Design, Synthesis, and Characterization of Novel CXCR4 Antagonists Featuring Cyclic Amines

Article abstract of DOI:10.1002/cmdc.202000268

Chemokine receptor CXCR4 and its natural ligand CXCL12 (also known as stromal cell-derived factor-1, or SDF-1) regulate a broad range of physiological functions. Dysregulation of the CXCL12/CXCR4 axis is involved in numerous pathological conditions such as HIV infection, inflammation and cancer. Herein, we report the design, synthesis, and characterization of novel CXCR4 antagonists based on cyclic amine scaffolds. Compound 24 was identified as a potent CXCR4 receptor antagonist (competitive inhibition of 12G5 binding, IC₅₀=24 nM; functional inhibition of CXCL12-induced cytosolic calcium increase, IC₅₀=0.1 nM). In addition, compound 24 potently inhibited cell migration in CXCR4/CXCL12-mediated chemotaxis in a matrigel invasion assay. The absolute configuration of compound 24 was elucidated by X-ray crystallography.

Full text of DOI:10.1002/cmdc.202000268

Accepted Article
Title: Design, Synthesis, and Characterization of Novel CXCR4
Antagonists Featuring Cyclic Amines
Authors: Xiaohu Zhang, Yu Lin, Zhanhui Li, Haikuo Ma, Yujie Wang,
Xu Wang, Shiwei Song, Li Zhao, Shuwei Wu, Sheng Tian,
Chunyan Fu, Lusong Luo, Fang Zhu, Sudan He, and Jiyue
Zheng
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000268
Link to VoR: https://doi.org/10.1002/cmdc.202000268
01/2020

10.1002/cmdc.202000268
ChemMedChem
Graphic Abstract
Compound 24 is a potent CXCR4 antagonist (competitive inhibition of 12G5 binding, IC50 = 24 nM;
functional inhibition of CXCL12 induced cytosolic calcium increase, IC50 = 0.1 nM). The absolute
configuration of compound 24 was elucidated by X-ray crystallography.
1
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ChemMedChem
Design, Synthesis, and Characterization of Novel CXCR4 Antagonists Featuring Cyclic Amines
Yu Lin,[a],# Zhanhui Li,[a],# Haikuo Ma,[a,b] Yujie Wang,[a] Xu Wang,[a] Shiwei Song,[a] Li Zhao,[a] Shuwei
Wu,[a] Sheng Tian, [a] Chunyan Fu,[c] Lusong Luo,[c] Fang, Zhu,[b,d] Sudan He,*[b,d] Jiyue Zheng,*[a] and
Xiaohu Zhang*[a]
[a] Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences,
Soochow University, Suzhou, Jiangsu 215123, China
[b] Cyrus Tang Hematology Center, Jiangsu Institute of Hematology and Collaborative Innovation
Center of Hematology, Soochow University, Suzhou 215123, P.R. China.
[c] BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park, Beijing
102206, 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
# 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: Chemokine receptor CXCR4 and its natural ligand CXCL12 (also known as stromal
cell-derived factor-1, or SDF-1) regulate a broad range of physiological functions. Dysregulation of the
CXCL12/CXCR4 axis is involved in numerous pathological conditions such as HIV infection,
inflammation and cancer. Herein, we report the design, synthesis, and characterization of novel CXCR4
antagonists based on cyclic amine scaffolds. Compound 24 was identified as a potent CXCR4 receptor
antagonist (competitive inhibition of 12G5 binding, IC50 = 24 nM; functional inhibition of CXCL12
induced cytosolic calcium increase, IC50 = 0.1 nM). In addition, compound 24 potently inhibited cell
migration in the CXCR4/CXCL12 mediated chemotaxis in a matrigel invasion assay. The absolute
configuration of compound 24 was elucidated by X-ray crystallography.
Keywords: Chemokine, CXCL12, CXCR4, GPCR
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ChemMedChem
Abbreviations: APC, allophycocyanin; CYP, cytochrome P450; DIPEA, N, N-diisopropylethylamine;
FLIPR, fluorescent imaging plate reader; GPCR, G protein-coupled receptor; HIV, human
immunodeficiency virus; MEF, mouse embryonic fibroblasts; SAR, structure-activity-relationship;
SDF-1, stromal cell-derived factor-1; SEM, standard error measurement; THQ, tetrahydroquinoline;
TLC, thin-layer chromatography.
Introduction
The chemokine receptor CXCR4 is a class A G-protein coupled receptor (GPCR). CXCR4 was
first identified as a coreceptor (along with CD4) used by T cell-tropic (X4) HIV-1 virus for cellular
entry.[1] Consequently, the majority of early research on CXCR4 antagonists centered on anti-HIV
application.[2] Recent studies demonstrated that CXCR4 and its natural ligand CXCL12 (also known as
stromal cell-derived factor-1, or SDF-1) regulate a broad range of physiological functions.[3-4]
Dysregulation of the CXCL12/CXCR4 axis is involved in many human diseases, such as
inflammatory/autoimmune diseases and cancer.[5-8] In addition, CXCR4 was reported to modulate
immune cell function by trafficking of key immune cells into tumor microenvironment.[9-10] Since
many of the immune cells were immunosuppressive (e.g., Treg, myeloid derived suppressor cells)
rather than immunoreactive, CXCL12/CXCR4 axis plays an important role in evasion of immune
surveillance.[11] Based on these results, inhibition of CXCR4 is proposed as a promising strategy for the
development of therapeutic agents.
Previous research on CXCR4 antagonists resulted in a number of small molecule CXCR4
antagonists (Figure 1).[2, 6, 12] The most advanced small molecule CXCR4 antagonist, AMD3100
(plerixafor, Figure 1), was originally developed for anti-HIV treatment.[13] Unfortunately, poor
bioavailability and dose-limiting toxicity hampered its development as an orally administered daily
drug for chronic HIV treatment.[14] Surprisingly, AMD3100 exhibited strong effect on mobilizing
hematopoietic stem cell during clinical investigation.[15-16] As a result, AMD3100 was approved by the
FDA for the treatment of non-Hodgkin’s lymphoma and multiple myeloma patients with autologous
stem cell transplants.[13] In order to address the poor oral bioavailability displayed by AMD3100, the
next generation CXCR4 antagonists were developed. AMD070 displayed improved bioavailability
along with enhanced potency and was progressed into clinical investigation.[17-18] However, AMD070
exhibited moderate inhibition of CYP450 isozymes (e.g., CYP3A4, 2D6).[19] Since the benzimidazole
of AMD070 was considered as the likely source of this potentially toxic off-target activity, significant
efforts were placed to substitute this functional group. As a result, GSK812397[20] and TIQ15[21] were
developed (Figure 1).
Figure 1. Examples of small molecule CXCR4 antagonists reported in the literature
In a similar vein, we have developed and reported a series of potent CXCR4 antagonists based on a
pyrimidine central scaffold.[19, 22-23] In order to further improve the potency of this scaffold, we decided
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10.1002/cmdc.202000268
ChemMedChem
to explore the “magic methyl” strategy[24-25] by addition of a methyl on the benzylic position of the
pyrimidine (Figure 2). Since this particular position is a connective point for the upper ring and the
lower ring systems, we hypothesized that the conformation of the whole molecule might be
significantly impacted by the addition of a methyl group. We next proposed to form a ring by tying the
two methyl groups (Figure 2). This configuration restriction may lock the molecule in a preferred
conformation to interact with CXCR4 receptor. Furthermore, the ring closure decreased rotatable bonds
and might have positive effects on absorption and metabolic stability.[26] Finally, since CXCR4 is an
important drug target and has been investigated extensively, the chemical space for intellectual property
of CXCR4 is very crowded. The central cyclic amine scaffolds, to the best of our knowledge, appeared
sparsely in the CXCR4 patent literature. Here we report our results on this novel series of CXCR4
antagonists.
Figure 2. Application of “magic methyl” and ring closure strategy for the novel cyclic amine scaffold
Results and Discussion
Chemistry
The original scaffold contains a chiral center on the tetrahydroquinoline (THQ) ring. Addition of
a methyl group on the benzylic position of the pyrimidine inevitably introduces a second chiral center.
This is also the case for the ring-closure analogues. With two chiral centers in the scaffold, the
synthetic chemistry can be challenging. We reasoned that we could fix the first chirality by using a
chirally pure starting material. The chirality of the second chiral center could be controlled by the first
chiral center in the subsequent chemical transformation. In addition, we decided to determine the
absolute configuration of the key compound by X-ray crystallography to ensure that the planned
transformation was indeed carried out correctly.
The synthesis of compounds 1-5, 18-21, and 24-34 was performed as shown in Scheme 1.
Cyclization of ester 6 with 2-methyl-2-thiopseudourea sulfate, followed by chlorination with POCl3
yielded intermediate 8. Substitution of 8 with (±)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine[19]
gave intermediate 9, which was reacted with N-methyl piperazine to afford compound 1. Condensation
of Boc-N-methyl-D-alanine (10a) with Meldrum's acid, followed by reaction with EtOH provided
β-ketoester 11a. Cyclization of 11a with 2-methyl-2-thiopseudourea sulfate led to pyrimidine 12a,
which was substituted with N-methyl piperazine in the presence of PyBOP, followed by de-Boc
protection to give amine 13a. Substitution of 13a with (±)-8-chloro-5,6,7,8-tetrahydroquinoline[19]
yielded compounds 2 and 3 as diastereoisomers. Similarly, using Boc-N-methyl-L-alanine (10b)
instead of 10a as start material, compounds 4 and 5 were obtained.
Condensation of acids 14a-h with Meldrum's acid, followed by reaction with EtOH afforded
β-ketoesters 15a-h. Cyclization of 15a or 15b with 2-methyl-2-thiopseudourea gave pyrimidines 16a
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10.1002/cmdc.202000268
ChemMedChem
and 16b, respectively. Treatment of 16a or 16b with N-methyl piperazine in the presence of PyBOP,
followed by de-Boc protection and alkylation with (±)-8-chloro-5,6,7,8-tetrahydroquinoline provided
compounds 18-21. The stereochemistry of compounds 18-21 was validated by reductive amination
(alkylation) of intermediate 17b with 6,7-dihydroquinolin-8(5H)-one[20]. Since intermediate 17b has an
existing chiral center, compound 20 was expected to be the major product. Indeed, compound 20 was
the only product isolated from the reductive amination reaction, thus establishing the stereochemistry
of compounds 18-21 with high confidence. Reaction of ketoesters 15b-h with acetamidine afforded
pyrimidines 22a-g, which were condensed with N-methyl piperazine and de-Boc protected with HCl to
give amines 23a-g. Reductive alkylation of 23a-e with 6,7-dihydroquinolin-8(5H)-one[20] (followed by
de-TBS protection in the case of 23c) yielded compounds 24-30 with moderate to good
diastereoselectivity (for example, compounds 29 vs 30, 4:1), while substitution of 23f or 23g with
(±)-8-chloro-5,6,7,8-tetrahydroquinoline furnished compounds 31-34. The structure of compound 24
was confirmed by X-ray crystallographic analysis (Figure 3). The stereochemistry of other compounds
was tentatively assigned by relating to compound 24 by TLC, LC-MS (retention time elution
order)[27-29]
.
N
N
N
N
Cl
Cl
Cl
N
N
a
c
d
b
O
N
N
O
OEt
S
N
OH
S
N
Cl
S
N
N
S
N
Cl
N
6
7
8
9
1
8
N
*
N
S
*
S
12
O
O
O
N
N
Boc
Boc
N
H
N
N
N
e
f
g
h
N
Boc
N
*
*
*
HO
EtO
N
N
*
S
N
N
HO
N
N
10a: * = (S)
10b: * = (R)
11a: * = (S)
11b: * = (R)
12a: * = (S)
12b: * = (R)
13a: * = (S)
13b: * = (R)
2: * = (8S, 12S)
3: * = (8R, 12S)
4: * = (8S, 12R)
5: * = (8R, 12R)
8
N
*
N
S
N
12
*
S
N
H
Boc
N
N
N
N
N
g
h or i
*
*
N
N
S
N
HO
N
N
f
16a: * = (S)
16b: * = (R)
17a: * = (S)
17b: * = (R)
18: * = (8S, 12S)
19: * = (8R, 12S)
20: * = (8S, 12R)
21: * = (8R, 12R)
O
O
Boc
N
Boc
O
N
e
*
*
HO
EtO
R1
R2
R1
R2
n
n
8
15a-h
14a-h
N
*
j
N
a: R1 = H, R2 = H, n = 1, * = (S)
b: R1 = H, R2 = H, n = 1, * = (R)
c: R1 = F, R2 = F, n = 1, * = (R)
d: R1 = H, R2 = OTBS, n = 1, * = (R)
e: R1 = H, R2 = OMe, n = 1, * = (R)
f: R1 = H, R2 = H, n = 0, * = (R)
g: R1 = H, R2 = H, n = 2, * = (S)
h: R1 = H, R2 = H, n = 2, * = (R)
12
N
*
R1
R2
N
N
n
Boc
N
H
N
N
N
g
k
*
*
HO
N
N
N
R1
R2
R1
R2
n
n
N
N
22a-g
23a-g
24: R1 = H, R2 = H, n = 1, * = (8S, 12R)
25: R1 = F, R2 = F, n = 1, * = (8S, 12R)
26: R1 = H, R2 = OH, n = 1, * = (8S, 12R)
27: R1 = H, R2 = OMe, n = 1, * = (8S, 12R)
a: R1 = H, R2 = H, n = 1, * = (R)
b: R1 = F, R2 = F, n = 1, * = (R)
c: R1 = H, R2 = OTBS, n = 1, * = (R)
d: R1 = H, R2 = OMe, n = 1, * = (R)
e: R1 = H, R2 = H, n = 0, * = (R)
f: R1 = H, R2 = H, n = 2, * = (S)
g: R1 = H, R2 = H, n = 2, * = (R)
28: R1 = H, R2 = OMe, n = 1, * = (8R, 12R)
29: R1 = H, R2 = H, n = 0, * = (8S, 12R)
30: R1 = H, R2 = H, n = 0, * = (8R, 12R)
31: R1 = H, R2 = H, n = 2, * = (8S, 12S)
32: R1 = H, R2 = H, n = 2, * = (8R, 12S)
33: R1 = H, R2 = H, n = 2, * = (8S, 12R)
34: R1 = H, R2 = H, n = 2, * = (8R, 12R)
5
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ChemMedChem
Scheme 1. Synthesis of Compounds 1-5, 18-21, and 24-34. Reagents and conditions: (a)
2-methyl-2-thiopseudourea sulfate, Na2CO3, H2O, rt, overnight; (b) POCl3, 100 °C, 1 h; (c)
(±)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine, KI, DIPEA, MeCN, rt, overnight; (d) N-methyl
piperazine, Et3N, EtOH, 80 °C, overnight; (e) Meldrum's acid, DCC, DMAP, CH2Cl2, rt, 48 h, then
EtOH, 80 °C, 2 h; (f) 2-methyl-2-thiopseudourea sulfate, K2CO3, H2O, rt; overnight; (g) i) N-methyl
piperazine, Et3N, PyBOP, MeCN, reflux, overnight, ii) HCl/EtOAc, rt, overnight; (h)
(±)-8-chloro-5,6,7,8-tetrahydroquinoline, KI, DIPEA, NMP, 120 °C, overnight; (i) for 20 from 17b,
6,7-dihydroquinolin-8(5H)-one, NaBH3CN, MeOH, rt, overnight; (j) acetamidine hydrochloride,
EtONa, EtOH, reflux, overnight; (k) for 24, 25, 27-30, 6,7-dihydroquinolin-8(5H)-one, NaBH3CN,
MeOH, rt, overnight; for 26, i) 6,7-dihydroquinolin-8(5H)-one, NaBH3CN, MeOH, rt, overnight; ii) 2
N HCl, rt, 2 h; for 31-34, (±)-8-chloro-5,6,7,8-tetrahydroquinoline, Et3N, EtOH, 80 °C, overnight.
Figure 3. Crystal structure of compound 24 (CCDC number 1957812).
Pharmacological Characterization of CXCR4 antagonists
The synthesis of racemic compound 1 was straightforward and efficient (Scheme 1). Compound 1
had been serving as a prototypical template for further modification. For example, the thioether can be
oxidized to sulfone and then substituted by nucleophiles. Alternatively, the sulfone can serve as an
anchor to mediate all kinds of coupling reactions (e.g., Suzuki coupling). Compounds 2-5 and 18-21
were synthesized from chiral starting materials. The chirality of the second chiral center was tentatively
assigned by relating to compound 24 via TLC/LC-MS (retention time elution order), or using
stereochemistry controlled methods such as reductive amination (alkylation) reactions.
Multiple assays have been developed to characterize the interaction of the CXCL12/CXCR4 axis.
Tom Van Loy’s group recently elegantly summarized most of the known CXCR4 ligands (including
peptides and small molecules) in a series of pharmacological and functional assays.[30] These assays
include the CXCL12 competitive binding assay, CXCL12-induced calcium mobilization, CXCR4
receptor internalization, CXCL12-guided chemotaxis and CXCR4-specific HIV entry and replication.
According to their results, we chose to use a cell base competitive binding assay as our primary
screening assay. The detailed description of assay development and validation had been reported.[19]
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 IC50. The
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10.1002/cmdc.202000268
ChemMedChem
structure-activity-relationship was summarized in Table 1. The prototypical compound 1 exhibited
excellent binding affinity with IC50 = 12 nM (Figure 2). Introduction of a methyl at the benzylic
position of pyrimidine added a second chiral center on the scaffold. As planned, we started with
chirally pure starting materials 10a and 10b (Scheme 1). The final alkylation with racemic
(±)-8-chloro-5,6,7,8-tetrahydroquinoline provided a pair of diastereomers (2 and 3 from 10a; 4 and 5
from 10b, respectively) with no appreciable selectivity. Compared with 1, compound 2 (348 nM) was
about 30 folds less potent in the 12G5 binding assay, while 3 was practically inactive. More
encouragingly, compounds 4 and 5 exhibited decent binding affinity at 66 nM and 106 nM,
respectively. The ring closure analogues demonstrated mostly deminished binding affinity to CXCR4,
with the exception of compound 20 (92 nM). Although 8 folds less active than 1, compound 20
represents a very unique template that appeared sparsely in the CXCR4 patent literature. This is
particularly valuable, because the patent space for CXCR4 is very crowded. The emerging role of
CXCR4 in oncology/immune-oncology and autoimmune/inflammatory diseases prompted us to further
investigate the cyclic amine template.
Table 1. SAR of the central connecting elements for the synthesized CXCR4 compounds
12G5
Binding IC50
(nM) [a]
12G5
Binding IC50
(nM) [a]
clogP
[c]
pKa
[d]
clogP
[c]
pKa
[d]
Cmpd
Linkage
Cmpd
18
Linkage
4.00
4.00
4.00
4.00
6.4
6.4
6.4
6.4
> 10000
2671 ± 347
92 ± 1
4.03
4.03
4.03
4.03
6.8
6.8
6.8
6.8
2
3
4
348 ± 80
> 10000
19
66 ± 19
106 ± 0.1
303 ± 24
20
> 10000
5
21
AMD
3100
[b]
[a] Inhibition of luminescence signaling in HPB-ALL CXCR4 competitive binding assay. Data are
expressed as geometric mean values of at least two runs ± the standard error measurement (SEM).
[b] AMD3100 was run as standard in each assay. Data are expressed as geometric mean values of nine
runs ± the standard error measurement (SEM).
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10.1002/cmdc.202000268
ChemMedChem
[c] Calculated by Molinspiration.
[d] Calculated by ACD/Labs 6.0.
Although the thioether can serve as a versatile intermediate for derivatization of the 2-pyrimidine
position, it is not suitable as a drug discovery lead due to its metabolic liability. We therefore replaced
the thioether with a simple methyl in our exploration of the cyclic amine template. We also introduced
substitutions on the cyclic amines (e.g., F, OMe, OH). More importantly, we expanded the scope of
cyclic amine from 5-membered ring to 4-membered and 6-membered rings (Scheme 1). The
structure-activity-relationship was summarized in Table 2. The methyl analogue compound 24
exhibited excellent binding affinity to CXCR4, displacing 12G5 with an IC50 of 24 nM. In addition, it
showed good physicochemical properties (MW 392, clogP 2.64, pKa 7.7), indicating its potential as a
lead compound. Introduction of fluorine atoms diminished binding activity (compound 25, 2910 nM).
Introduction of a hydroxyl was also detrimental to binding activity, albeit to a lesser degree (compound
26, 596 nM). The methoxy was well tolerated, as compound 27 maintained good binding affinity (86
nM). Compound 28, the diastereomer of 27, was practically inactive. This was consistent with the
previous SAR and reinforced the important role of the chirality connecting the cyclic amine with the
THQ moiety. Encouragingly, compound 29, the 4-membered cyclic amine with the preferred
configurations, displayed decent activity (94 nM). Conversely, compound 30, the diastereomer of 29,
was inactive. The most disappointing results came from the 6-membered cyclic amines. Only
compound 34 (4650 nM) showed marginal activity while all other isomers were inactive. AMD3100
was used as the positive standard throughout the SAR studies and it displayed an IC50 = 303 nM. In
summary, the current structure-activity investigation identified compound 24 as a potent CXCR4
antagonist based on the novel cyclic amine template. In addition to its excellent binding affinity to
CXCR4, compound 24 possesses good physicochemical properties (MW 392, clogP 2.64, pKa 7.7) and
was therefore selected for further functional testing.
Table 2. SAR of the modified central rings for the synthesized CXCR4 compounds
12G5
Binding
12G5
Binding
clogP
[c]
pKa
[d]
clogP
[c]
pKa
[d]
Cmpd
Linkage
Cmpd
Linkage
IC50 (nM) [a]
IC50 (nM) [a]
24 ± 4
2.64
3.01
7.7
7.7
> 10000
> 10000
2.37
3.15
7.7
8.3
24
30
2910 ± 1200
25
31
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596 ± 483
1.73
2.34
7.7
7.7
> 10000
> 10000
3.15
3.15
3.15
8.3
8.3
8.3
26
27
32
33
86 ± 4
> 10000
94 ± 32
2.34
2.37
7.7
7.7
4650 ± 1410
303 ± 24
28
29
34
AMD
3100
[b]
[a] Inhibition of luminescence signaling in HPB-ALL CXCR4 competitive binding assay. Data are
expressed as geometric mean values of at least two runs ± the standard error measurement (SEM).
[b] AMD3100 was run as standard in each assay. Data are expressed as geometric mean values of nine
runs ± the standard error measurement (SEM).
[c] Calculated by Molinspiration.
[d] Calculated by ACD/Labs 6.0.
Functional evaluation of compound 24
Binding of CXCL12 to CXCR4 triggers multiple cellular responses. A signature of CXCR4
activation is the transient increase of cytosolic calcium concentration. The calcium flux can be detected
efficiently by FLIPR Tetra.[31] Since compound 24 exhibited high binding affinity to CXCR4 in the
12G5 competitive binding assay, we next evaluated functional activity of 24 in the FLIPR Tetra based
calcium mobilization assay. The detailed experimental procedure was reported previously.[19]
The FLIPR Tetra based calcium mobilization assay validated compound 24 as a potent functional
CXCR4 antagonist (IC50 = 0.1 nM) while the positive control AMD3100 was less potent (IC50 = 6 nM)
under the same experimental conditions (Figure 4a). We next set to assess the preliminary functional
activities/specificities of compound 24. We selected CXCR1 as the representative within the CXCR
sub-family and CCR6 as the representative outside of the CXCR sub-family. The CXCR1 assay was a
cell-based assay using IL-8 as the stimulant and cAMP as the readout. The CCR6 assay was carried out
in a similar protocol to CXCR4 but using CCL20 as the stimulant. The detail experimental procedure
of these assays can be found in the SI. Compound 24 was inactive in both assays up to 10,000 nM
(Figure 4b, c).
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Figure 4. Compound 24 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
24/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 24/Navarixin (positive control for
CXCR1) based on a functional cAMP assay. (c) CCR6 dose response curve for CCL20 stimulation and
inhibition curves of compound 24/PF-9654-00 (positive control for CCR6) based on a calcium flux
assay.
CXCR4 is a chemokine receptor. One of its principal functions is chemotaxiing of cells towards
concentration gradient of CXCL12. We set up the matrigel invasion assay to evaluate compound 24 for
its inhibition of CXCL12/CXCR4 mediated chemotaxis. MDA-MB-231 cells which naturally express
CXCR4 and 24 (100 nM) were added to the upper chamber. Human CXCL12 (200 ng/mL) was added
to the lower chamber. By comparison to the cell invasion without treatment, the inhibitory effects of
cell invasion of 24 and AMD3100 can be determined (Figure 5a, b). Consistent with the 12G5 binding
and the calcium mobilization results, 24 displayed superior potency in the matrigel invasion assay than
AMD3100. Since general cellular toxicity can negatively affect cell mobility, leading to false positive
result in matrigel invasion assay, we therefore investigated the potential cytotoxicity effect of 24 using
the cell viability assay (ATP-based Cell Titer-Glo Luminescent Cell Viability Assay). As shown in
Figure 5c, 24 did not inhibit the proliferation of multiple human and murine cells at concentrations up
to 1000 nM.
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Figure 5. Effect of compound 24 on matrigel invasion of MDA-MB-231 cells and cytotoxicity
assessment of 24 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 24 on proliferation of human and murine cells.
Conclusion
Novel CXCR4 antagonists based on cyclic amine scaffolds were synthesized and characterized.
Compound 24 was identified as a potent CXCR4 receptor antagonist (competitive inhibition of 12G5
binding, IC50 = 24 nM; functional inhibition of CXCL12 induced cytosolic calcium increase, IC50 = 0.1
nM). In addition, compound 24 potently inhibited cell migration in the CXCR4/CXLC12 mediated
chemotaxis in a matrigel invasion assay. The absolute configuration of compound 24 was elucidated by
X-ray crystallography. Structure-activity-relationship study of other parts of this novel scaffold is
ongoing and results will be reported in due course.
Experimental Section
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. 1H NMR were obtained on 400 MHz (Varian) spectrometer, and
13C 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. Final compounds were purified with silica gel 100-200 mesh for
column chromatography. HPLC data were obtained using an Agilent 6120B single quadrupole LC/MS
system. HPLC method: 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
11
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10.1002/cmdc.202000268
ChemMedChem
a 1 min hold at 95% B. Flow: 1.2 mL/min. Detection: UV at 214 and 254 nm.
6-(Chloromethyl)-2-(methylthio)pyrimidin-4-ol (7): To a solution of ethyl 4-chloro-3-oxobutanoate
(13 g, 79 mmol) and 2-methyl-2-thiopseudourea sulfate (20 g, 72 mmol) in 200 mL of water was added
Na2CO3 (11.5 g, 108 mmol). The reaction mixture was stirred overnight at room temperature, before 6
N HCl was added carefully to adjust PH = 4. The resulting precipitate was filtered and dried in vacuum
to give the desired product (11.1 g, 73%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 12.90 (s, 1H),
6.42 (s, 1H), 4.35 (s, 2H), 2.59 (s, 3H).
4-Chloro-6-(chloromethyl)-2-(methylthio)pyrimidine (8): A solution of 7 (2.8 g, 14.7 mmol) in
POCl3 (10 mL) was stirred at 100 °C for 1 h. After cooling to room temperature, the reaction mixture
was concentrated in vacuum. The residue was dissolved in ethyl acetate (100 mL) and washed with
saturated NaHCO3 aqueous solution (100 mL). The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl
acetate = 100/3) to give the desired product (2.4 g, 80%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ
7.22 (s, 1H), 4.52 (s, 2H), 2.57 (s, 3H).
N-((6-Chloro-2-(methylthio)pyrimidin-4-yl)methyl)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine
(9): To a solution of 8 (540 mg, 2.6 mmol) and (±)-N-methyl-5,6,7,8-tetrahydroquinolin-8-amine (400
mg, 2.5 mmol) in MeCN (20 mL) was added KI (41 mg, 0.25 mmol) and DIPEA (800 mg, 6.2 mmol),
and the reaction mixture was stirred at room temperature overnight. The mixture was quenched with
saturated NaHCO3 aqueous solution and extracted with dichloromethane (50 mL x 3). The combined
organic layers were washed with brine, dried over Na2SO4 and concentrated. The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH = 200/1) to give the desired product (450 mg,
54%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 4.0 Hz, 1H), 7.51 (s, 1H), 7.37 (d, J =
7.6 Hz, 1H), 7.11-7.04 (m, 1H), 4.08-3.95 (m, 1H), 3.71 (s, 2H), 2.85-2.76 (m, 1H), 2.75-2.69 (m, 1H),
2.54 (s, 3H), 2.41 (s, 3H), 2.17-2.10 (m, 1H), 2.08-2.00 (m, 1H), 1.94-1.85 (m, 1H), 1.78-1.69 (m, 1H).
N-Methyl-N-((6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)methyl)-5,6,7,8-tetrahydr
oquinolin-8-amine (1): To a solution of 9 (450 mg, 1.3 mmol) in EtOH (10 mL) was added Et3N (1.3 g,
13 mmol) and N-methyl piperazine (150 mg, 1.4 mmol). The reaction mixture was stirred at 80 °C
overnight. The mixture was quenched with saturated NaHCO3 aqueous solution (10 mL) and extracted
with dichloromethane (20 mL x 3). The combined organic layers were washed with brine, dried over
Na2SO4 and concentrated. The residue was purified by silica gel column chromatography
1
(CH2Cl2/MeOH = 200/1) to give the desired product (450 mg, 54%) as a brown oil. H NMR (400
MHz, CDCl3) δ 8.48 (d, J = 3.6 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.06 (dd, J = 7.2, 4.8 Hz, 1H), 6.96
(s, 1H), 4.02 (d, J = 7.2 Hz, 1H), 3.72 (s, 4H), 3.58 (s, 2H), 2.86-2.75 (m, 1H), 2.74-2.65 (m, 1H), 2.48
(s, 3H), 2.47-2.43 (m, 4H), 2.39 (s, 3H), 2.33 (s, 3H), 2.16-2.07 (m, 1H), 2.05-1.97 (m, 1H), 1.96-1.85
(m, 1H), 1.69-1.63 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 170.3, 168.2, 162.3, 157.5, 147.4, 136.8,
134.3, 121.8, 96.6, 62.6, 58.9, 54.9, 46.3, 43.9, 39.4, 29.4, 23.0, 21.4, 14.1. HRMS (ESI): calcd for
C21H31N6S [M+H]+ 399.2325, found 399.2334.
General procedures for the synthesis of 11a and 11b: To a solution of 10a or 10b (1.0 eq.),
2,2-dimethyl-1,3-dioxane-4,6-dione (1.1 eq.), DMAP (1.5 eq.) in dichloromethane (20 mL) was added
12
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10.1002/cmdc.202000268
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DCC (1.1 eq.) at 0 °C. The mixture was stirred at room temperature for 48 h. The reaction solution was
filtered. And the filtrate was washed with saturated citric acid aqueous solution (50 mL). The organic
layer was dried over Na2SO4, filtered and evaporated. The residue was diluted with EtOH (20 mL) and
stirred at reflux for 2 h. The resulting solution was concentrated and purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 10:1) to give the desired product.
Ethyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-oxopentanoate (11a): Compound 11a was
obtained as a colorless oil (320 mg, 33 %). [α]2D5= -148 (c = 0.2, CHCl3). 1H NMR (400 MHz, CDCl3) δ
4.60-4.48 (m, 0.5H), 4.23-4.13 (m, 2H), 4.13-4.05 (m, 0.5H), 3.65-3.48 (m, 1H), 3.46-3.40 (m, 1H),
2.90-2.72 (m, 3H), 1.50-1.40 (m, 12H), 1.28-1.24 (m, 3H).
Ethyl (R)-4-((tert-butoxycarbonyl)(methyl)amino)-3-oxopentanoate (11b): Compound 11b was
obtained as a yellow oil (590 mg, 70 %). [α]2D5 = +122 (c = 0.2, CHCl3). 1H NMR (400 MHz, CDCl3) δ
4.62-4.48 (m, 0.5H), 4.22-4.14 (m, 2H), 4.14-4.06 (m, 0.5H), 3.62-3.48 (m, 1H), 3.46-3.38 (m, 1H),
2.90-2.73 (m, 3H), 1.49-1.41 (m, 9H), 1.33-1.25 (m, 6H).
General procedures for the synthesis of 12a and 12b: To a mixture of 11a or 11b (1.0 eq.) and
2-methyl-2-thiopseudourea sulfate (2.0 eq.) in water (10 mL) was added K2CO3 (4.0 eq.). The reaction
was stirred at room temperature overnight. The reaction mixture was quenched with AcOH to adjust pH
= 4 and extracted with dichloromethane (50 mL x 3). The combined organic layers were dried over
Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol = 20/1) to give the desired product.
tert-Butyl
(S)-(1-(6-hydroxy-2-(methylthio)pyrimidin-4-yl)ethyl)(methyl)carbamate
(12a):
Compound 12a was obtained as a white solid (270 mg, 75 %). [α]2D5= -9 (c = 0.2, CHCl3). 1H NMR (400
MHz, CDCl3) δ 6.09 (s, 1H), 5.23 (s, 0.5H), 4.91 (s, 0.5H), 2.83-2.72 (m, 3H), 2.55 (s, 3H), 1.46-1.42
(m, 9H), 1.34-1.27 (m, 3H).
tert-Butyl
(R)-(1-(6-hydroxy-2-(methylthio)pyrimidin-4-yl)ethyl)(methyl)carbamate
(12b):
25
1
Compound 12b was obtained as a white solid (380 mg, 60 %). [α]D= +18 (c = 0.2, CHCl3). H NMR
(400 MHz, CDCl3) δ 6.08 (s, 1H), 5.23 (s, 0.5H), 4.91 (s, 0.5H), 2.83-2.74 (m, 3H), 2.55 (s, 3H),
1.45-1.42 (m, 9H), 1.33-1.27 (m, 3H).
General procedures for the synthesis of 13a and 13b: To a solution of 12a or 12b (1.0 eq.), Et3N
(10.0 eq.) and N-methylpiperazine (1.5 eq.) in CH3CN (10 mL) was added PyBOP (530 mg, 0.98
mmol). The reaction mixture was stirred at reflux overnight and then concentrated. The resulting
residue was diluted with dichloromethane (50 mL), and washed with saturated NaHCO3 aqueous
solution (50 mL). The aqueous layer was extracted with dichloromethane (20 mL x 3). The combined
organic layers were dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel
column chromatography (dichloromethane/methanol/NH4OH = 100/1/1) to give a yellow oil. Then it
was diluted with ethyl acetate (5 mL), and 3 M HCl/ethyl acetate (5 mL) was added. The mixture was
stirred at room temperature overnight and then evaporated. The resulting residue was diluted with
dichloromethane (50 mL) and washed with saturated NaHCO3 aqueous solution (50 mL). The aqueous
layer was extracted with dichloromethane (20 mL x 3). The combined organic layers were dried over
Na2SO4, filtered and evaporated to give the desired product.
(S)-N-Methyl-1-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)ethanamine
(13a):
Compound 13a was obtained as a yellow oil (170 mg, 68 %). [α]2D5= -13 (c = 0.2, CHCl3). 1H NMR (400
MHz, CDCl3) δ 6.46 (s, 1H), 3.96-3.86 (m, 1H), 3.72 (s, 4H), 2.54 (s, 3H), 2.51 (s, 3H), 2.50-2.47 (m,
13
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10.1002/cmdc.202000268
ChemMedChem
4H), 2.35 (s, 3H), 1.61 (d, J = 6.8 Hz, 3H).
(R)-N-Methyl-1-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)ethanamine
(13b):
Compound 13b was obtained as a yellow oil (170 mg, 50 %). [α]2D5= +5 (c = 0.2, CHCl3). 1H NMR (400
MHz, CDCl3) δ 6.45 (s, 1H), 4.08-4.01 (m, 1H), 3.79-3.70 (m, 4H), 2.60 (s, 3H), 2.58-2.52 (m, 4H),
2.50 (s, 3H), 2.38 (s, 3H), 1.65 (d, J = 6.8 Hz, 3H).
(S)-N-Methyl-N-((S)-1-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)ethyl)-5,6,7,8-tet
rahydroquinolin-8-amine
(2)
and
(R)-N-methyl-N-((S)-1-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)ethyl)-5,6,7,8-tet
rahydroquinolin-8-amine (3): A mixture of (±)-8-chloro-5,6,7,8-tetrahydroquinoline (117 mg, 0.7
mmol), 13a (100 mg, 0.4 mmol), KI (20 mg, 0.12 mmol), and DIPEA (452 mg, 3.5 mmol) in 4 mL of
NMP was stirred at 120 °C overnight. The reaction mixture was diluted with EtOAc (50 mL) and
washed with brine (10 mL x 5). The organic layer was dried over Na2SO4, filtered and evaporated. The
residue was purified by silica gel column chromatography (dichloromethane/methanol/NH4OH =
100/1/1) to give the desired products 2 and 3. Compound 2 was obtained as a yellow oil (20 mg, 14 %).
25
1
Rf = 0.70 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 6.19 min. [α]D= +10 (c = 0.1, CHCl3). H
NMR (400 MHz, CDCl3) δ 8.53-8.46 (m, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.28-7.26 (m, 1H), 7.06-7.00
(m, 1H), 4.02-3.91 (m, 1H), 3.88-3.78 (m, 1H), 3.71 (s, 4H), 2.83-2.72 (m, 1H), 2.70-2.58 (m, 1H),
2.49 (s, 3H), 2.44-2.36 (m, 4H), 2.30 (s, 3H), 2.21 (s, 3H), 2.08-1.98 (m, 1H), 1.90-1.78 (m, 2H),
1.66-1.54 (m, 1H), 1.41 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 173.7, 170.2, 162.5, 158.2,
147.4, 136.7, 134.3, 121.5, 95.7, 62.1, 60.9, 54.8, 46.3, 43.9, 33.8, 29.6, 22.0, 21.6, 20.6, 14.2. HRMS
(ESI): calcd for C22H33N6S [M+H]+ 413.2487, found 413.2488. Compound 3 was obtained as a yellow
oil (10 mg, 7 %). Rf = 0.57 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 6.30 min. [α]25= -32 (c = 0.1,
D
CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 3.6 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.10-6.98 (m,
1H), 6.85 (s, 1H), 4.18-4.03 (m, 2H), 3.74-3.63 (m, 4H), 2.86-2.74 (m, 1H), 2.72-2.61 (m, 1H), 2.50 (s,
3H), 2.48-2.43 (m, 4H), 2.33 (s, 3H), 2.15 (s, 3H), 2.02-1.93 (m, 3H), 1.68-1.60 (m, 1H), 1.42 (d, J =
6.4 Hz, 3H). MS (ESI/APCI) m/z 412.8 [M+ H]+.
(S)-N-Methyl-N-((R)-1-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)ethyl)-5,6,7,8-tet
rahydroquinolin-8-amine
(4)
and
(R)-N-methyl-N-((R)-1-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)ethyl)-5,6,7,8-te
trahydroquinolin-8-amine (5): A mixture of (±)-8-chloro-5,6,7,8-tetrahydroquinoline (117 mg, 0.7
mmol), 13b (100 mg, 0.4 mmol), KI (20 mg, 0.12 mmol) and DIPEA (452 mg, 3.5 mmol) in 4 mL of
NMP was stirred at 120 °C overnight. The reaction was diluted with EtOAc (50 mL) and washed with
brine (10 mL x 5). The organic layer was dried over Na2SO4, filtered and evaporated. The residue was
purified by silica gel column chromatography (dichloromethane/methanol/NH4OH = 100/1/1) to give
the desired products 4and 5. Compound 4 was obtained as a yellow oil (10 mg, 7 %). Rf = 0.57
(CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 6.24 min. [α]25 = +6 (c = 0.1, CHCl3). H NMR (400
1
D
MHz, CDCl3) δ 8.45 (d, J = 4.0 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.09-7.00 (m, 1H), 6.83 (s, 1H),
4.16-4.02 (m, 2H), 3.73-3.65 (m, 4H), 2.84-2.75 (m, 1H), 2.72-2.63 (m, 1H), 2.50 (s, 3H), 2.47-2.43 (m,
4H), 2.33 (s, 3H), 2.13 (s, 3H), 2.05-1.90 (m, 3H), 1.72-1.60 (m, 1H), 1.42 (d, J = 6.8 Hz, 3H). MS
(ESI/APCI) m/z 412.8 [M+ H]+. Compound 5 was obtained as a yellow oil (20 mg, 14 %). Rf = 0.70
(CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 6.13 min. [α]25 = -4 (c = 0.1, CHCl3). H NMR (400
1
D
MHz, CDCl3) δ 8.52 (d, J = 3.6 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.30 (s, 1H), 7.08-7.01 (m, 1H),
14
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ChemMedChem
4.02-3.92 (m, 1H), 3.90-3.80 (m, 1H), 3.72 (s, 4H), 2.83-2.73 (m, 1H), 2.72-2.63 (m, 1H), 2.50 (s, 3H),
2.47-2.37 (m, 4H), 2.31 (s,3H), 2.22 (s, 3H), 2.09-1.75 (m, 4H), 1.42 (d, J = 6.8 Hz, 3H). MS
(ESI/APCI) m/z 412.8 [M+ H]+.
General procedures for the synthesis of 15a-h: To
a solution of 14a-h (1.0 eq.),
2,2-dimethyl-1,3-dioxane-4,6-dione (1.0 eq.), DMAP (2.0 eq.) in dichloromethane was added DCC (1.0
eq.) at 0 °C. The mixture was stirred at room temperature for 48 h. The reaction solution was filtered
and the filtrate was washed with 1 N HCl (100 mL). The organic layer was dried over Na2SO4, filtered
and evaporated. The residue was diluted with EtOH (100 mL) and stirred at reflux for 2 h. The
resulting solution was concentrated and purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 4:1) to give the desired products.
tert-Butyl (S)-2-(3-ethoxy-3-oxopropanoyl)pyrrolidine-1-carboxylate (15a): Compound 15a was
25
1
obtained as a yellow oil (4.0 g, 61 %). [α]D= -85 (c = 0.2, CHCl3). H NMR (400 MHz, CDCl3) δ
4.41-4.23 (m, 1H), 4.23-4.13 (m, 2H), 3.59-3.39 (m, 4H), 2.25-2.09 (m, 2H), 1.91-1.83 (m, 2H),
1.48-1.41 (m, 9H), 1.31-1.23 (m, 3H).
tert-Butyl (R)-2-(3-ethoxy-3-oxopropanoyl)pyrrolidine-1-carboxylate (15b): Compound 15b was
obtained as a yellow oil (4.4 g, 67 %). [α]2D5 = +87 (c = 0.2, CHCl3). H NMR (400 MHz, CDCl3) δ
1
4.43-4.23 (m, 1H), 4.23-4.13 (m, 2H), 3.68-3.32 (m, 4H), 2.28-1.80 (m, 4H), 1.50-1.38 (m, 9H), 1.27 (t,
J = 7.2 Hz, 3H).
tert-Butyl
(R)-2-(3-ethoxy-3-oxopropanoyl)-4,4-difluoropyrrolidine-1-carboxylate
(15c):
Compound 15c was obtained as a colorless oil (600 mg, 64 %). [α]2D5 = +56 (c = 0.2, CHCl3). 1H NMR
(400 MHz, CDCl3) δ 4.67-4.49 (m, 1H), 4.25-4.15 (m, 2H), 3.97-3.64 (m, 4H), 2.78-2.43 (m, 2H),
1.48-1.44 (m, 9H), 1.30-1.27 (m, 3H).
tert-Butyl
(2R,4R)-4-(tert-Butyldimethylsilyloxy)-2-(3-ethoxy-3-oxopropanoyl)pyrrolidine-1-carboxylate
25
1
(15d): Compound 15d was obtained as a colorless oil (1.0 g, 52 %). [α]D = +3.5 (c = 0.2, MeOH). H
NMR (400 MHz, CDCl3) δ 4.42-4.08 (m, 4H), 3.87-3.22 (m, 4H), 2.37-2.22 (m, 1H), 2.17-2.01 (m,
1H), 1.47 (s, 9H), 1.28-1.22 (m, 3H), 0.86 (s, 9H), 0.06 (d, J = 4.8 Hz, 6H).
tert-Butyl
(2R,4R)-2-(3-ethoxy-3-oxopropanoyl)-4-methoxypyrrolidine-1-carboxylate
(15e):
Compound 15e was obtained as a colorless oil (1.4 g, 70 %). [α]2D5= -6 (c = 0.2, CHCl3). 1H NMR (400
MHz, CDCl3) δ 4.53-4.35 (m, 1H), 4.21-4.17 (m, 2H), 3.94-3.90 (m, 1H), 3.84-3.74 (m, 1H), 3.61-3.45
(m, 3H), 3.33-3.20 (m, 3H), 2.52-2.15 (m, 2H), 1.47-1.41 (m, 9H), 1.30-1.25 (m, 3H).
tert-Butyl (R)-2-(3-ethoxy-3-oxopropanoyl)azetidine-1-carboxylate (15f): Compound 15f was
obtained as a colorless oil (1.2 g, 67 %). [α]2D5 = +135 (c = 0.2, CHCl3). 1H NMR (400 MHz, CDCl3) δ
4.71-4.64 (m, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.95-3.88 (m, 1H), 3.87-3.80 (m, 1H), 3.78-3.70 (m, 1H),
3.61-3.50 (m, 1H), 2.53-2.42 (m, 1H), 2.34-2.24 (m, 1H), 1.44 (s, 9H), 1.29-1.26 (m, 3H).
tert-Butyl (S)-2-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (15g): Compound 15g was
obtained as a yellow oil (1.8 g, 69 %). [α]2D5 = -114 (c = 0.2, CHCl3). H NMR (400 MHz, CDCl3) δ
1
4.88-4.60 (m, 1H), 4.25-4.10 (m, 2H), 4.05-3.80 (m, 1H), 3.53-3.35 (m, 2H), 2.92-2.68 (m, 1H),
2.20-2.08 (m, 1H), 1.66-1.59 (m, 3H), 1.46 (s, 9H), 1.41-1.36 (m, 2H), 1.28-1.23 (m, 3H).
tert-Butyl (R)-2-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (15h): Compound 15h was
obtained as a colorless oil (1.6 g, 61 %). [α]2D5 = +112 (c = 0.2, CHCl3). 1H NMR (400 MHz, CDCl3) δ
4.85-4.62 (m, 1H), 4.25-4.12 (m, 2H), 4.10-3.82 (m, 1H), 3.55-3.40 (m, 2H), 2.95-2.70 (m, 1H),
2.20-2.10 (m, 1H), 1.68-1.55 (m, 3H), 1.46 (s, 9H), 1.40-1.35 (m, 2H), 1.30-1.22 (m, 3H).
15
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ChemMedChem
General procedures for the synthesis of 16a and 16b: To a mixture of 15a or 15b (1.0 eq.) and
2-methyl-2-thiopseudourea sulfate (2.0 eq.) in water (25 mL) was added K2CO3 (4.5 eq.). The reaction
was stirred at room temperature overnight. The reaction mixture was quenched with 1 N HCl to adjust
pH = 4 and extracted with dichloromethane (50 mL x 3). The combined organic layers were dried over
Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol = 20/1) to give the desired products.
tert-Butyl
(S)-2-(6-hydroxy-2-(methylthio)pyrimidin-4-yl)pyrrolidine-1-carboxylate
(16a):
Compound 16a was obtained as a colorless oil (200 mg, 35 %). [α]2D5 = -95 (c = 0.2, CHCl3). 1H NMR
(400 MHz, CDCl3) δ 6.09-6.05 (m, 1H), 4.75-4.50 (m, 1H), 3.59-3.39 (m, 2H), 2.55 (s, 3H), 2.28-2.14
(m, 1H), 2.00-1.80 (m, 3H), 1.51-1.32 (m, 9H).
tert-Butyl
(R)-2-(6-hydroxy-2-(methylthio)pyrimidin-4-yl)pyrrolidine-1-carboxylate
(16b):
Compound 16b was obtained as a colorless oil (1 g, 91 %). [α]2D5 = +106 (c = 0.2, CHCl3). H NMR
(400 MHz, CDCl3) δ 6.06 (s, 1H), 4.75-4.48 (m, 1H), 3.59-3.46 (m, 2H), 2.54 (s, 3H), 2.29-2.18 (m,
1H), 2.00-1.80 (m, 3H), 1.47-1.30 (m, 9H).
1
General procedures for the synthesis of 17a and 17b: To a solution of 16a or 16b (1.0 eq.), Et3N (8.0
eq.) and N-methylpiperazine (1.2 eq.) in CH3CN (5 mL) was added PyBOP (1.2 eq.). The reaction
mixture was stirred at reflux overnight and then concentrated. The resulting residue was diluted with
dichloromethane, and washed with saturated NaHCO3 aqueous solution. The organic layer was dried
over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol = 100/1) to give a brown oil. Then it was diluted with ethyl acetate (3 mL),
and 3 M HCl/ethyl acetate (6.0 eq.) was added. The reaction mixture was stirred at room temperature
overnight before concentrated. The residue was diluted with dichloromethane (50 mL), and washed
with saturated NaHCO3 aqueous solution. The organic layer was dried over Na2SO4, filtered and
evaporated to give the desired products.
(S)-4-(4-Methylpiperazin-1-yl)-2-(methylthio)-6-(pyrrolidin-2-yl)pyrimidine (17a): Compound 17a
was obtained as a colorless oil (170 mg, 73 %). [α]2D5= -40 (c = 0.2, CHCl3). 1H NMR (400 MHz, CDCl3)
δ 6.29 (s, 1H), 3.98 (t, J = 7.2 Hz, 1H), 3.74-3.58 (m, 4H), 3.19-3.09 (m, 1H), 3.02-2.92 (m, 1H), 2.49
(s, 3H), 2.47-2.42 (m, 4H), 2.33 (s, 3H), 2.23-2.10 (m, 1H), 1.80-1.67 (m, 3H).
(R)-4-(4-Methylpiperazin-1-yl)-2-(methylthio)-6-(pyrrolidin-2-yl)pyrimidine (17b): Compound
25
1
17b was obtained as a yellow oil (570 mg, 52%). [α]D= +38 (c = 0.2, CHCl3). H NMR (400 MHz,
CDCl3) δ 6.29 (s, 1H), 4.00 (t, J = 7.2 Hz, 1H), 3.66 (s, 4H), 3.21-3.10 (m, 1H), 3.05-2.94 (m, 1H),
2.49 (s, 3H), 2.47-2.42 (m, 4H), 2.33 (s, 3H), 2.17-2.13 (m, 1H), 1.86-1.68 (m, 3H).
(S)-8-((S)-2-(6-(4-Methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)pyrrolidin-1-yl)-5,6,7,8-tetr
ahydroquinoline
(18)
and
(R)-8-((S)-2-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)pyrrolidin-1-yl)-5,6,7,8-tetr
ahydroquinoline (19): To a solution of (±)-8-chloro-5,6,7,8-tetrahydroquinoline (88 mg, 0.53 mmol)
and 17a (170 mg, 0.6 mmol) in EtOH (2 mL) was added Et3N (530 mg, 5.3 mmol). The reaction
mixture was stirred at reflux overnight, and then quenched with saturated NaHCO3 aqueous solution
(50 mL). The aqueous layer was extracted with dichloromethane (100 mL). The organic layer was
dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/NH4OH = 100/1/1 to 50/1/1) to give the desired products. Compound 18
16
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10.1002/cmdc.202000268
ChemMedChem
was obtained as a brown oil (20 mg, 9%). Rf = 0.27 (CH2Cl2:MeOH:NH4OH = 50:2:1). [α]25= -64 (c =
D
1
0.1, CHCl3). H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 4.4 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H),
7.04-6.97 (m, 1H), 6.57 (s, 1H), 4.57 (s, 1H), 4.01 (s, 1H), 3.67 (s, 4H), 2.98-2.90 (m, 1H), 2.85-2.74
(m, 1H), 2.69-2.58 (m, 2H), 2.51-2.45 (m, 7H), 2.38-2.23 (m, 5H), 2.00-1.91 (m, 3H), 1.77-1.68 (m,
3H). MS (ESI/APCI) m/z 424.8 [M+ H]+. Compound 19 was obtained as a brown oil (30 mg, 13 %). Rf
25
1
= 0.19 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 6.49 min. [α]D= -26 (c = 0.1, CHCl3). H NMR
(400 MHz, CDCl3) δ 8.39 (d, J = 4.4 Hz, 1H), 7.31-7.27 (m, 1H), 7.06-6.96 (m, 2H), 3.98 (s, 2H), 3.68
(s, 4H), 2.96 (s, 1H), 2.83-2.73 (m, 2H), 2.72-2.63 (m, 1H), 2.49 (s, 3H), 2.47-2.42 (m, 4H), 2.36-2.23
(m, 5H), 2.11-2.01 (m, 1H), 2.01-1.90 (m, 2H), 1.82-1.74 (m, 3H). 13C NMR (150 MHz, CDCl3) δ
170.1, 162.2, 146.9, 136.7, 133.6, 121.9, 95.6, 64.8, 59.4, 54.8, 48.8, 46.2, 43.8, 33.6, 29.0, 23.7, 23.6,
20.4, 14.2. HRMS (ESI): calcd for C23H33N6S [M+H]+ 425.2487, found 425.2468.
(S)-8-((R)-2-(6-(4-Methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)pyrrolidin-1-yl)-5,6,7,8-tet
rahydroquinoline
(20)
and
(R)-8-((R)-2-(6-(4-methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)pyrrolidin-1-yl)-5,6,7,8-tet
rahydroquinoline (21): To a solution of (±)-8-chloro-5,6,7,8-tetrahydroquinoline (100 mg, 0.6 mmol)
and 17b (210 mg, 0.7 mmol) in EtOH (5 mL) was added Et3N (610 mg, 6.0 mmol). The reaction
mixture was stirred at reflux overnight, and then quenched with saturated NaHCO3 aqueous solution
(50 mL). The aqueous layer was extracted with dichloromethane (100 mL). The organic layer was
dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/NH4OH = 100/1/1 to 50/1/1) to give the desired products. Compound 20
was obtained as a brown oil (34 mg, 13%). Rf = 0.19 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR =
25
1
6.49 min. [α]D= +32 (c = 0.1, CHCl3). H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 4.4 Hz, 1H),
7.30-7.26 (m, 1H), 7.07-6.95 (m, 2H), 4.05-3.93 (m, 2H), 3.74-3.61 (m, 4H), 3.01-2.90 (m, 1H),
2.82-2.72 (m, 2H), 2.72-2.64 (m, 1H), 2.48 (s, 3H), 2.47-2.40 (m, 4H), 2.38 -2.17 (m, 5H), 2.12-2.01
(m, 1H), 2.00-1.90 (m, 2H), 1.83-1.69 (m, 3H). HRMS (ESI): calcd for C23H33N6S [M+H]+ 425.2487,
found 425.2488. Compound 21 was obtained as a brown oil (50 mg, 20%). Rf = 0.27
(CH2Cl2:MeOH:NH4OH = 50:2:1). [α]2D5= +62 (c = 0.1, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.39 (d,
J = 4.8 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.10-7.00 (m, 1H), 6.80 (s, 1H), 4.86 (s, 1H), 4.16 (s, 1H),
3.73 (s, 4H), 2.87-2.60 (m, 4H), 2.54-2.48 (m, 7H), 2.38-2.30 (m, 5H), 2.06-1.96 (m, 3H), 1.90-1.77 (m,
3H). MS (ESI/APCI) m/z 424.8 [M+ H]+.
(S)-8-((R)-2-(6-(4-Methylpiperazin-1-yl)-2-(methylthio)pyrimidin-4-yl)pyrrolidin-1-yl)-5,6,7,8-tet
rahydroquinoline (20): To a solution of 17b (66 mg, 0.22 mmol) and 6,7-dihydroquinolin-8(5H)-one
(30 mg, 0.2 mmol) in MeOH (3 mL), was added AcOH (20 mg, 0.33 mmol). The mixture was stirred at
room temperature for 10 min, before NaBH3CN (26 mg, 0.4 mmol) was added. The resulting
suspension was stirred at room temperature for 2 h. The mixture was quenched with saturated NaHCO3
aqueous solution (100 mL) and extracted with dichloromethane (100 mL). The organic layer was dried
over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/NH4OH = 100/1/1) to give the desired product (20 mg, 23%) as a colorless
oil. The characterization matched well with compound 20 obtained by direct alkylation method shown
above.
General procedure for the synthesis of 22a-g: To a solution of 15a-h (1 g, 3.5 mmol) and
17
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000268
ChemMedChem
acetamidine hydrochloride (990 mg, 10.5 mmol) in EtOH (20 mL) was added EtONa (480 mg, 7.0
mmol). The reaction was stirred at reflux overnight. The reaction mixture was quenched with 1 N HCl
to adjust pH =4 and extracted with dichloromethane (30 mL x 3). The combined organic layers were
dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column
chromatography (dichloromethane/methanol = 20/1) to give the desired product.
tert-Butyl (R)-2-(6-hydroxy-2-methylpyrimidin-4-yl)pyrrolidine-1-carboxylate (22a): Compound
22a was obtained as a yellow oil (930 mg, 95 %). [α]2D5 = +55 (c = 0.2, CHCl3). H NMR (400 MHz,
1
CDCl3) δ 13.34 (s, 1H), 6.08 (s, 1H), 4.68-4.44 (m, 1H), 3.55-3.30 (m, 2H), 2.38-2.34 (m, 3H),
2.24-2.08 (m, 1H), 1.93-1.83 (m, 1H), 1.84-1.67 (m, 2H), 1.40-1.23 (m, 9H).
tert-Butyl
(R)-2-(2,2-Dimethyl-4,6-dioxo-1,3-dioxane-5-carbonyl)-4,4-difluoropyrrolidine-1-carboxylate
25
1
(22b): Compound 22b was obtained as a yellow oil (320 mg, 63 %). [α]D= +4 (c = 0.2, CHCl3). H
NMR (400 MHz, CDCl3) δ 13.09 (s, 1H), 6.23 (s, 1H), 4.98-4.73 (m, 1H), 4.07-3.87 (m, 1H),
3.86-3.73(m, 1H), 2.87-2.69 (m, 1H), 2.61-2.48 (m, 1H), 2.46 (s, 3H), 1.49-1.34 (m, 9H).
tert-Butyl
(2R,4R)-4-(tert-Butyldimethylsilyloxy)-2-(6-hydroxy-2-methylpyrimidin-4-yl)pyrrolidine-1-carbo
1
xylate (22c): Compound 22c was obtained as a pale yellow oil (580 mg, 62%). H NMR (400 MHz,
CDCl3) δ6.29 (d, J = 4.0 Hz, 1H), 4.83-4.57 (m, 1H), 4.40-4.29 (m, 1H), 3.78-3.63 (m, 1H), 3.46-3.29
(m, 1H), 2.46-2.40 (m, 3H), 2.39-2.30 (m, 1H), 2.21-2.14 (m, 1H), 1.49-1.33 (m, 9H), 0.72 (s, 9H),
-0.01 (s, 3H), -0.09 (s, 3H).
tert-Butyl
(2R,4R)-2-(6-hydroxy-2-methylpyrimidin-4-yl)-4-methoxypyrrolidine-1-carboxylate
(22d): Compound 22d was obtained as a yellow solid (570 mg, 45 %). [α]2D5= -4 (c = 0.2, CHCl3).1H
NMR (400 MHz, CDCl3) δ 12.82 (s, 1H), 6.20 (s, 1H), 4.79-4.58 (m, 1H), 3.96 (s, 1H), 3.79-3.55 (m,
2H), 3.33 (s, 3H), 2.45 (s, 3H), 2.43-2.32 (m, 1H), 2.15-2.00 (m, 1H), 1.51-1.28 (m, 9H).
tert-Butyl (R)-2-(6-hydroxy-2-methylpyrimidin-4-yl)azetidine-1-carboxylate (22e): Compound 22e
was obtained as a yellow oil (800 mg, 69 %). [α]2D5= +190 (c = 0.2, CHCl3).1H NMR (400 MHz, CDCl3)
δ 6.44 (s, 1H), 5.10-4.92 (m, 1H), 4.05-3.82 (m, 2H), 2.76-2.61 (m, 1H), 2.52 (s, 3H), 2.23-2.05 (m,
1H), 1.41 (s, 9H).
tert-Butyl (S)-2-(6-hydroxy-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (22f): Compound 22f
was obtained as a yellow oil (800 mg, crude). [α]2D5= -7 (c = 0.2, CHCl3). 1H NMR (400 MHz, CDCl3) δ
6.19 (s, 1H), 5.12 (s, 1H), 4.13-3.99 (m, 1H), 2.94-2.84 (m, 1H), 2.49 (s, 3H), 1.83-1.70 (m, 1H),
1.68-1.54 (m, 4H), 1.46-1.41 (m, 10H).
tert-Butyl (R)-2-(6-hydroxy-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (22g): Compound
22g was obtained as a colorless oil (1.0 g, 100 %). [α]2D5 = +11 (c = 0.2, CHCl3). H NMR (400 MHz,
1
CDCl3) δ 12.94 (s, 1H), 6.19 (s, 1H), 5.09 (s, 1H), 4.06 (s, 1H), 2.89 (s, 1H), 2.47 (s, 3H), 1.75 (s, 1H),
1.58 (s, 4H), 1.45 (s, 9H), 1.16 (s, 1H).
General procedure for the synthesis of 23a-g: To a solution of 22a-g (1.0 eq.), Et3N (10.0 eq.) and
N-methylpiperazine (1.5 eq.) in CH3CN was added PyBOP (1.1 eq.). The reaction mixture was stirred
at reflux overnight before concentrated. The resulting residue was diluted with dichloromethane and
washed with saturated NaHCO3 aqueous solution. The organic layer was dried over Na2SO4, filtered
and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/NH4OH = 100/1/1) to give the crude intermediate as a yellow oil. Then it
was diluted with ethyl acetate and 3 M HCl/ethyl acetate (4.0 eq.) was added. The reaction mixture was
18
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10.1002/cmdc.202000268
ChemMedChem
stirred at room temperature overnight before concentrated. The resulting residue was diluted with
dichloromethane and washed with saturated NaHCO3 aqueous solution. The organic layer was dried
over Na2SO4, filtered and evaporated to give the desired product.
(R)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-(pyrrolidin-2-yl)pyrimidine (23a): Compound 23a was
obtained as a colorless oil (660 mg, 77%). [α]2D5= +27 (c = 0.2, CHCl3). 1H NMR (400 MHz, CDCl3) δ
6.47 (s, 1H), 4.03 (t, J = 7.6 Hz, 1H), 3.66 (s, 4H), 3.20-3.10 (m, 1H), 3.07-2.96 (m, 1H), 2.50-2.43 (m,
7H), 2.33 (s, 3H), 2.29-2.16 (m, 2H), 1.83-1.79 (m, 1H), 1.74-1.64 (m, 1H).
(R)-4-(4,4-Difluoropyrrolidin-2-yl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine
(23b):
Compound 23b was obtained as a yellow oil (160 mg, 54 %). [α]2D5= +6 (c = 0.2, CHCl3). 1H NMR (400
MHz, CDCl3) δ 6.45 (s, 1H), 4.24 (t, J = 8.0 Hz, 1H), 3.67 (s, 4H), 3.48-3.35 (m, 1H), 3.33-3.20 (m,
1H), 2.69-2.55 (m, 1H), 2.50-2.43 (m, 7H), 2.37-2.25 (m, 4H).
4-((2R,4R)-4-(tert-butyldimethylsilyloxy)pyrrolidin-2-yl)-2-methyl-6-(4-methylpiperazin-1-yl)pyri
midine (23c): Compound 23c was obtained as a colorless oil (300 mg, 55%). [α]2D5 = +0.9 (c = 0.2,
1
MeOH). H NMR (400 MHz, CDCl3) δ 6.51 (s, 1H), 4.41 (s, 1H), 4.17-4.02 (m, 1H), 3.66 (s, 4H),
3.15-3.05 (m ,1H), 3.04-2.94 (m, 1H), 2.57-2.37 (m, 9H), 2.33 (s, 3H), 0.82 (s, 9H), -0.03 (s, 6H).
4-((2R,4R)-4-Methoxypyrrolidin-2-yl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine
(23d):
25
1
Compound 23d was obtained as a white solid (400 mg, 76 %). [α]D= -5 (c = 0.2, CHCl3). H NMR
(400 MHz, CDCl3) δ 6.49 (s, 1H), 4.21 (t, J = 7.2 Hz, 1H), 3.96 (s, 1H), 3.66 (s, 4H), 3.31 (s, 3H),
3.23-3.10 (m, 1H), 3.09-2.99 (m, 1H), 2.50-2.42 (m, 7H), 2.35-2.27 (m, 4H), 1.84-1.76 (m, 1H).
(R)-4-(Azetidin-2-yl)-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidine (23e): Compound 23e was
25
1
obtained as a yellow oil (210 mg, 28 %). [α]D = +66 (c = 0.2, CHCl3). H NMR (400 MHz, CDCl3) δ
6.61 (s, 1H), 4.80 (t, J = 8.0 Hz, 1H), 3.78 (q, J = 8.0 Hz, 1H), 3.72-3.66 (m, 4H), 3.41-3.34 (m, 1H),
2.73-2.62 (m, 1H), 2.50-2.42 (m, 7H), 2.35-2.30 (m, 4H).
(S)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-(piperidin-2-yl)pyrimidine (23f): Compound 23f was
25
1
obtained as a yellow solid (380 mg, 51%). [α]D= -8 (c = 0.2, CHCl3). H NMR (400 MHz, CDCl3) δ
6.41 (s, 1H), 4.78 (s, 2H), 3.65 (s, 4H), 3.50 (d, J = 10.8 Hz, 1H), 3.17 (d, J = 11.6 Hz, 1H), 2.83-2.69
(m, 1H), 2.48-2.40 (m, 7H), 2.32 (s, 3H), 1.87-1.81 (m, 1H), 1.68-1.59 (m, 1H), 1.54-1.44 (m, 2H).
(R)-2-Methyl-4-(4-methylpiperazin-1-yl)-6-(piperidin-2-yl)pyrimidine (23g): Compound 23g was
25
1
obtained as a colorless oil (320 mg, 89%). [α]D= +5 (c = 0.2, CHCl3). H NMR (400 MHz, CDCl3) δ
6.41 (s, 1H), 3.74-3.60 (m, 4H), 3.55-3.46 (m, 1H), 3.18 (d, J = 11.6 Hz, 1H), 2.85-2.72 (m, 1H), 2.48
(s, 3H), 2.47-2.41 (m, 4H), 2.32 (s, 3H), 1.96 (d, J = 12.8 Hz, 1H), 1.88 (s, 2H), 1.68-1.62 (m, 1H),
1.54-1.47 (m, 1H), 1.45-1.36 (m, 1H).
(S)-8-((R)-2-(2-Methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)pyrrolidin-1-yl)-5,6,7,8-tetrahyd
roquinoline (24): A mixture of 23a (200 mg, 0.8 mmol), 6,7-dihydroquinolin-8(5H)-one (135 mg, 0.9
mmol) in methanol (5 mL) was stirred at room temperature for 10 min, before NaBH3CN (97 mg, 1.5
mmol) was added. The resulting suspension was stirred at room temperature overnight. The mixture
was quenched with saturated NaHCO3 aqueous solution (100 mL) and extracted with dichloromethane
(100 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified
by silica gel column chromatography (dichloromethane/methanol/NH4OH = 200/1/1) to give the
desired product as a white solid (180 mg, 60 %). Rf = 0.21 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR
25
1
= 5.83 min. [α]D= +10 (c = 0.1, CHCl3). H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 4.4 Hz, 1H),
7.30-7.26 (m, 1H), 7.11 (s, 1H), 7.02-6.92 (m, 1H), 4.06-3.92 (m, 2H), 3.67 (s, 4H), 3.03-2.89 (m, 1H),
2.81-2.62 (m, 3H), 2.45 (s, 7H), 2.32 (s, 3H), 2.30-2.23 (m, 1H), 2.10-2.01 (m, 3H), 2.00-1.87 (m, 3H),
19
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10.1002/cmdc.202000268
ChemMedChem
1.71-1.60 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 172.7, 166.4, 163.0, 157.8, 146.9, 136.5, 133.5,
121.7, 96.7 64.8, 59.2, 55.0, 48.8, 46.3, 43.8, 33.9, 29.1, 26.1, 23.8, 23.7, 20.4. HRMS (ESI): calcd for
C23H33N6 [M+H]+ 393.2767, found 393.2761.
(S)-8-((R)-4,4-Difluoro-2-(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)pyrrolidin-1-yl)-5,6
,7,8-tetrahydroquinoline (25): A mixture of 23b (50 mg, 0.2 mmol), 6,7-dihydroquinolin-8(5H)-one
(30 mg, 0.2 mmol) in methanol (3 mL) was stirred at room temperature for 10 min, before NaBH3CN
(22 mg, 0.3 mmol) was added. The resulting suspension was stirred at room temperature overnight. The
mixture was quenched with saturated NaHCO3 aqueous solution (100 mL) and extracted with
dichloromethane (100 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The
residue was purified by silica gel column chromatography (dichloromethane/methanol/NH4OH =
100/1/1 to 100/3/1) to give the desired product as a colorless oil (50 mg, 58 %). Rf = 0.14
(CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 6.47 min. [α]25 = -6 (c = 0.1, CHCl3). H NMR (400
1
D
MHz, CDCl3) δ 8.40 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.07-6.99 (m, 1H), 4.26 (t, J = 8.4
Hz, 1H), 4.05-3.95 (m, 1H), 3.70 (s, 4H), 3.41-3.23 (m, 1H), 3.23-3.06 (m, 1H), 2.83-2.62 (m, 4H),
2.49-2.43 (m, 7H), 2.33 (s, 3H), 2.30-2.18 (m, 1H), 1.91-1.78 (m, 2H), 1.75-1.58 (m, 1H). 13C NMR
(150 MHz, CDCl3) δ 169.3, 166.9, 163.0, 156.3, 147.2, 136.8, 133.7, 128.1 (t, J = 246 Hz), 122.1, 96.8,
63.2, 58.5, 55.8 (t, J = 28 Hz), 54.9, 46.3, 43.8, 43.0, 29.0 (t, J = 25 Hz), 26.1, 23.2, 20.5. HRMS (ESI):
calcd for C23H31F2N6 [M+H]+ 429.2578, found 429.2556.
(3R,5R)-5-(2-Methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)-1-((S)-5,6,7,8-tetrahydroquinolin
-8-yl)pyrrolidin-3-ol (26): A mixture of 23c (80 mg, 0.2 mmol), 6,7-dihydroquinolin-8(5H)-one (36
mg, 0.24 mmol) in methanol (3 mL) was stirred at room temperature for 10 min, before NaBH3CN
(26 mg, 0.4 mmol) was added. The resulting suspension was stirred at room temperature overnight. The
mixture was quenched with saturated NaHCO3 aqueous solution (100 mL) and extracted with
dichloromethane (100 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The
residue was purified by silica gel column chromatography (dichloromethane/methanol/NH4OH =
200/1/1 to 100/1/1) to give a colorless oil (60 mg). It was then dissolved in 2 N HCl (5 mL) and stirred
at room temperature for 2 h. The aqueous layer was alkalized with NaOH and extracted with
dichloromethane (100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to give a
white solid (35 mg, 43%). [α]2D5 = +1.1 (c = 0.2, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 4.8
Hz, 1H), 7.11 (d, J = 7.2 Hz, 1H), 7.04-6.98 (m, 1H), 5.29 (d, J = 6.8 Hz, 1H), 4.28 (s, 1H), 4.03 (d, J =
10.0 Hz, 1H), 3.89 (s, 1H), 3.55-3.38 (m, 4H), 3.34-3.26 (m, 1H), 2.92-2.84 (m, 1H), 2.63-2.53 (m, 1H),
2.52-2.46 (m, 1H), 2.45-2.38 (m, 7H), 2.33 (s, 3H), 2.15-2.03 (m, 2H), 1.82-1.72 (m, 1H), 1.64-1.51 (m,
3H). 13C NMR (150 MHz, CDCl3) δ 170.2, 166.7, 162.0, 158.8, 145.4, 136.4, 134.9, 122.6, 96.4, 71.0,
63.0, 62.2, 61.7, 54.8, 46.2, 43.6, 40.7, 26.9, 26.8, 25.8, 17.0. HRMS (ESI): calcd for C23H32N6O
[M+H]+ 409.2710, found 409.2714.
(S)-8-((2R,4R)-4-Methoxy-2-(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)pyrrolidin-1-yl)-
5,6,7,8-tetrahydroquinoline
(R)-8-((2R,4R)-4-methoxy-2-(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)pyrrolidin-1-yl)-
5,6,7,8-tetrahydroquinoline (28): mixture of 23d (83 mg, 0.3 mmol),
(27)
and
A
6,7-dihydroquinolin-8(5H)-one (50 mg, 0.3 mmol) in methanol (3 mL) was stirred at room temperature
for 10 min, before NaBH3CN (36 mg, 0.6 mmol) was added. The resulting suspension was stirred at
20
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10.1002/cmdc.202000268
ChemMedChem
room temperature overnight. The mixture was quenched with saturated NaHCO3 aqueous solution (100
mL) and extracted with dichloromethane (100 mL). The organic layer was dried over Na2SO4, filtered
and evaporated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/NH4OH = 100/1/1 to 100/3/1) to give the desired products. Compound 27
was obtained as a white solid (10 mg, 8%). HPLC tR = 5.67 min. [α]25 = +18 (c = 0.1, CHCl3). 1H NMR
D
(400 MHz, CDCl3) δ 8.35 (d, J = 4.8 Hz, 1H), 7.30-7.26 (m, 1H), 7.17 (s, 1H), 7.03-6.97 (m, 1H),
4.07-3.96 (m, 1H), 3.95-3.87 (m, 2H), 3.73-3.65 (m, 4H), 3.21 (s, 3H), 3.08-3.01 (m, 1H), 2.93-2.84 (m,
1H), 2.82-2.56 (m, 4H), 2.46-2.43 (m, 8H), 2.32 (s, 3H), 2.12-2.02 (m, 1H), 1.87-1.78 (m, 1H),
1.70-1.61 (m, 1H). HRMS (ESI): calcd for C24H35N6O [M+H]+ 423.2872, found 423.2879. Compound
28 was obtained as a white solid (25 mg, 20%). Rf = 0.30 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR
= 5.92 min. [α]2D5= -10 (c = 0.1, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 4.8 Hz, 1H), 7.30 (d,
J = 7.6 Hz, 1H), 7.22 (s, 1H), 7.06-6.99 (m, 1H), 4.18 (t, J = 7.6 Hz, 1H), 4.03-3.96 (m, 1H), 3.95-3.87
(m, 1H), 3.70 (s, 4H), 3.27 (s, 3H), 3.25-3.16 (m, 1H), 2.85-2.71 (m, 1H), 2.71-2.61 (m, 2H), 2.48 (s,
3H), 2.46-2.41 (m, 4H), 2.41-2.34 (m, 1H), 2.32 (s, 3H), 2.23-2.06 (m, 1H), 1.97-1.80 (m, 3H),
1.72-1.59 (m, 1H). MS (ESI/APCI) m/z 422.9 [M+ H]+.
(S)-8-((R)-2-(2-Methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)azetidin-1-yl)-5,6,7,8-tetrahydro
quinoline
(29)
and
(R)-8-((R)-2-(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)azetidin-1-yl)-5,6,7,8-tetrahydro
quinoline (30): A mixture of 23e (100 mg, 0.4 mmol), 6,7-dihydroquinolin-8(5H)-one (72 mg, 0.5
mmol) in methanol (5 mL) was stirred at room temperature for 10 min before NaBH3CN (51 mg, 0.5
mmol) was added. The resulting suspension was stirred at room temperature overnight. The mixture
was quenched with saturated NaHCO3 aqueous solution (100 mL) and extracted with dichloromethane
(100 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified
by silica gel column chromatography (dichloromethane/methanol/NH4OH = 100/1/1) to give the
desired products. Compound 29 was obtained as a colorless oil (15 mg, 10 %). Rf = 0.31
(CH2Cl2:MeOH:NH4OH = 50:2:1). [α]25 = +4 (c = 0.1, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.94 (d,
D
J = 4.8 Hz, 1H), 7.26-7.23 (m, 1H), 6.90-6.81 (m, 1H), 5.84 (s, 1H), 4.66 (t, J = 8.4 Hz, 1H), 3.58 (s,
1H), 3.48-3.42 (m, 1H), 3.41-3.28 (m, 4H), 3.14 (q, J = 8.0 Hz, 1H), 2.89-2.72 (m, 2H), 2.52-2.43 (m,
1H), 2.40 (s, 3H), 2.39-2.33 (m, 4H), 2.33-2.30 (m, 4H), 2.13-2.05 (m, 1H), 1.92-1.83 (m, 1H),
1.75-1.65 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 171.4, 166.1, 162.3, 155.6, 146.2, 136.6, 132.4,
122.3, 95.1, 66.9, 64.7, 54.8, 49.8, 46.3, 43.5, 28.5, 26.0, 25.5, 25.2, 17.7. HRMS (ESI): calcd for
C22H31N6 [M+H]+ 379.2610, found 379.2604. Compound 30 was obtained as a colorless oil (4 mg,
3 %). Rf = 0.34 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 5.65 min. [α]25 = -15 (c = 0.1, CHCl3).
D
1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 4.4 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.13-7.07 (m, 1H),
6.81 (s, 1H), 4.25 (t, J = 8.2 Hz, 1H), 3.69 (s, 4H), 3.66-3.58 (m, 2H), 3.27 (t, J = 8.0 Hz, 1H),
2.88-2.75 (m, 1H), 2.75 -2.62 (m, 1H), 2.50 (s, 3H), 2.49-2.43 (m, 4H), 2.42-2.36 (m, 1H), 2.34 (s, 3H),
2.04-1.89 (m, 2H), 1.78-1.71 (m, 1H), 1.66-1.56 (m, 1H), 1.53-1.42 (m, 1H). MS (ESI/APCI) m/z
378.9 [M+ H]+
(S)-8-((S)-2-(2-Methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)piperidin-1-yl)-5,6,7,8-tetrahydr
oquinoline
(31)
and
(R)-8-((S)-2-(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)piperidin-1-yl)-5,6,7,8-tetrahydr
oquinoline (32): To a solution of (±)-8-chloro-5,6,7,8-tetrahydroquinoline (184 mg, 1.1 mmol) and 23f
21
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10.1002/cmdc.202000268
ChemMedChem
(150 mg, 0.5 mmol) in EtOH (5 mL) was added Et3N (556 mg, 5.5 mmol). The reaction mixture was
stirred at reflux overnight, and then quenched with saturated NaHCO3 aqueous solution (100 mL). The
aqueous layer was extracted with dichloromethane (100 mL). The organic layer was dried over Na2SO4
and concentrated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/NH4OH = 200/1/1) to give the desired products. Compound 31 was
obtained as a colorless oil (10 mg, 5%). Rf = 0.20 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 5.90
min. [α]2D5= -4 (c = 0.1, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 4.8 Hz, 1H), 7.38 (d, J = 7.6
Hz, 1H), 7.22 (s, 1H), 7.14-7.08 (m, 1H), 4.08-4.00 (m, 1H), 3.88-3.61 (m, 5H), 2.76-2.70 (m, 1H),
2.68-2.62 (m, 1H), 2.57-2.53 (m, 1H), 2.52-2.47 (m, 7H), 2.35 (s, 3H), 2.08-1.92 (m, 4H), 1.84-1.75 (m,
2H), 1.70-1.62 (m, 2H), 1.62-1.50 (m, 2H), 1.33-1.26 (m, 1H). MS (ESI/APCI) m/z 406.9 [M+ H]+.
Compound 32 was obtained as a colorless oil (10 mg, 5%). Rf = 0.33 (CH2Cl2:MeOH:NH4OH =
25
1
50:2:1). HPLC tR = 5.98 min. [α]D= -4 (c = 0.1, CHCl3). H NMR (400 MHz, CDCl3) δ 8.51-8.40 (m,
1H), 7.25-7.21 (m, 1H), 7.05-6.90 (m, 1H), 6.58 (s, 1H), 4.54 (br s, 1H), 3.94 (br s, 1H), 3.67 (s, 4H),
2.78-2.61 (m, 2H), 2.60-2.52 (m, 1H), 2.51-2.45 (m, 7H), 2.34 (s, 3H), 2.15-2.00 (m, 2H), 1.83-1.70 (m,
3H), 1.63-1.44 (m, 4H), 1.40-1.30 (m, 2H). MS (ESI/APCI) m/z 406.9 [M+ H]+.
(S)-8-((R)-2-(2-Methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)piperidin-1-yl)-5,6,7,8-tetrahydr
oquinoline
(33)
and
(R)-8-((R)-2-(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)piperidin-1-yl)-5,6,7,8-tetrahydr
oquinoline (34): To a solution of (±)-8-chloro-5,6,7,8-tetrahydroquinoline (50 mg, 0.3 mmol) and 23g
(90 mg, 0.33 mmol) in EtOH (5 mL) was added Et3N (303 mg, 3.0 mmol). The reaction mixture was
stirred at reflux overnight, and then quenched with saturated NaHCO3 aqueous solution (100 mL). The
aqueous layer was extracted with dichloromethane (100 mL). The organic layer was dried over Na2SO4
and concentrated. The residue was purified by silica gel column chromatography
(dichloromethane/methanol/NH4OH = 250/1/1) to give the desired products. Compound 33 was
obtained as a colorless oil (80 mg, 65%). Rf = 0.33 (CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 5.88
25
1
min. [α]D= +4 (c = 0.1, CHCl3). H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 4.4 Hz, 1H), 7.22 (d, J =
7.6 Hz, 1H), 7.01-6.91 (m, 1H), 6.55 (s, 1H), 4.54 (d, J = 10.8 Hz, 1H), 3.91 (t, J = 8.4 Hz, 1H), 3.63 (s,
4H), 2.75-2.60 (m, 2H), 2.57-2.53 (m, 1H), 2.51 (s, 3H), 2.48-2.44 (m, 4H), 2.34 (s, 3H), 2.16-2.02 (m,
2H), 1.93-1.84 (m, 2H), 1.82-1.67 (m, 2H), 1.67-1.44 (m, 4H), 1.38-1.28 (m, 1H). MS (ESI/APCI) m/z
406.9 [M+ H]+. Compound 34 was obtained as a colorless oil (30 mg, 24%). Rf = 0.20
25
1
(CH2Cl2:MeOH:NH4OH = 50:2:1). HPLC tR = 5.39 min. [α]D= +6 (c = 0.1, CHCl3). H NMR (400
MHz, CDCl3) δ 8.53-8.47 (m, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.05-6.95 (m, 2H), 4.08-3.97 (m, 1H),
3.69 (s, 4H), 3.58 (d, J = 10.4 Hz, 1H), 2.77-2.65 (m, 1H), 2.64-2.56 (m, 1H), 2.49 (s, 3H), 2.43 (s, 4H),
2.38-2.34 (m, 1H), 2.30 (s, 3H), 1.97-1.85 (m, 4H), 1.83-1.56 (m, 4H), 1.55-1.45 (m, 2H), 1.40-1.28 (m,
1H). 13C NMR (150 MHz, CDCl3) δ 171.4, 166.9, 162.8, 157.6, 147.5, 136.7, 134.7, 121.2, 97.5, 66.1,
59.6, 54.8, 46.5, 46.3, 43.9, 35.2, 29.8, 26.2, 25.8, 25.0, 21.9, 20.6. HRMS (ESI): calcd for C24H35N6
[M+H]+ 407.2923, found 407.2918.
In vitro biological assays
The detailed procedures of in vitro biological assays can be found in the supporting information.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No. 81973161,
22
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10.1002/cmdc.202000268
ChemMedChem
81773561, 21502133), the Priority Academic Program Development of the Jiangsu Higher Education
Institutes (PAPD) and the Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003).
Conflict of interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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