Development of Stem-Cell-Mobilizing Agents Targeting CXCR4 Receptor for Peripheral Blood Stem Cell Transplantation and beyond

Article abstract of DOI:10.1021/acs.jmedchem.7b01322

The function of the CXCR4/CXCL12 axis accounts for many disease indications, including tissue/nerve regeneration, cancer metastasis, and inflammation. Blocking CXCR4 signaling with its antagonists may lead to moving out CXCR4⁺ cell types from b

Full text of DOI:10.1021/acs.jmedchem.7b01322

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Article
Development of Stem Cell Mobilizing Agents Targeting CXCR4
Receptor for Peripheral Blood Stem Cell Transplantation and Beyond
Chien-Huang Wu, Jen-Shin Song, Hsuan-Hao Kuan, Szu-Huei Wu, Ming-Chen Chou, Jiing-Jyh Jan,
Lun Kelvin Tsou, Yi-Yu Ke, Chiung-Tong Chen, Kai-Chia Yeh, Sing-Yi Wang, Teng-Kuang Yeh, Chen-
Tso Tseng, Chen-Lung Huang, Mine-Hsine Wu, Po-Chu Kuo, Chia-Jui Lee, and Kak-Shan Shia
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01322 • Publication Date (Web): 09 Jan 2018
Downloaded from http://pubs.acs.org on January 10, 2018
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Development of Stem Cell Mobilizing Agents Targeting
CXCR4 Receptor for Peripheral Blood Stem Cell
Transplantation and Beyond
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†
†
†
ChienꢀHuang Wu, JenꢀShin Song, HsuanꢀHao Kuan, SzuꢀHuei Wu, MingꢀChen Chou,
JiingꢀJyh Jan, Lun K. Tsou, YiꢀYu Ke, ChiungꢀTong Chen, KaiꢀChia Yeh, SingꢀYi Wang,
TengꢀKuang Yeh, ChenꢀTso Tseng, ChenꢀLung Huang, MineꢀHsine Wu, PoꢀChu Kuo,
ChiaꢀJui Lee and KakꢀShan Shia*
Institute of Biotechnology and Pharmaceutical Research, National Health Research
Institutes, Miaoli County 35053, Taiwan, R.O.C.
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ABSTRACT:
The function of the CXCR4/CXCL12 axis accounts for many disease indications,
including tissue/nerve regeneration, cancer metastasis and inflammation. Blocking
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CXCR4 signaling with its antagonists may lead to moving out CXCR4 cell types from
bone marrow to peripheral circulation. We have discovered a novel series of pyrimidineꢀ
based CXCR4 antagonists, a representative (i.e., 16) of which was tolerated at a higher
dose and showed better HSCꢀmobilizing ability at the maximal response dose relative to
the approved drug 1 (AMD3100), and thus considered a potential drug candidate for
PBSCT indication. Docking compound 16 into the Xꢀray crystal structure of CXCR4
receptor revealed that it adopted a spiderꢀlike conformation striding over both major and
minor subpockets. This putative binding mode provides a new insight into CXCR4
receptorꢀligand interactions for further structural modifications.
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INTRODUCTION
The G proteinꢀcoupled CXC chemokine receptor 4 (CXCR4) is activated by CXCL12,
also known as stromal cellꢀderived factorꢀ1 or SDFꢀ1, and is constitutively expressed in
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–5
both CNS and peripheral systems. The interaction between CXCL12 and CXCR4 can
regulate the trafficking of many different cell types, such as hematopoietic stem cells
6
7
8
(HSCs), endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs).
Multiple functions have been found along the CXCR4/CXCL12 axis, including the
promotion of angiogenesis, metastasis, tumor growth and cancer survival, neurogenesis,
9ꢀ11
immunmodulation and protective/reparative effects in the lesioned tissues. Moreover,
both chemokine receptors CXCR4 and CCR5 act as coꢀreceptors in mediating HIVꢀ1
entry. The firstꢀinꢀclass CCR5 antagonist, maraviroc, was approved in 2007 as an antiꢀ
1
2
HIV drug. Unfortunately, clinical observation of HIVꢀinfected patients after maraviroc
administration resulted in a X4/R5 mixedꢀtropic viral population with poor prognosis,
13
suggesting that blocking both coꢀreceptors are desirable. To date, smallꢀmolecule
CXCR4 antagonists 1 (AMD3100, Plerixafor, Chart 1) and AMD11070 are not being
further developed for HIVꢀ1 infection due to lack of overall clinical benefit and
14–16
hepatotoxicity observed in longꢀterm animal studies, respectively.
Another advanced
CXCR4 blocker KRHꢀ3955, displaying high potency and oral bioavailability in a HIVꢀ1
1
7
replication model, is in early clinical development.
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Chart 1. Selected Small-Molecule CXCR4 Antagonists with anti-HIV-1 Activity
N
N
NH
N
N
NHHN
N HN
N
N
NH N
NHHN
NH2
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(AMD3100)
AMD11070
KRH-3955
Thus, a significant unmet medical need remains for treatment of AIDS by blocking the
18,19
CXCR4ꢀutilizing HIV strains. In addition to serving as antiꢀHIV agents,
increasing
evidence has also strongly supported that CXCR4 specific antagonists could effectively
induce functional recovery in various ischemic disease animal models associated with
ischemic stroke, acute kidney injury and myocardial infarction through autologous
2
0–25
migration of stem cells out of bone marrow into peripheral circulation.
CXCR4 may
become a versatile drug target in numerous therapeutic areas, including antiꢀcancer
2
6,27
28–31
32
metastasis,
antiꢀinflammation,
as well as tissue repair or nerve regeneration. A
specific CXCR4 antagonist 1 in combination with the granulocyte colonyꢀstimulating
factor (GꢀCSF; Filgrastim) has been approved by US/FDA in 2008 for use in
transplantation in nonꢀHodgkin’s lymphoma or multiple myeloma patients. It is utilized
in a medical procedure called peripheral blood stem cell transplantation (PBSCT) to help
33,34
cancer patients restore their immune system rapidly after chemoꢀ or radiotherapy.
More specifically, the procedure consists of an 8ꢀday treatment period, in which patients
receive a daily dose of GꢀCSF for four consecutive days to stimulate stem cell
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production, and beginning on day 5, a stem cell mobilizer 1 is coꢀinjected with GꢀCSF
6
+
daily for subsequent daily apheresis (day 5 to 8) until more than 6×10 CD34 cells/kg
35,36
are harvested.
More importantly, it was reported that HSCs collected in this manner
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also showed longꢀterm repopulating capacity, implying that the above PBSCT procedure
may replace a traditional surgical operation as a favorable source for adult stem cell
37,38
transplantation in the future.
Recent encouraging clinical evidence revealed that
when patients with acute ischemic stroke were transplantated intraꢀarterially with
+
autologous CD34 stem cells, they all showed significant improvement in functional
recovery and reduction in the brain infarct volumn in the following six months after
39
treatment. Though CXCR4 antagonists are structurally diverse as seen in many
7
,22,23,40
historical cases,
they appear to have a preference for incorporating multiple
nitrogen elements in a spiderꢀlike skeleton. This structural propensity is presumably due
to mimicking highly positively charged natural ligand CXCL12, comprising a sequence
40k
of 68 amino acids rich in Lys and Arg residues. In continuation of our longꢀterm
studies on CXCR4 antagonists, herein, we wish to report that out of 600 polyamine
analogues designed and experimentally realized via docking into the Xꢀray crystal
41,42
structure of CXCR4,
a selective and potent CXCR4 antagonist 16, named CX0714
in our inꢀhouse library, has been identified as a promising drug candidate for PBSCT and
43
also has potential utility in many other CXCR4ꢀmediated diseases. Results are
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presented as follows.
RESULTS AND DISCUSSION
The first and only marketed CXCR4 antagonist 1 is commonly considered toxic and
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particularly inappropriate for the chronic treatment (SI, S64 & see details in ref. 50, pp.
86–154). Therefore, we set out to identify a new generation of CXCR4ꢀtargeted
compounds with better safety profiles and HSC mobilization by optimizing a series of
23
quinazolineꢀcore analogues previously designed in our laboratories (e.g., 2 and 3).
NH
NH
HN
N
HN
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
N
N
2
3
We began by simplifying the original quinazoline ring into a pyrimidine unit while
maintaining the previously optimized triazole ring linker (e.g., 3). As illustrated in
Scheme 1 using compound 7 as a typical example, a general synthetic procedure was
implemented to prepare an array of analogues 7–13. Starting from 2,4ꢀdichloroꢀ6ꢀ
methylpyrimidine, C4ꢀsubstitution with 4ꢀaminoꢀ1ꢀbenzylꢀpiperidine was established at
room temperature in a chemoselective fashion to produce intermediate 4 in 62%, which
in turn underwent C2ꢀsubstitution with a NꢀBoc protected Linker 1 at elevated
o
temperature (140
C) in pentanol to afford intermediate 5 in 63%. Selective
hydrogenolysis was carried out under 1 atm of hydrogen with Pd/C as catalyst to give
amine 6 in 92% yield, in which NꢀBoc was subsequently deprotected under acidic
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conditions (1N HCl) to afford desired product 7 in 95% yield as a hydrochloride salt.
a
Scheme 1. The Synthesis of Representative Compound 7
NBn
NBn
Cl
N
HN
N
HN
N
1
1
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N
a
b
N
N
Cl
Cl
N
N
N
N
N
H
Boc
Boc
N
4
5
NH
NH
HN
HN
N
c
d
N
N
N
N
N
N
N
Boc
N
N
N
N
N
N
N
H
Boc
H
H
H
N
7
6
a
o
Reagents and conditions: (a) 4ꢀaminoꢀ1ꢀBenzylꢀpiperidine, TEA, CH
2
Cl
2
, 5 C to rt, 16 h, 62%;
o
o
(
b) Linker 1, 1ꢀpentanol, 140 C, 5 h, 63%; (c) Pd/C (10 %), 2ꢀpropanol, 60 C, 16 h, 92%; (d)
1
N HCl in diethylether, CH
2
Cl
2
, rt, 16 h, 95%.
1
As listed in Table 1, when R is a hydrogen or alkyl group, the corresponding
compounds 7–10 exhibit potent binding affinities (IC50 = 23–61 nM) toward CXCR4
receptors, suggesting that the pyrimidine unit can replace quinazoline ring as a
1
bioisosteric nucleus. However, as the neutral R group is replaced with either a strong
electronꢀdonating (OCH ) or electronꢀwithdrawing (CF ) group, a dramatic decrease in
3
3
binding affinities by 10ꢀfold is observed as demonstrated by compounds 11–13 (IC =
50
472–859 nM). In addition, an acute toxicity study revealed that all compounds in Table 1
remained toxic, showing a lower maximum tolerated dose (MTD = 10 mg/kg) than that
of the positive control 1 (MTD = 15 mg/kg) following the same subcutaneous (SC)
administration in mice.
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Table 1. Binding Affinity and Acute Toxicity of Polyamines 7–13
NH
HN
3
N
1
R1
N
N
N
N N
N
N
H
H
H
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a
b
Compd
R
Me
H
IC [nM]
MTD [mg/kg]
5
0
7
8
9
23.9 ± 3.0
34.1 ± 3.8
10
10
10
10
10
10
10
15
Et
46.8 ± 12.9
61.1 ± 4.4
10
iꢀPr
OMe
Cl
11
472.7 ± 136.3
708.3 ± 63.7
859.7 ± 255.6
12
13
1
CF
3
213.1 ± 26.0
a
125
Determined by 50% inhibition of radioligand [ I]CXCL12 binding to
hCXCR4ꢀtransfected HEK293 membrane; values represent the mean ± SD
b
of at least three independent experiments. Following SC administration in
C57BL/6 mice.
Further structural modifications intended for reducing acute toxicity were then attempted
by elongating the side arm at C4ꢀposition while Linker 1 at C2 remained intact. In fact,
the length of Linker 1 has been previously studied in the corresponding bioisosteric
2
3
quinazoline series as exemplified by compound 3. Linker 1 with a [1+3+3] methylene
unit was considered optimal and engrafted on the current pyrimidine series. According
to Scheme 2, it could be readily prepared using propargylamine as the starting material.
Propargylamine was first coupled with benzyl chloroformate to form a Cbz protected
intermediate in a quantitative yield (95%), which was then reacted with 3ꢀazidoꢀ
propanol via click chemistry under catalysis with Cu(I) to generate triazole alcohol in a
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regioselective manner in 83%. Triazole alcohol thus formed could further undergo
mesylation to afford the corresponding mesylate (90%), which was subsequently
substituted with propaneꢀ1,3ꢀdiamine followed by reductive amination with
1
1
1
1
1
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cyclohexanone and subsequent protection with Boc O to afford a fully Nꢀprotected
Linker 1 in 43% yield over three steps. Finally, the Cbz protecting group was selectively
a
Scheme 2. Preparation of N-Boc Protected Linker 1
a
b
H N
CbzHN
N
OH
2
CbzHN
N
N
N
c
d,e,f
CbzHN
N
N
OMs
CbzHN
N
N
Boc
Boc
N
N N
g
H
2
N
N
N
N
Boc
N
Boc
N
Linker 1
a
o
Reagents and conditions: (a) benzyl chloroformate, K
95%; (b) 3ꢀazidoꢀpropanol, CuSO , (+) sodium ꢀasorbate, K
83%; (c) MsCl, TEA, CH Cl , 5 C to rt, 15 h, 90%; (d) propaneꢀ1,3ꢀdiamine, THF, 65 C, 15 h;
2
CO
3
, THF/H
2
O = 1/2, 5 C to rt, 15 h,
4
L
2
CO , EtOH/H
3
2
O = 4/1, rt, 15 h,
o
o
2
2
o
o
(e) cyclohexanone, MeOH, 60 C, 6 h then NaBH
over three steps; (g) H
4
, 5 C, 1 h; (f) Boc
, Pd/C, 2ꢀpropanol, 60 C, 15 h, 93%.
2 2 2
O, CH Cl , rt, 15 h, 43%
o
2
removed via hydrogenolysis to afford the desired Linker 1 in 93% yield. As illustrated in
Scheme 3 using compound 16 as a typical example, compound 7 with fixed Linker 1 at
Cꢀ2 was subjected to further structural modifications via elongating the Cꢀ4 substituent
by coupling with an array of amino and phosphonic acids to generate compounds 16–26
in 51ꢀ60% yields over three steps. Intermediate 6 was first coupled with 3ꢀBocꢀ(2ꢀ
ethoxyꢀ2ꢀoxoethyl)amino) propionic acid under catalysis with EDCI/HOBt to provide
compound 14 in 71% yield, which in turn was subjected to ester hydrolysis under basic
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6
conditions to furnish the corresponding acid 15 in quantitative yield (99%). Compound
15 was hydrolyzed under acidic conditions to remove NꢀBoc to afford the desired 16 in
95% yield as a hydrochloride salt.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. The Synthesis of Representative Compound 16
O
O
N
N
CO
2
Et
N
N
CO H
2
Boc
Boc
a
HN
N
b
HN
N
6
N
N
N
N
N
N
N
N
N
N
N
N
N
H
Boc
Boc
H
Boc
Boc
N
1
5
14
O
N
N
H
2
CO H
c
HN
N
N
N
N
N
N
N
H
H
H
N
1
6
a
Reagents and conditions: (a) 3ꢀ((tertꢀbutoxycarbonyl)(2ꢀethoxyꢀ2ꢀoxoethyl)amino)propionic
acid, EDCI, HOBt, CH Cl , rt, 16 h, 71%; (b) LiOH, THF/H O, rt, 16 h, 99%; (c) 1N HCl in
diethylether, CH Cl , rt, 16 h, 95%.
2
2
2
2
2
Following a similar synthetic method starting from the same intermediate 6, compounds
7–32 were synthesized as a hydrochloride salt in 50ꢀ60% yield over a twoꢀstep
sequence, involving a typical S 2 substitution followed by hydrolysis of the NꢀBoc
2
N
protecting group under acidic conditions. As compiled in Table 2, both acute toxicity and
structureꢀactivity relationship (SAR) of these compounds are evaluated and elucidated as
follows.
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Table 2. Binding Affinity and Acute Toxicity of Polyamines 16–32
R3
N
R2
N
5
3N
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
N
N
N
N
H
N
H
N
H
N
1
2
3
a
MTD b
Compd
R
R
R
IC50 [nM]
[
mg/kg]
O
O
1
6
Me
H
34.2 ± 6.1
75
N
CO
2
H
H
H
H
N
1
1
7
8
Me
Me
H
H
82.6 ± 12.2
88.5 ± 16.0
100
75
CO
2
O
H
N
2
CO H
O
2
CO H
1
2
2
9
0
1
Me
Et
H
H
H
72.2 ± 13.8
72.0 ± 19.0
399.3 ± 73.8
50
50
N
H
O
N
H
CO
CO
CO
2
2
2
H
O
O
H
100
N
H
H
H
2
2
2
3
Me
Me
Me
H
97.6 ± 9.0
50
50
N
H
O
158.0 ± 19.8
CO
2
H
H
O
CO
2
24
Me
H
71.1 ± 9.3
50
NH
2
O
2
2
2
5
6
7
Me
Me
Me
H
H
H
91.6 ± 9.4
147.1 ± 33.2
47.8 ± 9.2
100
75
N
PO
3
H
2
H
O
PO
3
H
2
N
H
CH
3
10
Me
Me
H
H
CN
72.1 ± 21.1
25.6 ± 8.2
20
20
2
2
8
9
CONH
2
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1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
3
0
1
2
Me
Me
Me
H
H
H
CO
2
H
81.8 ± 4.9
93.0 ± 3.7
75
75
2
CO H
CO
2
H
70.4 ± 19.5
213.1 ± 26.0
100
15
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
a
125
Determined by 50% inhibition of radioligand [ I]CXCL12 binding to hCXCR4ꢀtransfected
HEK293 membrane; values represent the mean ± SD of at least three independent
experiments. Following SC administration in C57BL/6 mice.
b
3
In the beginning, compound 27 with an alkyl group as a R side arm was first
synthesized, of which binding affinity (IC = 47.8 ± 9.2 nM) and MTD (10 mg/kg) were
5
0
found comparable to its parent compound 7. When the terminal methyl moiety of 27 was
changed to the cyano group, however, the MTD of the resulting compound 28 was
increased by 2ꢀfold to 20 mg/kg though binding affinity (IC = 72.1 ± 21.1 nM) was
50
3
slightly decreased. This modest improvement in MTD prompted us to modify the R
spacer with more polar terminal groups. Changing the cyano to an amide group resulted
in compound 29 with little enhancement in potency (IC = 25.6 ± 8.2 nM), but acute
50
toxicity was retained (MTD = 20 mg/kg). When the amide group was further changed to
the carboxylic functionality, the resulting compounds 30–32 (IC50 = 70.4–93.0 nM,
MTD = 75–100 mg/kg) showed a sharp increase in MTD though their binding activities
decreased by 3ꢀfold relative to parent 7 (IC50 = 23.9 ± 3.0 nM, MTD = 10 mg/kg),
suggesting that a polar carboxylic group at the terminus of the Cꢀ4 substituent could
significantly reduce acute toxicity. As such, this functionality is kept constant while
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other portions of the pharmacophore are varied in further structural modifications. In the
meantime, to make the Cꢀ4 linker synthetically more variable and practical, two new
structural elements including a carbonyl moiety and nitrogen were appropriately
incorporated into the linker. As a result, compounds 16–19 (IC50 = 34.2–88.5 nM, MTD
= 50–100 mg/kg) were then prepared. As demonstrated with 16 (IC50 = 34.2 ± 6.1 nM,
MTD = 75 mg/kg), the Cꢀ4 linker containing [2+1] methylene units appeared optimal in
length in terms of potency and acute toxicity. Keeping it as a fixed theme, we slightly
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
altered R and R groups to afford structurally closely related analogues 20–24 (IC50
=
71.1–399.3 nM, 50–100 mg/kg). Unfortunately, these modifications led to losing
binding affinities by 2ꢀ to 10ꢀfold relative to 16 though their MTD remained at a
satisfactory level. Replacing the terminal carboxylic group with its equivalent,
phosphonic acid, was also explored; however, the resulting compounds 25 (IC50 = 91.6 ±
9.4 nM) and 26 (IC50 = 147.1 ± 33.2 nM) showed no improvement in binding affinities
though their MTD still maintained at a high level (50–100 mg/kg). The higher MTD
seen in this novel series of compounds than bicyclam 1 is presumably due to the
presence of the terminal carboxylic or phosphonic acid, allowing to increase protein
binding and decrease distribution (e.g., 16, 60~75% vs 1, 33~50% in rats). However, the
underlying cause remains to be determined. Compound 16 (IC50 = 34.2 ± 6.1 nM; MTD
=
75 mg/kg, SC) possessing a much stronger binding affinity and higher MTD than the
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
4
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4
4
4
4
4
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marketed drug 1 (IC50 = 213.1 ± 26.0 nM; MTD = 15 mg/kg, SC) was considered a
potential drug candidate and elected for various preclinical evaluations as shown below.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
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9
0
1
2
3
4
5
6
7
8
9
0
To examine compound 16’s specificity among chemokine family, it was extensively
screened against a panel of chemokine receptors in both CCR and CXCR types. As
listed in Table 3, compound 16 exhibited no crossꢀactivity up to 10 ꢁM in cAMP
functional assay toward all tested receptors (ꢀ11~0.5% inhibition) except CXCR4
(73.4% inhibition), indicating that it is a highly selective CXCR4 antagonist (see SI,
S10–S21).
Table 3. Selectivity of Compound 16 against Related Chemokine Receptors
Assay Target
CCR1
ꢀ1.2
CCR5
0.5
CCR6
ꢀ2.2
CCR7
ꢀ4.2
CCR10
0.5
XCR1
ꢀ2.8
a
Efficacy [%]
Assay Target
CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CXCR6
0.3 0.5 ꢀ7.6 73.4 ꢀ11 ꢀ3.9
a
Efficacy [%]
a
Percentage inhibition was determined in GPCR cAMP assay at a concentration of 10 ꢁM; very
weak inhibition (0.5~ꢀ11%) was observed for all tested chemokine receptors except CXCR4
73.4%).
(
Subsequently, the chemotaxis assay was conducted to evaluate compound 16’s role in
+
cellular migration of CXCR4 cells. As indicated in Figure 1, CXCL12ꢀinduced
movement of CCRFꢀCEM cells, a human cell line highly expressed with CXCR4, was
efficiently blocked at a very low concentration (EC50 = 13.7 ± 3.8 nM). Compound 16
was also subjected to the cytotoxicity assay against Detroit 551 (human normal skin
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fibroblast cells) with a CC50 value of more than 100 µM. Taken together, above in vitro
studies suggest that compound 16 is a highly selective CXCR4 antagonist and tolerated
up to high doses in mice.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
100
7
5
2
5
0
5
0
-9
-8
-7
-6
-5
log [Cpd 16] (M)
Figure 1. CXCL12ꢀinduced chemotaxis assay was conducted for compound 16. Data
were collected from three independent experiments and represent the mean ± SD.
For comparison purposes, pharmacokinetic studies on both control 1 and 16 were carried
out simultaneously prior to in vivo studies, results of which were summarized in Table 4.
.
Accordingly, compound 16’s blood exposure (AUC = 13515 ng/mL h) and Cmax (16400
.
ng/mL) are almost 2 times as high as drug 1 (AUC = 7152 ng/mL h; Cmax = 6200
ng/mL).
a
Table 4. Pharmacokinetic Studies of Control 1 and Compound 16
Compd
route
dose
mg/kg)
C
max
T
(h)
1/2
AUC(0ꢀ4h)
(ng/mL h)
.
(
(ng/mL)
1
SC
SC
6
6
6200 ± 394
1.4 ± 0.6
7152 ± 135
16
16400 ± 2858 0.5 ± 0.1 13515 ± 1618
a
Values indicate mean ± SD (n = 3) following SC in C57BL/6 mice.
Meanwhile, from the mechanistic point of view, CXCR4 antagonists are supposed to be
eliminated timely when stem cells circulating in peripheral blood are collected. Thus,
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
4
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4
4
4
4
4
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5
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5
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compound 16 with T1/2 = 30 min, presumably due to phase II metabolism by kidney,
apparently meets with this pharmacokinetic requirement for PBSCT indication. To
determine its maximal response dose, compound 16 was administered subcutaneously at
an escalating dose of 1, 6, 20, 35 and 50 mg/kg, respectively, in 8ꢀ to 10ꢀweekꢀold
0
1
2
3
4
5
6
7
8
9
0
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8
9
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9
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4
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8
9
0
+
C57BL/6 male mice. As a result, a dose dependent curve of the number of CXCR4
stem cells was observed via the flow cytometry analysis as illustrated in Figure 2.
1
1
500
000
**
*
*
*
5
00
0
C
1
6
20 35 50
[Cpd 16] (mg/kg)
+
Figure 2. CXCR4 cells were analyzed by flow cytometry. The blood samples
+
containing mobilized CXCR4 stem cells were collected from C57BL/6 mice and
measured by flow cytometry at a time point of 2 h after SC injection with indicated
concentrations of compound 16. Data represent the mean ± SEM (n = 3 per group).
Statistical analysis was performed by t test: *p < 0.05 and **p < 0.01 between control
and the indicated test group.
+
A 3ꢀfold (p < 0.01) increase in CXCR4 stem cells was observed at a dose of 35 mg/kg,
tentatively assigned as a maximal response dose, as compared to vehicle,. Since its
minimal effect dose (MED) occurred at 6 mg/kg, thus, the therapeutic window was
determined as MTD/MED = 12.5. More than the maximal response dose resulted in a
+
significant decrease in the number of CXCR4 stem cells as observed at a dose of 50
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+
+
mg/kg (p < 0.05). We are particularly interested in CD34 and CD133 as well as
+
+
+
+
+
VEGFR2 and VEGFR2 Scaꢀ1 cell types; the former (CD34 and CD133 ) are widely
44,45
46,47
recognized as biomarkers for HSCs
and the latter are for EPCs.
Thus, double
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
staining experiments (e.g., CXCR4 /CD34 ) were further performed and results were
analyzed as follows. A maximal response dose of positive control 1 (6 mg/kg) and test
compound 16 (35 mg/kg) was given via SC injection, respectively, to evaluate their
4
8
ceiling effects on moving out HSCs and EPCs stem cells. As displayed in Figures 3
(
A), (B), (C) and (D), treatment with compound 16 alone did exhibit greater ability than
marketed drug 1 as well as vehicle by about 2ꢀ and 7ꢀfold, respectively, to move out
HSCs and EPCs into the peripheral circulation. Following the clinical protocol of
positive control 1, however, compound 16 in combination with GꢀCSF was further
examined to evaluate its combination effects on moving out HSCs. Since the experiment
is critical for determining whether 16 is qualified as a drug candidate for PBSCT, instead
of the primary assay using flow cytometry, a more accurate but timeꢀconsuming CFU
assay was then conducted to analyze HSC counts between dosing with 1+GCSF and
16+GCSF.
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1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
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5
5
5
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5
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5
5
5
6
+
+
B. CXCR4 CD133+
+
A. CXCR4 CD34
*
*
2
1
1
0
5
0
5
0
4
3
2
1
00
00
00
00
0
*
**
*
**
**
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Control
1
16
Control
1
16
+
+
D. VEGFR2 Sca-1+
+
C. CXCR4 VEGFR2
*
3
5
2
1
1
0
5
0
5
0
**
*
**
30
25
**
2
0
**
15
10
5
0
Control
1
16
Control
1
16
+
+
Figure 3. Double staining analysis by flow cytometry. (A) CXCR4 CD34 cells, (B)
+
+
+
+
+
+
CXCR4 CD133 cells, (C) CXCR4 VEGFR2 cells, and (D) VEGFR2 Scaꢀ1 cells.
The blood samples were collected from C57BL/6 mice and measured by flow cytometry
at a time point of 2 h after SC injection of compound 1 (6 mg/kg) or 16 (35 mg/kg) at
the maximal response dose. Data represent the mean ± SEM (n = 3 per group).
Statistical analysis was performed by t test: *p < 0.05, **p < 0.01, and ***p < 0.001
between control and the indicated test group.
Peripheral blood mononuclear cells (PBMCs) were first isolated from blood samples and
incubated for 10–14 days until CFUꢀGM (colonyꢀforming unitꢀgranulocyte/monocyte)
colonies could be clearly formed and unambiguously counted under an inverted
microscope. In general, the aggregate equal to or more than 50 cells is recorded as a
colony. As seen in Figure 4, the number of CFUꢀGM colonies provided by 16+GCSF
was significantly increased by 1.6ꢀfold relative to 1+GCSF, interestingly which was in
+
+
good agreement with the CD34 and CD133 cell population ratio (1.7:1) obtained by
flow cytometry as illustrated in Figures 3A and 3B. Also emphasized is the fact that the
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number of HSCs, as demonstrated by CFUꢀGM colonies, was dramatically increased by
roughly 5.3ꢀfold in the combination regimen compared to control using GꢀCSF alone.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
***
***
6
5
4
3
2
1
0
F
F
F
S
S
S
-
C
-C
-C
G
G
G
+
+
6
1
1
Figure 4. In Vivo efficacy comparison in colonyꢀforming assay. C57BL/6 male mice
were treated with GꢀCSF (100 µg/kg) daily for 4 consecutive days by SC injection. On
day 5, GꢀCSF (100 µg/kg) in combination with 1 (6 mg/kg) or 16 (35 mg/kg) was given,
respectively. The heparinized blood samples containing mobilized stem cells were
collected 2 h after SC administration, and then peripheral blood mononuclear cells
(PBMCs) were isolated and cultured for CFUꢀGM colonyꢀforming assay. Data represent
the mean ± SEM with 8ꢀ12 mice per group. Statistical analysis was performed by t test:
***p < 0.001 between control (GꢀCSF alone) and the indicated test group.
Compound 16 was also subjected to 68 offꢀtarget standard assays (SI, S2–S9).
Accordingly, besides calcium channel Nꢀtype and muscarinic M2 sodium channel with
73% and 70% inhibition at 10 µM, respectively, compound 16 showed low binding and
inhibitory activity toward other 66 offꢀtargets (<50% inhibition at 10 µM). In addition,
no hERG liability was observed for compound 16 at a concentration up to 100 µM in the
patchꢀclamp assay (SI, S25–S35). Effects of compound 16 on 6 human liver microsomal
CYP enzymes, including 1A2, 2C9, 2C19, 2D6, 2E1 and 3A4, were also investigated
and results showed that IC50 values against above CYP enzymes were all greater than
100 µM, implying that drugꢀdrug interactions caused by coꢀadministration with
1
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compound 16 are not likely to be a major issue. In addition to moving out HSCs and
+
EPCs mentioned above, many CXCR4 mature immune cells such as white blood cells
(WBCs), also referred to as leukocytes, could be released by CXCR4 antagonists 1 and
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16 as shown in Figure 5A.
Figure 5. In Vivo efficacy comparison in WBC mobilization. The blood samples
containing mobilized white blood cells were collected from C57BL/6 mice and analyzed
for (A) total white blood cells, (B) neutrophils, (C) lymphocytes, and (D) monocytes at a
time point of 2 h after SC injection with compound 1 (6 mg/kg) or 16 (35 mg/kg). Data
represent the mean ± SEM (n = 5 per group). Statistical analysis was performed by t test:
*
p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated test group.
In general, WBCs are composed of neutrophils, eosinophils, basophils, lymphocytes and
49
monocytes. As seen in Figures 5B, 5C and 5D, compound 16 can mobilize out
neutrophils, lymphocytes and monocytes, which are mainly responsible for antiꢀ
infection, activating immunity and engulfing antigens in the living system, in cell counts
higher than drug 1 by 30~50%, and more than control by 2ꢀ to 4ꢀfold, again verifying
that it is a versatile CXCR4ꢀtargeted agent.
2
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To further support compound 16 as a suitable drug candidate, its nonꢀGLP 14ꢀday
repeated dose toxicology study (SD rats, 50 mg/kg/day, SC) has been carried out, results
of which revealed that all major organs, including liver, kidney, lung, heart, etc, are
normal in size, weight and color as compared to those in vehicle. As well, blood sample
analysis also showed that no significance difference was observed in both hematological
and biochemical data between vehicle and experimental rats (SI, S65–S67). Since
structurally compound 16 is a polyamine and highly suspected to be metabolized by
1
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kidney as seen with compound 1, an acute histology study on kidney was also
performed. Consequently, no specific histological difference was found in renal tissue
between the control and experimental groups at a dose up to 100 mg/kg as analyzed by
photomicrographs illustrated in Figures S1 and S2 (SI, S68–S69).
To better understand the SAR of this newly developed series, computational docking
studies were extensively undertaken during the course of development. As typified by
Figure 6, molecular docking of 16 to the Xꢀray structure of CXCR4 (PDB ID: 4RWS)
displayed that secondary amino and carboxylic groups of 16 could form hydrogen
bonding/polar interactions with Asn33, Asn37, Asp97, His203, Gly207 and Tyr256
(cyan) constituting the major subpocket, and the terminal cyclohexyl ring was anchored
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Figure 6. Docking compound 16 (Orange) to the CXCR4 crystal structure (PDB ID:
4RWS) after 20 ns of MD simulations.
at the minor subpocket surrounded by a series of hydrophobic residues Trp94, Trp102,
Val112, His113, Tyr116 and Cys186 (yellow). This putative binding mode, crossing both
major and minor binding pockets, could be further highlighted via superimposing the
above simulating complex structure on both Xꢀray coꢀcrystal structures IT1t/3ODU and
CVX15/3OE0. As illustrated in Figure 7, it clearly demonstrates that different from the
cyclic peptide CVX15 (pink) interacting with the major subpocket and small molecule
IT1t (green) with the minor subpocket, compound 16 (orange) assumes a stretchingꢀout
41
conformation to occupy both major and minor subpockets simultaneously. This spiderꢀ
like putative binding mode seems to interact favorably with the open and negatively
charged CXCR4 receptor, and provides a new insight into CXCR4 receptorꢀligand
interactions, whereby more effective CXCR4 antagonists with improved oral
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bioavailability might be developed in the future.
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Figure 7. Compound 16 (orange) is assumed to occupy both major and minor
subpockets displayed with a pink (CVX15) and green (IT1t) surface field.
CONCLUSION
Though currently autologous adult stem cell transplantation is the only therapeutic
utility for CXCR4ꢀtargeted agents (e.g., 1), CXCR4 might become an important drug
9
target in light of its versatile mechanistic pathways. We have discovered a novel series
of pyrimidineꢀbased CXCR4 antagonists, the representative (i.e., 16) of which
apparently exhibited a higher MTD dose and better HSC/EPCꢀmobilizing ability at the
maximal response dose than drug 1 in mice following SC administration, suggesting that
compound 16 could serve as a promising drug candidate for PBSCT and other disease
indications. In addition, inspired with the putative binding mode of this novel series of
compounds, further structural modifications will be carried out to generate a second
generation of CXCR4 antagonists with potential enhancement in safety profiles and oral
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bioavailability.
EXPERIMENTAL SECTION
General. Unless otherwise stated, all materials used were commercially available and
used as supplied. Reactions requiring anhydrous conditions were performed in flameꢀ
dried glassware and cooled under an argon or nitrogen atmosphere. Unless otherwise
stated, reactions were carried out under argon or nitrogen and monitored by analytical
thin layer chromatography performed on glassꢀbacked plates (5 × 10 cm) precoated with
silica gel 60 F254 as supplied by Merck. Visualization of the resulting chromatograms
was performed by looking under an ultraviolet lamp (λ = 254 nm) followed by dipping
in an ethanol solution of vanillin (5% w/v) containing sulfuric acid (3% v/v) or
phosphomolybdic acid (2.5% w/v) and charring with a heat gun. Flash chromatography
was used routinely for purification and separation of product mixtures using silica gel 60
of 230−400 mesh size as supplied by Merck. Eluent systems are given in
volume/volume concentrations. Melting points were determined using a KRUSS KIP1N
0
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1
13
melting point meter. H and C NMR spectra were recorded on a Varian Mercuryꢀ300
(300 MHz) and a Varian Mercuryꢀ400 (400 MHz). Chloroformꢀd, methanolꢀd4 or
deuterium oxideꢀd2 was used as the solvent and TMS (δ 0.00 ppm) as an internal
standard. Chemical shift values are reported in ppm relative to the TMS in delta (δ) units.
Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q
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(
quartet), dd (doublet of doublets), dt (doublet of triplets), and m (multiplet). Coupling
constants (J) are expressed in hertz. Electrospray mass spectra (ESMS) were recorded as
m/z values using an Agilent 1100 MSD mass spectrometer. All test compounds
displayed more than 95% purity as determined by an Agilent 1100 series HPLC system
using a C18 column (Thermo Golden, 4.6 mm × 250 mm). The gradient system for
1
1
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2
HPLC separation was composed of MeOH (mobile phase A) and H O solution
containing 0.1% trifluoroꢀacetic acid (mobile phase B). The starting flow rate was 0.5
mL/min and the injection volume was 10 ꢁL. During first 2 min the percentage of phase
A was 10%. At 6 min, the percentage of phase A was increased to 50%. At 16 min, the
percentage of phase A was increased to 90% over 9 min. The system was operated at 25
o
C. Peaks were detected at 254 nm. IUPAC nomenclature of compounds was determined
with ACD/Name Pro software. All novel compounds reported here were screened for
PAINS using KNIME: PAINSꢀIndigo (module) software, and results showed that no
51
PAINS liability was detected for them.
(1-Benzyl-piperidin-4-yl)-(2-chloro-6-methyl-pyrimidin-4-yl)-amine (4). A solution
of 2,4ꢀdichloroꢀ6ꢀmethylpyrimidine (1.00 g, 6.1 mmole), 1ꢀBenzylꢀpiperidinꢀ4ꢀylamine
(1.20 g, 6.3 mmole), and TEA (0.92 g, 9.1 mmole) in THF (72 mL) under an atmosphere
o
of nitrogen was stirred at 25 C for 15 h and then quenched with aqueous NH
4
Cl. The
resulting mixture was extracted with ethyl acetate. The combined organic extracts were
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washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The
residue thus obtained was purified by flash chromatography on silica gel with nꢀ
1
hexane/ethyl acetate (1:1) to give compound 4 (1.20 g, 62%) as sticky oil. H NMR (300
0
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3
MHz, CDCl ) δ 7.35−7.21 (m, 5H), 6.05 (s, 1H), 3.62 (m, 1H), 3.52 (s, 2H), 2.82 (m,
2H), 2.32 (s, 3H), 2.19 (m, 2H), 1.98 (m, 2H), 1.55 (m, 2H). ESMS m/z: 317.5 (M+1).
[3-(4-{[4-(1-Benzyl-piperidin-4-ylamino)-6-methyl-pyrimidin-2-ylamino]-methyl}-
[1,2,3]triazol-1-yl)-propyl]-[3-(tert-butoxycarbonyl-cyclohexyl-amino)-propyl]-
carbamic acid tert-butyl ester (5). A solution of compound 4 (1.01 g, 3.19 mmole) and
o
Linker 1 (1.74 g, 3.52 mmole) in 1ꢀpentanol (25 mL) was heated at 150 C for 15 h and
then concentrated. The residue thus obtained was purified by flash chromatography on
1
silica gel with MeOH/DCM (1:32) to afford compound 5 (1.55 g, 63%) as sticky oil. H
NMR (400 MHz, CDCl
3
) δ 7.61 (s, 1H), 7.34
.30 (t, J = 7.2 Hz, 2H), 3.87 (m, 1H), 3.52 (s, 2H), 3.26
.20 (s, 3H), 2.19 2.04 (m, 6H), 1.98 (m, 2H), 1.80 1.46 (m, 7H), 1.44 (s, 18H),
.40 1.22 (m, 5H), 1.05 (m, 1H). ESMS m/z: 775.6 (M+1).
−7.21 (m, 5H), 5.59 (s, 1H), 4.68 (m, 2H),
4
2
1
−2.98 (m, 6H), 2.84 (m, 2H),
−
−
−
[3-(tert-Butoxycarbonyl-cyclohexyl-amino)-propyl]-[3-(4-{[4-methyl-6-(piperidin-
4-ylamino)-pyrimidin-2-ylamino]-methyl}-[1,2,3]triazol-1-yl)-propyl]-carbamic
acid tert-butyl ester (6). A solution of 5 (1.50 g, 1.94 mmole) and 10% Pd/C (0.15 g) in
o
2
ꢀpropanol (30 mL) was stirred under H
2
(1 atm) at 60 C for 15 h. The resulting mixture
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was filtered and the filtrate was concentrated to give 6 (1.22 g, 92%) as a white solid:
o
1
3
mp 182.3ꢀ182.7 C. H NMR (400 MHz, CDCl ) δ 7.82 (s, 1H), 6.20 (s, 1H), 4.70 (s,
2H), 4.41
−
4.30 (m, 3H), 3.59 (m, 2H), 3.32
−
2.98 (m, 8H), 2.25 (s, 3H), 2.22
−2.00 (m,
1
1
1
1
1
1
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1
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2
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0
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6H), 1.82
−1.59 (m, 7H), 1.44 (s, 18H), 1.40−1.20 (m, 5H), 1.05 (m, 1H). ESMS m/z:
685.5 (M+1).
N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4-ylmethyl}-
-methyl-N4-piperidin-4-yl-pyrimidine-2,4-diamine hydrochloride salt (7). A
6
solution of 1N HCl/ether (5.13 mL, 5.13 mmole) was added to the solution of 6 (344 mg,
o
0
.51 mmole) in dichloromethane (6.88 mL). The mixture was stirred at 25 C for 15 h
1
and concentrated to afford 7 (300 mg, 95%). H NMR (300 MHz, D
2
O) δ 8.02 (s, 1H),
5.93 (s, 1H), 4.75 (s, 2H), 4.56 (t, J = 6.9 Hz, 2H), 4.17 (m, 1H), 3.46 (m, 2H),
3
1
9
2
.21
−
3.04 (m, 8H), 2.33 (m, 2H), 2.25 (s, 3H), 2.16
−
1.99 (m, 6H), 1.87
−1.62 (m, 5H),
13
.40
−1.17 (m, 6H). C NMR (100 MHz, D
2
O) δ 162.54, 153.85, 152.27, 145.46, 123.69,
6.56, 57.40, 47.38, 45.27, 44.71, 44.58, 42.60, 41.14, 36.14, 28.73, 27.18, 26.12, 24.36,
3.78, 22.79, 17.66. ESMS m/z: 485.5 (M+1). HPLC purity = 96.92%, tR = 13.04 min.
N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4-ylmethyl}-
N4-piperidin-4-yl-pyrimidine-2,4-diamine hydrochloride salt (8). Following a
similar synthetic procedure for compound 7, products 8−13 were prepared in a sequence
of four steps using various 2,4ꢀdichloropyrimidine derivatives as starting material.
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Starting from 2,4ꢀdichloropyrimidine (500 mg, 3.36 mmole), compound 8 (641 mg) was
1
obtained in 31% yield over four steps. H NMR (300 MHz, D
2
O) δ 8.06 (s, 1H), 7.56 (d,
J = 7.2 Hz, 1H), 6.11 (d, J = 7.2 Hz, 1H), 4.75 (s, 2H), 4.56 (t, J = 6.9 Hz, 2H), 4.19 (m,
H), 3.47 (m, 2H), 3.22 3.06 (m, 8H), 2.33 (m, 2H), 2.18 1.99 (m, 6H), 1.85 1.59 (m,
0
1
2
3
4
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8
9
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1
2
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3
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8
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1
−
−
−
13
5H), 1.40
123.73, 98.20, 57.38, 47.39, 45.33, 44.71, 44.58, 42.58, 41.15, 36.11, 28.72, 27.04,
6.14, 24.37, 23.79, 22.80. ESMS m/z: 471.4 (M+1). HPLC purity = 97.42%, tR = 12.71
2
−1.17 (m, 6H). C NMR (75 MHz, D O) δ 162.14, 153.36, 145.28, 140.34,
2
min.
N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4-ylmethyl}-
6-ethyl-N4-piperidin-4-yl-pyrimidine-2,4-diamine hydrochloride salt (9). Starting
from 2,4ꢀdichloroꢀ6ꢀethyl pyrimidine (503 mg, 2.84 mmole), compound 9 (513 mg) was
1
obtained in 28% yield over four steps. H NMR (300 MHz, D
2
O) δ 8.04 (s, 1H), 5.96 (s,
1
8
1
H), 4.77 (s, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.19 (m, 1H), 3.46 (m, 2H), 3.22
−3.08 (m,
H), 2.56 (q, J = 7.5 Hz, 2H), 2.36 (m, 2H), 2.18 2.00 (m, 6H), 1.88 1.60 (m, 5H),
−
−
13
2
.40−1.17 (m, 9H). C NMR (100 MHz, D O) δ 162.71, 157.40, 153.92, 145.49, 123.67,
95.12, 57.39, 47.38, 45.32, 44.72, 44.59, 42.60, 41.15, 36.18, 28.73, 27.18, 26.14, 25.10,
24.38, 23.79, 22.81, 10.84. ESMS m/z: 499.5 (M+1). HPLC purity = 97.92%, tR = 13.17
min.
N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4-ylmethyl}-
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-isopropyl-N4-piperidin-4-yl-pyrimidine-2,4-diamine hydrochloride salt (10).
Starting from 2,4ꢀdichloroꢀ6ꢀisopropyl pyrimidine (505 mg, 2.64 mmole), compound 10
1
(453 mg) was obtained in 26% yield over four steps. H NMR (400 MHz, D
2
O) δ 8.02 (s,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1H), 5.94 (s, 1H), 4.75 (s, 2H), 4.55 (t, J = 6.8 Hz, 2H), 4.18 (m, 1H), 3.46 (m, 2H),
3.21
1.40
−3.06 (m, 8H), 2.79 (m, 1H), 2.34 (m, 2H), 2.18
−
1.99 (m, 6H), 1.88
−1.62 (m, 5H),
13
−
1.17 (m, 12H). C NMR (100 MHz, D
2
O) δ 162.84, 161.28, 154.05, 145.65,
123.55, 93.73, 57.39, 47.29, 45.32, 44.72, 44.56, 42.60, 41.12, 36.22, 31.15, 28.73,
27.17, 26.14, 24.36, 23.78, 22.79, 19.68. ESMS m/z: 513.5 (M+1). HPLC purity =
98.68%, tR = 13.52 min.
N2-{1-[3-(3-Cyclohexylamino-propylamino)-propyl]-1H-[1,2,3]triazol-4-ylmethyl}-
6-methoxy-N4-piperidin-4-yl-pyrimidine-2,4-diamine trifluoroacetic acid salt (11).
Starting from 2,4,6ꢀtrichloro pyrimidine (501 mg, 2.73 mmole), compound 11 (470 mg)
1
was obtained in 18% yield over five steps. H NMR (400 MHz, D
2
O) δ 7.89 (s, 1H),
5
.25 (s, 1H), 4.53 (s, 2H), 4.36 (t, J = 6.4 Hz, 2H), 3.87 (m, 1H), 3.69 (s, 3H), 3.24 (m,
H), 2.98 2.81 (m, 8H), 2.12 (m, 2H), 1.83 1.76 (m, 6H), 1.63 1.40 (m, 5H), 1.19 0.86
2
−
−
−
−
13
2 3 2
(m, 6H). C NMR (100 MHz, D O) δ 164.36, 162.40 (q, J = 36.1 Hz, CF CO H),
3 2
153.35, 144.87, 143.56, 124.40, 116.24 (q, J = 289.8 Hz, CF CO H), 75.44, 57.59, 56.92,
48.03, 45.88, 44.78, 44.71, 42.71, 41.25, 35.83, 28.86, 27.54, 26.04, 24.50, 23.93, 22.92.
ESMS m/z: 501.5 (M+1). HPLC purity = 99.10%, tR = 13.18 min.
2
9
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