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

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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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

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Authors: Theresa Gainesa; Francisco Garciaa; Saniya Virania; Zhongxing Liangb,c; Younghyoun Yoonc; Oum, Yoon Hyeunb; Hyunsuk Shimb, c; Suazette Reid Mooringa* a Department of Chemistry, Georgia State University, Atlanta, GA, 30303, USA b Department of Radiology and Imaging Science, Emory University School of Medicine, Atlanta, GA, 30322, USA c Winship Cancer Institute, Emory University, Atlanta, GA, 30322, USA

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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

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Graphical Abstract:

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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 1

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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 proinflammatory 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 2aminopyridine 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-xylylenediamines. 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

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AMD 3100

N HN N NH

O R1 N R2

N

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N R2

N R2

N R1 R2

R1 N R2

N R2

N

R1 N

R2

S N R1 R2

R1 N R2 8

S

6

R1 N R2

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2

R1

R1

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R1

SC

WZ811

Figure 1: Development of furan-based and thiophene-based analogues from AMD3100.

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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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O

ZnCl2, NHR1R2, NaBH3CN

O

R2

MeOH, 2 hrs, rt (30-85%)

1

O 2

R1 = H R2 =

NH X

2u:

R1 N

2f: 2g: 2h: 2i: 2j:

N

=

R2

3-Et 4-Et 2-F 3-F 4-F

2k: 2-Cl 2l: 3-Cl 2m: 4-Cl 2n: 2-OMe 2o: 3-OMe

2p: 2q: 2r: 2s: 2t :

4-OMe 2-CF3 3-CF3 4-CF3 4-SMe

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X =

H 2-Me 3-Me 4-Me 2-Et

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2a: 2b: 2c: 2d: 2e:

R1 N R 2

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O

R1 N

N

2v:

R1

N

=

N

O

R2

2w:

R1

=

N

N

S

R2

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Scheme 1: Synthesis of 2,5-furan derivatives.

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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.

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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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Procedure A,or B

O

O

R2

(3-26%)

S

R1 N

R1 N R 2

S

7

8

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R1 = H H N

R2 =

X

8u:

8f: 8g: 8h: 8i: 8j: N

=

R2

3-Et 4-Et 2-F 3-F 4-F

8k: 2-Cl 8l: 3-Cl 8m: 4-Cl 8n: 2-OMe 8o: 3-OMe

8p: 8q: 8r: 8s: 8t:

4-OMe 2-CF3 3-CF3 4-CF3 4-SMe

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R1 N

H 2-Me 3-Me 4-Me 2-Et

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X =

8a: 8b: 8c: 8d: 8e:

N

8v:

N

=

N

O

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R1

8w:

=

N

N

S

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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

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Compounds synthesized were first evaluated using a binding affinity assay. Note, this is a semiquantitative 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

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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 7

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EC (nM)

Invasiona

Compd

R Group

EC (nM)

Invasiona

2a

Aniline

>1000

--

2m

4-Cl aniline

10

48%

2b

2-Me aniline

>1000

--

2n

2-OMe aniline

100

5%

2c

3-Me aniline

>1000

--

2o

3-OMe aniline

>1000

--

2d

4-Me aniline

100

75%

2p

4-OMe aniline

>1000

--

2e

2-Et aniline

1000

--

2q

2-CF3 aniline

100

24%

2f

3-Et aniline

>1000

--

2r

3-CF3 aniline

>1000

--

2g

4-Et aniline

>1000

--

2s

4-CF3 aniline

>1000

--

2h

2-F aniline

10

71%

2t

4-SMe aniline

>1000

--

2i

3-F aniline

>1000

--

2u

1-methylpiperazine

10

82%

2j

4-F aniline

100

53%

2v

Morpholine

1000

--

2k

2-Cl aniline

>1000

--

2w

Thiomorpholine

10

79%

2l

3-Cl aniline

>1000

--

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Compd

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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.

AMD3100[28, --1000 62% 32] Table 1: Binding and invasion assay results for 2,5-furan analogues synthesized. aThe 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.

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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

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6c

4-Me aniline

>1000

6d

4-Et aniline

>1000

6e

3-F aniline

AMD3100[28, 32]

---

R Group

EC (nM)

Invasiona

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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.

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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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Compd

R Group

EC (nM)

8a

Aniline

1

68%

8m

4-Cl aniline

1

22%

8b

2-Me aniline

10

61%

8n

2-OMe aniline

10

52%

8c

3-Me aniline

>1000

--

8o

3-OMe aniline

1000

--

8d

4-Me aniline

100

49%

8p

4-OMe aniline

10

84%

8e

2-Et aniline

1000

--

8q

2-CF3 aniline

>1000

--

8f

3-Et aniline

>1000

--

8g

4-Et aniline

>1000

--

8h

2-F aniline

1000

--

8i

3-F aniline

>1000

--

8j

4-F aniline

>1000

--

8k

2-Cl aniline

10

93%

8l

3-Cl aniline

100

51%

EC (nM)

Invasiona

SC

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R Group

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Invasiona Compd

3-CF3 aniline

100

77%

8s

4-CF3 aniline

>1000

--

8t

4-SMe aniline

100

88%

8u

1-methylpiperazine

100

86%

8v

Morpholine

>1000

--

8w

Thiomorpholine

1

88%

EP

TE D

8r

1000

62%

AC C

AMD3100[28, 32]

Table 3: Binding and invasion assay results for the 2,5-thiophene analogues synthesized. aThe 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 10

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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 2,5thiophene compounds that are not present in the 2,5-furan analogues or the 3,4-thiophene analogues.

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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.

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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-ofconcept 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.

EP

O

R

R

2

S

R

S R

R

R 8

6

R Group

EC (nM)

Invasiona

Carageenanb,c

2d

4-Me aniline

100

75%

15%

2h

2-F aniline

10

71%

--

2j

4-F aniline

100

53%

31%

2m

4-Cl aniline

10

48%

17%

2u

1-Methylpiperazine

10

82%

--

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Compd

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Aniline

10

72%

--

8a

Aniline

1

68%

30%

8b

2-Me aniline

10

61%

--

8d

4-Me aniline

100

49%

--

8k

2-Cl aniline

10

93%

>5%

8l

3-Cl aniline

100

51%

--

8n

2-OMe aniline

10

52%

--

8p

4-OMe aniline

10

8r

3-CF3 aniline

100

8w

Thiomorpholine

SC 84%

--

77%

--

1

88%

--

10

90%

40%

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WZ811[26]

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6a

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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. c Compounds with a dash in the carrageenan studies have not yet been tested.

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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.

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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

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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.

2d

AC C

2j

EC (nM)

EP

Active Compound

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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.

75%

100

100

2m

10

6a

10

Invasion

Key Residue Interactions TRP94b, TYR116b, ARG188b

53%

TRP94b, ASP97a

48%

TRP94b, ASP97a, CYS186b

72%

TRP94b, ASP97a

TRP94b, ASP97a, ARG188b Table 5: Effective concentrations and key residue interactions of hit compounds. aResidue required for CXCR4/CXCL12 signaling. bResidues that make up the active site of CXCR4. 8a

68%

1

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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.

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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.

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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.

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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.

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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-xylylenediamines 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

RI PT

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.

M AN U

SC

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

TE D

4.1 Chemistry The 1H NMR (400 MHz) and 13C 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.

EP

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.

AC C

2,5-bis(anilinomethyl)furan, (2a) Yellow oil, 38% yield. 1H 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. 2,5-bis(2-methylanilinomethyl)furan, (2b) Yellow solid, 35% yield. 1H 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

ACCEPTED MANUSCRIPT

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.

RI PT

2,5-bis(4-methylanilinomethyl)furan, (2d) Orange solid, 49% yield. 1H 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.

M AN U

SC

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.

TE D

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.

AC C

EP

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, 1JC,F = 237 Hz), 151.84. HRMS: m/z [M + H]+ calcd for C18H17ON2F2: 315.13, found: 315.1299. 2,5-bis(3-fluoroanilinomethyl)furan, (2i) Orange oil, 35% yield. 1H 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 7.13 (m, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 41.27, 99.82 (d, 2JC,F = 26 Hz), 104.42 (d, 2 JC,F = 22 Hz), 108.07, 108.99 (d, 4JC,F = 2 Hz), 130.30 (d, 3JC,F = 10 Hz), 149.30 (d, 3JC,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. 2,5-bis(4-fluoroanilinomethyl)furan, (2j) Orange oil, 52% yield. 1H NMR (400 MHz, CDCl3): 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); 13C NMR (100 MHz, CDCl3): δ ppm 42.10, 107.90, 114.15 (d, 3JC,F = 7 Hz), 115.66 (d, 2JC,F = 22 Hz), 143.86, 17

ACCEPTED MANUSCRIPT

143.88, 153.52 (d, 1JC,F = 235 Hz), 157.35. HRMS: m/z [M + H]+ calcd for C18H17ON2F2: 315.1294, found: 315.1303.

RI PT

2,5-bis(2-chloroanilinomethyl)furan, (2k) Orange oil, 6% yield. 1H 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 (br s, 2H), 7.26 (br s, 2H); 13C 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.

SC

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.

M AN U

2,5-bis(4-chloroanilinomethyl)furan, (2m) Orange oil, 71% yield. 1H NMR (400 MHz, CDCl3): 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 NMR (100 MHz, CDCl3): δ ppm 41.26, 107.80, 114.06, 122.32, 28.85, 146.01, 151.69. HRMS: m/z [M + H]+ calcd for C18H17ON2Cl2: 347.0712, found: 347.0696.

TE D

2,5-bis(2-methoxyanilinomethyl)furan, (2n) White solid, 29% yield. 1H NMR (400 MHz, CDCl3): δ 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, 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.

EP

2,5-bis(3-methoxyanilinomethyl)furan, (2o) Orange oil, 30% yield. 1H 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.

AC C

2,5-bis(4-methoxyanilinomethyl)furan, (2p) Brown solid, 35% yield. 1H 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), 7.31 - 7.41 (m, 2H), 7.45 (br d, J=7.58 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 41.06, 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. 2,5-bis(3-trifluoromethylanilinomethyl)furan, (2r) Orange oil, 60% yield. 1H 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, 3 JC,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

RI PT

2,5-bis(4-trifluoromethylanilinomethyl)furan, (2s) Orange oil, 33 % yield. 1H 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.

SC

2,5-bis(4-thiomethylanilinomethyl)furan, (2t) Orange oil, 25% yield. 1H 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.

M AN U

2,5-bis(4-methypiperazin-1-ylmethyl)furan, (2u) Yellow oil, 40% yield. 1H NMR (400 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 (100 MHz, CDCl3): 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.

TE D

2,5-bis(thiomorpholinomethyl)furan (2w) Clear oil, 6% yield. 1H 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): δ ppm 27.94, 51.88, 54.55, 55.68, 110.82, 118.85.

AC C

EP

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 3,4thiophenedicarbaldehyde 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

RI PT

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.

SC

3,4-bis(anilinomethyl)thiophene, (6a) Brown semisolid, 4% yield. 1H NMR (400 MHz, CDCl3): 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 MHz, CDCl3): δ ppm 42.89, 113.21, 117.98, 124.23, 129.20, 138.38, 147.94. HRMS: m/z [M + H]+ calcd for C18H19N2S: 291.0956, found: 291.0963.

M AN U

3,4-bis(3-methylanilinomethyl)thiophene, (6b) Orange solid, 26% yield. 1H NMR (400 MHz, CDCl3): δ 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), 7.18 - 7.28 (m, 4H); 13C 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.

TE D

3,4-bis(4-methylanilinomethyl)thiophene, (6c) Orange solid, 19% yield. 1H 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.

EP

3,4-bis(4-ethylanilinomethyl)thiophene, (6d) Orange oil, 3% yield. 1H 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.

AC C

3,4-bis(3-fluoroanilinomethyl)thiophene, (6e) Off-white semi solid, 12 % yield. 1H NMR (400 MHz, CDCl3): δ ppm 4.43 (br s, 4H), 6.21 - 6.66 (m, 8H), 7.14 (d, J = 7.07 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 49.13, 98.46 (d, 2JC,F= 26 Hz), 102.84 (d, 2JC,F= 21 Hz), 107.13, 114.68, 130.40 (d, 3JC,F= 10 Hz), 149.23 (d, 3JC,F= 11 Hz), 164.23 (d, 1JC,F= 241 Hz). HRMS: m/z [M + H]+ calcd for C18H17N2SF2: 331.1061, found: 331.1075. 3,4-bis(4-fluoroanilinomethyl)thiophene, (6f) Yellowish semisolid, 15% yield. 1H 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); 13 C NMR (100 MHz, CDCl3): δ ppm 43.58, 114.13 (d, 3JC,F = 7 Hz), 115.75 (d, 2JC,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

3,4-bis(3-chloroanilinomethyl)thiophene, (6g) Off-white oil, 31 % yield. 1H NMR (400 MHz, CDCl3): δ 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, 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.

RI PT

3,4-bis(4-chloroanilinomethyl)thiophene, (6h) Brown solid, 23% yield. 1H NMR (400 MHz, CDCl3): 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 NMR (100 MHz, CDCl3): δ ppm 42.97, 114.26, 122.67, 124.50, 129.15, 137.82, 146.39. HRMS: m/z [M + H]+ calcd for C18H17N2Cl2S: 363.0408, found: 363.0466.

M AN U

SC

3,4-bis(3-trifluoromethylanilinomethyl)thiophene, (6i) Orange oil, 20% yield. 1H NMR (400 MHz, CDCl3): δ ppm 4.32 (s, 4H), 6.74 (d, J=8.08 Hz, 2H), 6.82 (br s, 2H), 6.89 - 7.04 (m, 2H), 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. 3,4-bis(4-trifluoromethylanilinomethyl)thiophene, (6j) White semisolid, 6% yield. 1H 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.

EP

TE D

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 8a8p and 8t-8w.

AC C

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 8q8s. 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

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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.

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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, J=7.58 Hz, 2H); 13C 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.

SC

2,5-bis(4-methylanilinomethyl)thiophene (8d) 24 % yield. 1H NMR (400 MHz, CDCl3): δ ppm 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 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.

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2,5-bis(2-ethylanilinomethyl)thiophene (8e) 14 % yield. 1H 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 (t, J = 7.34 Hz, 7H), 6.86 (s, 7H), 7.04 - 7.15 (m, 14H); 13C 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.

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2,5-bis(3-ethylanilinomethyl)thiophene (8f) 18 % yield. 1H 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, J=7.07 Hz, 7H), 6.83 (br s, 7H), 7.09 (t, J=7.45 Hz, 7H); 13C 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.

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2,5-bis(4-ethylanilinomethyl)thiophene (8g) Yellow solid, 7 % yield. 1H NMR (400 MHz, CDCl3): 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 Hz, 4H), 6.83 (s, 2H), 7.02 (d, J=8.34 Hz, 4H); 13C NMR (100 MHz, CDCl3): δ ppm 15.92, 27.94, 44.01, 113.26, 124.64, 128.58, 133.93, 142.38, 145.53. HRMS: m/z [M + H]+ calcd for C22H27N2S: 351.1881, found: 351.1889.

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2,5-bis(2-fluoroanilinomethyl)thiophene (8h) Light yellow oil, 6% yield. 1H NMR (400 MHz, CDCl3): δ 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): δ 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 (d, 1JC,F=238 Hz). HRMS: m/z [M + H]+ calcd for C18H17N2F2S: 331.1070, found: 331.1075. 2,5-bis(3-fluoroanilinomethyl)thiophene (8i) Yellow oil, 3% yield. 1H 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 7.18 (m, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 43.52, 113.85 (d, 2JC,F=21 Hz), 114.92 (d, 2 JC,F=22 Hz), 123.62, 124.60, 129.81 (d, 3JC,F=8 Hz), 142.71 (d, 3JC,F=6 Hz), 143.02, 162.97 (d, 1 JC,F=249 Hz), 143.02. HRMS: m/z [M + H]+ calcd for C18H17N2F2S: 331.1070, found: 331.1075.

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2,5-bis(4-fluoroanilinomethyl)thiophene (8j) White solid, 6% yield. 1H NMR (400 MHz, CDCl3): δ ppm 4.36 (s, 4H), 6.49 - 6.63 (m, 4H), 6.76 - 6.93 (m, 6H); 13C NMR (100 MHz, CDCl3): δ ppm 43.32, 113.11 (d, 3JC,F=8 Hz), 116.75 (d, 2JC,F=22 Hz), 123.81, 141.19, 142.90, 155.2 (d, 1JC,F=234 Hz).

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2,5-bis(2-chloroanilinomethyl)thiophene (8k) Clear oil, 5% yield. 1H 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): δ ppm 43.27, 111.59, 117.91, 124.90, 127.77, 129.18, 141.63, 143.32.

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2,5-bis(3-chloroanilinomethyl)thiophene (8l) 3% yield. 1H 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 C18H17N2Cl2S: 363.0478, found: 363.0484.

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2,5-bis(4-chloroanilinomethyl)thiophene (8m) White solid, 5% yield. 1H NMR (400 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 (100 MHz, CDCl3): δ ppm 43.64, 114.19, 122.61, 124.85, 129.07, 141.88, 145.98 2,5-bis(2-methoxyanilinomethyl)thiophene (8n) 7 % yield. 1H 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.

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2,5-bis(3-methoxyanilinomethyl)thiophene (8o) 12 % yield. 1H NMR (400 MHz, CDCl3): δ 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); 13C 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.

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2,5-bis(4-methoxyanilinomethyl)thiophene (8p) Light yellow solid, 3 % yield. 1H 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); 13 C 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. 2,5-bis(2-trifluoromethylanilinomethyl)thiophene (8q) Clear oil, 20% yield. 1H 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), 7.34 (t, J=7.71 Hz, 2H), 7.45 (d, J=7.58 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 43.52, 99.73, 99.99, 104.52 (d, 2JC,F=22 Hz), 108.93, 124.98, 130.32 (d, 3JC,F=10 Hz), 141.74, 149.24 (d, 3JC,F=11 Hz). 2,5-bis(3-trifluoromethylanilinomethyl)thiophene (8r) Clear oil, 7% yield. 1H 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): δ ppm 43.70, 110.25, 113.97, 119.04, 124.74, 129.17, 139.08, 142.27, 147.62.

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2,5-bis(4-trifluoromethylanilinomethyl)thiophene (8s) Light yellow solid, 6% yield. 1H NMR (400 MHz, CDCl3): δ ppm 4.25 - 4.61 (m, 6H), 6.63 (d, J=8.34 Hz, 4H), 6.85 (s, 2H), 7.39 (d, J=8.59 Hz, 4H); 13C NMR (100 MHz, CDCl3): δ ppm 43.11, 112.24, 119.58 (d, 2JC,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.

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2,5-bis(4-thiomethylanilinomethyl)thiophene, (8t) Orange oil, 7% yield. 1H 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): δ ppm 18.58, 43.40, 113.57, 115.75, 124.61, 125.45, 130.82, 141.89, 144.99, 146.10.

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2,5-bis(4-methypiperazin-1-ylmethyl)thiophene, (8u) Pale yellow oil, 51% yield. 1H NMR (400 MHz, CDCl3): δ ppm 2.30 (s, 9H), 2.51 (br s, 15H), 3.67 (s, 4H), 6.73 (s, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 45.81, 52.52, 54.95, 57.28, 125.52, 140.91.

M AN U

2,5-bis(morpholinomethyl)thiophene (8v) Off-white/Light yellow solid, 10% yield. 1H NMR (400 MHz, CDCl3): δ ppm 2.49 (br s, 8H), 3.66 (s, 4H), 3.68 - 3.77 (m, 8H), 6.74 (s, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 53.27, 57.80, 66.98, 125.66, 140.76. HRMS: m/z [M + H]+ calcd for C14H23N2O2S ([M + H+]):283.1475, found: 283.1475.

Biology

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2,5-bis(thiomorpholinomethyl)thiophene (8w) White solid, 6% yield. 1H NMR (400 MHz, CDCl3): δ ppm 2.61 - 2.84 (m, 59H), 3.67 (s, 15H), 6.71 (s, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 28.00, 54.57, 58.18, 125.45, 141.05. HRMS: m/z [M + H]+ calcd for C14H23N2S3: 315.1018, found: 315.1023.

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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

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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]

Molecular Docking Simulation

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Paw Inflammation Suppression Test In this test, C57BL/6J does (Jackson Laboratories) are subcutaneously injected with λcarrageenan (50 µL 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)-β-cyclodextrin (CD) in PBS. Doses of the analogues were set at 10mg/kg and the dose for TN14003 was set at 300 µg/kg. The TN14003 dose was set lower for this experiment because it was found that 300 µg/kg gave the maximum efficacy at 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.

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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 cocrystalized 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

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

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6. References:

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