Symmetrical bis-tertiary amines as novel CXCR4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.04.040

CXCR4 inhibitors are promising agents for the treatment of cancer metastasis and inflammation. A series of novel tertiary amine derivatives targeting CXCR4 were designed, synthesized, and evaluated. The central benzene ring linker and side chains were mod

Full text of DOI:10.1016/j.ejmech.2016.04.040

European Journal of Medicinal Chemistry 118 (2016) 340e350
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Symmetrical bis-tertiary amines as novel CXCR4 inhibitors
,
Renren Bai a, Zhongxing Liang a b, Younghyoun Yoon a, Shuangping Liu c,
,
**
Theresa Gaines d, Yoonhyeun Oum a, Qi Shi e, Suazette Reid Mooring d
,
, b, *
Hyunsuk Shim a
a Department of Radiology and Imaging Science, Emory University School of Medicine, Atlanta, GA, 30322, USA
b Winship Cancer Institute, Emory University, Atlanta, GA, 30322, USA
c Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, 30322, USA
d Department of Chemistry, Georgia State University, Atlanta, GA, 30303, USA
e Department of Chemistry, Emory University, Atlanta, GA, 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
CXCR4 inhibitors are promising agents for the treatment of cancer metastasis and inflammation. A series
of novel tertiary amine derivatives targeting CXCR4 were designed, synthesized, and evaluated. The
central benzene ring linker and side chains were modified and optimized to study the structureeactivity
relationship. Seven compounds displayed much more potent activity than the reference drug, AMD3100,
in both the binding affinity assay and the blocking of Matrigel invasion functional assay. These com-
pounds exhibited effective concentration ranging from 1 to 100 nM in the binding affinity assay and
inhibited invasion from 65.3% to 100% compared to AMD3100 at 100 nM. Compound IIn showed a 50%
suppressive effect against carrageenan-induced paw inflammation in a mouse model, which was as
effective as the peptidic antagonist, TN14003 (48%). These data demonstrate that symmetrical bis-
tertiary amines are unique CXCR4 inhibitors with high potency.
Received 19 February 2016
Received in revised form
14 April 2016
Accepted 15 April 2016
Available online 19 April 2016
Keywords:
Tertiary amines
CXCR4 inhibitors
Binding affinity
Matrigel invasion
Anti-inflammatory activity
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
believed to be the rate-limiting step of the multi-step metastatic
process [7,8]. These suggest that the interaction between CXCL12
C-X-C chemokine receptor type 4 (CXCR4), also known as fusin
or cluster of differentiation 184 (CD184), is a seven transmembrane
G-protein coupled receptor (GPCR) belonging to the Class I GPCR or
rhodopsin-like GPCR family [1,2]. Stromal-derived-factor-1 (SDF-1)
or C-X-C chemokine ligand 12 (CXCL12) is the major ligand of
CXCR4 and the interaction recognition between CXCL12 and CXCR4
recruits cells to the organ sites with high levels of CXCL12 expres-
sion [3,4]. The CXCL12/CXCR4 axis has been shown to be involved in
a number of pathological conditions, including cancer and inflam-
mation [5,6]. CXCR4 plays a key role as a homing receptor to the
lymph nodes, lung, liver, and bone. Homing, the mechanism that
allows foreign tissue-origin cells to reside and proliferate, is
and CXCR4 plays a key role in the chemotaxis and homing of
metastatic cells [9]. CXCR4 is overexpressed in more than 23 human
cancer types [10,11], such as breast [12e14], leukemia [9], lung [15],
and prostate cancers [16].
Inflammation is inextricably associated with primary tumor
progression and contributes to metastatic outgrowth in distant
organs [17]. The COX pathway has long been used as the major
target for anti-inflammatory drugs [18]. Both traditional non-
steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2
inhibitors exhibit their anti-inflammatory activity by inhibiting
COX-2 [18]. Nevertheless, the prolonged use of traditional anti-
inflammatory drugs is associated with potential serious side ef-
fects such as kidney failure, ulcers and prolonged bleeding after an
injury or surgery [19e21]. Furthermore, rofecoxib and valdecoxib
were withdrawn from the market due to an increased risk of car-
diovascular complications [22,23].
* Corresponding author. Department of Radiology and Imaging Science, Emory
University School of Medicine, 1365C Clifton Road, NE, C5008, Atlanta, GA, 30322,
USA.
** Corresponding author. Department of Chemistry, Georgia State University, 519A
Science Annex, Atlanta, GA, 30302-4098, USA.
Accumulating evidence suggests the involvement of
CXCR4ꢀCXCL12 interaction in various inflammatory diseases,
including rheumatoid arthritis, autoimmune diseases, ischemic
injuries, inflammatory bowel disease, and pneumonia [24,25].
E-mail addresses: smooring@gsu.edu (S.R. Mooring), hshim@emory.edu
(H. Shim).
http://dx.doi.org/10.1016/j.ejmech.2016.04.040
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
341
Based on these findings, development of inhibitors blocking CXCR4
2.2. Primary binding affinity screening
presents a new avenue for complementary therapeutic strategy in
inflammatory diseases as well as cancer metastasis.
All the compounds were initially screened with a binding affinity
assay as described in our previous publications [31,34]. This assay
involves competition of a potent CXCR4 peptidic inhibitor, TN14003,
with the target compounds Ia-p and IIa-p. MDA-MB-231 cells were
pre-incubated with compounds at concentrations of 1, 10, 100, and
1000 nM, then incubated with biotinylated TN14003 and
streptavidin-conjugated rhodamine to determine the binding effi-
ciency of the new compounds to the CXCL12 binding domain of
CXCR4. The effective concentration (EC) is defined as the lowest
concentration at which a significant reduction in the rhodamine
fluorescentcolorisobservedascomparedtothecontrolreflectingthe
competitive displacement by peptide TN14003. Thus, this screening
is a semi-quantitative, first pass measure of the level of activity.
Just like the reported secondary amine derivatives, the sym-
metrical bis-tertiary amine framework also successfully retained
the significant CXCR4 antagonistic effect. Nineteen of the 32 syn-
thesized compounds exhibited moderate to good activity
(ꢁ1000 nM) in blocking the binding of peptide TN14003 to CXCR4
(Table 1 and Fig. 2). Ten substances displayed potent binding af-
finity, with EC values from 1 to 100 nM. They were all compara-
tively more effective than the reference drug, AMD3100 (1000 nM).
When only the benzene central ring was substituted with a pyr-
idine ring and the structure of side chains remained the same, most
of the compounds demonstrated better activity. This result indicates
that the central pyridine linker is well tolerated and further modi-
fication of the middle ring functionality is a reasonable strategy.
Surprisingly, if the terminal rings of the side chains were non-
aromatic instead of aromatic amines (for example, a pyrrolidine,
piperidine, or morpholine ring), the target compounds showed
improved binding affinity to the compounds possessing aromatic
side chains. Compounds IIa and IIb showed an EC of 1 nM, and
experienced 1000-fold improvement compared with AMD3100. In
addition, the substituent group introduced onto the 30 position of
the terminal ring may enhance the binding effect. By contrast, most
compounds substituted into the 20 or 40 positions are less active or
even inactive. For example, Ik, IIk and IIg all showed an EC of
10 nM, while compounds If, Ih, Ij, IIf, and IIk were not effective
even at 1000 nM.
The first CXCR4 inhibitor to enter clinical trials for treatment of
HIV infection is AMD3100 [25]. The development of AMD3100 has
followed a meandering course, starting with JM1657, an impurity in
commercial cyclam preparation with anti-HIV activity, and finally
leading to AMD3100 [26]. Because of the preorganisation and the
flexibility of the AMD3100 macrocyclic cyclam, it can bind to metal
ions and form highly stable metal complexes [3]. These chelating
properties are likely related to the cardiotoxicity reported in its
clinical trial against HIV [26e29]. In 2008, AMD3100 was approved
by FDA for stem cell mobilization [25]. Although it benefits patients
with certain diseases, long term treatment can introduce lung and
liver fibrosis [27]. Hence there is an urgent need to develop safe
CXCR4 inhibitors that can be used in long-term treatment.
Taking AMD3100 as the lead compound, we simplified and
optimized its bicyclam rings, leading to the design and synthesis of
a series of novel and potent CXCR4 inhibitors [30,31]. Previously, we
reported a novel small molecule, compound 2 (Fig. 1), which is now
under clinical trial (Phase II). Compound 2 is a partial CXCR4 in-
hibitor with properties quite unlike that of any other reported
CXCR4 antagonists. Compound 2 does not have stem cell mobilizing
capability nor calcium flux modulating activity, which can be safer
for long-term blockade of metastasis than other reported CXCR4
antagonists [27].
Most of our reported CXCR4 inhibitors are secondary amines.
Here we attempted to design and synthesize a new series of tertiary
amine compounds to explore whether these new structures will
maintain the CXCR4 inhibitory effect. Meanwhile, an extensive
structure-activity profile indicates that the central benzene ring is
critical for high CXCR4 affinity [31]. Since pyridine derivatives have
been explored for their potential as pharmaceutical agents [32], we
also substituted the middle benzene ring with a pyridine frame-
work. The structural formula was shown in Fig. 1.
2. Results and discussion
2.1. Chemistry
The synthetic route chosen to synthesize the title compounds
was outlined in Scheme 1. The previous method to efficiently pre-
pare symmetrical bis-secondary amine compounds was reductive
amination of 1,4-benzenedialdehyde with phenylamine derivatives
[30e32]. Since the reagents used in this preparation are secondary
amines like N-alkyl anilines or cyclic amines, the yield is no longer
satisfying. Herein, 1,4-bis(bromomethyl) benzene was used as the
starting material, which reacted with corresponding secondary
amines in anhydrous acetonitrile in the presence of potassium
carbonate, and afforded the target compounds Ia-p [33]. Similarly,
2,6-bis(chloromethyl)pyridine reacted with amine derivatives in
acetonitrile provided the title compounds IIa-p. Considering benzyl
chloride is less active than benzyl bromide, cesium carbonate was
used to shorten the reaction time.
2.3. Matrigel invasion assay
To model in vitro metastasis, a Matrigel invasion assay was used
as the functional assay [31,34]. This assay was performed for those
compounds with an EC value lower than 1000 nM in the binding
affinity assay to test whether they could block the CXCR4/CXCL12-
mediated chemotaxis and invasion at a single concentration of
100 nM. The compounds and cells were added to the upper
chamber and CXCL12 was added to the lower chamber as a che-
moattractant. A layer of Matrigel matrix-coated permeable support
(8 mm pore diameter) separates the upper and lower chambers. If
the compounds demonstrate a strong CXCR4 inhibitory effect,
fewer cells are able to move through the Matrigel. The results of
Fig. 1. Strategy for the design and optimization of anti-CXCR4 compounds.

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R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
Scheme 1. Reagents and conditions to make our compounds: (a) CH3CN, K2CO3, reflux, 3 h, 78e95%; (b) CH3CN, Cs2CO3, reflux, 5 h, 70e90%.
Table 1
been using TN14003 as the reference drug for our animal experi-
Preliminary effective concentration (EC) of anti-CXCR4 compounds in our binding
ments [27,35]. As illustrated in Fig. 5, although substances IIg and
IIm demonstrated excellent CXCR4 antagonistic activity, they
showed very weak in vivo potency (<20%), which may be attributed
to their metabolism or poor pharmacokinetic profile. Compound
IIk exhibited moderate inhibition (31.0%). IIn showed a 50%
inhibitory effect on inflammation, which was comparable to
TN14003 (48%). Compared with the low success rate and high cost
of the development of peptide drugs [36], small molecule CXCR4
inhibitors have practical advantages. After being treated with IIn,
the inflammation induced in the left paw was clearly suppressed
(Fig. 6). As shown in histological analysis in Fig. 7, the normal
mouse paw tissue exhibits a dermis tightly connected to the
epidermis through a basement membrane, and the papillary region
is composed of loose areolar connective tissue (A1-3). However, the
carrageenan-induced inflammatory tissue showed intense dermal
papillae edema, and a dense infiltration of inflammatory cells (B1-
3). Compound IIn significantly attenuated the mouse paw inflam-
mation and damage in histological assay. Both the edema volume
and the number of inflammatory cells decreased observably (C1-3).
These data confirm that this selected anti-CXCR4 candidate can
inhibit inflammation as anticipated.
affinity assay.
Compd
EC (nM) Compd EC (nM) Compd EC (nM) Compd EC (nM)
Ia
Ib
Ic
Id
Ie
If
Ig
Ih
1000
10
>1000
1000
100
>1000
1000
>1000
1000
Ii
Ij
>1000
>1000
10
1000
1000
>1000
>1000
>1000
IIa
IIb
IIc
IId
IIe
IIf
1
1
100
1000
1000
>1000
10
IIi
IIj
>1000
1000
10
>1000
1
Ik
Il
Im
In
Io
Ip
IIk
IIl
IIm
IIn
IIo
IIp
10
IIg
IIh
>1000
>1000
1000
AMD3100
Matrigel invasion were summarized in Fig. 3 and Fig. 4.
The invasion evaluation proved that most of the selected com-
pounds performed well in both the binding affinity assay and
blocking of Matrigel invasion assay. Only Ib, Ik and IIe exhibited an
inhibitory rate below 50%. The potency of Ie (66.4%), IIb (65.3%), IIc
(68.9%), IIg (88.6%), IIk (76.7%), IIm (100%) and IIn (72.6%) was
superior to the reference drug AMD3100 (62.0%).
Since compound IIn, which possesses a pyridine pharmaco-
phore, had the best suppression effect in vivo, we modified the
terminal ring and synthesized compounds Io, Ip, IIo and IIp,
bearing a nitrogen atom at the 30 or 40 position. Unfortunately, these
compounds exhibited poor ECs in the binding affinity assay
(>1000 nM). Hence we stopped the further investigation of these
compounds.
2.4. In vivo suppression against carrageenan-induced paw edema
CXCR4 plays a key role in the recruitment of inflammatory cells
to sites of inflammation. Blocking CXCR4 is a therapeutic strategy in
inflammatory diseases [24]. Previously, we reported utilizing a
carrageenan-induced mouse paw edema model as an efficient
model to evaluate the in vivo CXCR4 antagonistic activity for CXCR4
inhibitors [27,31]. It is
a widely used test to assess anti-
inflammatory activity in vivo. On the basis of the in vitro assay re-
sults, four of the best compounds (IIg, IIk, IIm and IIn) were
investigated. Because of the in vivo toxicity of AMD3100, we have
2.5. Discussion of paw edema results
Carrageenan-induced paw edema animal model is one of the

R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
343
Fig. 2. Fluorescence micrographs of binding affinity assay results of four selected compounds compared to AMD3100. The effective concentration (EC) of AMD3100 is 1000 nM,
while those of the compounds IIg, IIk, IIm and IIn are better, EC of 10, 10, 1, and 10 nM, respectively.
most popular in vivo tests to screen anti-inflammatory properties of
potential drugs. Complex sequential pathways for the inflamma-
tory response to inter dermal injection of carrageenan were
described by Vinergar R. et al. [37]. Briefly, it consisted of a non-
phagocytic inflammatory response followed by a phagocytic in-
flammatory response. The dermal nonphagocytic inflammatory
response comprised edema, hyperemia, and hyperalgesia followed
by hypoalgesia. The hyperalgesia and part of the edema were
sensitive to cyclooxygenase (COX) inhibitors. The dermal phago-
cytic inflammatory response consisted of mobilization of
neutrophils, monocytes, fibroblasts, and endothelial cells. While
COX inhibitors reduce edema formation and hyperalgesia by
blocking arachidonic acid metabolism, CXCR4 inhibitors reduce
carrageenan-induced paw edema by blocking the recruitment of
various inflammatory cells that are CXCR4-positive. In addition,
CXCR4 has been shown to modulate NFkB that is the master switch
of inflammation response [38].
Developing anti-CXCR4 drugs for inflammation indication pro-
vides an exciting avenue to target different pathways from COX
which has been the major, if not the only one, target of anti-

344
R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
Fig. 3. Matrigel invasion inhibition of AMD3100 and anti-CXCR4 compounds.
Fig. 4. Anti-Matrigel invasion effect of AMD3100 and four selected compounds. Fewer MDA-MB-231 cells are able to invade through the Matrigel after treatment with compounds
IIg, IIk, IIm and IIn than AMD3100.
inflammation drugs. By combining drugs targeting different path-
ways, the efficacy will be enhanced without increasing dosage of a
drug which is associated with unwanted side effects.
Previous reports on drug testing against inflammation using
carrageenan-induced paw edema model were geared toward pre-
vention by treating animals 30 min before carrageenan injection,
which is clinically irrelevant [18,39]. Patients present to hospital
after the fact. In our evaluation, the compounds were treated after
carrageenan injected.
2.6. Molecular modeling (docking) studies
To better understanding the possible interaction between IIn
and CXCR4, Schrodinger Maestro Package was performed based on
the available crystal structure of CXCR4 (PDB code: 3ODU [40]).
Fig. 8 illustrated the binding pose with the best docking score. A
hydrogen bond is formed between Asp97 and one of the nitrogen
Fig. 5. In vivo anti-inflammatory activity of compounds IIg, IIk, IIm and IIn.
atoms of IIn. One pyridine ring shows a
pep stacking with both
His113 and Trp94. The other pyridine ring demonstrates a partially

R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
345
Fig. 6. Suppression effect of anti-CXCR4 compound IIn on carrageenan-induced mouse paw inflammation. (A) Control mouse with left paw induced inflammation by carrageenan.
(B) IIn treated mouse with left paw induced inflammation by carrageenan with about 50% suppression.
Fig. 7. Compound IIn significantly attenuated the mouse paw inflammation and damage in histological assay. Paw tissue sections were stained with H&E. The whole tissue slices
were scanned/digitized by NanoZoomer 2.0 HT. Software NDP. view 2 was used to zoom in. Compared to the normal tissue (A1-3), carrageenan-induced skin inflammation exhibited
intense dermal papillae edema, and a dense infiltration of inflammatory cells (B1-3). After being treated with IIn, both the edema volume and the number of inflammatory cells
(dark purple) decreased observably (C1-3). (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this article.)
Fig. 8. A low energy predicted binding pose for compound IIn in the CXCR4 X-ray structure (grey). The best docking pose is illustrated by two different visualizations: ribbon (left)
and protein surface (right) representations.

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R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
hydrophobic interaction with Ile284. These favorable interactions
are likely contributing to the binding of IIn with CXCR4.
(100 MHz, CDCl3) d 137.31, 129.19, 63.87, 54.70, 26.19, 24.61. HRMS
calcd for C18H29N2 273.23253 [MþH]þ, found 273.23243.
3. Conclusion
4.1.2.3. 1,4-Bis(morpholinomethyl)benzene (Ic). White solid, yield
88%, m.p. 113e115 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.27 (s, 4H),
On the basis of the promising previously prepared CXCR4 in-
hibitor 2, a series of novel symmetrical bis-tertiary amines, non-
peptidic anti-CXCR4 small molecules, were designed and synthe-
sized. The central benzene ring linker and side chains were modi-
fied and optimized to study the structureeactivity relationship.
This symmetrical bis-tertiary amine framework successfully
maintained the significant CXCR4 antagonistic effect. Ten sub-
stances displayed potent binding affinity, with EC values from 1 to
100 nm. They were all comparatively more effective than the
reference drug, AMD3100 (1000 nM). Most of the compounds with
an EC lower than 1000 nM performed well in the invasion assay.
Compounds Ie, IIb, IIc, IIg, IIk, IIm and IIn significantly blocked the
tumor cell invasion. The inhibitory rates ranged from 65.3% to 100%,
which were superior to AMD3100 (62.0%). Compound IIn showed a
50.3% suppressive effect against an inflammation animal model,
comparable to the positive control, TN14003 (48.0%). These
promising results demonstrate that symmetrical bis-tertiary
amines are unique CXCR4 inhibitors with high potency.
3.70e3.72 (m, 8H), 3.48 (s, 4H), 2.43e2.45 (m, 8H). 13C NMR
(100 MHz, CDCl3)
d 136.83, 129.31, 67.20, 63.38, 53.80. HRMS calcd
for C16H25O2N2 277.19105 [MþH]þ, found 277.19089.
4.1.2.4. N,N0-(1,4-Phenylenebis(methylene))bis(N-methylaniline)
(Id). White solid, yield 83%, m.p. 77e79 ꢂC. 1H NMR (400 MHz,
CDCl3)
d
7.18e7.24 (m, 4H), 7.17 (s, 4H), 6.68e6.76 (m, 6H), 4.51 (s,
149.87,137.80,129.32,
4H), 3.00 (s, 6H). 13C NMR (100 MHz, CDCl3)
d
127.13, 116.67, 112.51, 56.51, 38.66. HRMS calcd for C22H25N2
317.20123 [MþH]þ, found 317.20070.
4.1.2.5. N,N0-(1,4-phenylenebis(methylene))bis(4-fluoro-N-methyl-
aniline) (Ie). White solid, yield 80%, m.p. 78e80 ꢂC. 1H NMR
(400 MHz, CDCl3)
1H), 4.45 (s, 4H), 2.96 (s, 6H). 13C NMR (100 MHz, CDCl3)
154.44, 146.65, 137.74, 127.26, 115.76, 115.54, 113.82, 113.74, 57.37,
d
7.16 (s, 4H), 6.88e6.95 (m, 4H), 6.63e6.69 (m,
d
156.78,
39.23. HRMS calcd for
352.17423.
C
22H22N2F2 352.17456 [M]þ, found
4. Experimental section
4.1.2.6. N,N0-(1,4-Phenylenebis(methylene))bis(1-(2-chlorophenyl)-
N-methylmethanamine) (If). Colorless liquid, yield 78%. 1H NMR
4.1. Chemistry
(400 MHz, CDCl3)
d
7.56 (dd, J ¼ 1.5, 0.9 Hz, 1H), 7.54 (dd, J ¼ 1.5,
0.9 Hz, 1H), 7.34 (d, J ¼ 1.5 Hz, 1H), 7.32 (s, 1H), 7.32 (s, 4H),
7.22e7.26 (m, 2H), 7.14e7.19 (m, 2H), 3.63 (s, 4H), 3.57 (s, 4H), 2.20
4.1.1. General information
Proton and carbon NMR spectra were recorded on INOVA-400
(400 MHz) or VNMR-400 spectrometers at Emory NMR Research
Center. The spectra obtained in deuteriochloroform (CDCl3) were
(s, 6H). 13C NMR (100 MHz, CDCl3)
d 138.14, 137.14, 134.43, 130.90,
129.58, 129.01, 128.17, 126.80, 62.21, 58.67, 42.46. HRMS calcd for
C
24H27N2Cl2 413.15458 [MþH]þ, found 413.15445.
referenced to the residual solvent peak. Chemical shifts (d) are re-
ported in parts per million (ppm) relative to residual undeuterated
solvent as an internal reference. The following abbreviations were
used to explain the multiplicities: s ¼ single; d ¼ doublet,
t ¼ triplet, q ¼ quartet, dd ¼ doublet of doublets, dt ¼ doublet of
triplets, m ¼ multiplet, br ¼ broad. Mass spectra were recorded on a
JEOL spectrometer at Emory University Mass Spectrometry Center.
Analytical thin layer chromatography (TLC) was performed on pre-
coated glass backed plates from Scientific Adsorbents Incorporated
(Silica Gel 60 F254; 0.25 mm thickness). Plates were visualized
using ultraviolet radiation.
4.1.2.7. N,N0-(1,4-Phenylenebis(methylene))bis(1-(3-chlorophenyl)-
N-methylmethanamine) (Ig). Colorless liquid, yield 80%. 1H NMR
(400 MHz, CDCl3)
d
7.37 (s, 2H), 7.31 (4, 1H), 7.20e7.25 (m, 6H), 3.52
141.73,
(s, 4H), 3.48 (s, 4H), 2.18 (s, 6H). 13C NMR (100 MHz, CDCl3)
d
137.93, 134.34, 129.67, 129.08, 127.32, 127.17, 110.22, 61.87, 61.38,
42.42. HRMS calcd for C24H27N2Cl2 413.15458 [MþH]þ, found
413.15482.
4.1.2.8. N,N0-(1,4-Phenylenebis(methylene))bis(1-(2-fluorophenyl)-
N-methylmethanamine) (Ih). Colorless liquid, yield 92%. 1H NMR
(400 MHz, CDCl3)
d
7.44 (td, J ¼ 7.5, 1.9 Hz, 2H), 7.31 (s, 4H),
4.1.2. General procedure for synthesis of compounds Iaep
7.19e7.25 (m, 2H), 7.11 (td, J ¼ 7.5, 1.3 Hz, 2H), 6.99e7.04 (m, 2H),
To a solution of 1,4-bis(bromomethyl)benzene (1 mmol) in
anhydrous acetonitrile (10 mL) was added potassium carbonate
(3 mmol) and the corresponding secondary amine (2.2 mmol). The
reaction mixture was heated at reflux for 3 h. The resulting mixture
was diluted with water (20 mL) and extracted with ethyl acetate
(15 mL) three times. The combined organic layers were washed
with brine, dried over anhydrous sodium sulfate, and concentrated
in vacuo. The crude was purified by column chromatography with
methanol/dichloromethane (150:1, v/v) to give the title compound
as a colorless liquid or a white solid.
3.59 (s, 3H), 3.54 (s, 3H), 2.20 (s, 6H). 13C NMR (100 MHz, CDCl3)
d
162.79, 160.35, 138.05, 131.52, 131.48, 129.01, 128.72, 128.63,
126.10, 125.95, 124.06, 124.02, 115.47, 115.24, 61.90, 54.30, 42.29.
HRMS calcd for C24H27N2F2 381.21368 [MþH]þ, found 381.21303.
4.1.2.9. N,N0-(1,4-Phenylenebis(methylene))bis(1-(3-fluorophenyl)-
N-methylmethanamine) (Ii). Colorless liquid, yield 85%. 1H NMR
(400 MHz, CDCl3)
4H), 6.90e6.95 (m, 2H), 3.51 (s, 4H), 3.49 (s, 4H), 2.18 (s, 6H). 13
NMR (100 MHz, CDCl3) 164.39, 161.94, 142.49, 142.42, 138.04,
d 7.31 (s, 4H), 7.24e7.29 (m, 2H), 7.09e7.13 (m,
C
d
129.80, 129.72, 129.00, 124.50, 124.47, 115.80, 115.59, 114.06, 113.84,
61.86, 61.43, 42.47. HRMS calcd for C24H27N2F2 381.21368 [MþH]þ,
found 381.21330.
4.1.2.1. 1,4-Bis(pyrrolidin-1-ylmethyl)benzene (Ia). Colorless liquid,
yield 95%. 1H NMR (400 MHz, CDCl3)
d
7.27 (s, 4H), 3.59 (s, 4H),
2.48e2.53 (m, 8H), 1.76e1.80 (m, 8H). 13C NMR (100 MHz, CDCl3)
d
137.95, 128.86, 60.54, 54.21, 23.49. HRMS calcd for C16H25N2
4.1.2.10. N,N0-(1,4-Phenylenebis(methylene))bis(1-(4-fluorophenyl)-
N-methylmethanamine) (Ij). White solid, yield 81%, mp 80e82 ꢂC.
245.20123 [MþH]þ, found 245.20084.
1H NMR (400 MHz, CDCl3)
d
7.28e7.33 (m, 8H), 6.97e7.03 (m, 4H),
4.1.2.2. 1,4-Bis(piperidin-1-ylmethyl)benzene (Ib). White solid, yield
3.49 (s, 4H), 3.47 (s, 4H), 2.16 (s, 6H). 13C NMR (100 MHz, CDCl3)
92%, m.p. 84e86 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.25 (s, 4H), 3.45 (s,
d 163.34, 160.91, 138.11, 135.27, 130.57, 130.49, 129.01, 115.28, 115.07,
4H), 2.37 (s, 8H), 1.54e1.59 (m, 8H), 1.40e1.45 (m, 4H). 13C NMR
61.78, 61.23, 42.37. HRMS calcd for C24H27N2F2 381.21368 [MþH]þ,

R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
347
found 381.21343.
4.1.3.1. 2,6-Bis(pyrrolidin-1-ylmethyl)pyridine
liquid, yield 86%. 1H NMR (400 MHz, CDCl3)
(IIa). Colorless
7.58 (t, J ¼ 7.7 Hz, 1H),
d
4.1. 2.11. N, N0-(1, 4-Phenylenebis(methylene))bis(1-(3-
7.25 (s, 1H), 7.24 (s, 1H), 3.75 (s, 4H), 2.52e2.58 (m, 8H), 1.73e1.81
(m, 8H). 13C NMR (100 MHz, CDCl3)
d 158.77, 136.89, 121.15, 62.41,
methoxyphenyl)-N-methylmethanamine) (Ik). Colorless liquid, yield
54.43, 23.69. HRMS calcd for C15H24N3 246.19647 [MþH]þ, found
87%. 1H NMR (400 MHz, CDCl3)
d 7.31 (s, 4H), 7.21e7.26 (m, 2H),
246.19644.
6.93e6.95 (m, 4H), 6.79e6.81 (m, 1H), 6.78 (dd, J ¼ 2.7, 1.0 Hz, 1H),
3.81 (s, 6H), 3.50 (s, 4H), 3.49 (s, 4H), 2.18 (s, 6H). 13C NMR
(101 MHz, CDCl3)
d
159.77, 141.22, 138.05, 129.30, 128.98, 121.41,
4.1.3.2. 2,6-Bis(piperidin-1-ylmethyl)pyridine (IIb). Colorless liquid,
114.41, 112.56, 61.98, 61.71, 55.33, 42.49. HRMS calcd for
yield 80%. 1H NMR (400 MHz, CDCl3)
d
7.59 (t, J ¼ 7.7 Hz,1H), 7.29 (s,
1H), 7.27 (s, 1H), 3.60 (s, 4H), 2.42e2.44 (m, 8H), 1.55e1.61 (m, 8H),
1.40e1.46 (m, 4H). 13C NMR (100 MHz, CDCl3)
158.71, 136.67,
C
26H33N2O2 405.25365 [MþH]þ, found 405.25331.
d
4.1.2.12. N,N0-(1,4-Phenylenebis(methylene))bis(N-benzylethan-
121.20, 65.57, 54.99, 26.20, 24.52. HRMS calcd for C17H28N3
274.22777 [MþH]þ, found 274.22757.
amine) (Il). Colorless liquid, yield 78%. 1H NMR (400 MHz, CDCl3)
d
7.36e7.39 (m, 4H), 7.29e7.33 (m, 8H), 7.20e7.24 (m, 2H), 3.57 (s,
4H), 3.55 (s, 4H), 2.50 (q, J ¼ 7.1 Hz, 4H), 1.07 (t, J ¼ 7.1 Hz, 6H). 13
C
4.1.3.3. 2,6-Bis(morpholinomethyl)pyridine (IIc). White solid, yield
NMR (100 MHz, CDCl3)
d
140.27, 138.55, 128.93, 128.73, 128.31,
90%, m.p. 93e95 ꢂC. 1H NMR (400 MHz, CDCl3)
d
7.63 (t, J ¼ 7.7 Hz,
126.86, 57.89, 57.65, 47.24, 12.07. HRMS calcd for
C26H33N2
1H), 7.32 (s,1H), 7.31 (s,1H), 3.73 (t, 8H), 3.66 (s, 4H), 2.51 (t, 8H). 13
C
373.26383 [MþH]þ, found 373.26385.
NMR (100 MHz, CDCl3) d 157.90, 136.86, 121.56, 67.14, 65.02, 53.92.
HRMS calcd for C15H24O2N3 278.18630 [MþH]þ, found 278.18562.
4.1.2.13. N,N0-(1,4-Phenylenebis(methylene))bis(N-benzyl-1-
cyclopropylmethanamine) (Im). Colorless liquid, yield 80%. 1H NMR
4.1.3.4. N,N0-(Pyridine-2,6-diylbis(methylene))bis(N-methylaniline)
(400 MHz, CDCl3)
4H), 7.19e7.24 (m, 2H), 3.65 (s, 4H), 3.64 (s, 4H), 2.33 (s, 2H), 2.31 (s,
2H), 0.88e0.97 (m, 2H), 0.43e0.48 (m, 4H), 0.01e0.05 (m, 4H). 13
NMR (100 MHz, CDCl3) 140.42, 138.68, 128.89, 128.68, 128.29,
d
7.38e7.41 (m, 4H), 7.32 (s, 4H), 7.28e7.32 (m,
(IId). White solid, yield 75%, m.p. 91e93 ꢂC. 1H NMR (400 MHz,
CDCl3)
d
7.49 (t, J ¼ 7.7 Hz, 1H), 7.20e7.25 (m, 4H), 7.02 (s, 1H), 7.00
C
(s, 1H), 6.70e6.75 (m, 6H), 4.65 (s, 4H), 3.13 (s, 6H). 13C NMR
d
(100 MHz, CDCl3) d 159.44, 149.41, 137.61, 129.41, 119.01, 116.82,
126.83, 58.52, 58.37, 58.16, 8.68, 4.17. HRMS calcd for C30H37N2
112.32, 59.00, 39.24. HRMS calcd for C21H24N3 318.19647 [MþH]þ,
425.29513 [MþH]þ, found 425.29508.
found 318.19664.
4.1.2.14. N,N0-(1,4-Phenylenebis(methylene))bis(N-methyl-1-(pyr-
4.1.3.5. N,N0-(Pyridine-2,6-diylbis(methylene))bis(4-fluoro-N-meth-
ylaniline) (IIe). Colorless liquid, yield 70%. 1H NMR (400 MHz,
idin-2-yl)methanamine) (In). Colorless liquid, yield 82%. 1H NMR
(400 MHz, CDCl3)
d
8.54 (dd, J ¼ 1.8, 0.9 Hz, 1H), 8.53 (dd, J ¼ 1.8,
CDCl3)
J ¼ 0.8 Hz, 1H), 6.90e6.95 (m, 4H), 6.63e6.66 (m, 4H), 4.59 (s, 4H),
3.08 (s, 6H). 13C NMR (101 MHz, CDCl3)
159.34, 156.83, 154.49,
d
7.52 (t, J ¼ 7.7 Hz, 1H), 7.03 (t, J ¼ 0.8 Hz, 1H), 7.01 (t,
1.0 Hz, 1H), 7.66 (td, J ¼ 7.6, 1.8 Hz, 2H), 7.50e7.52 (m, 2H), 7.33 (s,
4H), 7.13e7.16 (m, 2H), 3.69 (s, 4H), 3.57 (s, 4H), 2.24 (s, 8H). 13C
d
NMR (100 MHz, CDCl3)
d
159.87, 149.09, 137.83, 136.54, 128.99,
146.14, 137.64, 129.87, 128.55, 119.19, 115.86, 115.64, 113.45, 113.38,
59.63, 39.85. HRMS calcd for C21H22F2N3 354.17763 [MþH]þ, found
354.17776.
123.08, 122.02, 63.56, 61.98, 42.67. HRMS calcd for C22H27N4
347.22302 [MþH]þ, found 347.22275.
4.1.2.15. N,N0-(1,4-Phenylenebis(methylene))bis(N-methyl-1-(pyr-
4.1.3.6. N,N0-(Pyridine-2,6-diylbis(methylene))bis(1-(2-
idin-3-yl)methanamine) (Io). Colorless liquid, yield 79%. 1H NMR
chlorophenyl)-N-methylmethanamine) (IIf). Colorless liquid, yield
(400 MHz, CDCl3)
d
8.57 (d, 2H), 8.50 (d, J ¼ 1.7 Hz, 1H), 8.49 (d,
80%. 1H NMR (400 MHz, CDCl3)
d
7.63 (t, J ¼ 7.7 Hz, 1H), 7.57 (d,
J ¼ 1.7 Hz, 1H), 7.71e7.72 (m, 1H), 7.69e7.70 (m, 1H), 7.31 (s, 4H),
J ¼ 1.8 Hz, 1H), 7.55 (d, J ¼ 1.8 Hz, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.34
7.24e7.28 (m, 2H), 3.53 (s, 4H), 3.52 (s, 4H), 2.19 (s, 6H). 13C NMR
(d, J ¼ 1.5 Hz, 1H), 7.33 (d, J ¼ 1.5 Hz, 1H), 7.15e7.25 (m, 4H), 3.77 (s,
(100 MHz, CDCl3)
d
150.42, 148.63, 137.90, 136.78, 134.88, 129.04,
4H), 3.72 (s, 4H), 2.29 (s, 6H). 13C NMR (100 MHz, CDCl3)
d 158.88,
123.55, 61.82, 59.06, 42.40. HRMS calcd for C22H27N4 347.22302
137.10, 136.75, 134.51, 130.97, 129.63, 128.32, 126.79, 121.33, 63.86,
58.85, 42.77. HRMS calcd for C23H26N3Cl2 414.14983 [MþH]þ, found
414.14968.
[MþH]þ, found 347.22288.
4.1.2.16. N,N0-(1,4-Phenylenebis(methylene))bis(N-methyl-1-(pyr-
idin-4-yl)methanamine) (Ip). Colorless liquid, yield 80%. 1H NMR
4.1.3.7. N,N0-(Pyridine-2,6-diylbis(methylene))bis(1-(3-
(400 MHz, CDCl3)
d
8.54e8.55 (m, 4H), 7.31e7.33 (m, 8H), 3.53 (s,
149.80,
chlorophenyl)-N-methylmethanamine) (IIg). Colorless liquid, yield
4H), 3.51 (s, 4H), 2.20 (s, 6H). 13C NMR (100 MHz, CDCl3)
d
77%. 1H NMR (399 MHz, CDCl3)
d
7.68 (t, J ¼ 7.7 Hz, 1H), 7.39e7.41
(m, 4H), 7.20e7.25 (m, 6H), 3.70 (s, 4H), 3.55 (s, 4H), 2.24 (s, 6H). 13
NMR (100 MHz, CDCl3) 158.91, 141.55, 137.11, 134.34, 129.65,
149.02, 137.87, 129.01, 123.94, 61.96, 60.72, 42.61. HRMS calcd for
C
C
22H27N4 347.22302 [MþH]þ, found 347.22290.
d
129.08, 127.34, 127.16, 121.27, 63.69, 61.60, 42.76. HRMS calcd for
C
23H26N2Cl2 414.14983 [MþH]þ, found 414.14902.
4.1.3. General procedure for synthesis of compounds IIaep
To a solution of 2,6-bis(chloromethyl)pyridine (1 mmol) in
anhydrous acetonitrile (10 mL) was added cesium carbonate
(3 mmol) and the corresponding secondary amine (2.2 mmol). The
reaction mixture was heated at reflux for 5 h. The resulting mixture
was diluted with water (20 mL) and extracted with ethyl acetate
(15 mL) three times. The combined organic layers were washed
with brine, dried over anhydrous sodium sulfate, and concentrated
in vacuo. The crude was purified by column chromatography with
methanol/dichloromethane (100:1, v/v) to give the title compound
as a colorless liquid or a white solid.
4.1.3.8. N,N0-(Pyridine-2,6-diylbis(methylene))bis(1-(2-
fluorophenyl)-N-methylmethanamine) (IIh). Colorless liquid, yield
79%. 1H NMR (400 MHz, CDCl3)
d
7.65 (t, J ¼ 7.7 Hz, 1H), 7.45 (td,
J ¼ 7.5, 1.9 Hz, 2H), 7.41 (s, 1H), 7.39 (s, 1H), 7.20e7.25 (m, 2H), 7.10
(td, J ¼ 7.5, 1.3 Hz, 2H), 6.99e7.04 (m, 2H), 3.73 (s, 4H), 3.66 (s, 4H),
2.26 (s, 6H). 13C NMR (100 MHz, CDCl3)
d 162.76, 160.31, 158.86,
136.98, 131.57, 131.53, 128.81, 128.73, 125.67, 125.52, 123.98, 123.94,
121.21, 115.46, 115.24, 63.59, 54.60, 54.58, 42.47. HRMS calcd for
C
23H26N3F2 382.20893 [MþH]þ, found 382.20767.

348
R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
4.1.3.9. N,N0-(Pyridine-2,6-diylbis(methylene))bis(1-(3-
(100 MHz, CDCl3) d 158.80, 150.56, 148.73, 137.16, 136.75, 134.61,
fluorophenyl)-N-methylmethanamine) (IIi). Colorless liquid, yield
123.48, 121.34, 63.60, 59.38, 42.70. HRMS calcd for C21H26N5
80%. 1H NMR (400 MHz, CDCl3)
d
7.68 (t, J ¼ 7.7 Hz, 1H), 7.42 (s, 1H),
348.21827 [MþH]þ, found 348.21814.
7.40 (s, 1H), 7.23e7.29 (m, 2H), 7.13e7.15 (m, 2H), 7.11e7.12 (m, 2H),
6.90e6.95 (m, 2H), 3.70 (s, 4H), 3.57 (s, 4H), 2.25 (s, 6H). 13C NMR
4.1.3.16. N,N0-(Pyridine-2,6-diylbis(methylene))bis(N-methyl-1-(pyr-
(100 MHz, CDCl3)
d
164.31, 161.87, 158.89, 142.10, 142.03, 137.07,
idin-4-yl)methanamine) (IIp). Colorless liquid, yield 81%. 1H NMR
129.77, 129.69, 124.49, 124.47, 121.17, 115.78, 115.56, 114.08, 113.87,
63.58, 61.59, 61.58, 42.68. HRMS calcd for C23H26N3F2 382.20893
[MþH]þ, found 382.20915.
(400 MHz, CDCl3)
(s, 1H), 7.40 (s, 1H), 7.32e7.34 (m, 4H), 3.71 (s, 4H), 3.59 (s, 4H), 2.27
(s, 6H). 13C NMR (100 MHz, CDCl3)
158.70, 149.88, 148.62, 137.25,
d
8.53e8.55 (m, 4H), 7.69 (t, J ¼ 7.7 Hz, 1H), 7.42
d
123.92, 121.35, 63.72, 60.97, 42.91. HRMS calcd for C21H26N5
4.1.3.10. N,N0-(Pyridine-2,6-diylbis(methylene))bis(1-(4-
348.21827 [MþH]þ, found 348.21813.
fluorophenyl)-N-methylmethanamine) (IIj). Colorless liquid, yield
77%. 1H NMR (400 MHz, CDCl3)
d
7.66 (t, J ¼ 7.7 Hz, 1H), 7.39 (s, 1H),
7.37 (s, 1H), 7.30e7.35 (m, 4H), 6.96e7.02 (m, 4H), 3.68 (s, 4H), 3.54
(s, 4H), 2.22 (s, 6H). 13C NMR (100 MHz, CDCl3)
163.28, 160.84,
4.2. Primary binding affinity screening
d
For binding affinity assay, 2 ꢃ 104 MDA-MB-231 cells in 300
mL
158.94, 136.96, 134.86, 134.83, 130.55, 130.47, 121.14, 115.21, 115.00,
63.44, 61.34, 42.54. HRMS calcd for C23H26N3F2 382.20893 [MþH]þ,
found 382.20773.
of cell culture medium were seeded in an 8-well slide chamber 2
days before the experiments were conducted. Various concentra-
tions of different compounds (1, 10, 100, or 1000 nM) were added to
the separate wells and incubated for 10 min at room temperature,
and then the cells were fixed in 4% ice-cold paraformaldehyde. The
cells were rehydrated in phosphate-buffered saline (PBS). The
slides were subsequently incubated for 30 min at room tempera-
4.1.3.11. N,N0-(Pyridine-2,6-diylbis(methylene))bis(1-(3-
methoxyphenyl)-N-methylmethanamine) (IIk). Colorless liquid,
yield 90%. 1H NMR (400 MHz, CDCl3)
d
7.65 (dd, J ¼ 7.9, 7.4 Hz, 1H),
7.42 (s, 1H), 7.40 (s, 1H), 7.20e7.24 (m, 2H), 6.94e6.97 (m, 4H), 6.80
(dd, J ¼ 2.7, 1.4 Hz,1H), 6.78 (dd, J ¼ 2.7, 1.4 Hz,1H), 3.81 (s, 6H), 3.69
ture with 0.05 mg/mL biotinylated TN14003, washed three times
with PBS, and incubated in streptavidin-rhodamine (1:150 dilution;
Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min
at room temperature. Finally, the slides were washed with PBS and
mounted in an anti-fade mounting solution (Molecular Probes,
Eugene, OR), and the samples were analyzed on a Nikon Eclipse
E800 microscope [31,34].
(s, 4H), 3.56 (s, 4H), 2.25 (s, 6H). 13C NMR (100 MHz, CDCl3)
d 159.79,
159.13, 141.01, 137.03, 129.35, 121.45, 121.14, 114.49, 112.61, 63.63,
62.24, 55.38, 42.81. HRMS calcd for C25H32N3O2 406.24890 [MþH]þ,
found 406.24852.
4.1.3.12. N,N0-(Pyridine-2,6-diylbis(methylene))bis(N-benzylethan-
amine) (IIl). Colorless liquid, yield 86%. 1H NMR (400 MHz, CDCl3)
4.3. Matrigel invasion assay
d
7.62 (t, J ¼ 7.7 Hz, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 7.36e7.39 (m, 4H),
7.26e7.32 (m, 4H), 7.19e7.23 (m, 2H), 3.72 (s, 4H), 3.62 (s, 4H), 2.55
Matrigel invasion assay was performed by using a Matrigel in-
(q, J ¼ 7.1 Hz, 4H), 1.08 (t, J ¼ 7.1 Hz, 6H). 13C NMR (100 MHz, CDCl3)
vasion chamber from Corning Biocoat (Bedford MA). CXCL12
a
d
159.75, 139.86, 136.84, 128.88, 128.28, 126.89, 120.61, 59.87, 58.16,
(200 ng/mL; R & D Systems, Minneapolis, MN) was added to the
bottom chamber to induce the invasion of MDA-MB-231 cells
through the Matrigel. The selected compounds or AMD3100 were
added to the cells (100 nM) before the cells were seeded in the top
chamber. The Matrigel invasion chamber was incubated for 22 h in
a humidified cell culture incubator. First, non-invading cells were
removed from the top of the Matrigel with a cotton-tipped swab.
Invading cells on the filter at the bottom of the Matrigel were fixed
in methanol and stained with hematoxylin and eosin (H&E). The
percent of invasion was determined by counting the H&E stained
cells [31,34].
47.80, 12.15. HRMS calcd for C25H32N3 374.25907 [MþH]þ, found
374.25907.
4.1.3.13. N,N0-(Pyridine-2,6-diylbis(methylene))bis(N-benzyl-1-
cyclopropylmethanamine) (IIm). Colorless liquid, yield 72%. 1H NMR
(400 MHz, CDCl3)
d
7.64 (dd, J ¼ 8.1, 7.2 Hz, 1H), 7.50 (s, 1H), 7.48 (s,
1H), 7.40e7.42 (m, 4H), 7.28e7.33 (m, 4H), 7.20e7.24 (m, 2H), 3.84
(s, 4H), 3.71 (s, 4H), 2.40 (s, 2H), 2.38 (s, 2H), 0.89e0.99 (m, 2H),
0.43e0.48 (m, 4H), 0.01e0.07 (m, 4H). 13C NMR (100 MHz, CDCl3)
d
159.88, 140.08, 136.85, 128.88, 128.33, 126.92, 120.66, 60.34, 59.01,
58.71, 8.72, 4.15. HRMS calcd for C29H36N3 426.29037 [MþH]þ,
found 426.28990.
4.4. Paw inflammation suppression test
4.1.3.14. N,N0-(Pyridine-2,6-diylbis(methylene))bis(N-methyl-1-(pyr-
Acute inflammation was induced by subcutaneous injection of
idin-2-yl)methanamine) (IIn). Colorless liquid, yield 75%. 1H NMR
50
of the female C57BL/6J mice (Jackson Laboratories); the other hind
paw was injected with 50 L of saline, which was used as a non-
inflammation control. The selected compounds were dissolved in
10% DMSO and 90% of 45% (2-hydroxypropyl)- -cyclodextrin (CD)
mL of l-carrageenan (1% w/v in saline) into one of the hind paws
(400 MHz, CDCl3)
d
8.51 (dd, J ¼ 1.8, 0.9 Hz, 1H), 8.50 (dd, J ¼ 1.8,
0.9 Hz, 1H), 7.64 (d, J ¼ 1.7 Hz, 1H), 7.62 (d, J ¼ 1.7 Hz, 1H), 7.60 (d,
J ¼ 1.7 Hz, 1H), 7.49 (t, J ¼ 7.8, 1.1 Hz, 1H), 7.47 (t, J ¼ 7.8, 1.1 Hz, 1H),
7.38 (s, 1H), 7.36 (s, 1H), 7.13 (dd, J ¼ 4.9, 1.2 Hz, 1H), 7.11 (dd, J ¼ 4.9,
1.2 Hz,1H), 3.74 (s, 4H), 3.73 (s, 4H), 2.27 (s, 6H). 13C NMR (100 MHz,
m
b
in PBS. In the treatment group, compounds IIg, IIk, IIm and IIn
CDCl3)
d
159.31, 158.63, 149.17, 137.03, 136.60, 123.23, 122.15, 121.41,
were administered intraperitoneally (i.p.) at 10 mg/kg daily, while
63.70, 63.63, 42.83. HRMS calcd for C21H26N5 348.21827 [MþH]þ,
TN14003 was administered at 300
mg/kg, i.p., daily, 30 min
found 348.21800.
following carrageenan challenge. The rationale for using 300
mg/kg
for TN14003 was that we found this concentration to be the min-
imum concentration needed to achieve the maximum efficacy of
this compound in a breast cancer metastasis animal model [41].
Control animals received corresponding i.p. injections of the
vehicle. The animals were sacrificed 74 h after carrageenan chal-
lenge and 2 h after the last injection of the selected compounds. The
final paws were photographed and measured for thickness from
4.1.3.15. N,N0-(Pyridine-2,6-diylbis(methylene))bis(N-methyl-1-(pyr-
idin-3-yl)methanamine) (IIo). Colorless liquid, yield 74%. 1H NMR
(400 MHz, CDCl3)
d
8.59 (d, J ¼ 0.8 Hz, 1H), 8.59 (d, J ¼ 0.8 Hz, 1H),
8.50 (d, J ¼ 1.7 Hz,1H), 8.49 (d, J ¼ 1.7 Hz,1H), 7.72 (t, J ¼ 2.0 Hz,1H),
7.70 (t, J ¼ 2.0 Hz,1H), 7.67 (t, J ¼ 7.7 Hz,1H), 7.40 (s,1H), 7.38 (s,1H),
7.28e7.23 (m, 2H), 3.71 (s, 4H), 3.59 (s, 4H), 2.25 (s, 6H). 13C NMR

R. Bai et al. / European Journal of Medicinal Chemistry 118 (2016) 340e350
349
J.A. Burger, Small peptide inhibitors of the CXCR4 chemokine receptor
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the “palm” to the back of the paw with a caliper. These were
compared to the volume of carrageenan-untreated contralateral
paw to obtain the edema volume; the volume of the contralateral
paw was subtracted from the volume of the carrageenan injected
paw to obtain the edema volume. The inflammation-suppression
percentage was calculated by comparing the drug-treated group
to the control group. Five mice per group were used to determine
the effect of the CXCR4 inhibitors as previously described [27,31].
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4.5. Molecular modeling (docking) studies
The docking experiment was performed by Schrodinger
Maestro Package [42]. The crystal structure of CXCR4 (PDB code:
3ODU [40]) was processed following the established Protein Pre-
pare Wizard workflow to obtain the receptor structure suitable for
docking [43]. All water molecules were deleted and only Chain A
was used to prepare the receptor. All residues beyond 20 Å of the
ligand were removed to accelerate the calculation speed. The
binding site was selected using the default Receptor Grid Genera-
tion procedure and the docking grid was generated around the co-
crystalized small molecule ligand IT1t. The IIn ligand was initially
drawn by ChemDraw as a 2D structure and then prepared by the
Ligprep application to obtain the 3D conformers for docking [44].
The prepared ligand was then flexibly docked onto the receptor
using Glide [45] and the default setting parameters with no con-
strains. 10 docking poses were obtained and the one with the best
Glide score was chosen to study the interaction. The plot (Fig. 8)
was constructed using Pymol version 0.99 [46].
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gastrointestinal system, Ann. Clin. Lab. Sci. 28 (1998) 67e81.
Acknowledgments
[22] D.H. Solomon, Selective cyclooxygenase
2 inhibitors and cardiovascular
events, Arthritis Rheum. 52 (2005) 1968e1978.
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type 4: putative therapeutic approaches in inflammatory diseases, Curr. Opin.
Hematol. 21 (2014) 29e36.
This study was financially supported by a research grant from
NIH NCI (R01 CA165306). We thank Ms. Jessica Paulishen and Mr.
Andrew Teodorescu for proof-reading.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.040.
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