 1) [28, 29] and represents
a major improvement in drug candidate optimization.
Table 4. Preliminary in vitro safety evaluation of compound 46
CYP inhibition (%), test concentration (10 µM)
3A4 3A4
(midazolam) (testosterone)
hERG
IC50
Cmpd
1A2 2C9 2C19 2D6
(ꢀM)
46
> 30
-6
1
11
-5
7
-4
Table 5. Preliminary in vitro DMPK evaluation of compound 46
PPB (%)
RLMb
HLMc
Cmpda
Clint
t1/2
Clint
t1/2
mouse
33
rat
44
human
95
(mL/min/kg) (min) (mL/min/kg) (min)
46
123
20.2
66
26.2
a
b
c
The compound was tested at 1 µM concentration. RLM = Rat liver microsomes. HLM = Human
liver microsomes.
4.4. Evaluation of compound 46 in cancer metastasis model in mice
Cancer cells expressing CXCR4 can migrate to distant CXCL12-rich tissues such as bone marrow,
lungs, and lymph nodes. CXCR4 antagonists have the potential to blockade this process and therefore
inhibit cancer metastasis, which is a predominant factor for poor patient prognosis in many cancers. We
have established a lung metastasis mouse model using red florescence protein labeled Lewis lung
carcinoma LL/2 (RFP-LL/2) [38]. Compound 46 was evaluated in this model for its potential as an
anti-cancer metastasis agent. As shown in Figure 5a, C57BL/6 mice received tail intravenous injection
of RFP-LL/2 and developed lung metastasis. After 18 days, mice were sacrificed and lung tissues were
collected. The lung metastasis of LL/2 cells in mice was analyzed by measuring RFP fluorescence
intensities with the PerkinElmer IVIS Lumina II. Treatment by compound 46 (70 mg/kg D-tartaric acid
salt, equivalent to 30 mg/kg active pharmaceutical ingredient, i.p.) significantly reduced the lung
metastasis of LL/2 cells, indicating the potential of compound 46 as an anti-cancer metastasis agent
(Figure 5, b, c, and d).
13

Fig. 5. In vivo anti-metastasis evaluation of compound 46 salt. (a) Schematic of tumor metastasis assay.
(b) All images of the lungs taken by the camera. (c, d) Tumor metastasis of RFP-labeled LL/2 was
calculated by RFP luciferase intensity. Representative pictures and statistical diagram are present. *P <
0.05.
5. Conclusion
Herein we report the design, synthesis and evaluation of novel CXCR4 antagonists based on a
pyrrolidine scaffold. The structural exploration/optimization identified compound 46 as a potent
CXCR4 antagonist. Compound 46 competitively displaced fluorescent 12G5 antibody with IC50 = 79
nM and inhibited CXCL12 induced cytosolic calcium flux (IC50 = 0.25 nM). Moreover, in a transwell
invasion assay, compound 46 significantly mitigated CXLC12/CXCR4 mediated cell migration.
Compound 46 exhibited good physicochemical properties (MW 367, logD7.4 1.12, pKa 8.2) and
excellent in vitro safety profiles (e.g., hERG patch clamp IC50 > 30 ꢀM and minimal CYP isozyme
inhibition). Importantly, 46 displayed much improved metabolic stability in human and rat liver
microsomes. Lastly, 46 demonstrated marked efficacy in a mouse cancer metastasis model. These
results strongly support 46 as a prototypical lead for the development of promising CXCR4 antagonists
as clinical candidates.
6. Experimental protocols
6.1. Chemistry
General reaction progress was monitored by analytical thin layer chromatography performed on
silica gel HSGF254 pre-coated plates. Organic solutions were dried over anhydrous Na2SO4, and the
solvents were removed under reduced pressure. Final compounds were purified with silica gel 100-200
mesh for column chromatography. 1H NMR were obtained on 400 MHz (Varian) spectrometer, and 13
C
14

NMR were obtained on 600 MHz (Varian) spectrometer. Chemical shifts were given in ppm using
tetramethylsilane as internal standard. Mass spectra were obtained using an Agilent 1100 LC/MSD
Trap SL version Mass Spectrometer. HRMS analysis was recorded on an Agilent 6540 UHD
Accurate-Mass Q-TOF LC/MS. Values of optical rotation were measured on a Rudolph Automatic
Polarimeter A21101. HPLC method: Waters Acquity UPLC, BEH C18 2.1 mm × 50 mm, 1.7 ꢀm
particles. Mobile phase A: 5 mM aqueous ammonium acetate. Mobile phase B: MeOH. Temperature:
24 °C. Gradient: 5−40% B over 1 min, 40-70% B over 1 min, 70-95% B over 4 min, then a 1 min hold
at 95% B. Flow: 1.2 mL/min. Detection: UV at 214 and 254 nm.
6.1.1. 2-(Piperidin-2-yl)pyridine (15)
To a solution of 14 (3.1 g, 20 mmol), tin (9.4 g, 80 mmol) in ethanol (50 mL) was slowly added 6 N
o
HCl aqueous solution (40 mL) and stirred at 100 C for 12 h. The reaction mixture was slowly added
saturated NaOH aqueous solution to adjust pH 8-9. The water layer was extracted with
dichloromethane (100 mL * 3), dried over Na2SO4, filtered and concentrated. To a solution of the
resulting residue in methanol (40 mL) was added 10% Pd/C (300 mg) and stirred at room temperature
under H2 atmosphere for 16 h. The mixture was filtrated and concentrated. The residue was purified by
silica gel column chromatography (dichloromethane/methanol/ammonia = 100/1/1) to give the desired
product (200 mg, 6.3%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 4.0 Hz, 1H), 7.63
(t, J = 7.6 Hz, 1H), 7.65-7.59 (m, 1H), 7.19-7.10 (m, 1H), 4.25 (t, J = 7.6 Hz, 1H), 3.25-3.20 (m, 1H),
3.05-2.99 (m, 1H), 2.62-2.32 (m, 2H), 2.27-2.19 (m, 1H), 1.94-1.82 (m, 2H), 1.79-1.70 (m, 1H). MS
(ESI/APCI) m/z 163.0 [M+H]+.
6.1.2. 2-(3,4-Dihydro-2H-pyrrol-5-yl)pyridine (17)
To a solution of 60% NaH (3.2 g, 80 mmol) in dry THF (100 mL) was slowly added
o
1-vinylpyrrolidin-2-one (4.5 g, 40 mmol) and 16 (6.0 g, 40 mmol) at 0 C under N2 atmosphere. The
o
reaction mixture was heated to 60 C and stirred at the same temperature for 2 h. The mixture was
cooled to room temperature, 4 N HCl aqueous solution (50 mL) was added and stirred at room
temperature overnight. 20% NaOH aqueous solution (50 mL) was added and extracted with
dichloromethane (50 mL * 3). The combined organic layer was dried over Na2SO4, filtered and
concentrated. The residue was purified by silica gel column chromatography
1
(dichloromethane/methanol = 100/1) to give the desired product (1.1 g, 19%) as a colorless oil. H
NMR (400 MHz, CDCl3) δ 8.64 (d, J = 4.4 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.78-7.70 (m, 1H),
7.35-7.28 (m, 1H), 4.15-4.08 (m, 2H), 3.14-3.06 (m, 2H), 2.14-1.94 (m, 2H). MS (ESI/APCI) m/z
147.0 [M+H]+.
6.1.3. 2-(Pyrrolidin-2-yl)pyridine (18)
To a solution of 17 (290 mg, 2.0 mmol) and acetic acid (2 mL) in methanol (8 mL) was slowly added
NaBH4 (190 mg, 5 mmol) at -20 oC for 3 h. The reaction was quenched with water (20 mL) and washed
with ethyl acetate (30 mL). The water layer was adjusted pH to 9 by saturated NaHCO3 aqueous
solution and extracted with dichloromethane (20 mL * 4). The combined organic layer was dried over
1
Na2SO4, filtered and concentrated to give the desired product (126 mg, 43%) as a colorless oil. H
NMR (400 MHz, CDCl3) δ 8.56-8.47 (m, 1H), 7.67-7.59 (m, 1H), 7.40-7.30 (m, 1H), 7.19-7.10 (m,
1H), 4.27 (t, J = 7.2 Hz, 1H), 3.28-3.16 (m, 1H), 3.07-2.98 (m, 1H), 2.30-2.15 (m, 1H), 1.95-1.84 (m,
2H), 1.80-1.69 (m, 1H). MS (ESI/APCI) m/z 149.0 [M+H]+.
6.1.4. Ethyl 4-((diphenylmethylene)amino)-3-methyl-4-(pyridin-2-yl)butanoate (20)
A mixture of 19 (400 mg 3.7 mmol) and benzophenone (670 mg, 3.7 mmol) in toluene (10 mL) was
o
stirred at 120 C with Dean-Stark trap overnight. The mixture was cooled to room temperature and
15

washed with saturated NaHCO3 aqueous solution (10 mL * 2). The organic layer was dried over
Na2SO4, filtered and concentrated. To a solution of the resulting residue in acetonitrile (15 mL) was
added 50% NaOH aqueous solution (0.23 mL, 2.8 M), benzyltriethylammonium chloride (60 mg, 0.30
mmol) and stirred at room temperature for 30 min. Ethyl (E)-but-2-enoate (630 mg, 5.5 mmol) was
added and the mixture was stirred at room temperature overnight. The mixture was diluted with water
(20 mL) and extracted with dichloromethane (20 mL * 3). The combined organic phase was dried over
Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography
(petroleum ether/ethyl acetate = 10/1) to give the desired product (1.3 g, 92%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) δ 8.51 (d, J = 4.0 Hz, 1H), 7.75-7.68 (m, 2H), 7.67-7.58 (m, 1H), 7.45-7.32 (m, 7H),
7.16-7.10 (m, 1H), 7.03-6.90 (m, 2H), 4.53-4.38 (m, 1H), 4.08-3.98 (m, 2H), 2.89-2.70 (m, 1H),
2.58-2.05 (m, 2H), 1.25-1.11 (m, 3H), 1.00-0.80 (m, 3H). MS (ESI/APCI) m/z 386.9 [M+H]+.
6.1.5. 4-Methyl-5-(pyridin-2-yl)pyrrolidin-2-one (21)
To a solution of 20 (1.3 g, 3.3 mmol) in acetonitrile (15 mL) was added dropwise conc. HCl solution
(3 mL) and stirred at room temperature for 2 h. The mixture was diluted with water (20 mL) and
extracted with dichloromethane (20 mL * 3). The combined organic phase was dried over Na2SO4,
filtered and concentrated. The residue was purified by silica gel column chromatography
1
(dichloromethane/methanol = 100/1) to give the desired product (360 mg, 62%) as a yellow oil. H
NMR (400 MHz, CDCl3) δ 8.64-8.54 (m, 1H), 7.77-7.65 (m, 1H), 7.37-7.29 (m, 0.7H), 7.26-7.20 (m,
1.3H), 6.14-5.93 (m, 1H), 4.88 (d, J = 7.6 Hz, 0.3H), 4.39 (d, J = 6.0 Hz, 0.7H), 3.04-2.90 (m, 0.3H),
2.69-2.41 (m, 1.7 H), 2.25-2.06 (m, 1H), 1.27 (d, J = 6.8 Hz, 2.1H), 0.67 (d, J = 7.2 Hz, 0.9 H). MS
(ESI/APCI) m/z 177.1 [M+H]+.
6.1.6. 2-(3-Methylpyrrolidin-2-yl)pyridine (22)
To a solution of 21 (200 mg, 1.1 mmol) in THF (5 mL) was added LiAlH4 (170 mg, 4.5 mmol)
slowly and stirred at 50 oC overnight. The reaction mixture was cooled to room temperature. Water (0.2
mL), 25% NaOH solution (0.2 mL) and water (0.6 mL) were added in sequence to quench the reaction.
The mixture was filtrated, concentrated and purified by silica gel column chromatography
(dichloromethane/methanol/ammonia = 100/1/1) to give the desired product (89 mg, 50%) as a
1
colorless oil. H NMR (400 MHz, CDCl3) δ 8.61-8.48 (m, 1H), 7.70-7.60 (m, 1H), 7.34-7.28 (m, 1H),
7.21-7.14 (m, 1H), 4.49 (d, J = 7.2 Hz, 0.3H), 3.75 (d, J = 8.0 Hz, 0.7H), 3.32-3.10 (m, 2H), 2.63-2.53
(m, 1H), 2.17-2.03 (m, 2H), 1.64-1.55 (m, 1H), 1.08 (d, J = 6.0 Hz, 2.1H), 0.58 (d, J = 6.8 Hz, 0.9H).
MS (ESI/APCI) m/z 163.0 [M+H]+.
6.1.7. General procedure for the synthesis of 23-25
A mixture of 15/ 18/ 22 (1.0 mmol, 1.0 eq), 4-chloro-6-(chloromethyl)-2-methylpyrimidine (1.1
mmol, 1.1 eq), KI (0.10 mmol, 0.10 eq), and DIPEA (2.5 mmol, 2.5 eq) in acetonitrile (5 mL) was
stirred at room temperature overnight. The mixture was concentrated and purified by basic Al2O3
column chromatography (petroleum ether/ethyl acetate = 1/1) to give the desired product.
6.1.7.1. 4-Chloro-2-methyl-6-((2-(pyridin-2-yl)piperidin-1-yl)methyl)pyrimidine (23)
Compound 23 was obtained as a yellow oil (yield 73%). 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J =
4.4 Hz, 1H), 7.65-7.59 (m, 1H), 7.52-7.37 (m, 2H), 7.19-7.09 (m, 1H), 3.62 (d, J = 16.4 Hz, 1H),
3.52-3.42 (m, 1H), 3.20 (d, J = 16.4 Hz, 1H), 3.00-2.89 (m, 1H), 2.61 (s, 3H), 2.27-2.13 (m, 1H),
1.95-1.81 (m, 2H), 1.73-1.62 (m, 3H), 1.52-1.44 (m, 1H). MS (ESI/APCI) m/z 302.8 [M+H]+.
6.1.7.2. 4-Chloro-2-methyl-6-((2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (24)
Compound 24 was obtained as a colorless oil (yield 34%). 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J
= 4.4 Hz, 1H), 7.69-7.59 (m, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H), 7.19-7.11 (m, 1H), 3.83 (d, J =
16

16 Hz, 1H), 3.79-3.70 (m, 1H), 3.52 (d, J = 16.0 Hz, 1H), 3.34-3.23 (m, 1H), 2.64 (s, 3H), 2.50-2.30 (m,
2H), 2.10-1.81 (m, 3H). MS (ESI/APCI) m/z 288.8 [M+H]+.
6.1.7.3. 4-Chloro-2-methyl-6-((3-methyl-2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (25)
1
Compound 25 was obtained as a colorless oil (yield 13%). H NMR (400 MHz, CDCl3) δ 8.62-8.48
(m, 1H), 7.70-7.59 (m, 1H), 7.51-7.42 (m, 1H), 7.40-7.30 (m, 1H), 7.22-7.09 (m, 1H), 4.15-3.92 (m,
0.3H), 3.93-3.67 (m, 1H), 3.61-3.40 (m, 1H), 3.35-3.20 (m, 1.7H), 2.68-2.57 (m, 3H), 2.54-2.40 (m,
1H), 2.28-2.15 (m, 2H), 1.69-1.47 (m, 1H), 1.04 (d, J = 6.0 Hz, 2.5H), 0.58 (d, J = 7.2 Hz, 0.5H). MS
(ESI/APCI) m/z 302.9 [M+H]+.
6.1.8. General procedure for the synthesis of 26-28
A mixture of 23-25 (0.20 mmol, 1.0 eq), N-methylpiperazine (1.0 mmol, 5.0 eq) and TEA (2.0 mmol,
o
10 eq) in ethanol (5 mL) was stirred at 85 C for 3 h. The mixture was concentrated and purified by
basic Al2O3 column chromatography (dichloromethane/methanol = 100/1) to give the desired product.
6.1.8.1. 2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(pyridin-2-yl)piperidin-1-yl)methyl)pyrimidine (26)
1
Compound 26 was obtained as a colorless oil (yield 38%). H NMR (400 MHz, CDCl3) δ 8.48-8.41
(m, 1H), 7.60-7.52 (m, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.09-7.02 (m, 1H), 6.55 (s, 1H), 3.67-3.58 (m,
4H), 3.48 (d, J = 16.0 Hz, 1H), 3.42-3.33 (m, 1H), 3.09-2.95 (m, 2H), 2.47-2.41 (m, 4H), 2.37 (s, 3H),
2.30 (s, 3H), 2.15-2.05 (m, 1H), 1.88-1.72 (m, 2H), 1.68-1.56 (m, 3H), 1.47-1.33 (m, 1H). 13C NMR
(150 MHz, CDCl3) δ 166.9, 166.6, 164.1, 162.6, 149.0, 136.6, 122.0, 121.5, 97.1, 70.2, 61.0, 54.7, 54.0,
46.2, 43.7, 34.9, 26.0, 25.8, 24.6. HRMS (ESI): calcd for C21H30N6 [M+H]+ 367.2605, found 367.2606.
Purity: 99.1%.
6.1.8.2. 2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (27)
Compound 27 was obtained as a colorless oil (yield 73%). 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J
= 4.0 Hz, 1H ), 7.63-7.55 (m, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.14-7.06 (m, 1H), 6.44 (s, 1H), 3.74-3.66
(m, 2H), 3.64-3.58 (m, 4H), 3.38 (d, J = 15.2 Hz, 1H), 3.34-3.27 (m, 1H), 2.48-2.41 (m, 8H), 2.34-2.24
(m, 4H), 2.03-1.95 (m, 1H), 1.93-1.80 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 166.7, 166.6, 163.5,
162.6, 149.2, 136.6, 122.0, 121.5, 97.9, 71.0, 60.3, 54.8, 54.6, 46.2, 43.7, 33.5, 26.1, 23.2. HRMS (ESI):
calcd for C20H28N6 [M+H]+ 353.2448, found 353.2455. Purity: 99.0%.
6.1.8.3.
2-Methyl-4-((3-methyl-2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)-6-(4-methylpiperazin-1-yl)pyrimidine
(28)
1
Compound 28 was obtained as a colorless oil (yield 60%). H NMR (400 MHz, CDCl3) δ 8.60-8.48
(m, 1H), 7.65-7.54 (m, 1H), 7.47-7.38 (m, 1H), 7.18-7.08 (m, 1H), 6.48-6.38 (m, 1H), 3.93 (d, J = 8.8
Hz, 0.1H), 3.71-3.62 (m, 5H), 3.38-3.26 (m, 2H), 3.21 (d, J = 7.6 Hz, 0.9H), 2.51-2.38 (m, 8H), 2.34 (s,
3H), 2.27-2.15 (m, 2H), 1.58-1.47 (m, 1H), 1.04 (d, J = 5.6 Hz, 2.7H), 0.57 (d, J = 7.2 Hz, 0.3H). 13C
NMR (150 MHz, CDCl3) δ 166.7, 166.6, 162.6, 162.5, 149.2, 136.5, 122.2, 122.0, 97.9, 79.1, 60.6,
54.8, 53.3, 46.3, 43.8, 41.9, 31.9, 26.1, 18.4. HRMS (ESI): calcd for C21H30N6 [M+H]+ 367.2605, found
367.2621. Purity: 99.2%.
6.1.9. 3-(1,3-Dioxolan-2-yl)-1-(pyridin-2-yl)propan-1-one (30)
To a solution of Mg (1.1 g, 44 mmol) and I2 (20 mg) in dry THF (10 mL) at room temperature was
added the THF solution (2 mL) of 2-(2-bromoethyl)-1,3-dioxolane (696 mg, 4 mmol) under nitrogen
o
protection. The mixture was stirred at 40-50 C until the iodine color disappeared. The THF solution
(18 mL) of 2-(2-bromoethyl)-1,3-dioxolane solution (6.5 g, 36 mmol) was then added dropwise to the
o
turnings at room temperature to keep the inner temperature below 30 C. The reaction was stirred at
room temperature for an additional 1.5 h. This mixture was added dropwise to the solution of 29 (2.1 g,
17

o
20 mmol) in dry THF (50 mL) at 0 C and stirred at the same temperature for 2h. The saturated
ammonium chloride solution (20 mL) was added, extracted with ethyl acetate (30 mL * 3). The
combined organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 10/1) to give the desired product
1
(2.5 g, 61%) as a colorless oil. H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.12-7.95 (m, 1H),
7.87-7.72 (m, 1H), 7.52-7.40 (m, 1H), 5.02 (s, 1H), 4.00-3.92 (m, 2H), 3.88-3.82 (m, 2H), 3.36 (t, J =
7.2 Hz, 2H), 2.20-2.10 (m, 2H). MS (ESI/APCI) m/z 207.9 [M+H]+.
6.1.10. 4-Oxo-4-(pyridin-2-yl)butanal (31)
To a solution of 30 (1.3 g, 6.0 mmol) in acetone (20 mL) was added 3 N HCl aqueous solution (10
mL, 30 mmol) and stirred at room temperature overnight. The reaction mixture was added saturated
Na2CO3 aqueous solution to adjust pH to 7 and extracted with dichloromethane (20 mL * 3). The
combined organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 10/1) to give the desired product
(800 mg, 82%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 8.68 (d, J = 4.0 Hz, 1H),
8.02 (d, J = 7.6 Hz, 1H), 7.89-7.75 (m, 1H), 7.56-7.43 (m, 1H), 3.57 (t, J = 6.4 Hz, 2H), 2.91 (t, J = 6.4
Hz, 2H). MS (ESI/APCI) m/z 164.0 [M+H]+.
6.1.11. 2-((S)-1-((R)-1-(4-Methoxyphenyl)ethyl)pyrrolidin-2-yl)pyridine (32)
To a solution of 31 (490 mg, 3.0 mmol) and acetic acid (18 mg, 0.30 mmol) in dichloromethane (15
mL) was slowly added NaBH(OAc)3 (1.9 g, 9.0 mmol) at -70 oC and stirred at the same temperature for
30 min. (R)-1-(4-methoxyphenyl)ethan-1-amine (500 mg, 3.3 mmol) was added and stirred at room
temperature overnight. The mixture was quenched with water (5 mL), concentrated and purified by
basic Al2O3 column chromatography (petroleum ether/ethyl acetate = 1/1) to give the desired product
(410 mg, 48%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.49-8.35 (m, 1H), 7.63-7.48 (m, 1H),
7.44-7.33 (m, 1H), 7.20-7.10 (m, 2H), 7.10-7.00 (m, 1H), 6.79-6.65 (m, 2H), 4.05-3.92 (m, 1H),
3.80-3.71 (m, 4H), 3.11 (s, 1H), 2.63 (s, 1H), 2.24 (s, 1H), 1.99-1.85 (m, 1H), 1.76 (s, 2H), 1.35 (d, J =
4.8 Hz, 3H). MS (ESI/APCI) m/z 282.9 [M+H]+. [α]2D5 = -11 (c = 0.2, MeOH).
6.1.12. (S)-2-(Syrrolidin-2-yl)pyridine (33)
o
A mixture of 33 (410 mg, 1.5 mmol) in TFA (5 mL) was stirred at 50 C for 12 h. The mixture was
concentrated, dissolved in 1N HCl aqueous solution (10 mL) and then washed with dichloromethane
(10 mL * 3). The water layer was added saturated Na2CO3 aqueous solution to adjust pH to 9 and
extracted with dichloromethane (20 mL * 3). The combined organic phase was dried over Na2SO4,
1
filtered and concentrated to give the desired product (110 mg, 50%) as a colorless oil. H NMR (400
MHz, CDCl3) δ 8.50 (d, J = 4.4 Hz, 1H), 7.73-7.62 (m, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.23-7.16 (m,
1H), 4.67-4.52 (m, 1H), 3.39-3.24 (m, 2H), 2.45-2.32 (m, 1H), 2.06-1.82 (m, 3H). MS (ESI/APCI) m/z
149.0 [M+H]+.
6.1.13. (S)-4-Chloro-2-methyl-6-((2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (34)
A mixture of 33 (110 mg, 0.74 mmol), 4-chloro-6-(chloromethyl)-2-methylpyrimidine (150 mg,
0.81mmol), KI (12 mg, 0.074 mmol), and DIPEA (240 mg, 1.9 mmol) in acetonitrile (5 mL) was
stirred at room temperature overnight. The mixture was concentrated and purified by basic Al2O3
column chromatography (petroleum ether/ethyl acetate = 1/1) to give the desired product (68 mg, 32%)
as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.58-8.47 (m, 1H), 7.68-7.60 (m, 1H), 7.47 (d, J = 7.6
Hz, 1H), 7.38-7.32 (m, 1H), 7.17-7.09 (m, 1H), 3.82 (d, J = 16.0 Hz, 1H), 3.78-3.70 (m, 1H), 3.51 (d, J
= 15.6 Hz, 1H), 3.34-3.20 (m, 1H), 2.64 (s, 3H), 2.49-2.47 (m, 2H), 2.11-1.80 (m, 3H). MS (ESI/APCI)
m/z 288.8 [M+H]+. [α]2D5 = -2 (c = 0.2, MeOH).
18

6.1.14. (S)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine
(35)
A mixture of 34 (68 mg, 0.24 mmol), N-methylpiperazine (120 mg, 1.2 mmol) and TEA (240 mg,
2.4 mmol) in ethanol (5 mL) was stirred at 85 oC for 3 h. The mixture was concentrated and purified by
basic Al2O3 column chromatography (dichloromethane/methanol = 100/1) to give the desired product
(70 mg, 83 %) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.55-8.41 (m, 1H ), 7.62-7.51 (m, 1H),
7.41 (d, J = 7.6 Hz, 1H), 7.13-6.99 (m, 1H), 6.41 (s, 1H), 3.71-3.55 (m, 6H), 3.38-3.25 (m, 2H),
2.45-2.35 (m, 8H), 2.33-2.20 (m, 4H), 2.00-1.75 (m, 3H). 13C NMR (150 MHz, CDCl3) δ 166.6, 166.5,
163.5, 162.6, 149.1, 136.5, 121.9, 121.4, 97.8, 71.0, 60.3, 54.7, 54.6, 46.2, 43.6, 33.4, 26.0, 23.1.
HRMS (ESI): calcd for C20H28N6 [M+H]+ 353.2448, found 353.2450. Purity: 99.2%. [α]25 = +6 (c = 0.2,
D
MeOH).
6.1.15. 2-((R)-1-((S)-1-(4-Methoxyphenyl)ethyl)pyrrolidin-2-yl)pyridine (36)
To a solution of 31 (330 mg, 2.0 mmol) and acetic acid (18 mg, 0.30 mmol) in dichloromethane (15
o
mL) was added NaBH(OAc)3 (1.3 g, 6 mmol) slowly at -70 C and stirred at the same temperature for
30 min. (S)-1-(4-methoxyphenyl)ethan-1-amine (330 mg, 2.2 mmol) was added and stirred at room
temperature overnight. The mixture was quenched with water (5 mL), concentrated and purified by
basic Al2O3 column chromatography (petroleum ether/ethyl acetate = 1/1) to give the desired product
1
(400 mg, 71%) as a colorless oil. H NMR (400 MHz, DMSO-d6) δ 8.40-8.33 (m, 1H), 7.73-7.62 (m,
1H), 7.47 (d, J = 7.6 Hz, 1H), 7.21-7.06 (m, 3H), 6.80-6.68 (m, 2H), 3.97-3.86 (m, 1H), 3.68 (s, 3H),
3.66-3.60 (m, 1H), 2.93-2.76 (m, 1H), 2.65-2.51 (m, 1H), 2.20-2.03 (m, 1H), 1.80-1.55 (m, 3H), 1.22
(d, J = 6.0 Hz, 3H). MS (ESI/APCI) m/z 282.9 [M+H]+. [α]2D5 = +11 (c = 0.2, MeOH).
6.1.16. (R)-2-(Pyrrolidin-2-yl)pyridine (37)
o
A mixture of 36 (400 mg, 1.5 mmol) in TFA (5 mL) was stirred at 50 C for 12 h. The mixture was
concentrated, dissolved in 1 N HCl (10 mL) and washed with dichloromethane (10 mL * 3). The water
layer was added saturated Na2CO3 aqueous solution to adjust pH to 9 and extracted with
dichloromethane (20 mL * 3). The combined organic phase was dried over Na2SO4, filtered and
concentrated to give the desired product (100 mg, 45%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
δ 8.57-8.47 (m, 1H), 7.70-7.54 (m, 1H), 7.40-7.30 (m, 1H), 7.19-7.08 (m, 1H), 4.30-4.17 (m, 1H),
3.27-3.15 (m, 1H), 3.08-2.92 (m, 1H), 2.30-2.15 (m, 1H), 1.90-1.70 (m, 3H). MS (ESI/APCI) m/z
149.0 [M+H]+.
6.1.17. (R)-4-Chloro-2-methyl-6-((2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (38)
A mixture of 37 (100 mg, 0.67 mmol), 4-chloro-6-(chloromethyl)-2-methylpyrimidine (130 mg, 0.74
mmol), KI (11 mg, 0.067 mmol), and DIPEA (220 mg, 1.7 mmol) in acetonitrile (5 mL) was stirred at
room temperature overnight. The mixture was concentrated and purified by basic Al2O3 column
chromatography (petroleum ether/ethyl acetate = 1/1) to give the desired product (60 mg, 31%) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 4.0 Hz, 1H), 7.68-7.60 (m, 1H), 7.51-7.45 (m,
1H), 7.35 (s, 1H), 7.18-7.09 (m, 1H), 3.82 (d, J = 16.0 Hz, 1H), 3.79-3.70 (m, 1H), 3.51 (d, J = 15.6 Hz,
1H), 3.34-3.23 (m, 1H), 2.64 (s, 3H), 2.48-2.30 (m, 2H), 2.08-1.81 (m, 3H). MS (ESI/APCI) m/z 288.8
[M+H]+. [α]2D5 = +2 (c = 0.2, MeOH).
6.1.18. (R)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(pyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine
(39)
A mixture of 38 (60 mg, 0.21 mmol), N-methylpiperazine (104 mg, 1.0 mmol) and TEA (210 mg,
2.1 mmol) in ethanol (5 mL) was stirred at 85 oC for 3 h. The mixture was concentrated and purified by
basic Al2O3 column chromatography (dichloromethane/methanol = 100/1) to give the desired product
19

(50 mg, 68%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 4.0 Hz, 1H ), 7.65-7.54 (m,
1H), 7.45 (d, J = 8.0 Hz, 1H), 7.15-7.08 (m, 1H), 6.45 (s, 1H), 3.72-3.68 (m, 2H), 3.65-3.58 (m, 4H),
3.40-3.31 (m, 2H), 2.50-2.40 (m, 8H), 2.36-2.26 (m, 4H), 2.05-1.83 (m, 3H). 13C NMR (150 MHz,
CDCl3) δ 166.7, 166.6, 163.5, 162.6, 149.2, 136.6, 122.0, 121.5, 97.9, 71.0, 60.3, 54.8, 54.6, 46.2, 43.7,
33.5, 26.1, 23.2. HRMS (ESI): calcd for C20H28N6 [M+H]+ 353.2448, found 353.2450. Purity: 96.2%.
[α]2D5 = -6 (c = 0.2, MeOH).
6.1.19. General procedure for the synthesis of 41a-d
To a solution of Mg (44 mmol, 2.2 eq) and I2 (20 mg) in dry THF (10 mL) at room temperature was
added the THF solution (2 mL) of 2-(2-bromoethyl)-1,3-dioxolane (4.0 mmol, 0.20 eq) under nitrogen
o
protection. The mixture was stirred at 40-50 C until the iodine color disappeared. The THF solution
(18 mL) of 2-(2-bromoethyl)-1,3-dioxolane (36 mmol, 1.8 eq) was then added dropwise to the turnings
o
at room temperature to keep the inner temperature below 30 C. The reaction was stirred at room
temperature for an additional 1.5 h. This mixture was added dropwise to the solution of 40a-d (20
o
mmol, 1.0 eq) in dry THF (50 mL) at 0 C and stirred at the same temperature for 2 h. The saturated
ammonium chloride solution (20 mL) was added, extracted with ethyl acetate (30 mL * 3). The
combined organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 10/1) to give the desired product.
6.1.19.1. 3-(1,3-Dioxolan-2-yl)-1-(3-methylpyridin-2-yl)propan-1-one (41a)
1
Compound 41a was obtained as a colorless oil (yield 49%). H NMR (400 MHz, DMSO-d6) δ
8.55-8.45 (m, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.54-7.45 (m, 1H), 4.92-4.80 (m, 1H), 3.87-3.80 (m, 2H),
3.77-3.71 (m, 2H), 3.17 (t, J = 7.2 Hz, 2H), 2.46 (s, 3H), 1.98-1.85 (m, 2H). MS (ESI/APCI) m/z 221.9
[M+H]+.
6.1.19.2. 3-(1,3-Dioxolan-2-yl)-1-(4-methylpyridin-2-yl)propan-1-one (41b)
Compound 41b was obtained as a colorless oil (yield 31%). 1H NMR (400 MHz, CDCl3) δ 8.52 (d, J
= 4.8 Hz, 1H), 7.85 (s, 1H), 7.32-7.26 (m, 1H),5.02 (t, J = 4.0 Hz, 1H), 4.00-3.92 (m, 2H), 3.88-3.80
(m, 2H), 3.34 (t, J = 7.6 Hz, 2H), 2.41 (s, 3H), 2.18-2.10 (m, 2H). MS (ESI/APCI) m/z 221.9 [M+H]+.
6.1.19.3. 3-(1,3-Dioxolan-2-yl)-1-(5-methylpyridin-2-yl)propan-1-one (41c)
1
Compound 41c was obtained as a colorless oil (yield 79%). H NMR (400 MHz, CDCl3) δ 8.47 (s,
1H), 7.92 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 5.05-4.98 (m, 1H), 3.97-3.92 (m, 2H), 3.86-3.82
(m, 2H), 3.32 (t, J = 7.2 Hz, 2H), 2.39 (s, 3H), 2.20-2.10 (m, 2H). MS (ESI/APCI) m/z 221.9 [M+H]+.
6.1.19.4. 3-(1,3-Dioxolan-2-yl)-1-(6-methylpyridin-2-yl)propan-1-one (41d)
Compound 41d was obtained as a colorless oil (yield 70%). 1H NMR (400 MHz, CDCl3) δ 7.82 (d, J
= 7.6 Hz, 1H), 7.72-7.65 (m, 1H), 7.30 (d, J = 7.6 Hz, 1H), 5.03 (t, J = 4.0 Hz, 1H), 3.99-3.93 (m, 2H),
3.87-3.84 (m, 2H), 3.36 (t, J = 7.6 Hz, 2H), 2.60 (s, 3H), 2.19-2.06 (m, 2H). MS (ESI/APCI) m/z 221.9
[M+H]+.
6.1.20. General procedure for the synthesis of 42a-d
To a solution of 41a-d (6.0 mmol, 1.0 eq) in acetone (20 mL) was added 3 N HCl aqueous solution
(30 mmol, 5.0 eq) and stirred at room temperature overnight. The reaction mixture was added saturated
Na2CO3 aqueous solution to adjust pH to 7 and extracted with dichloromethane (20 mL * 3). The
combined organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 10/1) to give the desired product.
6.1.20.1. 4-(3-Methylpyridin-2-yl)-4-oxobutanal (42a)
1
Compound 42a was obtained as a colorless oil (yield 87%). H NMR (400 MHz, CDCl3) δ 9.89 (s,
1H), 8.60-8.38 (m, 1H), 7.65-7.46 (m, 1H), 7.39-7.26 (m, 1H), 3.61-3.35 (m, 2H), 2.95-2.72 (m, 2H),
20

2.56 (s, 3H). MS (ESI/APCI) m/z 177.9 [M+H]+.
6.1.20.2. 4-(4-Methylpyridin-2-yl)-4-oxobutanal (42b)
1
Compound 42b was obtained as a colorless oil (yield 90%). H NMR (400 MHz, CDCl3) δ 9.88 (s,
1H), 8.53 (d, J = 4.8 Hz, 1H), 7.85 (s, 1H), 7.29 (d, J = 4.4 Hz, 1H), 3.55 (t, J = 6.4 Hz, 2H), 2.90 (t, J
= 6.4 Hz, 2H), 2.42 (s, 3H). MS (ESI/APCI) m/z 177.9 [M+H]+.
6.1.20.3. 4-(5-Methylpyridin-2-yl)-4-oxobutanal (42c)
1
Compound 42c was obtained as a colorless oil (yield 67%). H NMR (400 MHz, CDCl3) δ 9.88 (s,
1H), 8.48 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 3.54 (t, J = 7.2 Hz, 2H), 2.89 (t, J
= 6.4 Hz, 2H), 2.41 (s, 3H). MS (ESI/APCI) m/z 177.9 [M+H]+.
6.1.20.4. 4-(6-Methylpyridin-2-yl)-4-oxobutanal (42d)
1
Compound 42d was obtained as a colorless oil (yield 94%). H NMR (400 MHz, CDCl3) δ 9.90 (s,
1H), 7.83 (d, J = 8.0 Hz, 1H), 7.73-7.67 (m, 1H), 7.33 (d, J = 7.6 Hz, 1H), 3.58 (t, J = 6.4 Hz, 2H), 2.90
(t, J = 6.4 Hz, 2H), 2.61 (s, 3H). MS (ESI/APCI) m/z 178.0 [M+H]+.
6.1.21. General procedure for the synthesis of 43a-d
To a solution of 42a-d (2.0 mmol, 1.0 eq) and acetic acid (0.20 mmol, 0.10 eq) in dichloromethane
o
(15 mL) was slowly added NaBH(OAc)3 (6.0 mmol, 3.0 eq) at -70 C and stirred at the same
temperature for 30 min. (R)-1-(4-methoxyphenyl)ethan-1-amine (2.2 mmol, 1.1 eq) was added and
stirred at room temperature overnight. The mixture was quenched with water (5 mL), concentrated and
purified by basic Al2O3 column chromatography (petroleum ether/ethyl acetate = 1/1) to give the
desired product.
6.1.21.1. 2-((S)-1-((R)-1-(4-Methoxyphenyl)ethyl)pyrrolidin-2-yl)-3-methylpyridine (43a)
Compound 43a was obtained as a colorless oil (yield 25%). 1H NMR (400 MHz, CDCl3) δ 8.43-8.33
(m, 1H), 7.15-7.06 (m, 1H), 7.05-6.98 (m, 2H), 6.90-6.80 (m, 1H), 6.60-6.47 (m, 2H), 3.99-3.87 (m,
1H), 3.69 (s, 3H), 3.59-3.48 (m, 1H), 3.42-3.29 (m, 1H), 2.60-2.47 (m, 1H), 2.18-2.02 (m, 5H),
1.86-1.72 (m, 2H), 1.31 (d, J = 6.8 Hz, 3H). MS (ESI/APCI) m/z 296.9 [M+H]+. [α]2D5 = -6 (c = 0.2,
MeOH).
6.1.21.2. 2-((S)-1-((R)-1-(4-Methoxyphenyl)ethyl)pyrrolidin-2-yl)-4-methylpyridine (43b)
Compound 43b was obtained as a colorless oil (yield 55%). 1H NMR (400 MHz, CDCl3) δ 8.26 (d,
J = 4.4 Hz, 1H), 7.13 (d, J = 7.6 Hz, 2H), 7.05 (s, 1H), 6.82 (d, J = 4.4 Hz, 1H), 6.67 (d, J = 8.0 Hz,
2H), 3.89-3.75 (m, 1H), 3.74 (s, 3H), 3.70-3.61 (m, 1H), 3.18-3.04 (m, 1H), 2.66- 2.52 (m, 1H),
2.28-2.13 (m, 4H), 1.98-1.84 (m, 1H), 1.87-1.75 (m, 2H), 1.33 (d, J = 6.4 Hz, 3H). MS (ESI/APCI) m/z
296.9 [M+H]+. [α]2D5 = -16 (c = 0.2, MeOH).
6.1.21.3. 2-((S)-1-((R)-1-(4-Methoxyphenyl)ethyl)pyrrolidin-2-yl)-5-methylpyridine (43c)
1
Compound 43c was obtained as a colorless oil (yield 21%). H NMR (400 MHz, CDCl3) δ 8.26 (s,
1H), 7.35 (d, J = 7.2 Hz, 1H), 7.30-7.26 (m, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H),
3.93-3.86 (m, 1H), 3.77-3.68 (m, 4H), 3.09-2.92 (m, 1H), 2.67-2.52 (m, 1H),2.26 (s, 3H), 2.22-2.11 (m,
1H), 1.94-1.82 (m, 1H), 1.81-1.68 (m, 2H), 1.31 (d, J = 6.8 Hz, 3H). MS (ESI/APCI) m/z 297.0
[M+H]+. [α]2D5 = -15 (c = 0.2, MeOH).
6.1.21.4. 2-((S)-1-((R)-1-(4-Methoxyphenyl)ethyl)pyrrolidin-2-yl)-6-methylpyridine (43d)
Compound 43d was obtained as a colorless oil (yield 27%). 1H NMR (400 MHz, CDCl3) δ 7.51-7.42
(m, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 7.2 Hz, 1H), 6.72 (d, J = 8.4 Hz,
2H), 4.00-3.85 (m, 1H), 3.80-3.70 (m, 4H), 3.09-2.95 (m, 1H), 2.69-2.56 (m, 1H), 2.46 (s, 3H),
2.30-2.15 (m, 1H), 1.96-1.70 (m, 3H), 1.33 (d, J = 6.4 Hz, 3H). MS (ESI/APCI) m/z 296.9 [M+H]+.
[α]2D5 = -5 (c = 0.2, MeOH).
21

6.1.22. General procedure for the synthesis of 44a-d
o
A mixture of 43a-d (1.5 mmol) in TFA (5 mL) was stirred at 50 C for 12 h. The mixture was
concentrated, dissolved in 1N HCl aqueous solution (10 mL) and washed with dichloromethane (10 mL
* 3). The water layer was added saturated Na2CO3 aqueous solution to adjust pH to 9 and extracted
with dichloromethane (20 mL * 3). The combined organic phase was dried over Na2SO4, filtered and
concentrated to give the desired product.
6.1.22.1. (S)-3-Methyl-2-(pyrrolidin-2-yl)pyridine (44a)
Compound 44a was obtained as a colorless oil (yield 82%). 1H NMR (400 MHz, CDCl3) δ 8.42-8.30
(m, 1H), 7.48-7.36 (m, 1H), 7.13-7.00 (m, 1H), 4.46-4.27 (m, 1H), 3.40-3.24 (m, 1H), 3.03-2.88 (m,
1H), 2.34 (s, 3H), 2.27-2.21 (m, 1H), 1.97-1.77 (m, 2H), 1.62-1.46 (m, 1H). MS (ESI/APCI) m/z 163.0
[M+H]+. [α]2D5 = -25 (c = 0.2, MeOH).
6.1.22.2. (S)-4-Methyl-2-(pyrrolidin-2-yl)pyridine (44b)
Compound 44b was obtained as a colorless oil (yield 43%). 1H NMR (400 MHz, CDCl3) δ 8.40-8.31
(m, 1H), 7.15 (s, 1H), 7.07-6.95 (m, 1H), 4.57-4.46 (m, 1H), 3.32-3.19 (m, 2H), 2.40-3.30 (m, 4H),
2.02-1.77 (m, 3H). MS (ESI/APCI) m/z 163.0 [M+H]+.
6.1.22.3. (S)-5-Methyl-2-(pyrrolidin-2-yl)pyridine (44c)
1
Compound 44c was obtained as a colorless oil (yield 75%). H NMR (400 MHz, CDCl3) δ 8.35 (s,
1H), 7.44 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 4.27-4.11 (m, 1H), 3.28-3.15 (m, 1H), 3.06-2.93
(m, 1H), 2.30 (s, 3H), 2.24-2.16 (m, 1H), 1.93-1.79 (m, 2H), 1.79-1.65 (m, 1H). MS (ESI/APCI) m/z
163.0 [M+H]+.
6.1.22.4. (S)-6-Methyl-2-(pyrrolidin-2-yl)pyridine (44d)
Compound 44d was obtained as a colorless oil (yield 90%). 1H NMR (400 MHz, CDCl3) δ 7.53-7.45
(t, J = 7.6 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 4.20 (t, J = 7.6 Hz, 1H),
3.23-3.13 (m, 1H), 3.01-2.97 (m, 1H), 2.50 (s, 3H), 2.25-2.16 (m, 1H), 1.91-1.79 (m, 2H), 1.75-1.66 (m,
1H). MS (ESI/APCI) m/z 163.1 [M+H]+.
6.1.23. General procedure for the synthesis of 45a-d
A mixture of 44a-d (0.67 mmol, 1.0 eq), 4-chloro-6-(chloromethyl)-2-methylpyrimidine (0.74 mmol,
1.1 eq), KI (0.067 mmol, 0.1 eq), and DIPEA (1.7 mmol, 2.5 eq) in acetonitrile (5 mL) was stirred at
room temperature overnight. The mixture was concentrated and purified by basic Al2O3 column
chromatography (petroleum ether/ethyl acetate = 1/1) to give the desired product.
6.1.23.1. (S)-4-Chloro-2-methyl-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (45a)
Compound 45a was obtained as a colorless oil (yield 32%). 1H NMR (400 MHz, CDCl3) δ 8.53-8.39
(m, 1H), 7.38-7.32 (m, 1H), 7.31 (s, 1H), 7.06-6.97 (m, 1H), 3.94-3.81 (m, 1H), 3.72 (d, J = 14.8 Hz,
1H), 3.52 (d, J = 15.2 Hz, 1H), 3.36-3.27 (m, 1H), 2.61 (s, 3H), 2.48-2.30 (m, 4H), 2.27-2.16 (m ,1H),
2.13-1.96 (m, 2H), 1.95-1.85 (m, 1H). MS (ESI/APCI) m/z 302.8 [M+H]+. [α]2D5 = -2 (c = 0.2, MeOH).
6.1.23.2. (S)-4-Chloro-2-methyl-6-((2-(4-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (45b)
Compound 45b was obtained as a colorless oil (yield 59%). 1H NMR (400 MHz, CDCl3) δ 8.42-8.27
(m, 1H), 7.38-7.25 (m, 1H), 7.20-7.08 (m, 1H), 6.99-6.89 (m, 1H), 3.87-3.63 (m, 2H), 3.59-3.45 (m,
1H), 3.34-3.18 (m, 1H), 2.64 (s, 3H), 2.49-2.37 (m, 1H), 2.37-2.17 (m, 4H), 2.08-1.77 (m, 3H). MS
(ESI/APCI) m/z 302.9 [M+H]+. [α]2D5 = -3 (c = 0.2, MeOH).
6.1.23.3. (S)-4-Chloro-2-methyl-6-((2-(5-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (45c)
Compound 45c was obtained as a colorless oil (yield 60%). 1H NMR (400 MHz, CDCl3) δ 8.38-8.30
(m, 1H), 7.48-7.40 (m, 1H), 7.38-7.29 (m, 2H), 3.80 (d, J = 16.0 Hz, 1H), 3.74-3.63 (m, 1H), 3.49 (d, J
= 16.0 Hz, 1H), 3.31-3.20 (m, 1H), 2.63 (s, 3H), 2.43-2.36 (m, 1H), 2.34-2.22 (m ,4H), 2.08-1.96 (m,
22

1H), 1.95-1.85 (m, 2H). MS (ESI/APCI) m/z 302.9 [M+H]+. [α]2D5 = -6 (c = 0.2, MeOH).
6.1.23.4. (S)-4-Chloro-2-methyl-6-((2-(6-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine (45d)
Compound 45d was obtained as a colorless oil (yield 55%). 1H NMR (400 MHz, CDCl3) δ 7.56-7.48
(m, 1H), 7.36 (s, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H), 3.83 (d, J = 16.0 Hz, 1H),
3.75-3.65 (m, 1H), 3.49 (d, J = 16.0 Hz, 1H), 3.31-3.20 (m, 1H), 2.63 (s, 3H), 2.52 (s, 3H), 2.45-2.29
(m, 2H), 2.05-1.93 (m, 1H), 1.93-1.78 (m, 2H). MS (ESI/APCI) m/z 303.0 [M+H]+. [α]2D5 = +2 (c = 0.2,
MeOH).
6.1.24. General procedure for the synthesis of 46-49
A mixture of 45a-d (0.20 mmol, 1.0 eq), N-methylpiperazine (1.0 mmol, 5.0 eq) and TEA (2.0 mmol,
o
10 eq) in ethanol (5 mL) was stirred at 85 C for 3 h. The mixture was concentrated and purified by
basic Al2O3 column chromatography (dichloromethane/methanol = 100/1) to give the desired product.
6.1.24.1.
(S)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine
(46)
1
Compound 46 was obtained as a colorless oil (yield 23%). H NMR (400 MHz, CDCl3) δ 8.53-8.41
(m, 1H), 7.37-7.30 (m, 1H), 7.06-6.96 (m, 1H), 6.42 (s, 1H), 3.99-3.81 (m, 1H), 3.73-3.50 (m, 5H),
3.48-3.29 (m, 2H), 2.53-2.38 (m, 8H), 2.36-2.30 (m, 6H), 2.25-2.14 (m, 1H), 2.11-2.00 (m, 2H),
1.93-1.82 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 166.4, 166.3, 162.5, 159.9, 147.3, 138.0, 131.7,
121.7, 98.3, 67.1, 60.6, 54.8, 54.7, 46.2, 43.7, 31.4, 26.0, 23.1, 18.8. HRMS (ESI): calcd for C21H30N6
[M+H]+ 367.2605, found 367.2608. Purity: 99.4%. [α]2D5 = +13 (c = 0.2, MeOH).
6.1.24.2.
(S)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(4-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine
(47)
Compound 47 was obtained as a colorless oil (yield 43%). 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J
= 4.8 Hz, 1H), 7.26-7.23 (m, 1H), 6.93 (d, J = 4.4 Hz, 1H), 6.46 (s, 1H), 3.74-3.58 (m, 6H), 3.50-3.40
(m, 1H), 3.39-3.29 (m, 1H), 2.54-2.43 (m, 8H), 2.36 (s, 3H), 2.30 (s, 3H), 2.05-1.96 (m, 1H), 1.93-1.86
(m, 3H). 13C NMR (150 MHz, CDCl3) δ 166.7, 166.6, 163.1, 162.6, 148.8, 147.6, 123.0, 122.5, 98.1,
71.0, 60.4, 54.8, 54.7, 46.2, 43.6, 33.4, 26.0, 23.2, 21.2. HRMS (ESI): calcd for C21H30N6 [M+H]+
367.2605, found 367.2609. Purity: 97.7%. [α]2D5 = +5 (c = 0.2, MeOH).
6.1.24.3.
(S)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(5-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine
(48)
1
Compound 48 was obtained as a colorless oil (yield 24%). H NMR (400 MHz, CDCl3) δ 8.35 (s,
1H), 7.41 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 6.46 (s, 1H), 3.71-3.54 (m, 6H), 3.37-3.25 (m,
2H), 2.54-2.38 (m, 8H), 2.34 (s, 3H), 2.31-2.24 (m, 4H), 2.05-1.94 (m, 1H), 1.89-1.78 (m, 2H). 13C
NMR (150 MHz, CDCl3) δ 166.8, 166.7, 162.7, 160.4, 149.6, 137.2, 131.4, 121.1, 97.9, 70.8, 60.3,
54.8, 54.6, 46.3, 43.8, 33.4, 26.1, 23.1, 18.2. HRMS (ESI): calcd for C21H30N6 [M+H]+ 367.2605, found
367.2609. Purity: 98.8%. [α]2D5 = +2 (c = 0.2, MeOH).
6.1.24.4.
(S)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(6-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine
(49)
1
Compound 49 was obtained as a colorless oil (yield 45%). H NMR (400 MHz, CDCl3) δ 7.53-7.47
(m, 1H), 7.31 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1 H), 6.48 (s, 1H), 3.74-3.60 (m, 6H), 3.38-3.20
(m, 2H), 2.51 (s, 3H), 2.49-2.30 (m, 8H), 2.30-2.26 (m, 4H), 1.99-1.75 (m, 3H). 13C NMR (150 MHz,
23

CDCl3) δ 166.7, 166.6, 162.9, 162.6, 157.5, 136.9, 121.6, 118.1, 97.9, 71.1, 60.1, 54.7, 54.4, 46.2, 43.7,
33.6, 26.0, 24.5, 23.1. HRMS (ESI): calcd for C21H30N6 [M+H]+ 367.2605, found 367.2609. Purity:
98.9%. [α]2D5 = +6 (c = 0.2, MeOH).
6.1.25. Procedure for the synthesis of 46-salt
Compound 46 (1.1 g, 3.0 mmol) was added in one portion to a solution of D-tartaric acid (1.7 g, 11
o
mmol) in IPA/EA (10 mL/40 mL) at 60 C for 10 min. The reaction was cooled to room temperature
naturally and filtrated. The filter cake was dried in vacuum to give the desired product (2.0 g, 83%) as a
1
white solid. H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 3.6 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H),
7.30-7.18 (m, 1H), 6.56 (s, 1H), 4.50-4.38 (m, 1H), 4.24 (s, 6H), 3.90-3.71 (m, 2H), 3.66-3.43 (m, 5H),
2.97-2.77 (m, 1H), 2.53 (s, 4H), 2.42-2.20 (m, 10H), 2.07-1.93 (m, 2H), 1.93-1.81 (m, 1H). Purity:
99.0%.
6.1.26. General procedure for the synthesis of 53-55
A mixture of 45a (0.10 mmol, 1.0 eq), corresponding amine (0.50 mmol, 5.0 eq) and TEA (1.0 mmol,
o
10 eq) in ethanol (5 mL) was stirred at 85 C for 3 h. The mixture was concentrated and purified by
basic Al2O3 column chromatography (dichloromethane/methanol = 100/1) to give the desired product.
6.1.26.1.
(S)-1-Methyl-4-(2-methyl-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidin-4-yl)-1,4-diaze
pane (53)
Compound 53 was obtained as a yellow oil (yield 68%). 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J =
4.0 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.05-6.93 (m, 1H), 6.29 (s, 1H), 3.98-3.19 (m, 8H), 2.70-2.46 (m,
5H), 2.43-2.30 (m, 9H), 2.24-2.12 (m, 1H), 2.09-1.79 (m, 5H). 13C NMR (150 MHz, CDCl3) δ 166.2,
165.7, 162.0, 159.6, 147.2, 138.1, 131.8, 121.7, 97.7, 67.3, 60.4, 58.3, 57.5, 54.5, 46.6, 45.8, 45.2, 31.2,
27.3, 26.1, 23.1, 18.7. HRMS (ESI): calcd for C22H32N6 [M+H]+ 381.2761, found 381.2773. Purity:
99.1%. [α]2D5 = +8 (c = 0.2, MeOH).
6.1.26.2.
(S)-N,N-Dimethyl-1-(2-methyl-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidin-4-yl)piperi
din-4-amine (54)
Compound 54 was obtained as a yellow oil (yield 62%). 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J =
4.0 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.00-6.92 (m, 1H), 6.36 (s, 1H), 4.47-4.27 (m, 2H), 3.81 (t, J =
8.0 Hz, 1H), 3.61 (d, J = 14.4 Hz, 1H), 3.36-3.26 (m, 2H), 2.79-2.64 (m, 2H), 2.42-2.28 (m, 8H), 2.25
(s, 6H), 2.18-1.76 (m, 6H), 1.44-1.29 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 166.4, 166.1, 162.1,
159.6, 147.2, 137.9, 131.6, 121.6, 98.1, 67.0, 62.3, 60.5, 54.6, 43.2, 43.1, 41.6, 31.2, 28.1, 28.0, 26.0,
23.0, 18.7. HRMS (ESI): calcd for C23H34N6 [M+H]+ 395.2918, found 395.2926. Purity: 99.5%. [α]25
=
D
+17 (c = 0.2, MeOH).
6.1.26.3.
(S)-2-Methyl-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)-N-(2-morpholinoethyl)pyrimidin-4-a
mine (55)
Compound 55 was obtained as a yellow oil (yield 38%). 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J =
4.0 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.02-6.93 (m, 1H), 6.30 (s, 1H), 5.42 (s, 1H), 3.84 (t, J = 8.0 Hz,
1H), 3.74-3.65 (m, 4H), 3.60 (d, J = 14.8 Hz, 1H), 3.40-3.15 (m, 4H), 2.53 (t, J = 6.0 Hz, 2H),
2.50-2.41 (m, 4H), 2.39 (s, 3H), 2.36-2.29 (m, 4H), 2.22-1.80 (m, 4H). 13C NMR (150 MHz, CDCl3) δ
166.6, 165.8, 162.7, 159.3, 147.3, 138.0, 131.8, 121.7, 99.0, 66.9, 59.8, 56.8, 54.4, 53.2, 37.2, 31.2,
25.8, 23.0, 18.7. HRMS (ESI): calcd for C22H32N6O [M+H]+ 397.2710, found 397.2725. Purity: 99.8%.
[α]2D5 = +15 (c = 0.2, MeOH).
24

6.1.29.
(S)-5-Fluoro-2-methyl-4-(4-methylpiperazin-1-yl)-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)p
yrimidine (50)
A
mixture
of
44a
(24
mg,
0.15
mmol),
5-fluoro-4-(fluoromethyl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine (24 mg, 0.15 mmol),
2-ethyl-2-(hydroxymethyl)propane-1,3-diol (53 mg, 0.3 mmol) and KI (8.3 mg, 0.050 mmol) in H2O (2
mL) was stirred at 120 oC overnight. The mixture was concentrated and purified by basic Al2O3 column
chromatography (dichloromethane/methanol = 100/1) to give the desired product (20 mg, 35%) as a
1
colorless oil. H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 3.6 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H),
7.03-6.94 (m, 1H), 3.87 (t, J = 8.0 Hz, 1H), 3.77-3.64 (m, 5H), 3.54-3.45 (m, 1H), 3.33-3.23 (m, 1H),
2.55-2.47 (m, 1H), 2.47-2.41 (m, 4H), 2.38 (s, 3H), 2.36 (s, 3H), 2.30 (s, 3H), 2.19-2.08 (m, 1H),
2.05-1.95 (m, 1H), 1.93-1.76 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 161.1 (d, J = 7.5 Hz), 159.6,
151.1 (d, J = 4.5 Hz), 150.6 (d, J = 15 Hz), 147.2, 144.1 (d, J = 256.5 Hz), 137.8, 131.5, 121.5, 66.3,
55.1, 54.1, 52.4, 46.1, 45.9 (d, J = 7.5 Hz), 31.4, 25.3, 22.7, 18.6. HRMS (ESI): calcd for C21H29FN6
[M+H]+ 385.2510, found 385.2518. Purity: 99.1%. [α]2D5 = -3 (c = 0.2, MeOH).
6.1.30.
tert-Butyl
(S)-4-(5-cyano-2-methyl-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidin-4-yl)piperazine-
1-carboxylate (51)
A
mixture
of
44a
(32
mg,
0.20
mmol),
tert-butyl
4-(6-(chloromethyl)-5-cyano-2-methylpyrimidin-4-yl)piperazine-1-carboxylate (70 mg, 0.20 mmol),
KI (3.3 mg, 0.020 mmol), and DIPEA (65 mg, 0.50 mmol) in acetonitrile (5 mL) was stirred at room
temperature overnight. The mixture was concentrated and purified by basic Al2O3 column
chromatography (petroleum ether/ethyl acetate = 1/1) to give the desired product (60 mg, 63%) as a
1
colorless oil. H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 7.57-7.36 (m, 1H), 7.18-6.97 (m, 1H),
4.24-3.67 (m, 7H), 3.63-3.38 (m, 5H), 2.56-2.27 (m, 7H), 2.12-2.01 (m, 2H), 2.00-1.92 (m, 2H), 1.48 (s,
9H). MS (ESI/APCI) m/z 477.9 [M+H]+.
6.1.31.
(S)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-((2-(3-methylpyridin-2-yl)pyrrolidin-1-yl)methyl)pyrimidine
-5-carbonitrile (52)
A mixture of 51 (57 mg, 0.12 mmol) and TFA (1 mL) in DCM (2 mL) was stirred at room
temperature for 2 h. The mixture was concentrated in vacuum to give the residue. To a solution of this
crude product in methanol (2 mL) was added formaldehyde (37 wt percent in water, 0.5 mL) and
NaBH3CN (15 mg, 0.24 mmol). The reaction was stirred at room temperature for 2 h, and quenched
with saturated NaHCO3 aqueous solution (5 mL). The aqueous layer was extracted with
dichloromethane (30 mL * 3), dried over Na2SO4, filtered and concentrated. The residue was purified
by basic Al2O3 column chromatography (petroleum ether/ethyl acetate = 1/10) to give the desired
product (30 mg, 64%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 3.6 Hz, 1H), 7.34 (d,
J = 7.6 Hz, 1H), 7.04-6.94 (m, 1H), 4.05-3.96 (m, 1H), 3.95-3.86 (m, 4H), 3.82 (d, J = 13.2 Hz, 1H),
3.63 (d, J = 13.2 Hz, 1H), 3.44-3.34 (m, 1H), 2.64-2.54 (m, 1H), 2.51-2.45 (m, 4H), 2.43 (s, 3H), 2.40
(s, 3H), 2.31 (s, 3H), 2.34-2.12 (m, 1H), 2.10-1.81 (m, 3H). 13C NMR (150 MHz, CDCl3) δ 172.2,
168.3, 161.8, 159.3, 147.2, 138.0, 131.7, 121.7, 117.0, 87.5, 67.0, 57.8, 54.9, 54.5, 46.3, 46.0, 31.3,
26.4, 23.0, 18.8. HRMS (ESI): calcd for C22H29N7 [M+H]+ 392.2557., found 392.2564. Purity: 98.9%.
[α]2D5 = +3 (c = 0.2, MeOH).
25

6.2. In vitro biological assays
6.2.1. HPB-ALL CXCR4 competitive binding assay
HPB-ALL cells were maintained in RPMI-1640 (Gibico) supplemented with 10% FBS (Hyclone).
APC-conjugated anti-human CXCR4 was from Sungene. EC80 was first determined for 12G5 binding
to CXCR4. Then the compounds for testing were added into 96-well plates serially diluted at a ratio of
1:3. Cells were washed once with ice-cold assay buffer (DPBS+2% HI-FBS) and then re-suspended in
the same buffer at a final concentration of 1 × 106/mL. Cell suspension was then added into the wells
and with the addition of APC-conjugated anti-human CXCR4 clone 12G5 at its EC80 determined. The
mix of cell, compounds and APC-conjugated anti-human CXCR4 were incubated at 4 ºC for 3 h before
addition of 100 µL of 4% PFA. Cells were then washed once and resuspended in assay buffer and
examined by FACS.
6.2.2. FLIPR Tetra calcium mobilization assay
The FLIPR Tetra calcium mobilization assay was performed by HD Bioscience. Briefly, The
Molecular Devices, Fluorescent Imaging Plate Reader (FLIPR) Tetra was used in this assay. Excitation
was achieved through unique placement of LED’s within the instrument and emission captured by a
CCD camera (EMCCD camera for FI and ICCD camera for luminescence). The homogeneous FLIPR
Calcium 4 assay kit from Molecular Devices was used as the fluorescence reagent. Compounds were
solubilized in 100% dimethyl sulfoxide (DMSO) to a concentration of 30 mM. A 10-point, 4-fold,
intermediate dilution series was created in 100% DMSO with a top concentration of 4 mM and a
bottom concentration of 0.01 µM. A near assay ready, direct dilution plate (ddNARP) was prepared
from this compound dilution plate by transferring 1 µL of each dilution of compound in 100% DMSO
to a Greiner#781201 plate. In addition, each ddNARP plate also contained positive and negative
control wells to define the upper and lower limits for the assay signal. The final assay concentration
range of compound was 10 µM to 0.035 nM in 0.5% DMSO. Human CD4+ T-Cells were isolated from
human whole blood and subsequently activated and expanded using a CD3/CD28 expansion kit (Life
Technologies). The cells were frozen in ThermoFisher-formulated Recovery Cell Culture Freezing
Medium containing 10% Dimethyl sulfoxide (DMSO) and 10% Fetal Bovine Serum (FBS)
(ThermoFisher Catalog No. 10100147). When used, cells were resuspended using room temperature
1X HBSS/20 mM HEPES/0.005% P-104 assay buffer, adjusted the volume of the suspension to
achieve a cell concentration of 2.5 × 106 cells/mL. 2X Calcium 4 dye (20 µL/well) were added and the
mixture were centrifuged briefly (~10 s) and stopped when it reached 1000 rpm. The plates were
allowed to equilibrate before compounds and CXCL12 were added to the plates. The raw data were
analyzed using Abase. The percent (%) effect at each concentration of compound was calculated by
Abase and was based on and relative to the amount of calcium produced in the positive and negative
control wells contained within each assay plate. The concentrations and % effect values for tested
compounds were plotted by Abase and the concentration of compound required for 50% effect (IC50)
was determined with a four-parameter logistic dose response equation.
6.2.3. Matrigel invasion assay
The human breast cancer cell line MDA-MB-231 was purchased from ATCC (Manassas, VA).
MDA-MB-231 cell line was cultured in DMEM medium supplemented with 10% heat-inactivated fetal
bovine serum, 100 units/ml of penicillin and 100 mg/mL of streptomycin. Cells were cultured at 37 oC
26

in a humidified atmosphere of 20% O2/5% CO2. All cultures were monitored routinely and found to be
free of contamination by mycoplasma or fungi, discarded after three months, and new lines propagated
from frozen stocks.
Matrigel invasion assays were carried out in modified Boyden chambers with filter inserts with 8-ꢀm
pores in 24-well plates (Corning, NY, USA). The surfaces of the filters were coated with 50mg/L
ice-cold Matrigel (Matrigel basement membrane matrix, BD Bioscience, NJ, USA). The lower chamber
was filled with medium containing 10% serum. The target compounds (100 nM) and the human breast
cancer cell line MDA-MB-231 cells (1 × 105 cells/well) were added to in the upper chamber of a vessel
and CXCL12 was added in the lower chamber as a chemoattractant in serum free medium. After 24 h
incubation, the filters were gently removed from the chambers, and the cells on the upper surface were
removed by wiping with a cotton swab. Cells that had invaded to the lower surface areas were fixed
with ice cold methanol, stained with crystal violet, and counted in 10 randomly selected fields under a
microscope (100×). Results shown are representative of three independent experiments.
6.2.4. Cell viability (MTT) assay
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Briefly, cells were plated at a density of 5 × 103 cells/well on 96-well plates and subjected to compound
3 treatment. Following 48 h incubation at 37 ºC in a humidified atmosphere containing 5% CO2/95%
air,
the
samples
were
incubated
for
another
4
h
with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent. The formazan product was
dissolved in dimethyl sulfoxide and read at 570 nm on a Victor3 Multi Label plate reader (PerkinElmer,
Boston, MA, USA).
6.2.5. Other assays
CXCR1 functional assay, CCR6 functional assay, these assay procedures have been reported
previously[29].
6.3. Preliminary in vitro safety test
CYP inhibitory potency and human/mouse liver microsomes metabolic stability were evaluated as
previously reported [39]. hERG inhibition and Caco-2 assays were reported before [40].
6.4. In vivo tumor metastasis test
Red fluorescence protein (RFP)-labeled lung carcinoma LL/2 cells (4 × 105 cells) were injected into
C57BL/6 mice in a volume of 200 ꢀL PBS via the tail intravenous injection. Vehicle, compound 14 (70
mg/kg) were intraperitoneal injected to these mice 30min before tumor cells injection, and 3 h after
tumor cells injection. Then, these mice were given the same dose of vehicle, compound 14 (70 mg/kg)
every day for 18 days. Eighteen days after injection of tumor cells, mice were sacrificed and lung
tissues were collected. The lung metastasis of LL/2 cells in mice was analyzed by measuring RFP
fluorescence intensities with the PerkinElmer IVIS Lumina II.
6.5 Procedure for pKa and log D7.4 measurements
The pKa was determined by the automated Sirius T3 system. The Sirius T3 can measure one sample
in around 4 min, using 5 ꢀL of 10 mM DMSO stock solution and collecting up to 50 data points from
pH 2.0 to pH 12.0 by UV detection.
The 1-octanol/water partition coefficient at pH7.4 was determined using the shake flask technique. A
27

phosphate pH7.4 aqueous buffer solution has been prepared and then saturated with n-octanol. Likewise,
an n-octanol solution saturated with pH7.4 aqueous buffer has been also prepared. Approximately 1 mg
of sample was dispensed into a 3 ml glass screw-top vial to which 1 ml octanol was added. Compounds
that dissolved incompletely were diluted and further sonicated until completely dissolved. A final
volume of 1ml of octanol solution and 1 ml of phosphate buffer (PBS) pH7.4 were shaken for 24 h. The
resulting solution was centrifuged and the octanol and water layers were extracted separated. The
amount of sample present in the octanol and PBS solutions was determined by HPLC. The logD7.4 was
calculated from the area under the curve obtain from the 2 samples, corrected for the dilution factors
and injection volume. Results shown are average value of three independent experiments.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No. 81973161,
81773561, 21502133), the Priority Academic Program Development of the Jiangsu Higher Education
Institutes (PAPD) and the Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003). The
CAMS Innovation Fund for Medical Sciences (CIFMS; 2019-I2M-1-004, 2016-I2M-1-005 and
2019-I2M-1-003).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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