Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based derivatives as CXCR4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111562

The interaction between G-Protein coupled receptor CXCR4 and its natural ligand CXCL12 has been linked to inflammation experienced by patients with Irritable Bowel Disease (IBD). Blocking this interaction could potentially reduce inflammatory symptoms in

Full text of DOI:10.1016/j.ejmech.2019.111562

Accepted Manuscript
Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based as
CXCR4 inhibitors
Theresa Gaines, Francisco Garcia, Saniya Virani, Zhongxing Liang, Younghyoun
Yoon, Yoon Hyeun Oum, Hyunsuk Shim, Suazette Reid Mooring
PII:
S0223-5234(19)30686-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111562
Article Number: 111562
Reference:
EJMECH 111562
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 April 2019
Revised Date: 22 July 2019
Accepted Date: 23 July 2019
Please cite this article as: T. Gaines, F. Garcia, S. Virani, Z. Liang, Y. Yoon, Y.H. Oum, H. Shim,
S.R. Mooring, Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based as
CXCR4 inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111562.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based as CXCR4
inhibitors
Authors: Theresa Gainesa; Francisco Garciaa; Saniya Virania; Zhongxing Liangb,c; Younghyoun
Yoonc; Oum, Yoon Hyeunb; Hyunsuk Shimb, c; Suazette Reid Mooringa*
aDepartment of Chemistry, Georgia State University, Atlanta, GA, 30303, USA
bDepartment of Radiology and Imaging Science, Emory University School of Medicine, Atlanta,
GA, 30322, USA
cWinship Cancer Institute, Emory University, Atlanta, GA, 30322, USA
Keywords: furan, thiophene, CXCR4 inhibitor, Matrigel Invasion, Binding affinity, cancer
metastasis, anti-inflammatory
Highlights:
•
•
•
•
Fifty-six potential furan and thiophene based CXCR4/CXCL12 modulators synthesized
Sixteen hit compounds inhibited metastasis of breast cancer cells by 50% or more
Two hit compounds reduced carrageenan induced inflammation by 30%
In silico analysis suggests hit compounds interact with key CXCR4/CXCL12 residues
Graphical Abstract:
Abstract:
The interaction between G-Protein coupled receptor CXCR4 and its natural ligand CXCL12 has
been linked to inflammation experienced by patients with Irritable Bowel Disease (IBD).
Blocking this interaction could potentially reduce inflammatory symptoms in IBD patients. In
this work, several thiophene-based and furan-based compounds modeled after AMD3100 and
WZ811—two known antagonists that interrupt the CXCR4-CXCL12 interaction—were
synthesized and analyzed. Fifteen hit compounds were identified; these compounds exhibited
effective concentrations (EC) lower than 1000 nM (AMD3100) and inhibited invasion of
metastatic cells by at least 45%. Selected compounds (2d, 2j, 8a) that inhibited metastatic
invasion at a higher rate than WZ811 (62%) were submitted for a carrageenan inflammation test,
where both 8a and 2j reduced inflammation in the same range as WZ811 (40%) but did not
reduce inflammation more than 40%. Select compounds were also modeled in silico to show key
residue interactions. These preliminary results with furan-based and thiophene-based analogues
contribute to the new class on heterocyclic aromatic-based CXCR4 antagonists.
1. Introduction:
IBD is comprised of several different diseases—including Crohn’s disease (CD) and
ulcerative colitis (UC)—which are environmentally triggered and appear in genetically
susceptible individuals. The trigger causes an unusual immune response to the individual’s
intestinal flora resulting in gastrointestinal inflammation[1, 2]. Individuals with IBD have
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exhibited altered chemokines and receptors in their epithelial cells, which could disrupt the
body’s recognition of their gut bacteria. Not only are individuals with IBD, unable to recognize
enteric microbiota, but it is also possible that they are unable to regulate the amount of pro-
inflammatory chemokines that are sent as the immune response [3, 4].
CXCL12 is a chemokine ligand, which is expressed in healthy intestinal epithelial cells
(IEC)[5-7]. CXCR4, a specific receptor for CXCL12, is also expressed by IEC’s in healthy GI
membranes[6, 7]. Both CXCR4 and CXCL12 are needed for necessary physiological functions
including electrolyte secretion [8], migration, and upkeep of the epithelial mucosal membrane.[9]
CXCR4 and CXCL12 expression, however, are both upregulated in the IEC’s of patients with
IBD[10, 11]. CXCL12 is a strong chemoattractant of CXCR4. The presence of extra CXCL12
chemoattracts CD45RO+ T cells, which are rich with CXCR4. This interaction triggers
inflammation in the intestinal membrane and disrupts the intestinal membrane’s homeostasis
[11].
The interaction between CXCR4 and CXCL12 has been identified as a potential target in
a host of other inflammatory diseases including: rheumatoid arthritis,[12] atherosclerosis[13],
allergic airway disease[14], psoriasis[15], and anxiety[16]. Several studies have also suggested
that disruption of the CXCR4/CXCL12 axis can reduce inflammation in these diseases when
AMD3100, a CXCR4 antagonist is used[15-19]. AMD3100 (Fig. 1) is the first CXCR4
antagonist to be FDA approved [20]. AMD3100 exhibits cardiotoxicity[21, 22] and poor
bioavalibility [22, 23], which prevents it from being used for therapeutic purposes; however, it
was approved for one time use for patients with multiple myolma.[20] AMD3100 is comprised
of a benzene ring connected to two bicyclam rings. Preliminary structure activity relationship
(SAR) studies determined that activity disappears when the central ring is aliphatic[24, 25].
Other SAR studies have explored modifications of the bicyclam rings, creating a class of
CXCR4 antagonists called p-xylyl-enediamines [26, 27].
One of these analogues, WZ811 (Fig. 1), replaced the bicyclam rings with 2-
aminopyridine rings and exhibited impressive antagonist activity against CXCR4 [26].
Unfortunately, WZ811 did not perform well in clinical trials due to poor bioavilability and
toxicity issues [27]. Many p-xylyl-enediamine analogues have been synthesized with sidechain
modifications; however, little research has centered on modification of the central ring using
heterocyclic aromatic structures.
Our previous work has included synthesizing pyridine-based analogues (Fig. 1),
mimicking p-xylyl-enediamines [28-30], as well as well as diamino and dianinlinomethyl
pyridine derivatives [31]. In one of the previously published works, docking studies suggested
that the primary interaction between the active antagonists and CXCR4 occurred between the
lone pairs on the nitrogen in the central pyridine ring and the residue ASP97 [30]. This residue is
important for the binding of CXCL12 in the active site of CXCR4. The promising assay results
of these pyridine-based analogues and the following docking analyses have set an important
precedent for more SAR studies of various heterocyclic aromatic derivatives of p-xylyl-
enediamines. This work will focus on 2,5-furan, 2,5-thiophene, and 3,4-thiophene derivatives
shown in Figure 1.
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NH HN
NH
N
N
HN
NH HN
AMD 3100
N
HN
N
NH
WZ811
R1
N
R1
N
R1
N
R1
N
N
N
R2
R2
R2
R2
S
8
R1
R1
N
N
O
2
R1
R1
R2
R2
N
N
R2
R2
S
6
R1
N
R2
R1
N
R2
Figure 1: Development of furan-based and thiophene-based analogues from AMD3100.
2. Results and Discussion
2.1 Chemistry
All furan compounds (2a-2w) were synthesized using a one-pot reductive amination reaction
between 2,5-furanodicarbaldehyde (1) a substituted amine. Zinc chloride was used as a catalyst
and sodium cyanoborohydride was used as the reducing agent (Scheme 1).
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ZnCl2,
NHR1R2,
NaBH3CN
R1
R1
N
O
N
O
R2
R2
MeOH,
2 hrs, rt
(30-85%)
O
1
O
2
R1 = H
R2 =
NH
X
2a: H
2b: 2-Me
X = 2c: 3-Me
2d: 4-Me
2f: 3-Et
2g: 4-Et
2h: 2-F
2i: 3-F
2j: 4-F
2k: 2-Cl
2l: 3-Cl
2m: 4-Cl
2n: 2-OMe
2o: 3-OMe
2p: 4-OMe
2q: 2-CF3
2r: 3-CF3
2s: 4-CF3
2t : 4-SMe
2e: 2-Et
R1
=
R2
R1
N
N
N
O
=
2u:
2v:
N
N
R2
R1
N
N
S
=
2w:
R2
Scheme 1: Synthesis of 2,5-furan derivatives.
For the 3,4-thiophene derivatives, 3,4-thiophenedicarbaldehyde (5) was synthesized in two steps,
as shown in Scheme 2. First, 3,4-thiophenedicarboxylic acid (3) was reduced to an alcohol using
diisobutylaluminium hydride. Second, the resulting dialcohol (4) was quenched and isolated
before it was oxidized over manganese dioxide to give the dicarbaldehyde product (5).
Scheme 2: Synthesis of 3,4-thiophenedicarbaldehyde
The 3,4-thiophene analogues (6a-j) were synthesized via a reductive amination reaction between
3,4-thiophenedicarbaldehyde (5) and a substituted amine (Scheme 3). Zinc chloride was used as
a catalyst in the first step. Sodium borohydride was used as the reducing agent in the second step.
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Scheme 3: Synthesis of 3,4-thiophene derivatives.
The 2,5-thiophene derivatives (8a-8w) were synthesized in a similar manner using a reductive
amination procedure with 2,5-thiophenedicarbaldehyde (7) and a substituted amine (Scheme 4).
The final product (8) was synthesized using one of two methods. Method A involved the use of
acetic acid as a catalyst and sodium triacetoxyborohydride as the reducing agent. In method B,
the substituted amine and the 2,5-thiophenedicarbaldehyde (7) were first reacted at room
temperature to form the imine, then sodium borohydride was used to reduce the imine to the final
product (8).
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R1
R1
N
Procedure A,or B
O
N
O
R2
R2
(3-26%)
S
7
S
8
R1 = H
R2 =
H
N
X
8a: H
8b: 2-Me
X = 8c: 3-Me
8d: 4-Me
8f: 3-Et
8g: 4-Et
8h: 2-F
8i: 3-F
8j: 4-F
8k: 2-Cl
8l: 3-Cl
8m: 4-Cl
8n: 2-OMe
8o: 3-OMe
8p: 4-OMe
8q: 2-CF3
8r: 3-CF3
8s: 4-CF3
8t: 4-SMe
8e: 2-Et
R1
=
R2
R1
R2
N
N
N
O
=
8u:
8v:
N
N
R1
N
N
S
=
8w:
R2
Scheme 4: Synthesis of 2,5-thiophene derivatives. Method A was used to synthesize 8a-8p and
8t-8w. Method B was used to synthesize 8q-8s.
2.2 Binding Affinity Assay
Compounds synthesized were first evaluated using a binding affinity assay. Note, this is a semi-
quantitative assay that primarily functions as a preliminary screen to identify compounds that
should be tested further. In this assay, MDA-MB-231 breast cancer cells, which over-express
CXCR4, are incubated with the analogues at 1 nM, 10 nM, 100 nM, and 1000 nM
concentrations. TN14003, a biotinylated peptide and known CXCR4 antagonist, and
streptavidin-rhodiamine are added to the solution and the cells are incubated again. Fluorescence
of each solution is measured to obtain the effective concentration (EC). The EC value is the
lowest concentration, by order of magnitude, where a significant reduction in the fluorescence
was observed compared to the control (Figure 2). As this assay is a preliminary screen, all
compounds synthesized were screened in this assay.
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Figure 2: Reduction of fluorescence observed for selected derivatives. 2h had an EC of 10 nM.
2j had an EC of 100 nM and 2e had an EC of 1000 nM.
2.3 Matrigel Invasion Assay
The Matrigel invasion assay probes the compound’s effectiveness in blocking the
CXCR4/CXCL12 interaction to prevent chemotaxis and invasion. This assay uses a special
double chambered apparatus that is separated by a Matrigel matrix that MDA-MB-231 cells can
pass through. MDA-MB-231 cells are incubated in 100 nM concentrations of the analogue and
are placed into the top chamber. A solution containing CXCL12 is added to the bottom chamber
as the chemoattractant. If the analogue successfully blocks the CXCR4-CXCL12 interaction, the
cells will not migrate to the bottom chamber. When the assay is complete, the cells in the bottom
chamber are stained and counted. The results of this assay are given as a percentage of cells that
were prevented from migrating compared to the negative control or a percentage of the inhibition
of chemotaxis. Compounds that inhibited chemotaxis will have a higher percentage value
because less cells migrated to the bottom chamber.
Only compounds that showed promise in the binding assay (EC ≤ 100 nM) were tested in the
Matrigel invasion assay. AMD 3100 is used as a benchmark in the results shown.
The results for this Matrigel invasion assay and the binding affinity assay can be found in Tables
1 through Table 3 below, where Table 1 shows the data for the 2,5-furan analogues. Table 2
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shows the data for the 3,4-thiophene analogues, and Table 3 shows the data for the 2,5-thiophene
analogues.
EC
(nM)
EC
(nM)
Compd
2a
2b
2c
R Group
Aniline
Invasiona Compd
R Group
4-Cl aniline
Invasiona
>1000
--
--
10
48%
5%
--
2m
2n
2o
2p
2q
2r
2-Me aniline >1000
3-Me aniline >1000
2-OMe aniline
3-OMe aniline
4-OMe aniline
2-CF3 aniline
3-CF3 aniline
4-CF3 aniline
4-SMe aniline
1-methylpiperazine
Morpholine
100
--
>1000
>1000
100
4-Me aniline
2-Et aniline
100
75%
--
--
2d
2e
1000
24%
--
3-Et aniline >1000
4-Et aniline >1000
--
>1000
>1000
>1000
10
2f
--
--
2g
2h
2i
2s
2-F aniline
3-F aniline
4-F aniline
10
>1000
100
71%
--
--
2t
82%
--
2u
2v
2w
53%
--
1000
10
2j
2-Cl aniline >1000
3-Cl aniline >1000
Thiomorpholine
79%
2k
2l
--
AMD3100[28,
32]
---
1000
62%
a
Table 1: Binding and invasion assay results for 2,5-furan analogues synthesized. The invasion
assay concentration used for all compounds tested was 100 nM.
Of the twenty-three 2,5-furan analogues synthesized, eight showed improved activity over
AMD3100 in the binding affinity assay: 2d, 2h, 2j, 2m, 2n, 2q, 2u and 2w. Of these compounds,
four showed favorable activity in the Matrigel invasion assay, where 60% inhibition or greater
was observed. These analogues included, the 4-methyl aniline (2d) at 75% inhibition of invasion,
the 2-fluoro (2h) and 4-fluoro (2j) derivatives with invasion inhibition of 71% and 53%
respectively. The 1-methylpiperazine analogue (2u) at 82% and the thiomorpholine analogue
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(2w) at 79% inhibition of invasion. These results show the potential of these 2,5-furan analogues
as a novel CXCR4 modulator.
Out of all the 2,5-furano-based compounds, active analogues were either -ortho or -para
substituted. Weakly electron donating groups like methyl and weakly electron withdrawing
groups like the fluorine and chlorine seem to be well tolerated. Interestingly, the 2-CF3 analogue
(2q) with a strong electron withdrawing group, had some activity with an effective concentration
of 10 nM and an invasion inhibition of 24%.
R
S
R
6
EC
(nM)
Compd
R Group
Aniline
EC (nM) Invasiona Compd
R Group
4-F aniline
Invasiona
10
72%
--
>1000
--
--
--
--
--
6a
6f
6g
6h
6i
3-Me aniline
4-Me aniline
4-Et aniline
3-F aniline
---
>1000
>1000
>1000
>1000
1000
3-Cl aniline
4-Cl aniline
3-CF3 aniline
4-CF3 aniline
>1000
>1000
>1000
>1000
6b
--
6c
--
6d
6e
--
6j
62%
AMD3100[28, 32]
Table 2: Binding and invasion assay results for the 3,4-thiophene analogues synthesized. aThe
invasion assay concentration used for all compounds tested was 100 nM.
The 3,4-thiophene analogues (Table 2) were the least active out of any of the heterocyclic
analogues synthesized. Only the aniline analogue (6a) showed favorable activity with an
effective concentration of 10 nM and an invasion inhibition of 72%. All other compounds had an
effective concentration over 1000 nM and showed no significant activity. Since these compounds
showed little to no activity, additional analogues were not synthesized.
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EC
(nM)
EC
(nM)
Compd
8a
8b
8c
R Group
Aniline
Invasiona Compd
R Group
Invasiona
22%
52%
--
1
68%
61%
--
4-Cl aniline
2-OMe aniline
3-OMe aniline
4-OMe aniline
2-CF3 aniline
3-CF3 aniline
4-CF3 aniline
4-SMe aniline
1-methylpiperazine
Morpholine
1
10
8m
8n
8o
8p
8q
8r
2-Me aniline
3-Me aniline
4-Me aniline
2-Et aniline
3-Et aniline
4-Et aniline
2-F aniline
3-F aniline
4-F aniline
2-Cl aniline
3-Cl aniline
10
>1000
100
1000
10
49%
--
84%
--
8d
8e
1000
>1000
>1000
1000
>1000
>1000
10
>1000
100
--
77%
--
8f
--
>1000
100
8g
8h
8i
8s
--
88%
86%
--
8t
--
100
8u
8v
8w
--
>1000
1
8j
93%
51%
62%
Thiomorpholine
88%
8k
8l
100
AMD3100[28,
32]
1000
a
Table 3: Binding and invasion assay results for the 2,5-thiophene analogues synthesized. The
invasion assay concentration used for all compounds tested was 100 nM.
Of the compounds synthesized, the 2,5-thiophene analogues had the best activity among all the
analogues. Twelve analogues had better binding affinity than AMD 3100 and ten exhibited
inhibition of invasion above 50% in the Matrigel invasion assay. All aniline derivatives with a
chlorine (8k, 8l and 8m) had activity. In general, the ortho and meta analogues had the most
activity; however, the para chloroaniline (8m) only inhibited invasion by 22%. The ortho and
para methoxy (8n and 8p) derivatives both had an effective concentration of 10 nM and inhibited
invasion by 52% and 84% respectively. Two of the methyl analogues, also ortho and para (8b
and 8d) exhibited effective concentrations of 10 nM and 100 nM respectively and invasion assay
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results of 61% and 49%. The aniline derivative (8a) which had an effective concentration of 1
nM and inhibited 68% of invasion; the 3-trifluoromethyl (8r) analogue’s effective concentration
was 100 nM and inhibited invasion by 77%. The thiomethylaniline (8t) and 1-methylpiperazine
(8u) analogs has an effective concentration of 100 nM and inhibited invasion by 88% and 86%,
respectively. Lastly, the thiomorpholine analog (8w) had an effective concentration of 1 nM and
inhibited invasion by 88%. There is a greater diversity of active substituents for the 2,5-
thiophene compounds that are not present in the 2,5-furan analogues or the 3,4-thiophene
analogues.
Similar to the 2,5-furan analogues, ortho and para weak electron donating and weak electron
withdrawing groups seem to have the best activity in the 2,5-thiophene analogues with a few
exceptions. None of the fluoro-substituted 2,5-thiophene analogues had significant activity,
whereas, these were active with the furan analogues. In addition, the methoxy substituted
thiophene analogues (8n and 8p) also had activity, whereas, they did not show any activity in the
furan analogues.
2.4 In vivo carrageenan suppression
Select compounds that scored a 35% prevention of inhibition in the Matrigel invasion assay were
subjected to the in vivo carrageenan suppression test. The mouse paw edema model, is used to
determine if the compounds have anti-inflammatory activity. This is done by inflaming a
mouse’s paw and treating it with the analogue. In the presence of a potent antagonist,
inflammation, which is triggered by the CXCR4/CXCL12 interaction, would be reduced. All
compounds that performed well in both the binding assay and the Matrigel invasion assay were
tested in the paw edema model. The mouse paw edema model, therefore, is a good proof-of-
concept test [26, 32]. This test also gives preliminary insight into the toxicity of the analogues.
The mice were monitored during the test and none of them showed any signs of toxicity when
the compounds were administered. The results from this test can be found in Table 4.
O
S
R
R
S
R
R
R
R
2
8
6
Compd
2d
R Group
EC (nM)
100
10
Invasiona
Carageenanb,c
4-Me aniline
2-F aniline
4-F aniline
4-Cl aniline
75%
15%
--
71%
2h
100
10
53%
31%
17%
--
2j
48%
2m
2u
1-Methylpiperazine
10
82%
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Aniline
10
1
72%
68%
61%
49%
93%
51%
52%
84%
77%
88%
90%
--
30%
--
6a
Aniline
8a
2-Me aniline
4-Me aniline
2-Cl aniline
10
100
10
100
10
10
100
1
8b
--
8d
8k
>5%
--
3-Cl aniline
8l
2-OMe aniline
4-OMe aniline
3-CF3 aniline
Thiomorpholine
--
8n
--
8p
8r
--
--
8w
WZ811[26]
10
40%
Table 4: Assay and test results for hit compounds. aThe concentration used in the invasion assay
was 100 nM. bMice were dosed used 10mg of compound for every kg the mouse weighed.
cCompounds with a dash in the carrageenan studies have not yet been tested.
If these analogues can disrupt the CXCR4/CXCL12 interaction, then it also has an effect on
inflammation. If inflammation were to be induced in the presence of these compounds, a
reduction in said inflammation would be observed for potent CXCR4 antagonists. All
compounds that performed well in both the binding assay and Matrigel invasion assay were
submitted for the paw edema test.
In this test, the hind paws of mice are inflamed using carrageenan, and one paw is treated with a
solution of the analogue (10 mg of analogue per kg), and the other is treated with a saline
solution. Both the saline solution and analogue solution are delivered via injection into the paw.
The paws are then measured at the end of the test using calipers to determine the percent
reduction in swelling. Compounds that score 100 nM or below in the binding assay and above a
35% in the invasion assay were considered hit compounds and were submitted for further
analysis in the paw edema test. Of the compounds synthesized, fifteen qualified for the mouse
paw edema test (shown in Table 4).
AMD3100 is not used as a benchmark for this test due to its toxicity. However, WZ811 (Figure
1) is used as the benchmark in this assay. WZ811 reduced inflammation by 40% compared to the
control. The 4-fluoro furan analogue (2j), and the aniline 2,5-thiophene (8a) showed the best
activity of 31% and 30%, respectively. None of these compounds surpassed WZ811 in this group
of compounds, whereas a few of the previously reported for pyridine analogues did [30].
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The other remaining compounds did not have a reduction in inflammation above 20% compared
to the control, but some reduction was observed. Surprisingly, several compounds that scored
well in the preliminary assays showed little to no edema reduction. Compounds 2d and 8k are
prime examples of this. It is possible that compounds such as 2d and 8k have poor
pharmacokinetic profiles and additional studies would be needed discern if this is an issue.
3. Molecular Modeling
A brief molecular modeling experiment was conducted to probe possible residue interactions of
five selected hit compounds with CXCR4. These compounds were selected primarily because of
their low effective concentrations, and their ability to hinder invasion in the Matrigel invasion
assay; however, the final criteria prioritization of modeling compounds for which carrageenan
test data was available. Due to these criteria, compounds 2d, 2j, 2m, 6a, and 8a were selected for
in silico analysis. The key residue interactions for each compound are shown in Table 5 below.
Figure 3 and 4 show the key residue interactions and the orientation of two of these compounds
in the active site.
In the literature, several key residues have been identified for binding between the natural ligand
(CXCL12) and CXCR4. These residues include ASP97, GLU288, ASP187, PHE87, ASP171,
and PHE292. ASP97, GLU288 and ASP187, have found to be necessary for triggering the
CXCR4/CXCL12 signaling pathway[33, 34]. There are several other residues that have been
identified in other CXCR4 analyses—including CYS186, TRP102, TRP94, and TYR116—that
form the active site in CXCR4 [35]. Analogues that have significant interactions with these
residues can still block the CXCR4/CXCL12 interaction, as they still partially block the active
site.
Invasion
75%
Key Residue
Interactions
Active Compound
EC (nM)
TRP94b, TYR116b,
100
100
10
10
1
2d
2j
ARG188b
TRP94b, ASP97a
53%
48%
TRP94b, ASP97a,
2m
6a
8a
CYS186b
TRP94b, ASP97a
TRP94b, ASP97a,
72%
68%
ARG188b
a
Table 5: Effective concentrations and key residue interactions of hit compounds. Residue
required for CXCR4/CXCL12 signaling. bResidues that make up the active site of CXCR4.
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Of the five hit compounds analyzed through docking, four of them had a significant interaction
with ASP97, a key residue in blocking the CXCR4/CXCL12 signaling interaction. The only
residue that did not have an interaction with ASP97 was compound 2d. This analogue has
significant pi-pi interactions with TRP94 and TYR116, and a pi-cation interaction ARG188. All
three of the aforementioned residues comprise the active site of CXCR4. This suggests that even
though 2d is not interacting directly with any of the residues that facilitate the signaling pathway,
it is still effectively blocking the active site of CXCR4.
All other hit compounds (2j, 2m, 6a and 8a) had a polar interaction with ASP97—one of the key
residues which facilitate the CXCR4/CXCL12 signaling pathway. In addition to the interaction
with ASP97, they have all interact with TRP94 in the active site through a pi-pi interaction.
Compound 2m has an additional polar interaction with CYS186, and compound 8a has an
additional pi-cation interaction with ARG188.
Again, only the top five hit compounds were submitted for in silico analysis, but the results so
far have shown that all of these active compounds interact exclusively with residues in the active
site of CXCR4 (TRP94, TYR116, CYS186 and ARG188) and most of them interact with
ASP97, a residue that plays a key role in CXCR4/CXCL12 signaling and transduction pathways.
.
14

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Figure 3: This figure shows active analogue 2d’s pi-pi interactions with TRP94 and TYR116
and a pi-cation interaction with ARG188.
Figure 4: This figure shows active analogue 6a’s pi-pi interaction with TRP94 and hydrogen
bonding interaction with ASP97.
Figure 4: This figure shows active analogue 8a’s pi-pi interaction with TRP94 and hydrogen
bonding interaction with ASP97 and a pi-cation interaction with ARG188.
Conclusions
In this work, 56 furan-based and thiophene-based modulators of CXCR4, that mimic p-xylyl-
enediamines have been synthesized and evaluated for their potential as CXCR4 modulators.
These compounds were analyzed in binding assays, Matrigel invasion assays and an in vivo paw
edema test. Overall the 3,4-thiophene analogues were largely inactive. Four of the 2,5-furan
15

ACCEPTED MANUSCRIPT
analogs (2d, 2h, 2u and 2w) showed better activity than AMD3100 in the Matrigel invasion
assay and compound 2j inhibited paw inflammation about 30% compared to the control. Many
of 2,5-thiophene compounds outperformed AMD3100 in the Matrigel invasion assay, with one
compound 8k, showing an inhibition of invasion of over 90% in the Matrigel invasion assay.
Overall, the 2,5-furan analog 2j and the 2,5-thiophene analogue 8a, showed good activity in both
assays and showed inhibition of inflammation at around 30%. These results are promising that
some of these compounds have potential as CXCR4 modulators that could be used as a treatment
for inflammatory disease or for metastatic cancer.
Further pharmacokinetic studies will be necessary to determine if compounds that perform well
in in vitro assays but not as well in vivo (2d, 2m and 8k) are metabolized too quickly or if there
is some other problem. Additional optimization of the 2,5-furan, 3,4-thiophene and 2,5-thiophene
analogues to improve outcomes in vitro and in vivo are anticipated through synthesizing more
compounds with modified side chains based on previous hit compounds as well as benzylamine
derivatives instead of aniline based derivatives to expand the current SAR library.
4. Experimental
4.1 Chemistry
1
13
The H NMR (400 MHz) and C NMR (100 MHz) spectra were recorded on a Bruker Ac 400
FT NMR spectrometer in deuterated chloroform (CDCl3). All chemical shifts were reported
using parts per million (ppm). Mass spectra were recorded on a JEOL spectrometer at Georgia
State University Mass Spectrometry Center.
General Procedure for the Synthesis of the 2,5-furan-based analogues (2). To a solution of
methanol, 50 mg (0.4029 mmol) of furan-2,5-dicarbaldehyde was combined in a dry vial with
the aniline derivative of choice (0.8865 mmol) and 75.963 mg (1.2088 mmol) of sodium
cyanoborohydride (NaBH3CN). The solution was stirred for five minutes at room temperature
before 164.578 mg (1.2088 mmol) of zinc chloride (ZnCl2) was added. The solution was then
stirred for two hours and purified by flash chromatography.
1
2,5-bis(anilinomethyl)furan, (2a) Yellow oil, 38% yield. H NMR (400 MHz, CDCl3):
δ ppm
ppm 4.27 (s, 4H), 6.15 (s, 2H), 6.66 (d, J=7.58 Hz, 4H), 6.74 (t, J=7.33 Hz, 2H), 7.18 (t, J=7.96
Hz, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 41.49, 107.80, 113.18, 118.04, 129.22, 147.58,
152.11. HRMS: m/z [M + H]+ calcd for C18H19ON2: 279.1492, found: 279.1487.
1
2,5-bis(2-methylanilinomethyl)furan, (2b) Yellow solid, 35% yield. H NMR (400 MHz,
CDCl3):
δ ppm 2.14 (s, H), 3.84 (br s, H), 4.33 (s, 4H), 6.17 (s, 2H), 6.63 - 6.75 (m, 4H), 7.00 -
7.17 (m, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 17.49, 41.50, 107.83, 110.13, 117.61, 112.34,
127.07, 130.14, 145.56, 152.22. HRMS: m/z [M + H]+ calcd for C20H23ON2: 307.1805, found:
307.1792.
16

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2,5-bis(3-methylanilinomethyl)furan, (2c) Light orange solid, 49% yield. 1H NMR (400 MHz,
CDCl3):
δ ppm 2.27 (s, 6H), 4.25 (s, 4H), 6.13 (s, 2H), 6.42 - 6.50 (m, 4H), 6.56 (d, J=7.58 Hz,
2H), 7.06 (t, J=1.00 Hz, 2H); 13C NMR (100 MHz, CDCl3):
δ ppm 21.61, 41.53, 107.73, 110.27,
114.01, 118.97, 129.90, 138.98, 147.64, 152.17. HRMS: m/z [M + H]+ calcd for C20H23ON2:
307.1817, found: 307.1810.
1
2,5-bis(4-methylanilinomethyl)furan, (2d) Orange solid, 49% yield. H NMR (400 MHz,
CDCl3):
δ ppm 2.22 (s, 6H), 4.21 (s, 4H), 6.10 (s, 2H), 6.56 (d, J=8.08 Hz, 4H), 6.97 (d, J=7.83
Hz, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 20.40, 41.82, 107.67, 113.38, 127.21, 129.68,
145.33, 152.25. HRMS: m/z [M+ Z]+ calcd for C20H22ON2Na: 329.1617, found: 329.1624.
2,5-bis(2-ethylanilinomethyl)furan, (2e) Yellow oil, 44% yield. 1H NMR (400 MHz, CDCl3):
δ
ppm 1.24 (br d, J=5.56 Hz, 6H), 2.44 - 2.56 (m, 4H), 3.94 (br s, 2H), 4.33 (br s, 4H), 6.16 (br s,
2H), 6.66 - 6.78 (m, 4H), 7.05 - 7.16 (m, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 12.90, 23.79,
41.59, 107.79, 110.52, 117.81, 126.93, 127.92, 128.02, 144.98, 152.28. HRMS: m/z [M + H]+
calcd for C22H27ON2: 335.2118, found: 335.2103.
2,5-bis(3-ethylanilinomethyl)furan, (2f) Orange oil, 42% yield. 1H NMR (400 MHz, CDCl3):
δ
ppm 1.21 (br d, J=5.31 Hz, 6H), 2.51 - 2.63 (m, 4H), 3.93 (br s, 2H), 4.26 (br s, 4H), 6.14 (br s,
2H), 6.45 - 6.54 (m, 4H), 6.54 - 6.65 (m, 2H), 7.05 - 7.14 (m, 2H); 13C NMR (100 MHz, CDCl3):
δ
ppm 15.51, 29.00, 41.57, 107.75, 110.47, 112.93, 117.78, 129.15, 145.40, 147.70, 152.20.
HRMS: m/z [M + H]+ calcd for C22H27ON2: 335.2118, found: 335.2103.
2,5-bis(4-ethylanilinomethyl)furan, (2g) Orange oil 27 % yield. 1H NMR (400 MHz, CDCl3):
δ
ppm 1.18 (t, J=7.58 Hz, 6H), 2.54 (q, J=7.41 Hz, 4H), 4.24 (s, 4H), 6.12 (s, 2H), 6.60 (d, J=8.34
Hz, 4H), 7.01 (d, J=8.08 Hz, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 15.91, 27.93, 41.83,
107.69, 113.35, 128.52, 113.88, 145.58, 152.28. HRMS: m/z [M + H]+ calcd for C22H27ON2:
335.2118, found: 335.2111.
2,5-bis(2-fluoroanilinomethyl)furan, (2h) Yellow oil, 40% yield. 1H NMR (400 MHz, CDCl3):
δ
ppm 4.28 (br s, 4H), 6.14 (s, 2H), 6.59 - 6.78 (m, 4H), 6.90 - 7.05 (m, 4H); 13C NMR (100
MHz, CDCl3):
δ =
ppm 41.02, 107.94, 112.51, 113.54, 114.50 (d, 2JC,F = 19 Hz), 117.35 (d, 3JC,F
7 Hz), 118.58, 122.29, 124.52 (d, 3JC,F = 3 Hz), 126.93, 130.41, 135.99 (d, 2JC,F = 11 Hz), 151.66
(d, JC,F = 237 Hz), 151.84. HRMS: m/z [M + H]+ calcd for C18H17ON2F2: 315.13, found:
1
315.1299.
1
2,5-bis(3-fluoroanilinomethyl)furan, (2i) Orange oil, 35% yield. H NMR (400 MHz, CDCl3):
δ
ppm 4.23 (s, 4H), 6.15 (s, 2H), 6.34 (dd, J=11.49, 1.64 Hz, 2H), 6.37 - 6.45 (m, 4H), 7.05 -
13
2
7.13 (m, 2H); C NMR (100 MHz, CDCl3):
δ ppm 41.27, 99.82 (d, JC,F = 26 Hz), 104.42 (d,
2JC,F = 22 Hz), 108.07, 108.99 (d, JC,F = 2 Hz), 130.30 (d, JC,F = 10 Hz), 149.30 (d, JC,F = 10
Hz), 151.69, 164.01 (d, 1JC,F = 242 Hz). HRMS: m/z [M + H]+ calcd for C18H17ON2F2: 315.1303,
found: 315.1292.
4
3
3
1
2,5-bis(4-fluoroanilinomethyl)furan, (2j) Orange oil, 52% yield. H NMR (400 MHz, CDCl3):
13
δ
4.22 (s, 4H), 6.13 (s, 2H), 6.53 - 6.64 (m, 4H), 6.88 (m, J=8.70, 8.70 Hz, 4H); C NMR (100
3
2
MHz, CDCl3):
δ
ppm 42.10, 107.90, 114.15 (d, JC,F = 7 Hz), 115.66 (d, JC,F = 22 Hz), 143.86,
17

ACCEPTED MANUSCRIPT
1
143.88, 153.52 (d, JC,F = 235 Hz), 157.35. HRMS: m/z [M + H]+ calcd for C18H17ON2F2:
315.1294, found: 315.1303.
1
2,5-bis(2-chloroanilinomethyl)furan, (2k) Orange oil, 6% yield. H NMR (400 MHz, CDCl3):
δ
ppm 4.35 (br s, 4H), 4.67 (br s, 2H), 6.17 (br s, 2H), 6.63 - 6.69 (m, 2H), 6.73 (br s, 2H), 7.12
13
(br s, 2H), 7.26 (br s, 2H); C NMR (100 MHz, CDCl3):
δ ppm 41.16, 107.98, 111.59, 117.89,
119.46, 127.76, 129.19, 143.42, 151.72. HRMS: m/z [M + H]+ calcd for C18H17ON2Cl2:
347.0712, found: 347.0509.
2,5-bis(3-chloroanilinomethyl)furan, (2l) Orange oil, 59% yield. 1H NMR (400 MHz, CDCl3):
δ
ppm 4.21 (s, 4H), 6.13 (s, 2H), 6.49 (d, J=8.34 Hz, 2H), 6.61 (br s, 2H), 6.69 (d, J=6.82 Hz,
2H); 13C NMR (100 MHz, CDCl3):
δ ppm 41.18, 108.10, 111.43, 112.79, 114.89, 117.83,
130.21, 148.69, 151.67. HRMS: m/z [M + H]+ calcd for C18H17ON2Cl2: 347.0712, found:
347.0685.
1
2,5-bis(4-chloroanilinomethyl)furan, (2m) Orange oil, 71% yield. H NMR (400 MHz,
CDCl3):
NMR (100 MHz, CDCl3):
m/z [M + H]+ calcd for C18H17ON2Cl2: 347.0712, found: 347.0696.
δ
ppm 4.21 (s, 4H), 6.12 (s, 2H), 6.54 (d, J=8.59 Hz, 4H), 7.09 (d, J=8.59 Hz, 4H). 13C
δ
ppm 41.26, 107.80, 114.06, 122.32, 28.85, 146.01, 151.69. HRMS:
1
2,5-bis(2-methoxyanilinomethyl)furan, (2n) White solid, 29% yield. H NMR (400 MHz,
CDCl3): ppm 7.12 (t, J = 7.71 Hz, 2H), 7.06 (d, J = 7.07 Hz, 2H), 6.64 - 6.72 (m, 4H), 6.17 (s,
δ
2H), 4.33 (s, 4H), 2.14 (s, 6H); 13C NMR (100 MHz, CDCl3):
δ ppm 17.49, 41.50, 107.83,
110.13, 117.61, 122.34, 127.07, 130.14, 145.56, 152.22. HRMS: m/z [M + Z]+ calcd for
C20H22N2O3Na: 361.1528, found: 361.1537.
1
2,5-bis(3-methoxyanilinomethyl)furan, (2o) Orange oil, 30% yield. H NMR (400 MHz,
CDCl3):
δ ppm 7.00 - 7.13 (m, 2H), 6.19 - 6.35 (m, 6H), 6.15 (s, 2H), 4.25 (s, 4H), 3.65 - 3.79
(m, 6H); 13C NMR (100 MHz, CDCl3):
δ ppm 41.45, 55.07, 99.20, 103.09, 106.23, 107.87,
129.96, 148.98, 151.99, 160.74. HRMS: m/z [M + H]+ calcd for C20H23N2O3: 339.1676, found:
339.1687.
1
2,5-bis(4-methoxyanilinomethyl)furan, (2p) Brown solid, 35% yield. H NMR (400 MHz,
CDCl3):
δ ppm 3.74 (6H, s), 4.22 (4H, s), 6.13 (2H, s), 6.63 (4H, d, J=8.84 Hz), 6.78 (4H, d,
J=8.84 Hz); 13C NMR (100 MHz, CDCl3):
δ ppm 42.52, 55.73, 107.72, 114.81, 116.42, 141.77,
152.34, 152.58. HRMS: m/z [M + H]+ calcd for C20H23N2O3 : 339.1701, found: 339.1703.
2,5-bis(2-trifluoromethylanilinomethyl)furan, (2q) Yellow oil, 9% yield. 1H NMR (400 MHz,
CDCl3):
δ
ppm 4.37 (br d, J=4.55 Hz, 4H), 4.72 (br s, 2H), 6.17 (s, 2H), 6.70 - 6.88 (m, 4H),
ppm 41.06,
7.31 - 7.41 (m, 2H), 7.45 (br d, J=7.58 Hz, 2H); 13C NMR (100 MHz, CDCl3):
δ
108.02, 112.18, 113.96 (m, 2JC,F = 29 Hz), 116.66, 125.10 (d, 1JC,F = 270 Hz), 126.68 (m, 3JC,F
=
5 Hz), 133.06, 144.97, 151.48. HRMS: m/z [M + H]+ calcd for C20H17F6N2O: 415.1240, found:
415.1246.
1
2,5-bis(3-trifluoromethylanilinomethyl)furan, (2r) Orange oil, 60% yield. H NMR (400
MHz, CDCl3):
δ
ppm 4.29 (s, 4H), 6.17 (s, 2H), 6.76 - 6.80 (m, 2H), 6.86 (br s, 2H), 6.95 - 6.98
18

ACCEPTED MANUSCRIPT
(m, 2H), 7.20 - 7.24 (m, 2H); 13C NMR (100 MHz, CDCl3):
δ ppm 41.14, 108.23, 109.31 (q,
3JC,F = 4 Hz), 114.44 (q, 3JC,F = 4 Hz), 116.11, 117.97, 124.32 (d, 1JC,F = 271 Hz), 129.65, 128.73,
131.56 (d, 2JC,F = 31 Hz), 146.74, 147.67. MS (ESI): m/z [M]+: 413.11
1
2,5-bis(4-trifluoromethylanilinomethyl)furan, (2s) Orange oil, 33 % yield. H NMR (400
MHz, CDCl3):
δ ppm 4.32 (s, 4H), 6.18 (s, 2H), 6.66 (d, J=8.34 Hz, 4H), 7.41 (d, J=8.08 Hz,
4H); 13C NMR (100 MHz, CDCl3):
δ ppm 40.94, 108.23, 112.23, 119.80, 123.53, 126.64,
149.93, 151.54. HRMS: m/z [M+ Z]+ calcd for C20H16F6N2ONa: 437.1066, found: 437.1059.
1
2,5-bis(4-thiomethylanilinomethyl)furan, (2t) Orange oil, 25% yield. H NMR (400 MHz,
CDCl3):
δ ppm 2.40 (s, 6H), 4.25 (s, 4H), 6.14 (s, 2H), 6.60 (d, J=8.59 Hz, 4H), 7.20 (d, J=8.59
Hz, 4H). 13C NMR (100 MHz, CDCl3):
δ ppm 18.87, 41.48, 107.92, 113.81, 125.17, 131.12,
146.31, 151.94. HRMS: m/z [M + H]+ calcd for C20H23N2OS2: 371.1238, found: 371.1246.
1
2,5-bis(4-methypiperazin-1-ylmethyl)furan, (2u) Yellow oil, 40% yield. H NMR (400 MHz,
CDCl3):
(100 MHz, CDCl3):
δ
ppm 2.30 (s, 6H), 2.53 (m, J=16.17 Hz, 16H), 3.54 (s, 4H), 6.13 (s, 2H); 13C NMR
δ
ppm 45.84, 52.48, 54.76, 54.89, 109.48, 151.09. HRMS: m/z [M + H]+
calcd for C16H29N4O: 293.2336, found: 293.2323.
2,5-bis(morpholinomethyl)furan (2v) Clear oil, 8% yield. 1H NMR (400 MHz, CDCl3):
δ ppm
1.61 (br s, 5H), 2.50 (br s, 5H), 3.54 - 3.76 (m, 19H), 3.88 (br s, 4H), 6.35 (br s, 1H), 7.13 (br s,
3H); 13C NMR (100 MHz, CDCl3):
δ
ppm 51.93, 55.21, 66.78, 110.86, 118.67.
1
2,5-bis(thiomorpholinomethyl)furan (2w) Clear oil, 6% yield. H NMR (400 MHz, CDCl3):
δ
ppm 2.65 - 2.72 (m, 8H), 2.73 - 2.81 (m, 8H), 3.63 (s, 4H), 6.34 (s, 1H), 7.14 (s, 1H); 13C NMR
(100 MHz, CDCl3): ppm 27.94, 51.88, 54.55, 55.68, 110.82, 118.85.
δ
1.1.1 General Procedure for the Synthesis of the 3,4-Thiophene Analogues (6)
Procedure for the Synthesis of Thiophene-3,4-dimethanol (4).
The dimethanol compound was prepared according to a literature procedure[36] where 500 mg
of thiophene-3,4-dicarboxylicacid (2.9mmol) was added to 10mL of dry THF in a dry round
bottom flask and was chilled to 0°C. 17.375mL (17.375mmol) of diisobutylaluminimum hydride
(139mL in 1M hexanes) was added to the flask and the solution was stirred at room temperature
for 16 hours. The reaction was quenched with methanol and water and then 25mL of HCl was
added to break up the solid chunks in the reaction. The solution as then extracted with ethyl
acetate, washed with brine and dried with MgSO4. The solution was then evaporated under
reduced pressure to give an orange oil in 77% yield.
Procedure for the Synthesis of Thiophene-3,4-dicarbaldehyde (5). The 3,4-
thiophenedicarbaldehyde was prepared using an adapted literature procedure[37] where 2.0 g of
Compound 4 (13.9 mmol) was added to a 100 mL solution of dry 1,4-dioxane. 6.0 g of dry
activated MnO2 (69.00 mmol) was added and the mixture was refluxed under N2 for forty-five
minutes at 96oC. The solution was filtered through a fritted filter funnel and was concentrated
19

ACCEPTED MANUSCRIPT
under reduced pressure to yield a light yellow solid in 90% yield. 1H NMR was used to confirm
the structure with the literature characterization
General Procedure for the Synthesis of the 3,4-bis(anilino)thiophene analogues (6). To a
solution of methanol, 50mg (0.3568 mmol) of thiophene-3,4-dicarbaldehyde was combined in a
dry vial with the aniline derivative of choice (0.7849 mmol), 145.68 mg (1.070 mmol) of zinc
chloride (ZnCl2). The solution was stirred at room temperature overnight. The solution was
reduced to dryness and was then dissolved in ethanol, where 67.24 mg (1.070 mmol) of sodium
cyanoborohydride (NaBH3CN) was added. The solution was then stirred overnight and purified
by flash chromatography.
1
3,4-bis(anilinomethyl)thiophene, (6a) Brown semisolid, 4% yield. H NMR (400 MHz,
CDCl3):
MHz, CDCl3):
H]+ calcd for C18H19N2S: 291.0956, found: 291.0963.
δ
ppm 4.31 (s, 4H), 6.44 (d, 2H), 6.65 (d, 4H), 7.18 (t, 4H), 7.24 (s, 2H); 13C NMR (100
δ
ppm 42.89, 113.21, 117.98, 124.23, 129.20, 138.38, 147.94. HRMS: m/z [M +
1
3,4-bis(3-methylanilinomethyl)thiophene, (6b) Orange solid, 26% yield. H NMR (400 MHz,
CDCl3): ppm 2.34 (s, 6H), 4.77 (s, 4H), 6.81 (d, J=7.83 Hz, 2H), 6.84 (s, 2H), 7.00 (s, 2H),
δ
13
7.18 - 7.28 (m, 4H); C NMR (100 MHz, CDCl3):
δ ppm 21.83, 49.59, 114.71, 116.91, 118.75,
120.29, 128.74, 138.62, 140.60, 151.66. HRMS: m/z [M + H]+ calcd for C20H23N2S: 323.1576,
found: 323.1561.
1
3,4-bis(4-methylanilinomethyl)thiophene, (6c) Orange solid, 19% yield. H NMR (400 MHz,
CDCl3):
δ ppm 2.33 (s, 6H), 4.77 (s, 4H), 6.90 (d, J=8.08 Hz, 4H), 7.01 (s, 2H), 7.80 (d, J=8.34
Hz, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 20.82, 49.67, 114.69, 120.05, 121.71, 123.21,
129.47. HRMS: m/z [M + H]+ calcd for C20H23N2S: 323.1576, found: 323.1565.
1
3,4-bis(4-ethylanilinomethyl)thiophene, (6d) Orange oil, 3% yield. H NMR (400 MHz,
CDCl3):
δ ppm 1.19 (t, J = 7.45 Hz, 6H), 2.54 (q, J = 7.58 Hz, 4H), 4.28 (s, 4H), 6.60 (d, J =
8.08 Hz, 4H), 7.02 (d, J = 8.08 Hz, 4H), 7.23 (s, 2H); 13C NMR (100 MHz, CDCl3):
δ ppm
42.89, 113.21, 117.98, 124.23, 129.30, 138.38, 147.94. HRMS: m/z [M + H]+ calcd for
C22H27N2S: 351.1889, found: 351.1888.
3,4-bis(3-fluoroanilinomethyl)thiophene, (6e) Off-white semi solid, 12 % yield. 1H NMR (400
13
MHz, CDCl3):
(100 MHz, CDCl3):
δ
ppm 4.43 (br s, 4H), 6.21 - 6.66 (m, 8H), 7.14 (d, J = 7.07 Hz, 2H); C NMR
2
2
δ
ppm 49.13, 98.46 (d, JC,F= 26 Hz), 102.84 (d, JC,F= 21 Hz), 107.13,
114.68, 130.40 (d, JC,F= 10 Hz), 149.23 (d, JC,F= 11 Hz), 164.23 (d, JC,F= 241 Hz). HRMS:
3
3
1
m/z [M + H]+ calcd for C18H17N2SF2: 331.1061, found: 331.1075.
1
3,4-bis(4-fluoroanilinomethyl)thiophene, (6f) Yellowish semisolid, 15% yield. H NMR (400
MHz, CDCl3):
δ ppm 4.25 (s, 4H), 6.49 - 6.61 (m, 4H), 6.88 (t, J = 8.72 Hz, 4H), 7.23 (s, 2H);
3
2
13C NMR (100 MHz, CDCl3):
δ
ppm 43.58, 114.13 (d, JC,F = 7 Hz), 115.75 (d, JC,F = 22 Hz),
124.40, 138.22, 144.26, 156.16 (d, 1JC,F = 233 Hz). HRMS: m/z [M + H]+ calcd for C18H17N2SF2:
331.1072, found: 331.1075.
20

ACCEPTED MANUSCRIPT
1
3,4-bis(3-chloroanilinomethyl)thiophene, (6g) Off-white oil, 31 % yield. H NMR (400 MHz,
CDCl3): ppm 4.26 (s, 4H), 6.48 (dd, J=8.21, 1.39 Hz, 2H), 6.60 (s, 2H), 6.69 (dd, J=0.80 Hz,
δ
2H), 7.06 (t, J=7.96 Hz, 2H); 13C NMR (100 MHz, CDCl3):
δ ppm 42.60, 111.47, 112.76,
117.85, 124.53, 130.27, 135.05, 137.64, 148.95. HRMS: m/z [M + H]+ calcd for C18H17N2Cl2S:
363.0484, found: 363.0466.
1
3,4-bis(4-chloroanilinomethyl)thiophene, (6h) Brown solid, 23% yield. H NMR (400 MHz,
CDCl3):
NMR (100 MHz, CDCl3):
m/z [M + H]+ calcd for C18H17N2Cl2S: 363.0408, found: 363.0466.
δ
ppm 4.26 (s, 4H), 6.54 (d, J=8.84 Hz, 4H), 7.11 (d, J=8.59 Hz, 4H), 7.22 (s, 2H); 13C
δ
ppm 42.97, 114.26, 122.67, 124.50, 129.15, 137.82, 146.39. HRMS:
1
3,4-bis(3-trifluoromethylanilinomethyl)thiophene, (6i) Orange oil, 20% yield. H NMR (400
MHz, CDCl3): ppm 4.32 (s, 4H), 6.74 (d, J=8.08 Hz, 2H), 6.82 (br s, 2H), 6.89 - 7.04 (m, 2H),
δ
7.23 - 7.33 (m, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 42.61, 109.28, 114.50, 116.09, 125.77,
125.63, 129.76, 131.48, 137.47, 147.95. HRMS: m/z [M + H]+ calcd for C20H17F6N2S: 431.1011,
found: 431.0994.
1
3,4-bis(4-trifluoromethylanilinomethyl)thiophene, (6j) White semisolid, 6% yield. H NMR
(400 MHz, CDCl3):
δ ppm 4.33 (br s, 4H), 6.62 (d, J=8.08 Hz, 4H), 7.25 (s, 2H), 7.40 (d, J=8.34
Hz, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 42.36, 112.21, 124.77, 126.66, 126.69, 126.73,
137.26, 150.17. HRMS: m/z [M + H]+ calcd for C20H17F6N2S: 431.1011, found: 431.0995.
General Procedure for the Synthesis of the 2,5-bis(anilino)thiophene analogues (8).
Procedure A: To a solution of 60 mg of thiophene-2,5-dicarbaldehyde (0.4281 mmol) in DCE
(4.3 mL), a substituted amine (2.3 equivalents), and acetic acid (0.8562 mmol) were added. The
solution was stirred for 5 min at room temperature, and then treated with sodium
triacetoxyborohydride (NaBH(OAc)3) (1.2843 mmol). The solution was then stirred from for 5
hrs to overnight depending on the amine. The product was purified via flash column
chromatography, or preparative thin layer chromatography. Procedure A used to synthesize 8a-
8p and 8t-8w.
Procedure B: To a solution of 60 mg of thiophene-2,5-dicarbaldehyde (0.4281 mmol) in
Methanol (4.3 mL), a substituted amine (2.0-2.3 equivalents) was added. The solution was stirred
for 1 h at room temperature, and then treated with sodium brorohydride (NaBH4) (1.2843 mmol).
The solution was then stirred overnight. The product was purified via flash column
chromatography, or preparative thin layer chromatography. Procedure B used to synthesize 8q-
8s.
2,5-bis(anilinomethyl)thiophene (8a) Off-white solid, 26% yield. 1H NMR (400 MHz, CDCl3):
δ
ppm 4.42 (s, 4H), 6.65 (d, =7.83 Hz, 4H), 6.73 (t, J=7.33 Hz, 2H), 6.83 (s, 2H), 7.17 (t, J=7.83
Hz, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 43.64, 113.12, 118.06, 124.75, 129.26, 142.19,
147.53. HRMS: m/z [M + H]+ calcd for C18H18N2SNa: 317.1088, found: 317.1088.
2,5-bis(2-methylanilinomethyl)thiophene (8b) 15 % yield. 1H NMR (400 MHz, CDCl3):
δ ppm
2.15 (s, 6H), 4.49 (s, 4H), 6.63 - 6.74 (m, 4H), 6.87 (s, 2H), 7.02 - 7.16 (m, 4H); 13C NMR (100
21

ACCEPTED MANUSCRIPT
MHz, CDCl3):
δ
ppm 17.52, 43.66, 110.14, 117.66, 122.28, 124.80, 127.11, 130.15, 142.26,
145.53. HRMS: m/z [M + H]+ calcd for C20H22N2S: 323.1576, found: 323.1576.
2,5-bis(3-methylanilinomethyl)thiophene (8c) 14 % yield. 1H NMR (400 MHz, CDCl3):
δ ppm
2.27 (s, 6H), 4.42 (s, 4H), 6.39 - 6.52 (m, 4H), 6.56 (d, J=7.58 Hz, 2H), 6.83 (s, 2H), 7.07 (t,
13
J=7.58 Hz, 2H); C NMR (100 MHz, CDCl3):
δ ppm 21.62, 43.67, 110.20, 113.94, 119.01,
124.71, 129.14, 139.05, 142.24, 147.59. HRMS: m/z [M + H]+ calcd for C20H18N2SNa:
345.1401, found: 345.1401.
2,5-bis(4-methylanilinomethyl)thiophene (8d) 24 % yield. 1H NMR (400 MHz, CDCl3):
2.23 (s, 6H), 4.39 (s, 4H), 6.57 (d, J=8.34 Hz, 4H), 6.81 (s, 2H), 6.98 (d, J=8.34 Hz, 4H); 13C
δ ppm
NMR (100 MHz, CDCl3):
δ ppm 20.42, 43.98, 113.27, 124.61, 129.75, 142.34, 145.28. HRMS:
m/z [M + H]+ calcd for C20H23N2S: 323.1576, found: 323.1576.
1
2,5-bis(2-ethylanilinomethyl)thiophene (8e) 14 % yield. H NMR (400 MHz, CDCl3):
δ ppm
1.24 (t, J = 7.53 Hz, 16H), 2.49 (q, J = 7.50 Hz, 4H), 4.48 (s, 4H), 6.69 (d, J = 8.02 Hz, 7H), 6.74
13
(t, J = 7.34 Hz, 7H), 6.86 (s, 7H), 7.04 - 7.15 (m, 14H); C NMR (100 MHz, CDCl3):
δ ppm
12.88, 23.88, 43.68, 110.47, 117.84, 124.75, 126.96, 127.95, 142.33, 144.90. HRMS: m/z [M +
H]+ calcd for C22H25N2S: 349.1744, found: 349.1733.
1
2,5-bis(3-ethylanilinomethyl)thiophene (8f) 18 % yield. H NMR (400 MHz, CDCl3):
δ ppm
1.12 - 1.27 (m, 21H), 2.56 (q, J=7.07 Hz, 14H), 4.42 (br s, 4H), 6.42 - 6.54 (m, 14H), 6.60 (d,
13
J=7.07 Hz, 7H), 6.83 (br s, 7H), 7.09 (t, J=7.45 Hz, 7H); C NMR (100 MHz, CDCl3):
δ ppm
15.51, 28.99, 43.69, 110.38, 112.84, 117.79, 124.73, 129.19, 142.22, 145.45, 147.63. HRMS:
m/z [M + H]+ calcd for C22H27N2S: 351.1889, found: 351.1889.
1
2,5-bis(4-ethylanilinomethyl)thiophene (8g) Yellow solid, 7 % yield. H NMR (400 MHz,
CDCl3):
Hz, 4H), 6.83 (s, 2H), 7.02 (d, J=8.34 Hz, 4H); 13C NMR (100 MHz, CDCl3):
27.94, 44.01, 113.26, 124.64, 128.58, 133.93, 142.38, 145.53. HRMS: m/z [M + H]+ calcd for
S: 351.1881, found: 351.1889.
δ
ppm 1.18 (t, J=7.58 Hz, 6H), 2.54 (q, J=7.58 Hz, 4H), 4.41 (s, 4H), 6.61 (d, J=8.34
δ
ppm 15.92,
C
22
H
27
N2
2,5-bis(2-fluoroanilinomethyl)thiophene (8h) Light yellow oil, 6% yield. 1H NMR (400 MHz,
CDCl3): ppm 4.48 (s, 4H), 6.66 (dd, J=3.28, 4.55 Hz, 5H), 6.71 - 6.79 (m, 5H), 6.86 (s, 5H),
6.93 - 7.03 (m, 10H); 13C NMR (100 MHz, CDCl3): ppm 43.25, 112.51 (d, 3JC,F=3 Hz), 114.54
(d, 2JC,F=18 Hz), 117.41 (d, 3JC,F=7 Hz), 124.56, 124.93, 135.98 (d, 2JC,F=11 Hz), 141.85, 151.57
δ
δ
(d, 1JC,F=238 Hz). HRMS: m/z [M + H]+ calcd for C18
H17
N2
F2
S: 331.1070, found: 331.1075.
1
2,5-bis(3-fluoroanilinomethyl)thiophene (8i) Yellow oil, 3% yield. H NMR (400 MHz,
CDCl3):
δ ppm 4.43 (s, 4H), 6.28 - 6.38 (m, 2H), 6.42 (d, J=7.83 Hz, 4H), 6.86 (s, 2H), 7.03 -
2
7.18 (m, 2H); 13C NMR (100 MHz, CDCl3):
δ ppm 43.52, 113.85 (d, JC,F=21 Hz), 114.92 (d,
2JC,F=22 Hz), 123.62, 124.60, 129.81 (d, JC,F=8 Hz), 142.71 (d, JC,F=6 Hz), 143.02, 162.97 (d,
3
3
1JC,F=249 Hz), 143.02. HRMS: m/z [M + H]+ calcd for C18
H17
N2
F2S: 331.1070, found:
331.1075.
22

ACCEPTED MANUSCRIPT
1
2,5-bis(4-fluoroanilinomethyl)thiophene (8j) White solid, 6% yield. H NMR (400 MHz,
CDCl3):
CDCl3):
δ
δ
ppm 4.36 (s, 4H), 6.49 - 6.63 (m, 4H), 6.76 - 6.93 (m, 6H); 13C NMR (100 MHz,
3
2
ppm 43.32, 113.11 (d, JC,F=8 Hz), 116.75 (d, JC,F=22 Hz), 123.81, 141.19, 142.90,
155.2 (d, 1JC,F=234 Hz).
1
2,5-bis(2-chloroanilinomethyl)thiophene (8k) Clear oil, 5% yield. H NMR (400 MHz,
CDCl3): δ ppm 4.51 (br s, 4H), 6.60 - 6.77 (m, 4H), 6.87 (br s, 2H), 7.12 (t, J=7.45 Hz, 2H), 7.22
- 7.31 (m, 3H); 13C NMR (100 MHz, CDCl3):
129.18, 141.63, 143.32.
δ
ppm 43.27, 111.59, 117.91, 124.90, 127.77,
1
2,5-bis(3-chloroanilinomethyl)thiophene (8l) 3% yield. H NMR (400 MHz, CDCl3):
δ ppm
4.42 (s, 4H), 6.52 (dd, J=1.76, 8.22 Hz, 7H), 6.63 (s, 7H), 6.70 (d, J=7.82 Hz, 6H), 6.85 (s, 7H),
7.08 (t, J=8.12 Hz, 7H); 13C NMR (100 MHz, CDCl3):
δ ppm 43.41, 111.39, 112.79, 125.05,
130.24, 135.03, 141.68, 148.57. HRMS: m/z [M + H]+ calcd for C18
363.0484.
H17
N
2Cl2
S: 363.0478, found:
1
2,5-bis(4-chloroanilinomethyl)thiophene (8m) White solid, 5% yield. H NMR (400 MHz,
CDCl3):
(100 MHz, CDCl3):
δ
ppm 4.38 (s, 4H), 6.51 - 6.57 (m, 4H), 6.81 (s, 2H), 7.07 - 7.13 (m, 4H); 13C NMR
δ
ppm 43.64, 114.19, 122.61, 124.85, 129.07, 141.88, 145.98
1
2,5-bis(2-methoxyanilinomethyl)thiophene (8n) 7 % yield. H NMR (400 MHz, CDCl3):
δ
ppm 3.83 (s, 6H), 4.46 (s, 4H), 6.63 - 6.74 (m, 4H), 6.75 - 6.81 (m, 2H), 6.82 - 6.90 (m, 4H); 13C
NMR (100 MHz, CDCl3):
δ ppm 43.44, 55.44, 109.56, 110.37, 117.20, 121.23, 124.70, 137.53,
142.20, 146.94. HRMS: m/z [M + Z]+ calcd for C20H22N2O2SNa: 377.1300, found: 377.1300.
1
2,5-bis(3-methoxyanilinomethyl)thiophene (8o) 12 % yield. H NMR (400 MHz, CDCl3):
δ
13
ppm 3.74 (s, 6H), 4.41 (s, 4H), 6.14 - 6.36 (m, 6H), 6.83 (s, 2H), 7.08 (t, J=8.08 Hz, 2H); C
NMR (100 MHz, CDCl3):
δ ppm 43.64, 55.08, 99.21, 103.22, 106.21, 124.83, 130.02, 142.08,
148.90, 160.79. HRMS: m/z [M + Z]+ calcd for C20H22N2O2SNa: 377.1300, found: 377.1300.
1
2,5-bis(4-methoxyanilinomethyl)thiophene (8p) Light yellow solid, 3 % yield. H NMR (400
MHz, CDCl3):
δ ppm 3.74 (s, 6H), 4.39 (s, 4H), 6.63 (d, J=8.84 Hz, 4H), 6.75 - 6.85 (m, 6H);
13C NMR (100 MHz, CDCl3):
δ ppm 44.66, 55.75, 114.56, 114.86, 124.61, 141.75, 142.41,
152.55. HRMS: m/z [M + H]+ calcd for C20H22N2O2SNa: 377.1300, found: 377.1300.
1
2,5-bis(2-trifluoromethylanilinomethyl)thiophene (8q) Clear oil, 20% yield. H NMR (400
MHz, CDCl3):
δ
ppm 4.51 (d, J=5.31 Hz, 4H), 4.77 (br s, 2H), 6.70 - 6.81 (m, 4H), 6.85 (s, 2H),
ppm 43.52,
7.34 (t, J=7.71 Hz, 2H), 7.45 (d, J=7.58 Hz, 2H); 13C NMR (100 MHz, CDCl3):
δ
2
3
99.73, 99.99, 104.52 (d, JC,F=22 Hz), 108.93, 124.98, 130.32 (d, JC,F=10 Hz), 141.74, 149.24
(d, 3JC,F=11 Hz).
1
2,5-bis(3-trifluoromethylanilinomethyl)thiophene (8r) Clear oil, 7% yield. H NMR (400
MHz, CDCl3):
δ ppm 4.24 (br s, 2H), 6.72 - 6.82 (m, 2H), 6.82 - 6.92 (m, 4H), 6.97 (d, J=7.83
Hz, 2H), 7.20 - 7.33 (m, 2H); 13C NMR (100 MHz, CDCl3):
119.04, 124.74, 129.17, 139.08, 142.27, 147.62.
δ ppm 43.70, 110.25, 113.97,
23

ACCEPTED MANUSCRIPT
1
2,5-bis(4-trifluoromethylanilinomethyl)thiophene (8s) Light yellow solid, 6% yield. H NMR
(400 MHz, CDCl3): ppm 4.25 - 4.61 (m, 6H), 6.63 (d, J=8.34 Hz, 4H), 6.85 (s, 2H), 7.39 (d,
δ
2
J=8.59 Hz, 4H); 13C NMR (100 MHz, CDCl3):
δ ppm 43.11, 112.24, 119.58 (d, JC,F=32 Hz),
124.91 (d, 1JC,F = 269 Hz), 125.10, 125.15, 126.66 (q, 3JC,F=4 Hz), 141.57, 149.87.
1
2,5-bis(4-thiomethylanilinomethyl)thiophene, (8t) Orange oil, 7% yield. H NMR (400 MHz,
CDCl3):
δ ppm 2.36 - 2.40 (m, 12H), 4.37 (s, 2H), 6.56 - 6.61 (m, 7H), 6.80 (s, 1H), 7.13 - 7.18
(m, 7H), 7.18 - 7.20 (m, 1H);13C NMR (100 MHz, CDCl3):
124.61, 125.45, 130.82, 141.89, 144.99, 146.10.
δ
ppm 18.58, 43.40, 113.57, 115.75,
1
2,5-bis(4-methypiperazin-1-ylmethyl)thiophene, (8u) Pale yellow oil, 51% yield. H NMR
(400 MHz, CDCl3):
(100 MHz, CDCl3):
δ
δ
ppm 2.30 (s, 9H), 2.51 (br s, 15H), 3.67 (s, 4H), 6.73 (s, 2H); 13C NMR
ppm 45.81, 52.52, 54.95, 57.28, 125.52, 140.91.
1
2,5-bis(morpholinomethyl)thiophene (8v) Off-white/Light yellow solid, 10% yield. H NMR
13
(400 MHz, CDCl3):
NMR (100 MHz, CDCl3):
calcd for C14H23N2O2S ([M + H+]):283.1475, found: 283.1475.
δ
ppm 2.49 (br s, 8H), 3.66 (s, 4H), 3.68 - 3.77 (m, 8H), 6.74 (s, 2H); C
δ
ppm 53.27, 57.80, 66.98, 125.66, 140.76. HRMS: m/z [M + H]+
1
2,5-bis(thiomorpholinomethyl)thiophene (8w) White solid, 6% yield. H NMR (400 MHz,
CDCl3): δ δ
ppm 2.61 - 2.84 (m, 59H), 3.67 (s, 15H), 6.71 (s, 2H); 13C NMR (100 MHz, CDCl3):
ppm 28.00, 54.57, 58.18, 125.45, 141.05. HRMS: m/z [M + H]+ calcd for C14H23N2S3: 315.1018,
found: 315.1023.
Biology
Primary binding affinity screening
This assay is performed by incubating twenty thousand MDA-MB-231 breast cancer cells in an
8-well slide chamber for two days in 300 µL of medium. The compounds were incubated in
separate wells at concentrations of 1, 10, 100, and 1000 nM for ten minutes at room temperature.
The cells were fixed in a chilled solution of 4% paraformaldehyde and were rehydrated in
phosphate-buffered-saline (PBS). The slides were incubated with 0.05 µg/mL of biotinylated
TN14003 for thirty minutes at room temperature and washed three times with the PBS solution
and incubated in streptavidin-rhodamine (1:150 dilution; Jackson ImmunoResearch Laboratories,
West Grove, PA) for thirty minutes at room temperature. The slides were washed with the PBS
solution and were mounted in an antifade mounting solution (Molecular Probes, Eugene, OR).
All samples were analyzed using a Nikon Eclipse E800 microscope.[26, 38]
Matrigel invasion assay
The Matrigel invasion assay was performed using a Matrigel invasion chamber (Corning
Biocoat; Bedford, MA). A solution of CXCL12 (200 ng/mL; R&D Systems, Minneapolis, MN)
was added to the bottom chamber of the apparatus. 100 nM solutions of the selected compounds
were added to MDA-MB-231 cells and AMD3100 was used as a positive control. The cells were
placed into the top chamber and the apparatus was incubated for 22 hours in a humidified
incubator. After 22 hours, the remaining cells in the top chamber were removed using a cotton
24

ACCEPTED MANUSCRIPT
swab and the cells in the bottom chamber were stained with hematoxylin and eosin (H&E) and
fixed in methanol. The stained cells were counted to calculate the rate of invasion.[26, 38]
Paw Inflammation Suppression Test
In this test, C57BL/6J does (Jackson Laboratories) are subcutaneously injected with
carrageenan (50 L in 1% w/v in saline) in the right hind paw to trigger inflammation; the other
hind paw is used as the non-inflammation control. The selected analogues were prepared in 10%
DMSO and 90% of 45% (2-hydroxypropyl)- -cyclodextrin (CD) in PBS. Doses of the analogues
were set at 10mg/kg and the dose for TN14003 was set at 300 g/kg. The TN14003 dose was set
lower for this experiment because it was found that 300 g/kg gave the maximum efficacy at
λ-
ꢀ
β
ꢀ
ꢀ
minimum concentration in breast cancer metastasis in an animal model. The mice were dosed 30
minutes after the carrageenan injection and then once a day following the initial dose. The mice
were sacrificed 74 hours after inflammation was induced and two hours after the last injection of
the selected analogues. The hind paws of the mice were photographed and calipers were used to
measure the thickness of the paw from front to back. To quantify the edema, the measurement
from the untreated paw was subtracted from the volume of the treated paw. The inflammation
suppression percentage was determined by comparing the analogue treated groups to the control
group. Each analogue was tested in quintuplicate using the above procedure.[26, 32] Paw tissue
slices were also collected and stained with H&E. Tissue slices were scanned and digitized by
NanoZoomer 2.0 HT. The software NDP.view 2 was used to view the slices in detail.
Molecular Docking Simulation
The default parameters in the Maestro docking module of Schrödinger Suite (v. 9.3) were used
unless otherwise noted. The receptor protein (CXCR4) was prepared using the Protein
Preparation Wizard in the Schrödinger Suite that assigns bond orders, adds missing hydrogens,
partial charges, side chains, and creates disulfide bonds. The hydrogen-bonding network was
optimized at neutral pH 7.0. Receptor grid was constructed with Glide using a (10 × 10 × 10) Å3
boundary box spanning the entire ligand binding pocket and centered on the centroid of the co-
crystalized ligand (IT1t from 3ODU: PDB ID). The 3-D structures of ligands were generated
using LigPrep and docked into the CXCR4 grid using Glide Extra Precision (Glide XP) mode
and the Epic state-penalties were added to the Glide score. Our docking model was validated by
redocking study using co-crystalized ligand (IT1t) and RMSD between original structure and
redocking is 1.4443 Å. After docking simulation, the lowest energy pose was selected and
examined by manual review. In the case of unreasonable docking pose, Induced Fit Docking
study was performed using IFD module of Maestro as followings; Standard IFD protocol,
OPLS3 force field, and B-factor based automatic side chain trim options were selected with the
default parameter set-ups (Receptor VDW scaling: 0.70, Ligand VDW scaling 0.50, and max. #
pose: 20).
5. Acknowledgements
We thank the Department of Education GAANN #P200A150308 (TG), Georgia State
University, Department of Chemistry (SRM)
25

ACCEPTED MANUSCRIPT
We thank Dr. Jeremiah Harden for useful discussions and input on the reductive amination
reaction methods
26

ACCEPTED MANUSCRIPT
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