Anti-inflammatory hybrids of secondary amines and amide-sulfamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.02.085

The CXCR4/CXCL12 chemokine axis can chemotactically accumulate inflammatory cells to local tissues and regulate the release of inflammatory factors. Developing novel CXCR4 modulators may provide a desirable strategy to control the development of inflammation. A series of novel hybrids were designed by integrating the key pharmacophores of three CXCR4 modulators. The majority of compounds displayed potent CXCR4 binding affinity. Compound 7a exhibited 1000-fold greater affinity than AMD3100 and significantly inhibited invasion of CXCR4-positive tumor cells. Additionally, compound 7a blocked mice ear inflammation by 67% and suppressed the accumulation of inflammatory cells in an in vivo mouse ear edema evaluation. Western blot analyses revealed that 7a inhibited the CXCR4/CXCL12-mediated phosphorylation of Akt and p44 in a dose-dependent manner. Moreover, compound 7a had no observable cytotoxicity and displayed a favorable plasma stability in our preliminary pharmacokinetic study. These results confirmed that this is a feasible method to develop CXCR4 modulators for the regulation and reduction of inflammation.

Full text of DOI:10.1016/j.ejmech.2018.02.085

European Journal of Medicinal Chemistry 150 (2018) 195e205
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Anti-inflammatory hybrids of secondary amines and amide-sulfamide
derivatives
, b,
Renren Bai a **, Jian Sun a, Zhongxing Liang b, Younghyoun Yoon b, Eric Salgado b,
, c, d, *
Amber Feng b, Yoonhyeun Oum b, Yuanyuan Xie a, Hyunsuk Shim b
a College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
b Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia, USA
c Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
d Department of Radiology and Imaging Sciences, Emory University School of Medicine, Atlanta, Georgia, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The CXCR4/CXCL12 chemokine axis can chemotactically accumulate inflammatory cells to local tissues
and regulate the release of inflammatory factors. Developing novel CXCR4 modulators may provide a
desirable strategy to control the development of inflammation. A series of novel hybrids were designed
by integrating the key pharmacophores of three CXCR4 modulators. The majority of compounds dis-
played potent CXCR4 binding affinity. Compound 7a exhibited 1000-fold greater affinity than AMD3100
and significantly inhibited invasion of CXCR4-positive tumor cells. Additionally, compound 7a blocked
mice ear inflammation by 67% and suppressed the accumulation of inflammatory cells in an in vivo
mouse ear edema evaluation. Western blot analyses revealed that 7a inhibited the CXCR4/CXCL12-
mediated phosphorylation of Akt and p44 in a dose-dependent manner. Moreover, compound 7a had
no observable cytotoxicity and displayed a favorable plasma stability in our preliminary pharmacokinetic
study. These results confirmed that this is a feasible method to develop CXCR4 modulators for the
regulation and reduction of inflammation.
Received 12 January 2018
Received in revised form
15 February 2018
Accepted 27 February 2018
Available online 2 March 2018
Keywords:
CXCR4
Hybrids
Amide-sulfamide
Inflammation
Inflammatory cell accumulation
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
chemokine ligand 12 (CXCL12) axis has been shown to be involved
in various pathological conditions, including HIV infection, cancer
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the
most frequently administered drugs due to their anti-
inflammatory, analgesic, and antipyretic properties [1]. Nonselec-
tive NSAIDs show unfavorable dose-dependent gastric mucosal
injury side effects due to their inhibition of biosynthesis of neces-
sary PGs by COX-1 in the stomach. Selective COX-2 inhibitors
reduce the risk of gastric ulcers compared with nonselective
NSAIDs, but they have been associated with an increased risk of
cardiovascular side effects, including myocardial infarction, stroke,
and hypertension [1-3].
and inflammation [4, 5]. Evidence has shown that the CXCR4/
CXCL12 chemokine axis can chemotactically accumulate inflam-
matory cells (neutrophils, monocytes, and lymphocytes) to local
tissues and regulate the release of inflammatory factors that cause
inflammatory responses [6, 7]. Therefore, developing novel anti-
inflammatory agents targeting CXCR4 may be a desirable strategy
to avoid the side effects of NSAIDs.
As shown in Fig. 1, AMD3100 is the first small molecule CXCR4
antagonist to enter clinical trials for the treatment of HIV infection
[8]. However, AMD3100 was not approved due to poor oral
bioavailability and serious cardiotoxicity [9-11]. Taking AMD3100
as the lead compound, our research group discovered a promising
bis-secondary amine drug candidate, Q-122, which is under Phase II
clinical trials. Subsequent work mainly focused on modifying the
two amino groups of the secondary amine structure to obtain
compound RB-55 with better CXCR4 inhibitory activity, proving
that the amide- sulfamide structure is a new, promising scaffold
targeting CXCR4 [12].
The C-X-C chemokine receptor type
4
(CXCR4)/C-X-C
* Corresponding author. Department of Radiation Oncology, Emory University
School of Medicine, 1701 Uppergate Drive, C5018, Atlanta, GA, 30322, USA.
** Corresponding author. College of Pharmaceutical Sciences, Zhejiang University
of Technology, 18 Chaowang Road, Hangzhou, Zhejiang, 310014, China.
E-mail addresses: renrenbai@zjut.edu.cn, renren.bai@emory.edu (R. Bai),
hshim@emory.edu (H. Shim).
https://doi.org/10.1016/j.ejmech.2018.02.085
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

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Fig. 1. Strategy for the discovery of novel amide-sulfamide anti-inflammatory agents.
The key pharmacophores of compounds AMD3100, Q-122, and
exhibited 1000-fold more potent activity than AMD3100. In terms
of the benzenesulfonyl side chain, receptor affinity was obviously
increased with no substitution or substitution with an electron-
donating group (-CH3). Moreover, when substituted with
electron-withdrawing groups (-F, -NO2, -CF3), binding affinity
remained weak, as all the ECs were no less than 1000 nM. Only 3-
Cl, 4-Cl and 3,4-difluoro-substituted derivatives showed favorable
activity. More importantly, when a nitrogen atom, heterocyclic, or
aromatic ring were introduced to the benzenesulfonyl moiety, the
affinity weakened significantly.
RB-55 were incorporated into a single structure and a series of
long-chain hybrids were designed and synthesized in the following
study. The anti-CXCR4 effect, in vivo anti-inflammatory activity,
pharmacokinetic properties, and cytotoxicity were also systemati-
cally screened.
2. Results and discussion
2.1. Chemistry
The synthetic route chosen to synthesize the targeted com-
pounds was outlined in Scheme 1. Compounds 2a-q were synthe-
sized by sulfonylation of the starting material 4-(Boc-aminomethyl)
benzylamine (1) with the corresponding sulfonyl chlorides in
dichloromethane (DCM). The protective group Boc was subse-
quently removed in the presence of trifluoroacetic acid (TFA) pro-
ducing the benzylamine intermediates 3a-q. Compound 5 was
prepared by reductive amination of another starting material ter-
ephthaldicarboxaldehyde (4) with 2-amino-pyrimidine. The sub-
sequent oxidation reaction of compound 5 with Jones reagent
afforded the key intermediate 6. The final compounds 7a-q were
synthesized by the acylation of intermediate 3a-q with acid 6.
2.3. Matrigel invasion assay
Activation of CXCR4 through its ligand CXCL12 mediates
migration and invasion. Thus, a Matrigel invasion assay was per-
formed to probe whether the selected compounds with better
binding affinities to CXCR4 can also block CXCR4/CXCR12 mediated
chemotaxis and invasion [13, 15]. The target compounds and cells
were added to the upper chamber of a vessel and CXCL12 was
added to the lower chamber as a chemoattractant in serum-free
medium. Binding of CXCR4 at the cell surface with the selected
compounds would block CXCL12 chemotaxis to the cells. Therefore,
MDA-MB-231 human breast cancer cells at the top chamber treated
with the compounds would be inhibited to migrate from the top
chamber through the Matrigel-coated filter pores to the bottom of
the filter. Inhibition of cell invasion with each tested compound was
calculated by comparing results to the cell invasion without treat-
ment. The results of Matrigel invasion are summarized in Fig. 3.
All selected compounds (EC ꢀ 100 nM) effectively inhibited tu-
mor cell invasion. Compared to the negative control group, the
number of cells invading through the chamber decreased signifi-
cantly, exceeding 65% inhibitory activity (Fig. 4). The compounds’
anti-invasive activities was improved when sulfonamide side chain
was substituted. Compounds 7d (4-CH3) and 7i (3-Cl) showed the
most effective anti-invasive activity, reaching 87% and 88% inhibi-
tion, respectively, which is superior to the reference drug AMD3100
(55%). The above invasion results give eloquent proof that the
amide-sulfamide structure is a potent scaffold to block CXCR4
function.
2.2. Primary binding affinity screening
All of the prepared compounds were first screened with a
binding affinity assay as described in our previous publications [5,
12-15]. The screening protocol is a competitive CXCR4 binding
assay between biotinylated TN14003, a potent CXCR4 peptidic in-
hibitor, and the target compounds 7a-q at concentrations of 1, 10,
100, and 1000 nM. The effective concentration (EC) is used to
measure the affinity, which is defined as the lowest concentration
at which a significant reduction in the rhodamine fluorescent color
is observed as compared to control (Fig. 3, without CXCR4 modu-
lators). Thus, this initial screening is a semi-quantitative, primary
screening of the level of activity, which is different from IC50
.
The vast majority of compounds showed comparable or even
better CXCR4 binding affinity than that of AMD3100 (Table 1 and
Fig. 2). Compound 7b displayed 100-fold, and compounds 7a and 7i

R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
197
Scheme 1. Reagents and conditions: (a) DCM, TEA, ice bath to r.t., 6 h, 70e90%; (b) i. DCM, TFA, r.t., 8 h; ii. NaHCO3, 90e95%; (c) NaBH(OAc)3, HOAc, ClCH2CH2Cl, r.t., 12 h, 82%; (d)
Jones reagent (2 M CrO3 in aqueous H2SO4), ice bath, 1 h, 75%; (e) DCM, TEA, HOBT, EDCI, r.t., 16 h, 68e85%.
2.4. In vivo suppression against xylene-induced ear edema
A xylene-induced ear edema experiment was used to evaluate
the in vivo anti-inflammatory activity of the amide-sulfamide
compounds [16]. This xylene-induced ear edema model is widely
used in the evaluation of inflammatory activity. The application of
xylene induces the release of substance P from sensory neurons,
leading to vasodilatation and plasma extravasations, which subse-
quently causes ear swelling in mice [17].
Compounds achieving greater than 70% inhibition in the
Matrigel assay were evaluated in the ear edema test. Although
AMD3100 is the best investigated small molecule CXCR4 antago-
nist, its bicyclam structure leads to serious toxicity in this animal
model. Therefore, AMD3100 was not selected as the reference drug
in this test [18]. Compounds 7d, 7i and 7l were not effective in the
Table 1
Preliminary effective concentration (EC) of anti-CXCR4 compounds.
Compd
EC (nM)
Compd
EC (nM)
7a
7b
7c
7d
7e
7f
7g
7h
7i
1
10
7j
7k
7l
7m
7n
7o
7p
7q
100
1000
100
1000
>1000
1000
1000
1000
1000
100
100
1000
1000
>1000
1000
1
AMD3100

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R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
Fig. 2. Representative immunofluorescence images of competitive-binding affinity assay of three selected compounds compared to AMD3100. CXCR4 receptors on the cell surface
are illustrated via the red fluorescent color in this binding affinity assay using biotinylated TN14003 that binds to CXCR4. When our test compounds are preincubated with the cells
and block the binding of biotinylated TN14003, the red fluorescent color is reduced. The EC of AMD3100 was 1000 nM, while compounds 7a, 7b, 7i and 7j showed EC of only 1, 10, 1
and 100 nM, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
treatment of inflammation (Fig. 5), while 7d and 7j showed mod-
erate anti-inflammatory activity with 37% and 30% inhibition,
respectively. Interestingly, the unsubstituted compound 7a proved
to demonstrate the most potent anti-inflammatory activity with
67% inhibition.
2.5. Evaluation of anti-CXCR4 activity of compound 7a at the
molecular level
2.5.1. Compound 7a suppressed CXCL12 induced phosphorylation of
Akt
Histological analysis was performed to better study the micro-
cosmic effect of compound 7a on inflammatory tissues. After
treatment with compound 7a, the swollen volume and thickness of
the mouse ear were significantly reduced (Fig. 6). Notably, the
number of inflammatory cells in inflammatory tissues was also
significantly decreased, which further propagates the anti-
inflammatory mechanism whereby blocking CXCR4 could effec-
tively inhibit the recruitment of inflammatory cells to the site of
inflammation.
Phosphoinositide 3-kinase (PI3K) activates Akt, a serine threo-
nine kinase which plays a key role in tumor cell survival and
possibly proliferation. PI3K/Akt pathways are independently
involved in the proliferative signal mediated by CXCL12 [19]. Our
previous results demonstrated that CXCR4/CXCL12 activation in-
duces Akt phosphorylation, which results in tumor angiogenesis
and progression of tumors by increasing expression of vascular
endothelial growth factor (VEGF) [20]. The ability of 7a to block the
PI3K/Akt pathway was investigated via Western blot analysis. As

R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
199
Compound 7a formed two hydrogen bonds with CXCR4, with the N
atom in sulfonamide to Gln200 and with the N atom in amide to
Glu288, respectively. In addition, the benzene ring in the amide
side chain showed a
pyrimidine ring displayed another
p
-
p
stacking with Trp94 and Tyr116, and the
stacking with Trp102 and
p- p
His113. These favorable interactions are likely contributing to the
binding of 7a with CXCR4.
2.7. Preliminary pharmacokinetic study of compound 7a
The plasma stability of a drug has a significant influence on the
concentration of active drug available in circulation. To determine
the pharmacokinetics of this novel amide-sulfamide structure, the
concentration and stability of compound 7a in plasma were eval-
uated in a mouse model. The peak area of 7a in plasma was
detected by high-performance liquid chromatography (HPLC) at
three time points: 5, 20 and 50 min. Generally, compound 7a
demonstrated a moderate plasma stability (Fig. 9). The approxi-
mate half-life (t1/2) was calculated to be ~20 min. However, the
basal metabolic rate per gram of body weight (the mass-specific
rate) is seven times greater in mice than in humans [23]. There-
fore, we inferred that t1/2 of 7a in human is about 2.5 h, exhibiting
acceptable plasma stability.
Fig. 3. Matrigel invasion assay results induced by CXCR4/CXCL12-mediated interaction
using MDA-MB-231 cells. CXCL12 was added to the bottom chamber, and the com-
pounds were added to the top chamber. After incubating for 22 h, the invading cells
were fixed in methanol and stained with hematoxylin and eosin. The percent of in-
vasion was determined by counting the stained cells.
a
shown in Fig. 7A, CXCR4 modulator 7a significantly inhibited
phosphorylation of Akt in a dose-dependent manner, with an active
concentration of only 1 nM.
2.5.2. Compound 7a suppressed CXCL12 induced phosphorylation of
p44/42
The interaction between CXCL12 and CXCR4 has also been
shown to activate the extracellular signal-regulated kinase-1/2
(ERK-1/2 or also known as p44/42) in various cell types [21]. The
CXCR4 inhibitory activity of compound 7a was verified by exam-
ining its ability to block phosphorylation of p44/42. Western blot
analysis proved that compound 7a suppressed the phosphorylation
of p44 effectively with a concentration of only 0.1 nM (Fig. 7B).
2.6. Molecular modeling (docking) study
To better understand the possible interaction between 7a and
CXCR4, Schrodinger Maestro Package was performed based on the
available crystal structure of CXCR4 (PDB code: 3ODU [22]), the
docking procedure was reported in our previous publication [13].
Fig. 8 illustrated the binding pose with the best docking score.
Fig. 5. In vivo anti-inflammatory test using a xylene-induced ear edema model. The
right ear of each mouse was treated with 30 mL xylene. The selected compounds were
administered intraperitoneally (i.p.) 30 min later at 10 mg/kg. Two hours later, one ear
plug was removed from both ears and weighed to calculate the percentage of in-
flammatory suppression.
Fig. 4. Micrographs of Matrigel invasion assay induced by CXCR4/CXCL12-mediated interaction using MDA-MB-231 cells in the presence of CXCR4 modulators. The number of
invading cells to the bottom chamber decreased when cells were incubated with our compounds in the top chamber.

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R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
Fig. 6. Histological analysis of the anti-inflammatory activity of compound 7a. Whole tissue slices were scanned/digitized by NanoZoomer 2.0 HT. Software NDP.view 2 was used to
zoom in.
MDA-MB-231and MCF-10A cells even at concentrations as high as
10 mM (Fig. 10), a 1000-fold increase to the working concentrations.
Generally, 7a had no observable cytotoxicity.
3. Conclusion
Taking the first small molecule CXCR4 inhibitor entering clinical
trials, AMD3100, together with two drug candidates discovered by
our research group, Q-122 and RB-55, as the lead compounds, a
series of hybrids were designed by integrating the key pharmaco-
phores of these three CXCR4 modulators. This novel amide-
sulfamide scaffold maintained significant CXCR4 inhibitory capac-
itively, and the majority of compounds displayed potent CXCR4
binding affinity. Compounds 7a and 7i exhibited 1000-fold greater
affinity than AMD3100. All of the compounds with favorable
binding affinity significantly suppressed invasion of CXCR4 positive
tumor cells. In the in vivo mouse ear edema evaluation, compound
7a demonstrated the best anti-inflammatory activity, effectively
blocking the aggregation of inflammatory cells to the site of
inflammation. Western blot analyses revealed that 7a inhibited the
CXCR4/CXCL12-mediated phosphorylation of Akt and p44 in a
dose-dependent manner. Compound 7a also exhibited acceptable
plasma stability in the preliminary pharmacokinetic study. In the
cytotoxicity screening, 7a did not inhibit the proliferation of all the
Fig. 7. Compound 7a blocked the phosphorylation of Akt and p44/42 mediated by
CXCR4/CXCL12 axis.
2.8. Cytotoxicity evaluation of compound 7a
Cytotoxic agents have the potential to cause serious destruction
to healthy and normal cells. To preliminarily evaluate the safety of
compound 7a, its cytotoxicity was evaluated on two representative
cell lines, MDA-MB-231 (CXCR4-positive), SKBR3 (CXCR4-positive),
MCF-7 (CXCR4-positive), AC1-ExR (CXCR4-positive) and 184A1N4
(CXCR4-negative) cells, by a cell viability (MTT) assay. Of note,
blocking CXCR4 should not impact adherent cell proliferation on
petri dish [24]. Compound 7a displayed potent CXCR4 binding af-
finity at only 1 nM, however, it did not inhibit the proliferation of
tumor cell lines even at 10 mM, showing no observable cytotoxicity.
In summary, the sulfamide pharmacophore was proved to be an
effective isostere of amine and amide groups because of the simi-
larity in chemical properties. This modification is a promising
strategy for the further design of CXCR4 modulators. The novel
hybrids exhibited potent CXCR4 inhibitory activity both in vitro and
in vivo, and showed reasonable metabolic stability with no cyto-
toxicity. The designed modulators could effectively inhibit the ac-
tivity of CXCR4, significantly suppressing the accumulation of the

R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
201
Fig. 8. A low energy predicted binding pose for compound 7a in the CXCR4 X-ray structure.
Fig. 9. The plasma stability of compound 7a in mouse serum. Nude mice received a single dose (30 mg/kg) of compound 7a via intravenous injection (i.v.). Blood samples were
collected at 0, 5, 20 and 50 min. The blood sample was centrifuged and filtrated and analyzed by HPLC to estimate the amount of compound in serum.
Fig. 10. Cytotoxicity evaluation of compound 7a in MDA-MB-231, SKBR3, MCF-7, AC1-ExR and 184A1N4 cell lines. The antiproliferative activity of the compounds was determined
using MTT assay. Cells were treated with Compound 7a or vehicle control for 72 h. The results showed no statistically significant differences, which suggests that 7a does not have
antiproliferative activity.

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R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
inflammatory cells to the inflammatory sites. These results
confirmed that developing CXCR4 modulators to control and
reduce inflammation is a feasible and effective strategy for the
treatment of inflammation.
added to adjust to pH ¼ 7. Then the mixture was filtered and
extracted with ethyl acetate (3 ꢂ 10 mL). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude was purified by column chro-
matography with DCM/methanol (150:1, v/v) to give the interme-
diate 6 as a white solid.
4. Experimental section
4.1. Chemistry
4.1.6. General procedure for synthesis of target compounds 7a-q
To a solution of intermediate 3 (1.0 mmol), hydroxybenzo-
triazole (HOBT, 1.2 mmol) , 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCI, 12 mmol) and TEA (3.0 mmol) in anhydrous
DCM (8 mL), acid intermediate 6 (1 mmol) was added. The reaction
mixture was stirred for 16 h at room temperature, then diluted with
saturated aqueous NaHCO3 and extracted with DCM (10 mL) three
times. The combined DCM layer was sequentially washed with
water and brine, dried with anhydrous Na2SO4, and concentrated in
vacuo. The crude was purified by column chromatography with
DCM/methanol to give the compound as a white solid.
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 DMSO-d6 were referenced to the
residual solvent peak. Chemical shifts (d) are reported in parts per
million (ppm) relative to residual undeuterated solvent as an in-
ternal reference. Mass spectra were recorded on a JEOL spectrom-
eter at Emory University Mass Spectrometry Center. Analytical thin
layer chromatography (TLC) was performed on precoated glass
backed plates from Scientific Adsorbents Incorporated (Silica Gel 60
F254; 0.25 mm thickness).
4.1.6.1. N-(4-(phenylsulfonamidomethyl)benzyl)-4-((pyrimidin-2-
ylamino)methyl)benzamide (7a). White solid, yield 85%, m.p.
4.1.2. General procedure for synthesis of intermediate 2a-q
189e191 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.95 (t, J ¼ 6.0 Hz, 1H),
A solution of 4-(Boc-aminomethyl)benzylamine (1) (1.0 mmol)
and TEA (3.0 mmol) in anhydrous DCM (8 mL) was cooled with an
ice bath, then the corresponding sulfochloride (1.1 mmol, dissolved
in 2 mL anhydrous DCM) was added dropwise. The reaction
mixture was allowed to stir at 0 ꢁC for 1 h. After removing the
cooling bath, the resulting mixture was stirred for 5 h at room
temperature, then diluted with saturated aqueous NaHCO3 and
extracted with DCM (10 mL) for three times. The combined organic
layer was sequentially washed with water and brine, dried with
anhydrous Na2SO4, and concentrated in vacuo. The crude was pu-
rified by column chromatography with DCM/methanol (150:1, v/v)
to yield the product as a white solid [5].
8.26 (d, J ¼ 4.8 Hz, 2H), 8.12 (t, J ¼ 6.3 Hz, 1H), 7.75e7.81 (m, 5H),
7.54e7.61 (m, 3H), 7.37 (d, J ¼ 8.3 Hz, 2H), 7.20 (d, J ¼ 8.3 Hz, 2H),
7.16 (d, J ¼ 8.2 Hz, 2H), 6.58 (t, J ¼ 4.8 Hz,1H), 4.53 (d, J ¼ 6.4 Hz, 2H),
4.41 (d, J ¼ 6.0 Hz, 2H), 3.93 (d, J ¼ 6.2 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6)
d 166.00, 162.20, 157.96, 143.85, 140.66, 138.69, 135.99,
132.65, 132.27, 129.13, 127.51, 127.15, 127.06, 126.71, 126.40, 110.33,
45.89, 43.67, 42.25. HRMS calcd for C26H26O3N5S 488.17509 [M þ
H]þ, found 488.17597.
4.1.6.2. N-(4-(((2-methylphenyl)sulfonamido)methyl)benzyl)-4-
((pyrimidin-2-ylamino)methyl)benzamide (7b). White solid, yield
81%, m.p. 156e158 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 8.95 (t,
J ¼ 6.1 Hz, 1H), 8.26 (d, J ¼ 4.7 Hz, 2H), 8.20 (s, 1H), 7.76e7.82 (m,
4H), 7.45e7.50 (m, 1H), 7.32e7.38 (m, 4H), 7.12e7.20 (m, 4H), 6.58
(t, J ¼ 4.8 Hz, 1H), 4.54 (d, J ¼ 6.4 Hz, 2H), 4.41 (d, J ¼ 6.0 Hz, 2H),
4.1.3. General procedure for synthesis of intermediate 3a-k
A solution of intermediates 2 (1.0 mmol) in DCM (10 mL) was
treated with trifluoroacetic acid (4 mmol) at room temperature.
The resulting mixture was stirred for 8 h. The solvent was removed
under reduced pressure. The residue was dissolved in saturated
aqueous NaHCO3 (2 mL) followed by adding more saturated
aqueous NaHCO3 to adjust to pH ¼ 10. Then, the mixture was
filtered and the intermediate 5 was obtained as the filter cake
without further purification [5].
3.97 (s, 2H), 2.55 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
d 165.98,
162.19, 157.93, 143.83, 138.88, 138.60, 136.39, 136.27, 132.65, 132.38,
132.26, 128.28, 127.41, 127.14, 126.99, 126.69, 126.10, 110.32, 45.58,
43.66, 42.24, 19.77. HRMS calcd for C27H28O3N5S 502.19074 [M þ
H]þ, found 502.19088.
4.1.6.3. N-(4-(((3-methylphenyl)sulfonamido)methyl)benzyl)-4-
((pyrimidin-2-ylamino)methyl)benzamide (7c). White solid, yield
4.1.4. Procedure for synthesis of intermediate 5
80%, m.p. 126e128 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 8.95 (t,
To a solution of terephthaldicarboxaldehyde (4, 1.0 mmol) and
2-amino-pyrimidine (1 mmol) in 1,2-dichloroethane (10 mL), acetic
acid (2 mmol) and 4 Å molecular sieves (0.5 g) were added, and the
mixture was stirred at room temperature until 2-amino-pyrimidine
completely dissolved. The resulting mixture was then treated with
sodium triacetoxyborohydride (3 mmol). After being stirred for
12 h at room temperature under an argon atmosphere, the reaction
was quenched by adding aqueous NaOH (10 mL, 1.0 N). The
resulting mixture was extracted with ethyl acetate (3 ꢂ 10 mL), and
the combined organic layers were washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude was pu-
rified by column chromatography with DCM/methanol (200:1, v/v)
to give the product 5 as a white solid [25].
J ¼ 6.0 Hz, 1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 8.07 (t, J ¼ 6.2 Hz, 1H),
7.76e7.82 (m, 3H), 7.58 (d, J ¼ 5.1 Hz, 2H), 7.36e7.47 (m, 4H), 7.21 (d,
J ¼ 8.1 Hz, 2H), 7.16 (d, J ¼ 7.3 Hz, 2H), 6.57 (t, J ¼ 4.8 Hz, 1H), 4.53 (d,
J ¼ 6.4 Hz, 2H), 4.41 (d, J ¼ 5.9 Hz, 2H), 3.93 (d, J ¼ 5.9 Hz, 2H), 2.36
(s, 3H). 13C NMR (100 MHz, DMSO-d6)
d 165.99, 162.20, 157.95,
143.84, 140.58, 138.66, 136.04,132.85,132.65,128.98, 127.52, 127.48,
127.15, 127.03, 126.82, 126.70, 123.56, 110.33, 45.89, 43.66, 42.25,
20.81. HRMS calcd for C27H28O3N5S 502.19074 [M þ H]þ, found
502.18989.
4.1.6.4. N-(4-(((4-methylphenyl)sulfonamido)methyl)benzyl)-4-
((pyrimidin-2-ylamino)methyl)benzamide (7d). White solid, yield
83%, m.p. 194e196 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 8.96 (t,
J ¼ 6.0 Hz, 1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 8.03 (t, J ¼ 6.3 Hz, 1H),
7.76e7.82 (m, 3H), 7.68 (d, J ¼ 8.3 Hz, 2H), 7.37 (d, J ¼ 8.0 Hz, 4H),
7.21 (d, J ¼ 8.2 Hz, 2H), 7.17 (d, J ¼ 8.2 Hz, 2H), 6.57 (t, J ¼ 4.8 Hz, 1H),
4.53 (d, J ¼ 6.4 Hz, 2H), 4.42 (d, J ¼ 6.0 Hz, 2H), 3.90 (d, J ¼ 6.3 Hz,
4.1.5. Procedure for synthesis of intermediate 6
A solution of intermediate 5 (1.0 mmol) in acetone (8 mL) was
cooled with an ice bath. Jones reagent (4 mL, 2 M CrO3 in aqueous
H2SO4), ice bath was added dropwise. The reaction mixture was
stirred at 0 ꢁC for 30 min. Saturated aqueous NaHCO3 was then
2H), 2.37 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
d 166.00, 162.20,
157.95, 143.85, 142.53, 138.68, 137.75, 136.05, 132.65, 129.56, 127.51,

R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
203
127.16, 127.05, 126.71, 126.50, 110.33, 45.88, 43.67, 42.25, 20.92.
4.1.6.10. N-(4-(((4-chlorophenyl)sulfonamido)methyl)benzyl)-4-
HRMS calcd for C27H28O3N5S 502.19074 [M þ H]þ, found 502.19090.
((pyrimidin-2-ylamino)methyl)benzamide (7j). White solid, yield
75%, m.p. 196e198 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 8.95 (t,
J ¼ 6.0 Hz, 1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 8.23 (t, J ¼ 6.2 Hz, 1H),
7.76e7.82 (m, 5H), 7.64 (d, J ¼ 8.6 Hz, 2H), 7.37 (d, J ¼ 8.0 Hz, 2H),
7.21 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 8.2 Hz, 2H), 6.57 (t, J ¼ 4.8 Hz, 1H),
4.53 (d, J ¼ 6.1 Hz, 2H), 4.41 (d, J ¼ 5.7 Hz, 2H), 3.95 (d, J ¼ 6.2 Hz,
4.1.6.5. N-(4-(((2-fluorophenyl)sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7e). White solid, yield 78%,
m.p. 155e157 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.94 (t, J ¼ 6.0 Hz,
1H), 8.47 (s, 1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 7.71e7.82 (m, 4H),
7.58e7.64 (m, 1H), 7.29e7.38 (m, 4H), 7.18 (d, J ¼ 8.2 Hz, 2H), 7.14 (d,
J ¼ 8.4 Hz, 2H), 6.57 (t, J ¼ 4.7 Hz, 1H), 4.53 (d, J ¼ 6.3 Hz, 2H), 4.39
(d, J ¼ 6.0 Hz, 2H), 4.07 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
2H). 13C NMR (100 MHz, DMSO-d6)
d 166.01, 162.20, 157.96, 143.85,
139.57, 138.78, 137.13, 135.78, 132.64, 129.25, 128.40, 127.56, 127.16,
127.07, 126.70, 110.33, 45.88, 43.66, 42.25. HRMS calcd for
C
26H25O3N5SCl 522.13611 [M þ H]þ, found 522.13567.
d
165.99, 162.20, 159.30, 157.96, 156.78, 143.85, 138.64, 135.97,
135.02, 134.94, 132.65, 129.48, 127.38, 127.15, 127.00, 126.71, 124.72,
124.68, 117.17, 116.96, 110.33, 45.68, 43.66, 42.23. HRMS calcd for
C
4.1.6.11. N-(4-(((4-nitrophenyl)sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7k). White solid, yield 77%,
26H25O3N5SF 506.16567 [M þ H]þ, found 506.16474.
m.p. 208e210 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.94 (t, J ¼ 6.0 Hz,
1H), 8.52 (s, 1H), 8.36e8.39 (m, 2H), 8.26 (d, J ¼ 4.8 Hz, 2H),
7.99e8.02 (m, 2H), 7.75e7.81 (m, 3H), 7.37 (d, J ¼ 8.6 Hz, 2H), 7.20
(d, J ¼ 8.4 Hz, 2H), 7.15 (d, J ¼ 8.5 Hz, 2H), 6.58 (t, J ¼ 4.8 Hz,1H), 4.53
(d, J ¼ 6.4 Hz, 2H), 4.39 (d, J ¼ 6.0 Hz, 2H), 4.01 (s, 2H). 13C NMR
4.1.6.6. N-(4-(((3-fluorophenyl)sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7f). White solid, yield 75%,
m.p. 158e160 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.95 (t, J ¼ 6.1 Hz,
1H), 8.26 (d, J ¼ 4.9 Hz, 3H), 7.75e7.82 (m, 3H), 7.60e7.63 (m, 2H),
7.53e7.56 (m,1H), 7.44e7.49 (m,1H), 7.37 (d, J ¼ 8.3 Hz, 2H), 7.20 (d,
J ¼ 8.3 Hz, 2H), 7.16 (d, J ¼ 7.9 Hz, 2H), 6.58 (t, J ¼ 4.8 Hz,1H), 4.53 (d,
J ¼ 6.4 Hz, 2H), 4.41 (d, J ¼ 6.0 Hz, 2H), 3.98 (d, J ¼ 5.7 Hz, 2H). 13C
(100 MHz, DMSO-d6)
d 166.00, 162.19, 157.90, 149.42, 146.30,
143.85, 138.88, 135.53, 132.62, 128.02, 127.61, 127.14, 127.06, 126.69,
124.46, 110.32, 45.92, 43.66, 42.22. HRMS calcd for C26H25O5N6S
533.16017 [M þ H]þ, found 533.15952.
NMR (100 MHz, DMSO-d6)
d 165.99, 162.90, 162.20, 160.43, 157.95,
143.85, 138.78, 135.75, 132.64, 131.56, 131.48, 127.56, 127.15, 127.07,
126.70, 122.68, 122.65, 119.50, 119.30, 113.57, 113.33, 110.32, 45.91,
43.66, 42.23. HRMS calcd for C26H25O3N5SF 506.16567 [M þ H]þ,
found 506.16471.
4.1.6.12. N-(4-(((3,4-difluorophenyl)sulfonamido)methyl)benzyl)-4-
((pyrimidin-2-ylamino)methyl)benzamide (7l). White solid, yield
75%, m.p. 215e217 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 8.96 (t,
J ¼ 6.0 Hz, 1H), 8.20e8.31 (m, 3H), 7.76e7.82 (m, 4H), 7.62e7.66 (m,
2H), 7.37 (d, J ¼ 8.3 Hz, 2H), 7.22 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 8.1 Hz,
2H), 6.57 (t, J ¼ 4.8 Hz, 1H), 4.54 (d, J ¼ 6.4 Hz, 2H), 4.41 (d,
J ¼ 5.9 Hz, 2H), 3.99 (d, J ¼ 5.8 Hz, 2H). 13C NMR (100 MHz, DMSO-
4.1.6.7. N-(4-(((4-fluorophenyl)sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7g). White solid, yield 73%,
m.p. 196e198 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.96 (t, J ¼ 6.0 Hz,
d6)
d 166.03, 162.21, 157.97, 153.05, 152.93, 150.54, 150.42, 150.29,
147.92, 147.79, 143.86, 138.84, 138.01, 137.97, 137.93, 135.64, 132.66,
127.63, 127.17, 127.08, 126.71, 124.33, 124.30, 124.25, 124.22, 118.62,
118.44, 116.43, 116.24, 110.33, 45.93, 43.68, 42.25. HRMS calcd for
1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 8.17 (s, 1H), 7.76e7.85 (m, 5H),
7.36e7.43 (m, 4H), 7.21 (d, J ¼ 8.4 Hz, 2H), 7.16 (d, J ¼ 8.5 Hz, 2H),
6.57 (t, J ¼ 4.8 Hz, 1H), 4.53 (d, J ¼ 6.4 Hz, 2H), 4.41 (d, J ¼ 6.0 Hz,
C
26H24O3N5SF2 524.15624 [M þ H]þ, found 524.15619.
2H), 3.95 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
d 166.03, 165.26,
162.76, 162.21, 157.97, 143.85, 138.75, 137.10, 135.84, 132.65, 129.49,
129.40, 127.56, 127.16, 127.07, 126.71, 116.35, 116.13, 110.34, 45.89,
43.67, 42.25. HRMS calcd for C26H25O3N5SF 506.16567 [M þ H]þ,
found 506.16537.
4.1.6.13. 4-((Pyrimidin-2-ylamino)methyl)-N-(4-(((3-(tri-
fluoromethyl)phenyl)sulfonamido)methyl)benzyl)benzamide (7m).
White solid, yield 72%, m.p. 160e162 ꢁC. 1H NMR (400 MHz, DMSO-
d6)
d
8.93 (t, J ¼ 6.0 Hz, 1H), 8.39 (t, J ¼ 6.0 Hz, 1H), 8.26 (d,
J ¼ 4.8 Hz, 2H), 7.95e8.04 (m, 3H), 7.75e7.81 (m, 4H), 7.37 (d,
J ¼ 8.0 Hz, 2H), 7.17 (d, J ¼ 8.1 Hz, 2H), 7.12 (d, J ¼ 8.3 Hz, 2H), 6.57 (t,
J ¼ 4.8 Hz, 1H), 4.53 (d, J ¼ 6.4 Hz, 2H), 4.38 (d, J ¼ 6.0 Hz, 2H), 4.01
4.1.6.8. N-(4-(((2-chlorophenyl)sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7h). White solid, yield 80%,
m.p. 170e172 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.93 (t, J ¼ 6.0 Hz,
(d, J ¼ 5.9 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
d 165.98, 162.20,
1H), 8.42 (s, 1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 7.88e7.90 (m, 1H),
7.76e7.82 (m, 3H), 7.52e7.59 (m, 2H), 7.45 (ddd, J ¼ 7.9, 7.0, 1.7 Hz,
1H), 7.37 (d, J ¼ 7.7 Hz, 2H), 7.15 (s, 4H), 6.58 (t, J ¼ 4.8 Hz, 1H), 4.53
(d, J ¼ 6.4 Hz, 2H), 4.39 (d, J ¼ 6.0 Hz, 2H), 4.06 (s, 2H). 13C NMR
157.95,143.85,142.05,138.79,135.48,132.64,130.69,130.46,129.52,
128.95, 128.91, 127.58, 127.14, 127.06, 126.69, 122.98, 122.95, 110.32,
45.91, 43.66, 42.21. HRMS calcd for C27H25O3N5SF3 556.16247 [M þ
H]þ, found 556.16193.
(100 MHz, DMSO-d6)
d 165.98, 162.20, 157.94, 143.85, 138.62,
138.24, 135.96, 133.71, 132.66, 131.58, 130.54, 130.26, 127.47, 127.41,
127.15, 126.95, 126.71, 110.33, 45.78, 43.66, 42.22. HRMS calcd for
C
4.1.6.14. N-(4-((pyridine-3-sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7n). White solid, yield 68%,
26H25O3N5SCl 522.13611 [M þ H]þ, found 522.13623.
m.p. 213e215 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.95 (t, J ¼ 6.0 Hz,
1H), 8.91 (s, 1H), 8.74 (d, J ¼ 4.9 Hz, 1H), 8.40 (s, 1H), 8.26 (d,
J ¼ 4.8 Hz, 2H), 8.09 (ddd, J ¼ 8.0, 2.4, 1.6 Hz, 1H), 7.82 (d, J ¼ 8.3 Hz,
2H), 7.77 (t, J ¼ 6.4 Hz, 1H), 7.55 (ddd, J ¼ 8.1, 4.8, 0.8 Hz, 1H), 7.38 (d,
J ¼ 8.2 Hz, 2H), 7.19 (d, J ¼ 8.2 Hz, 2H), 7.15 (d, J ¼ 8.3 Hz, 2H), 6.57 (t,
J ¼ 4.8 Hz, 1H), 4.54 (d, J ¼ 6.3 Hz, 2H), 4.40 (d, J ¼ 5.9 Hz, 2H), 4.02
4.1.6.9. N-(4-(((3-chlorophenyl)sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7i). White solid, yield 78%,
m.p. 167e169 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.95 (t, J ¼ 6.0 Hz,
1H), 8.28 (s, 1H), 8.26 (d, J ¼ 4.7 Hz, 2H), 7.66e7.82 (m, 6H), 7.58 (t,
J ¼ 7.9 Hz,1H), 7.37 (d, J ¼ 8.2 Hz, 2H), 7.20 (d, J ¼ 8.3 Hz, 2H), 7.15 (d,
J ¼ 8.1 Hz, 2H), 6.58 (t, J ¼ 4.8 Hz, 1H), 4.53 (d, J ¼ 6.4 Hz, 2H), 4.41
(d, J ¼ 5.9 Hz, 2H), 3.98 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
(s, 2H). 13C NMR (100 MHz, DMSO-d6)
d 166.01, 162.21, 157.96,
152.77, 146.95, 143.86, 138.80, 137.18, 135.58, 134.36, 132.65, 127.59,
127.16, 127.10, 126.72, 124.12, 110.33, 45.88, 43.68, 42.24. HRMS
calcd for C25H25O3N6S 489.17034 [M þ H]þ, found 489.17011.
d
165.99, 162.19, 157.94, 143.83, 142.65, 138.78, 135.65, 133.69,
132.64, 132.19, 131.15, 127.57, 127.14, 127.06, 126.69, 126.06, 125.10,
110.31, 45.91, 43.66, 42.24. HRMS calcd for
C26H25O3N5SCl
4.1.6.15. N-(4-(((2,3-dihydrobenzofuran)-5-sulfonamido)methyl)
522.13611 [M þ H]þ, found 522.13631.
benzyl)-4-((pyrimidin-2-ylamino)methyl)benzamide
(7o).

204
R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
White solid, yield 75%, m.p. 192e194 ꢁC. 1H NMR (400 MHz, DMSO-
d6)
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 [13, 15].
d
8.96 (t, J ¼ 6.0 Hz, 1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 7.89 (t,
J ¼ 6.4 Hz, 1H), 7.76e7.82 (m, 3H), 7.53e7.60 (m, 2H), 7.37 (d,
J ¼ 8.3 Hz, 2H), 7.21 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 8.3 Hz, 2H), 6.90
(d, J ¼ 8.4 Hz,1H), 6.58 (t, J ¼ 4.8 Hz,1H), 4.62 (t, J ¼ 8.8 Hz, 2H), 4.53
(d, J ¼ 6.4 Hz, 2H), 4.42 (d, J ¼ 6.0 Hz, 2H), 3.89 (d, J ¼ 6.3 Hz, 2H),
3.21 (t, J ¼ 8.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
d 166.01,
162.75, 162.20, 157.96, 143.85, 138.61, 136.12, 132.64, 132.24, 128.60,
127.74, 127.54, 127.15, 126.99, 126.70, 123.93, 110.33, 108.91, 72.07,
45.90, 43.66, 42.24, 28.44. HRMS calcd for C28H28O4N5 530.18565
[M þ H]þ, found 530.18544.
4.4. Xylene-induced ear inflammation suppression test
Five mice per group were used to determine the effect of the
CXCR4 modulators. The inner and outer surfaces of the right ear of
4.1.6.16. N-(4-(((40-fluoro-[1,10-biphenyl])-4-sulfonamido)methyl)
each mouse were treated with a total 30
duction of ear edema, whereas the left ear surfaces were treated
with 30 L of saline, which was used as a non-inflammation con-
trol. The selected compounds were dissolved in 10% DMSO and 90%
of 45% (2-hydroxypropyl)- -cyclodextrin (CD) in PBS. 30 min after
mL of xylene for the in-
benzyl)-4-((pyrimidin-2-ylamino)methyl)benzamide
White solid, yield 80%, m.p. 172e174 ꢁC. 1H NMR (400 MHz, DMSO-
d6)
(7p).
m
d
8.96 (t, J ¼ 6.0 Hz, 1H), 8.26 (d, J ¼ 4.8 Hz, 2H), 8.18 (t,
J ¼ 6.3 Hz, 1H), 7.86 (s, 4H), 7.76e7.81 (m, 5H), 7.32e7.38 (m, 4H),
7.22 (d, J ¼ 8.4 Hz, 2H), 7.19 (d, J ¼ 8.4 Hz, 2H), 6.57 (t, J ¼ 4.8 Hz, 1H),
4.53 (d, J ¼ 6.4 Hz, 2H), 4.41 (d, J ¼ 5.9 Hz, 2H), 3.97 (d, J ¼ 6.2 Hz,
b
the application of xylene, 14 selected compounds were adminis-
tered intraperitoneally (i.p.) at 10 mg/kg. Control animals received
corresponding i.p. injections of the vehicle. The animals were
sacrificed 2 h later, and two ear plugs (7 mm in diameter) were
removed from both the treated ear and the untreated ears. Weights
of treated and untreated ear plugs were measured. The difference
in weight of the two ear plugs was taken as a measure of edematous
response. The inflammation-suppression percentage was calcu-
lated by comparing the drug-treated group to the control group
[16].
2H). 13C NMR (100 MHz, DMSO-d6)
d 166.02, 163.63, 162.20, 161.19,
157.96, 143.85, 142.77, 139.40, 138.72, 136.01, 135.05, 135.02, 132.65,
129.23, 129.14, 127.55, 127.32, 127.15, 127.07, 126.70, 116.05, 115.84,
110.33, 45.91, 43.67, 42.26. HRMS calcd for C32H29O3N5FS 582.19697
[M þ H]þ, found 582.19752.
4.1.6.17. N-(4-((naphthalene-1-sulfonamido)methyl)benzyl)-4-((pyr-
imidin-2-ylamino)methyl)benzamide (7q). White solid, yield 82%,
m.p. 159e161 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.92 (t, J ¼ 6.0 Hz,
1H), 8.66 (d, J ¼ 9.0 Hz, 1H), 8.50 (t, J ¼ 6.1 Hz, 1H), 8.26 (d,
J ¼ 4.8 Hz, 2H), 8.17 (d, J ¼ 8.2 Hz, 1H), 8.04e8.11 (m, 2H), 7.76e7.81
(m, 3H), 7.58e7.72 (m, 3H), 7.37 (d, J ¼ 8.1 Hz, 2H), 7.11 (d, J ¼ 8.3 Hz,
2H), 7.07 (d, J ¼ 8.3 Hz, 2H), 6.57 (t, J ¼ 4.8 Hz, 1H), 4.53 (d,
J ¼ 6.4 Hz, 2H), 4.37 (d, J ¼ 5.9 Hz, 2H), 3.98 (d, J ¼ 6.1 Hz, 2H). 13C
4.5. Western blot analysis
Forty micrograms of protein were separated by SDS-PAGE and
transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The
membrane was blocked for 30 min in a blocking solution (5% milk
in Trisbuffered saline containing 0.1% Tween-20) and incubated
overnight at 4 ꢁC using monoclonal rabbit anti-phospho-Akt
(Ser473) antibody (Cat No., 9271)/monoclonal rabbit anti-Akt
(pan) antibody (cat No., 4691) or monoclonal rabbit anti-
phospho-p44/42 MAPK (Thr202/Tyr204) antibody (cat No., 4376)/
monoclonal rabbit anti-p44/42 MAPK antibody (cat No., 4695) at
1:500 in blocking solution. All antibodies were purchased from Cell
Signaling Technology (Danvers, USA). The membrane was incu-
bated for 1 h with goat anti-rabbit IgG (H þ L)-HRP conjugated
secondary antibody at 1:10000 (Cat No. 1706515; Bio-rad, Hercules,
USA) after washing. Enzyme-linked chemiluminescence was per-
formed to detect hybridized protein bands.
NMR (100 MHz, DMSO-d6)
d 165.97, 162.20, 157.96, 143.84, 138.56,
136.08, 135.76, 133.81, 133.59, 132.66, 128.86, 128.35, 127.74, 127.49,
127.35, 127.15, 126.90, 126.75, 126.70, 124.72, 124.44, 110.33, 45.69,
43.67, 42.21. HRMS calcd for C30H28O3N5S 538.19074 [M þ H]þ,
found 538.19150.
4.2. Primary binding affinity screening
For binding affinity assay, 2 ꢂ 104 MDA-MB-231 cells in 300
mL
of cell culture medium were seeded in an 8-well slide chamber two
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-
4.6. Preliminary pharmacokinetic study of compound 7a
ture with 0.05
m
g/mL biotinylated TN14003, washed three times
Nude mice with body weight about 20 g were used in the PK
study. The mice were anaesthetized with Ketamine hydrochloride
(90 mg/kg) and Xylazine (4.5 mg/kg). Compound 7a was dissolved
in 10% DMSO and 90% of 45% (2-hydroxypropyl)-b-cyclodextrin
(CD) in PBS. The mice were warmed by the heat lamp for 2 min and
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 [13, 15, 25].
then received a single dose (30 mg/kg) of compound 7a via intra-
venous injection (i.v.). Blood samples (100 mL) were collected at 0, 5,
20 and 50 min post-dose from orbital venous sinus with heparin-
ized capillary tubes (I.D. 1.1e1.2 mm). After collection, the blood
sample was centrifuged at 13000 g for 4 min at 4 ꢁC. The super-
natant was then filtrated by microcon centrifugal filter (10000
NMWL) at 13000 g for 30 min at 4 ꢁC. The filtrate was frozen
at ꢃ80 ꢁC until HPLC analysis. The HPLC analysis of the blood
sample was performed on a C18 column (250*4.6 mm). Acetonitrile
(0.1%TFA)-Water (0.1%TFA) (50:50, V/V) was applied as the mobile
phase and the detective wavelength was set at 254 nm.
4.3. Matrigel invasion assay
Matrigel invasion assay was performed by using a Matrigel in-
vasion chamber from Corning Biocoat (Bedford, MA). CXCL12
(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 (100 nM) or
AMD3100 were added to the cells before the cells were seeded in
a

R. Bai et al. / European Journal of Medicinal Chemistry 150 (2018) 195e205
205
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medicinal chemistry 24 (2016) 5052e5060.
The antiproliferative activity of the compounds was determined
using MTT assay. Cells were seeded in 96-well microculture plates
at 3000 cells/well in 100
37 ꢁC in CO2 incubator. Following the incubation for 24 h, these cells
were treated with Compound 7a for 24 h at 37 ꢁC. Finally, 20
L of
mL of medium and incubated for 24 h at
m
CellTiter 96AQ reagent (Promega, Madison, WI) was added into
each well and incubated for an additional 2 h, and the absorbance at
490 nm was measured.
Acknowledgments
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.02.085.
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