N-Sulfonyl-aminobiaryls as Antitubulin Agents and Inhibitors of Signal Transducers and Activators of Transcription 3 (STAT3) Signaling

Article abstract of DOI:10.1021/acs.jmedchem.5b00659

A series of N-sulfonyl-aminobiaryl derivatives have been examined as novel antitubulin agents. Compound 21 [N-(4′-cyano-3′-fluoro-biphenyl-2-yl)-4-methoxy-benzenesulfonamide] exhibits remarkable antiproliferative activity against four cancer cell lines (pancreatic AsPC-1, lung A549, liver Hep3B, and prostate PC-3) with a mean GI₅₀ value of 57.5 nM. Additional assays reveal that 21 inhibits not only tubulin polymerization but also the phosphorylation of STAT3 inhibition with an IC₅₀ value of 0.2 μM. Four additional compounds (8, 10, 19, and 35) are also able to inhibit this phosphorylation. This study describes novel N-sulfonyl-aminobiaryl (biaryl-benzenesulfonamides) as potent anticancer agents targeting both STAT3 and tubulin. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00659

Page 1 of 30
Journal of Medicinal Chemistry
Revised Manuscript for Article
1
2
3
4
5
N-Sulfonyl-aminobiaryls as Antitubulin Agents and Inhibitors of Signal Transducer
6
7
8
9
and Activator of Transcription 3 (STAT3) Signaling
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
＆
＆
Mei-Jung Lai,‡, Hsueh-Yun Lee,†, Hsun-Yueh Chuang, Li-Hsun Chang,# An-Chi Tsai,¶ Mei-Chuan
Chen,§ Han-Lin Huang,¶ Yi-Wen Wu,¢ Che-Ming Teng,# Shiow-Lin Pan,¶,¢ Yi-Min Liu,† Samir
Mehndiratta,† Jing-Ping Liou*,†,¥
School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031,
Taiwan. Ph.D Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy,
Taipei Medical University. Center for Translational Medicine, Taipei Medical University. School of
Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan. The Ph.D program for
Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical
University. School of Pharmacy, National Defense Medical Center, Taipei, Taiwan. Department of
Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan.
＆ Contributed equally to this work.
† School of Pharmacy, College of Pharmacy, Taipei Medical University.
§ Ph.D Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy, Taipei Medical University.
‡ Center for Translational Medicine, Taipei Medical University.
# Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan.
¶ The Ph.D Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical
University.
¥ School of Pharmacy, National Defense Medical Center, Taipei, Taiwan.
¢
Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan.
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 30
ABSTRACT. A series of N-sulfonyl-aminobiaryl derivatives have been examined as novel antitubulin
agents. Compound 21 [N-(4'-cyano-3'-fluoro-biphenyl-2-yl)-4-methoxy-benzenesulfonamide] exhibits
remarkable antiproliferative activity against four cancer cell lines (pancreatic AsPC-1, lung A549, liver
Hep3B, and prostate PC-3) with a mean GI50 value of 57.5 nM. Additional assays reveal that 21 inhibits
not only tubulin polymerization but also the phosphorylation of STAT3 inhibitory with an IC50 value of
0.2 μM. Four additional compounds (8, 10, 19, and 35) are also able to inhibit this phosphorylation.
This study describes novel N-sulfonyl-aminobiaryl (biaryl-benzenesulfonamides) as potent anticancer
agents targeting both STAT3 and tubulin.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Introduction
Microtubules are assembled by the aggregation of α- and β-tubulin heterodimers in a head-to-tail
manner to form hollow cylindrical filaments.1 In the cell cycle, specifically the M-phase, microtubules
assembled as a spindle act as guides that direct chromosomes into individual cells; they therefore are a
crucial target for development of anticancer agents. Natural products such as paclitaxel (1), vinca
alkaloids (2), and colchicine (3) were found to bind to microtubules at specific binding sites and thus
display remarkable microtubule-targeting activity. These microtubule-targeting compounds are
functionally categorized into microtubule-stabilizers (1) and microtubule-destabilizers (2 and 3).2 In
addition to their involvement in the proliferation process, microtubules are associated with several
functions including cell migration and cell skeleton development. These alternative functions are
probably correlated with post-translational modifications such as acetylation, polyglycylation,
polyglutamylation, and tyrosination of tubulin.3
Signal transducers and activators of transcription 3 (STAT3) proteins are transcription factors
normally resident in the cytoplasm. They are activated by phosphorylation by the JAK family of kinases
and subsequently translocate into the nucleus where they regulate gene transcription.4,5 This signaling
pathway is transient in normal cells but in cancer cells, STAT3 is constitutively activated, triggering
ACS Paragon Plus Environment
2

Page 3 of 30
Journal of Medicinal Chemistry
proliferation and oncogenesis.6-9 The poorly understood association of STAT3 protein with tubulin
correlates promotion of cell migration10, but is probably destroyed by microtubule targeting agents. For
example, paclitaxel (1), a tubulin stabilizing agent, has been reported to be capable of triggering the
inhibition of STAT3 expression.11 Consequently, development of multifunctional bioactive molecules
can support clinical indications in the treatment of diverse cancers.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
OH
O
O
OH
N
H CO
3
O
NH O
H
NHCOCH
O
N
H
3
N
O
H
H CO
3
O
H
O
HO
O
O
MeO C
2
CH O
3
OH
H
N
R
O
MeO
OAc
HO CO Me
OCH
2
3
2a: R = Me, vinblastine
2b: R = CHO, vincristine
1: Paclitaxel
3: Colchicine
OCH
3
Cl
H
N
S
O
2
N
N
O
N
N
NH
NH
H
O S
2
O S
2
OCH
3
OH
4: ABT-751
H CO
3
5: Indisulam
6
Figure 1. Natural microtubule-targeting molecules and synthetic benzensulfonamides.
The benzenesulfonamide moiety has been observed in numerous antitubulin agents such as ABT-
751 (4) and indisulam (5), which are currently undergoing clinical trials.12 Our earlier work on the
modification of compound ABT-751 revealed that the benzenesulfonamide moiety is crucial for the
compound’s anticancer activity.13-15 In addition, biaryl moieties have been frequently observed in the
development of anti-tubulin agents.15-17 Compound 6, with both benzenesulfonamide and biaryl
moieties, was found in our previous study to have remarkable antiproliferative activity.15 Literature
surveys indicate that the N-sulfonyl-aminobiaryl moiety has been comprehensively included in various
heterocycles such as carbazole18, phenanthridinones, and phenanthridines19,20; but little investigation of
its biological potential has been reported., This study therefore, is aimed at synthesis of a series of N-
sulfonyl-aminobiaryl derivatives (7-26) and assays of their activity against the growth of cancer cells. In
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 30
view of the known correlation of microtubule-targeting molecules with inhibition of the STAT3 protein,
the synthetic compounds (7-26) have also been examined for their inhibitory activities in tubulin
polymerization and STAT3 phosphorylation.
1
2
3
4
5
6
7
8
9
O2
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
N
H
1
1'
O2
S
2'
3'
6'
5'
N
H
OMe
R
Het
OMe
4'
15: R = 3'-F
16: R = 3'-Cl
17: R = 3'-CN
18: R = 3'-CF3
19: R = 3',4'-diF
20: R = 3'-Cl-4'-F
21: R = 3'-F-4'-CN
22: R = 3',4'-diOMe
23: R = 3',4',5'-triOMe
7: R = H
24: Het = 4-pyridinyl
25: Het = 3-pyridinyl
26: Het = 2-furanyl
8: R = 4'-F
9: R = 4'-Cl
10: R = 4'-CN
11: R = 4'-OH
12: R = 4'-OMe
13: R = 4'-COOMe
14: R = 4'-COOH
Figure 2. Synthetic N-sulfonyl-aminobiaryl compounds (7-26).
Results and Discussion
Chemistry
Scheme 1 shows the synthetic routes to N-sulfonyl-aminobiaryl compounds (7-26 and 29-36). 1-
Bromo-2-nitrobenzene (27) was allowed to react with various phenylboronic acids under conditions of
the Suzuki reaction, yielding the corresponding 2-nitrobiphenyls (28a-s). The nitro groups were reduced
by iron powder in the presence of NH4Cl and refluxing EtOH to afford the corresponding amines and
this was followed by reaction with 4-methoxybenzenesulfonyl chloride to provide compounds 7-26. To
explore the effect of N-substitution on biological activity, compound 10 was reacted with various alkyl
halides in the presence of K2CO3, yielding compounds 29-35. The ester group of compound 34 was
hydrolyzed by LiOH to afford the corresponding carboxylic acid 36.
Scheme 1. Synthetic Approaches to Compounds 7-26 and 29-36a
ACS Paragon Plus Environment
4

Page 5 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
a
NO
2
or
NO
2
NO
2
Br
27
R
Het
28a: R = H
28b: R = 4-F
28c: R = 4-Cl
28d: R = 4-CN
28e: R = 4-OH
28f: R = 4-OCH
28h: R = 3-F
28i: R = 3-Cl
28j: R = 3-CN
28k: R = 3-CF
28l: R = 3,4-diF
28m: R = 3-Cl-4-F
28q: Het = 4-pyridinyl
28r: Het = 3-pyridinyl
28s: Het = 2-furanyl
3
3
28g: R = 4-COOCH 28n: R = 3-F-4-CN
3
28o: R = 3,4-diOCH
28p: R = 3,4,5-triOCH
3
9
3
10
11
12
13
14
15
16
17
18
19
20
21
22
O
S
2
O
S
2
O
S
N
R
2
b
c
N
or
H
N
OMe
H
OMe
Het
R
OMe
CN
29 R = CH
30: R = CH CH
31 R = (CH ) CH
32: R = (CH ) Cl
33: R = (CH ) OH
34: R = CH COOCH
35: R = COCH
15: R = 3'-F
16: R = 3'-Cl
17: R = 3'-CN
18: R = 3'-CF
24: Het = 4-pyridinyl
25: Het = 3-pyridinyl
26: Het = 2-furanyl
7: R = H
8: R = 4'-F
9: R = 4'-Cl
3
2
3
2 2
3
10: R = 4'-CN
11: R = 4'-OH
2 2
3
19: R = 3',4'-diF
b
2 2
20: R = 3'-Cl-4'-F
21: R = 3'-F-4'-CN
22: R = 3',4'-diOMe
23: R = 3',4',5'-triOMe
36: R = CH COOH
2
12: R = 4'-OMe
13: R = 4'-COOMe
14: R = 4'-COOH
2
3
3
aReagents and conditions: (a) substituted phenylboronic acid, Pd(PPh3)4, 2M K2CO3(aq), EtOH, toluene, reflux; (b) i. iron
powder, NH4Cl, i-PrOH, H2O; ii. 4-methoxybenzensulfonyl chloride, pyridine, 50 oC; (c) for 10: K2CO3, alkyl halides, DMF
or MeCN, rt or heating; (d) 1M LiOH(aq), 1,4-dioxane, 40 oC.
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2 describes the synthesis of compounds 37-38 and 41 which possess alternative linking
patterns between the biaryl and the benzene ring. Compound 28d was reduced by iron powder and
NH4Cl to afford the corresponding amine product, which was reacted with 4-methoxybenzyl bromide,
or chloride, to give compound 37. Alternatively, the resulting amine products were reacted with p-
anisoyl chloride to yield the corresponding amide 38. Benzyl derivative 41, in which a carbon atom is
inserted between the sulfonamide moiety and the biphenyl ring, was synthesized by the reaction of
compound 39 with 4-methoxybenzenesulfonyl chloride, and subsequent Suzuki arylation with 4-
cyanophenylboronic acid.
Scheme 2. Synthetic Approaches to Compounds 37-38 and 41a
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 30
1
2
NO2
N
H
a
OMe
3
4
5
CN
28d
CN
37
6
7
O
8
9
N
H
b
OMe
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CN
38
OMe
H
OMe
N
c
d
S
H
N
NH2
O2
S
O2
Br
Br
39
40
41
CN
aReagents and conditions: (a) i. Fe, NH4Cl, H2O, i-PrOH, reflux; ii. anisaldehyde, NaBH3CN, acetic acid, MeOH; (b) i. Fe,
NH4Cl, H2O, i-PrOH, reflux; ii. p-anisoyl chloride, pyridine or THF, rt; (c) 4-methoxybenzenesulfonyl chloride, pyridine, 90
oC; (d) 4-cyanophenylboronic acid, Pd(PPh3)4, 2M K2CO3(aq), EtOH, toluene, reflux.
Biological Evaluation.
A. In Vitro Cell Growth Inhibitory Activity.
Table 1 summarizes the structure-activity relationships in the synthetic compounds, together with
reference compound 4 on the inhibition of cancer cells. Specifically, the antiproliferative activity against
four human cancer cell lines, pancreatic carcinoma AsPC-1, lung carcinoma A549, hepatocellular
carcinoma Hep3B, and prostatic carcinoma PC-3 (Table 1) was examined.
Table 1. Antiproliferative activity of Tested Compounds and Reference Compound 4.
OMe
O
O
S
2
H
N
N
N
S
N
H
O
H
2
R
2
OMe
OMe
OMe
R
1
CN
CN
CN
7-26, 29-36
37
41
38
AsPC-1
A549
Hep3B
GI50 ± SD (μM)a
PC-3
R1
H
R2
compd
H
H
0.60±0.09 0.36±0.02
7
0.88±0.07
0.44±0.01
4-F
0.30±0.05
8
0.36±0.01 0.34±0.01 0.25±0.01
ACS Paragon Plus Environment
6

Page 7 of 30
Journal of Medicinal Chemistry
4-Cl
4-CN
H
0.63±0.24 0.60±0.05 0.42±0.05
9
0.59±0.08
0.15±0.01 0.19±0.01
2.31±0.01 1.60±0.02
1
2
3
4
5
6
7
8
H
0.10±0.01
4.77±0.71 1.21±0.12
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
29
30
31
32
33
34
35
36
0.03±0.01
4-OH
H
4-OCH3
4-COOCH3
4-COOH
3-F
H
2.23±0.82
>10
>10
>10
>10
1.19±0.13
1.15±0.02
H
>10
>10
>10
>10
H
>10
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
H
0.79±0.15 0.54±0.44 0.31±0.02
2.16±1.68
0.52±0.04
3-Cl
H
1.13±0.03 0.65±0.02 0.60±0.03
3-CN
H
0.51±0.05 0.78±0.21 0.51±0.05
0.55±0.03
3-CF3
H
1.79±0.20
3.02±0.13 2.96±0.03 3.43±0.07
3,4-F
H
0.28±0.05 0.30±0.02 0.15±0.03
0.34±0.04
0.19±0.02
3-Cl, 4-F
3-F, 4-CN
3,4-OCH3
3,4,5-OCH3
4-pyridine
3-pyridine
2-furan
4-CN
H
0.53±0.08 0.40±0.01 0.24±0.02
H
0.02±0.01
4.65±0.67
>10
0.03±0.01
0.10±0.01
>10
0.08±0.01
2.17±0.29
>10
H
2.19±0.17
>10
H
>10
H
0.66±0.19 2.25±0.39
2.10±0.03 1.93±0.01
H
1.70±0.42
1.84±0.39 1.00±0.26
1.91±0.01
H
6.13±0.57 3.26±0.21
0.74±0.15 0.39±0.03
3.85±0.09
1.74±0.19
1.5±0.02
5.21±0.93
4.79±0.04
1.52±0.28
>10
1.75±0.04
Me
0.73±0.01
1.84±0.04
1.88±0.05
6.52±0.07
0.51±0.04
>10
4-CN
Et
0.77±0.06 1.10±0.11
1.14±0.07 1.41±0.17
1.50±0.23 1.55±0.06
0.86±0.07 0.66±0.01
4-CN
Pr
2-Cl-Et
2-OH-Et
-CH2COOMe
Ac
4-CN
4-CN
4-CN
>10
>10
4-CN
0.49±0.06
>10
0.30±0.01 0.21±0.01 0.48±0.02
4-CN
-CH2COOH
–
>10
>10
>10
–
1.65±0.06
>10
1.52±0.04
>10
0.93±0.06 0.87±0.06
37
38
41
4
–
–
>10
>10
>10
>10
–
–
>10
>10
–
–
4.11±0.32 5.33±1.14 0.84±0.03 0.62±0.02
aSD: standard deviation, all experiments were independently performed at least three times.
Compound 7 with no substitution, exhibits activity that is slightly improved over that of compound
4, suggesting that the biphenyl-containing sulfonamide provides a useful skeleton for the development
59
60
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 30
of bioactive molecules. Compound 7 inhibits the growth of AsPC-1, A549, Hep3B, and PC-3 cells with
GI50 values of 0.88, 0.60, 0.36, and 0.44 μM, respectively. Replacement of a hydrogen atom with
fluorine or chlorine at C3’ or C4’ position (8, 9, and 15) is tolerated but the C3’-Cl in compound 16
leads to a slight decrease in activity. Among mono-halogenated compounds (8, 9, 15, and 16),
compound 8 with a fluorine at C4’ position showed about 2-fold improved activity over that of 7 for
AsPC-1, A549, and PC-3 cells. Comparison of the mono-halogenated compounds indicated that the
order of influence of halogen atoms on the antiproliferative activity is F > Cl; and a C4’-halogen is
favored. The additional halogen atoms at the C3’ position (19 and 20) have only minimal influence on
the antiproliferative activity when compared to compound 8. A variety of functional groups such as
hydroxyl, methoxy, methoxycarbonyl, and aminocarbonyl groups lead to marked decrease of
antiproliferative activity but the cyano group in compounds 10, 17, and 21 contributes to an
improvement in the biological activity. Compound 10 possessing a C4’-CN is 2- to 10-fold more potent
than compound 8 which has a C4’-F and inhibits the growth of AsPC-1, A549, Hep3B, and PC-3 cells
with GI50 values of 0.03, 0.10, 0.15, and 0.19 μM, respectively. Moving the cyano group from C4’ to
C3’, as in 17, leads to a 3- to 17-fold decrease of antiproliferative activity as compared to its
regioisomer 10, and this is consistent with the observation that C4’-substitution is favored. The slight
influence of an additional C3’-F of compound 21 was observed to lead to a 5-fold increase of inhibitory
activity of Hep3B cells when compared to compound 10. The replacement of phenyl ring with pyridine
(24 and 25) or furan (26) resulted in a significant decrease of activity compared to 7. The introduction
of substituents at the nitrogen atom of the sulfonamide group (29-36) caused a remarkable decrease of
activity when compared to 10, and this appears to be independent of steric and electronic effects.
Modification of the sulfonamide (37, 38, and 41) leads to decrease of potency, indicating that the
sulfonamide moiety is crucial for antiproliferative activity.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B. Inhibition of Tubulin Polymerization.
ACS Paragon Plus Environment
8

Page 9 of 30
Journal of Medicinal Chemistry
Compound 21 was tested for tubulin polymerization inhibitory activity, using paclitaxel and
1
2
vincristine as reference compounds (Figure 3), and was found to inhibit tubulin polymerization in a
dose-dependent manner. As shown in Figure 3A, both compound 21 and vincristine destroy the tubulin
assembly at the same concentration of 10 μM. The result of immunofluorescence staining revealed that
compound 21 acts as a microtubule-destabilizing agent, similar to vincristine (Figure 3B). Generally,
microtubule-destabilizing agents bind to -tubulin either at vinca binding site or colchicine binding
site.21 Since compound 21 acts as a microtubule-destabilizing agent, we further identify the binding site
of compound 21 using [3H]-colchicine competitive binding assay. As shown in Figure 3C, both
compound 21 and colchicine can inhibit the binding of [3H]-colchicine to tubulin significantly, with IC50
values of 0.67 and 2.21 M respectively. This result indicated that compound 21 could directly bind to
tubulin at colchicine binding site.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C. Inhibition of STAT3 tyrosine phosphorylation.
Five compounds (8, 10, 19, 21, and 35) were examined for their ability to inhibit the phosphorylation of
tyrosine residues of STAT3 in human non-small cell lung A549 cancer cells. The results are presented
as IC50 values in Table 2. Compound 21, which exhibits the most potent antiproliferative activity,
inhibits the phosphorylation of STAT3 with an IC50 value of 0.2 μM. Removal of C3’-F (10) led to a 2-
fold decrease of activity as compared with compound 21. This phenomenon, that a C3’-F weakens the
inhibitory activity of STAT3 phosphorylation is also observed in the comparison of 8 with 19. Although
compound 35 exhibits weaker antiproliferative activity, it displays slight better inhibitory activity of
STAT3 phosphorylation than compound 10. This result indicates that the C4’-CN plays a significant
role in inhibition of the tyrosine phosphorylation of STAT3. We also conducted Western blot analysis to
confirm the inhibitory activity of the synthetic compounds on STAT3 phosphorylation. As shown in
Figure 4, compound 21, at 0.1 μM, inhibits STAT3 constitutive activation, consistent with the results in
Table 2. Compounds 35 and 10, which exhibited slightly higher IC50 values in STAT3 tyrosine
phosphorylation than 21, suppressed STAT3 constitutive activation at the dose of 1 μM. Evidence has
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 30
shown that microtubule targeting agents may suppress STAT3 signaling10,11, which may not exclude the
fact that STAT3 inhibition may be the consequence of microtubule-destabilizing effect. However, in our
case, compound 21 may need 3 M to cause tubulin depolymerization and 0.67 μM to bind to
colchicine binding site (Figure 3A and Figure 3C), whereas the IC50 value of STAT3 inhibition is only
around 0.2 M (Table. 2), which indicated that STAT3 inhibition effect of compound 21 may be
stronger than tubulin binding effect. We also used STAT3 inhibitor S3I-201 to prove that whether
STAT3 inhibition may cause G2/M arrest. As shown in Figure 5, STAT3 inhibition did not affect the
percentage of G2/M cells. Therefore, we may conclude that the inhibition effect of compound 21 on
STAT3 phosphorylation may be independent of the effect on microtubule destabilization.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
10

Page 11 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
Figure 3. Compound 21 inhibits tubulin polymerization. (A) Tubulin in reaction buffer was incubated at 37 C in the
presence of control (DMSO), the indicated concentration of 21 (3 and 10 μM), 0.3 μM paclitaxel, and 1 μM vincristine. The
microtubule assembly was measured by spectrophotometry. (B) A549 cells were incubated with DMSO (Control; CTL), 3
μM 21, 1 μM paclitaxel, and 1 μM vincristine. The cellular microtubule network was analyzed by a confocal microscopy
using monoclonal anti-β-tubulin antibody, FITC-conjugated anti-mouse antibody, and counterstaining with DAPI. (C)
Tubulin in reaction buffer was incubated at 37 C in the presence of compound 21 or colchicine followed by the addition of
[3H]-colchicine to compete with colchicine binding site.
Table 2. Inhibition of STAT3 Tyrosine Phosphorylation of Compounds 8, 10, 19, 21, and 35.
compd
8
IC50 (µM ± SD)
4.02 ± 0.75
10
0.44 ± 0.04
19
2.75 ± 0.22
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 30
21
35
0.20 ± 0.03
0.32 ± 0.03
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. N-Sulfonyl-aminobiaryl analogues (8, 10, 19, 21, and 35) inhibit STAT3 tyrosine phosphorylation. A549 cells
were treated with various concentrations of tested compounds for 24 hr. Then, whole cell lysates of A549 were subjected to
Western blot analysis. Similar resultes were obtained in at least three independent experiments.
Figure 5. A549 cells were treated with S3I-201 (STAT3 inhibitor) for 24 h and then cell cycle was analyzed by flow
cytometry.
Conclusion
ACS Paragon Plus Environment
12

Page 13 of 30
Journal of Medicinal Chemistry
This study describes novel N-sulfonyl-aminobiaryl derivatives acting as antitubulin agents with
1
2
STAT3 inhibitory activity. A series of N-sulfonyl-aminobiaryl compounds has been synthesized and the
relevant pharmacological assays have been conducted. Compound 21 exhibits remarkable
antiproliferative activity against four cancer cell lines with a mean GI50 value of 57.5 nM. It interrupts
tubulin assembly acting, like vincristine as a microtubule-destabilizing agent. Compound 21 reduces the
intracellular level of phosphorylated STAT3 with an IC50 value of 0.2 μM. Compounds 35 and 10 are
also able to inhibit the STAT3 activation in both IC50 value evaluation and Western blot analysis.
Interestingly, the cyano substitution seems to be necessary for inhibition of STAT3 phosphorylation.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental Section
(A) Chemistry. Nuclear magnetic resonance (1H NMR) spectra were obtained with Bruker DRX-500
spectrometer (operating at 500 MHz), with chemical shift in parts per million (ppm, δ) downfield from
TMS as an internal standard. High-resolution mass spectra (HRMS) were measured with a JEOL (JMS-
700) electron impact (EI) mass spectrometer. Elemental analyses were performed on a Heraeus varioIII-
NCH. Purity of the final compounds were determined using an Agilent 1100 series HPLC system using
C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm × 150 mm) and were found to be ≥
95%. Flash column chromatography was done using silica gel (Merck Kieselgel 60, no. 9385, 230-400
mesh ASTM). All reactions were carried out under an atmosphere of dry N2.
General procedures for the synthesis of N-sulfonyl-2-aminobiaryl analogues (7-26): N-(4'-
hydroxy-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (11).
A mixture of compound 27 (0.5 g, 2.48 mmol), Pd(PPh3)4 (0.2 g, 0.17 mmol), K2CO3 (aq) (2M, 3 mL),
and toluene (9 mL) was stirred under N2 at room temperature for 10 min. To this mixture was added a
solution of 4-hydroxyphenylboronic acid (0.38 g, 2.72 mmol) in EtOH (9 mL) and then it was heated to
reflux until the reaction was complete. The reaction mixture was filtrated through a pad of Celite and the
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 30
filtrate was extracted with H2O (10 mL) and EtOAc (3 × 10 mL). The organic layer was collected and
purified by flash chromatography over silica gel (1:20 EtOAc/n-hexane) to yield the corresponding 2-
nitrobiaryl product. To the resulting product (0.33 g, 1.52 mmol) was added NH4Cl (0.16 g, 2.97 mmol),
iron powder (0.25 g, 4.46 mmol), H2O (3 mL), and i-PrOH (15 mL) and then the mixture was heated to
reflux until the reaction was complete. The reaction mixture was cooled and filtrated through a pad of
Celite. The filtrate was extracted by H2O (10 mL) and EtOAc (3 × 10 mL). The organic layer was dried
over anhydrous MgSO4 and evaporated to give the corresponding biphenyl-2-amine. A solution of the
resulting product (0.23 g, 1.24 mmol), 4-methoxybenzenesulfonyl chloride (0.18 g, 0.89 mmol), and
pyridine (2 mL) was stirred at 50 °C overnight. After the reaction completed, the mixture was purified
by flash chromatography over silica gel (1:2 EtOAc/n-hexane) to afford compound 11 as a yellow solid
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(0.4 g, 91% three steps); H NMR (500 MHz, CDCl3) δ 3.84 (s, 3H), 6.56 (s, 1H), 6.76 (d, J = 8.5 Hz,
2H), 6.81 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 9.0 Hz, 2H), 7.07 (dd, J = 2.0, 7.5 Hz, 1H), 7.11 (td, J = 1.0,
7.5 Hz, 1H), 7.29 (dt, J = 1.5, 8.5 Hz, 1H), 7.54 (d, J = 7.0 Hz, 2H), 7.67 (d, J = 8.0 Hz, 1H); MS (EI)
m/z 355 (M+); HRMS (EI) for C19H17NO4S (M+) calcd 355.0878, found 355.0878.
N-(Biphenyl-2-yl)-4-methoxybenzenesulfonamide (7)
The title compound was obtained in 89% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 100.0-102.0 C; H NMR (500 MHz, CDCl3) δ 3.84 (s, 3H),
6.57 (s, 1H), 6.84 (d, J = 9.0 Hz, 2H), 6.88-6.90 (m, 2H), 7.09 (dd, J = 1.5, 7.5 Hz, 1H), 7.14 (td, J = 1.0,
7.5 Hz, 1H), 7.30-7.36 (m, 4H), 7.51 (d, J = 7.0 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H); MS (EI) m/z 339
(M+); HRMS (EI) for C19H17NO3S (M+) calcd 339.0929, found 339.0930.
N-(4’-Fluorobiphenyl-2-yl)-4-methoxybenzenesulfonamide (8)
The title compound was obtained in 81% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 113.0-114.0 C; H NMR (500 MHz, CDCl3) δ 3.84 (s, 3H),
6.43 (s, 1H), 6.84-6.89 (m, 4H), 7.02-7.09 (m, 3H), 7.13-7.17 (m, 1H), 7.31-7.36 (m, 1H), 7.51-7.4 (m,
ACS Paragon Plus Environment
14

Page 15 of 30
Journal of Medicinal Chemistry
1H), 7.67 (s, 1H); MS (EI) m/z 357 (M+); HRMS (EI) for C19H16FNO3S (M+) calcd 357.0835, found
1
2
3
357.0832.
4
5
N-(4'-Chloro-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (9)
6
7
8
9
The title compound was obtained in 87% overall yield from compound 27 in a manner similar to that
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
1
described for the preparation of 11: mp 124.0-126.0 C; H NMR (500 MHz, CDCl3) δ 3.84 (s, 3H),
6.39 (s, 1H), 6.81 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 7.06 (d, J = 9.0 Hz, 1H), 7.14 (t, J = 7.0
Hz, 1H), 7.29-7.36 (m, 3H), 7.51 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H); MS (EI) m/z 373 (M+);
HRMS (EI) for C19H16ClNO3S (M+) calcd 373.0539, found 373.0542.
N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (10)
The title compound was obtained in 57% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 167.0-169.0 C; H NMR (500 MHz, CDCl3) δ 3.86 (s, 3H),
6.29 (s, 1H), 6.87 (dd, J = 1.5, 7.5 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 7.10 (dd, J = 1.5, 7.5 Hz, 1H), 7.21
(td, J = 1.0, 7.5 Hz, 1H), 7.39 (td, J = 1.5 7.5 Hz, 1H), 7.52 (dd, J = 1.5, 7.0 Hz, 2H), 7.62-7.65 (m, 3H);
MS (EI) m/z 364 (M+); HRMS (EI) for C20H16N2O3S (M+) calcd 364.0882, found 364.0885.
4-Methoxy-N-(4'-methoxy-(1,1'-biphenyl)-2-yl)benzenesulfonamide (12)
The title compound was obtained in 29% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 102.1-104.0 C; H NMR (500 MHz, CDCl3) δ 3.83 (s, 3H),
3.84 (s, 3H), 6.58 (s, 1H), 6.80-6.89 (m, 6H), 7.06-7.12 (m, 2H), 7.27-7.31 (m, 1H), 7.52 (d, J = 7.0 Hz,
2H), 7.67 (d, J = 8.0 Hz, 1H); MS (EI) m/z 369 (M+); HRMS (EI) for C20H19NO4S (M+) calcd 369.1035,
found 369.1032.
Methyl 2'-(4-methoxyphenylsulfonamido)-(1,1'-biphenyl)-4-carboxylate (13)
The title compound was obtained in 43% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 136.0-138.0 C; H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H),
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 30
3.96 (s, 3H), 6.44 (s, 1H), 6.84 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 7.10 (dd, J = 1.0, 7.5 Hz,
1H), 7.18 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 7.0 Hz, 1H), 7.48 (d, J = 9.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 1H),
8.00 (d, J = 8.0 Hz, 2H); MS (EI) m/z 397 (M+); HRMS (EI) for C21H19NO5S (M+) calcd 397.0984,
found 397.0982.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2'-(4-Methoxyphenylsulfonamido)-(1,1'-biphenyl)-4-carboxylic acid (14)
LiOH(aq) (1M, 1 mL, 1.0 mmol) was added to a solution of compound 13 (0.2 g, 0.5 mmol) in 1,4-
dioxane (3 mL) and stirred at 40 °C overnight. The reaction was concentrated in vacuo and the resulting
residue was acidified to pH value of 5~6, and then was extracted by H2O (10 mL) and EtOAc (3 × 10
mL). The organic layer was purified by flash chromatography (10:1 CH2Cl2/MeOH) to yield the desired
o
1
compound 14 as a yellow solid (0.15 g, 78%): mp 197.0-200.0 C; H NMR (500 MHz, CDCl3) δ 3.75
(s, 3H), 6.77 (dd, J = 1.5, 7.0 Hz, 2H), 6.87 (d, J = 8.0 Hz, 2H), 7.06 (dd, J = 1.5, 7.5 Hz, 1H), 7.14 (td,
J = 1.0, 7.5 Hz, 1H), 7.21 (td, J = 1.5, 8.0 Hz, 1H), 7.29 (dd, J = 2.0, 7.5 Hz, 2H), 7.36 (d, J = 8.0 Hz,
1H), 7.79 (d, J = 8.0 Hz, 2H); MS (EI) m/z 383 (M+); HRMS (EI) for C20H17NO5S (M+) calcd 383.0827,
found 383.0828.
N-(3'-Fluoro-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (15)
The title compound was obtained in 55% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 118.3-119.5 C; H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H),
6.41 (d, J = 9.0 Hz, 1H), 6.46 (s, 1H), 6.74 (d, J = 7.5 Hz, 1H), 6.84 (dd, J = 2.0, 7.5 Hz, 2H), 7.05-7.10
(m, 2H), 7.16 (t, J = 7.0 Hz, 1H), 7.32-7.37 (m, 2H), 7.48 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 8.5 Hz, 1H);
MS (EI) m/z 357 (M+); HRMS (EI) for C19H16FNO3S (M+) calcd 357.0835, found 357.0835.
N-(3'-Chloro-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (16)
The title compound was obtained in 78% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 93.0-95.0 C; H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H), 6.41
ACS Paragon Plus Environment
16

Page 17 of 30
Journal of Medicinal Chemistry
(s, 1H), 6.66 (s, 1H), 6.85-6.88 (m, 3H), 7.06 (dd, J = 1.5, 7.5 Hz, 1H), 7.17 (td, J = 1.0, 7.5, Hz, 1H),
7.28-7.39 (m, 3H), 7.46 (d, J = 7.0 Hz, 2H), 7.74 (d, J = 8.5 Hz, 1H); MS (EI) m/z 357 (M+); HRMS (EI)
for C19H16ClNO3S (M+) calcd 373.0539, found 373.0540.
1
2
3
4
5
6
7
8
9
N-(3'-Cyano-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (17)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The title compound was obtained in 52% overall yield from compound 27 in a manner similar to that
described for the preparation of 11: mp 97.0-100.0 oC; 1H NMR (500 MHz, CDCl3) δ 3.89 (s, 3H), 6.21
(s, 1H), 6.80-6.81 (m, 1H), 6.90-6.93 (m, 2H), 7.06 (dd, J = 1.5, 7.5 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H),
7.26-7.29 (m, 1H), 7.397.43 (m, 1H), 7.46-7.51 (m, 3H), 7.63 (dt, J = 1.0, 8.0 Hz, 1H), 7.75 (d, J = 8.0
Hz, 1H); MS (EI) m/z 364 (M+); HRMS (EI) for C20H16N2O3S (M+) calcd 364.0882, found 364.0885.
4-Methoxy-N-(3'-(trifluoromethyl)-(1,1'-biphenyl)-2-yl)benzenesulfonamide (18)
The title compound was obtained in 67% overall yield from compound 27 in a manner similar to that
1
described for the preparation of 11: mp 96.0-98.0 oC; H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H), 6.35
(s, 1H), 6.85 (d, J = 8.5 Hz, 2H), 7.03 (s, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 2H), 7.39 (t,
J = 7.5 Hz, 1H), 7.48-7.53 (m, 3H), 7.63 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H); MS (EI) m/z 407
(M+); HRMS (EI) for C20H16F3NO3S (M+) calcd 407.0803, found 407.0801.
N-(3',4'-Difluoro-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (19)
The title compound was obtained in 66% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 93.0-95.0 C; H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H), 6.33
(s, 1H), 6.49-6.53 (m, 1H), 6.67-6.70 (m, 1H), 6.87 (d, J = 8.5 Hz, 2H), 7.05 (dd, J = 1.0, 7.5 Hz, 1H),
7.12-7.18 (m, 2H), 7.36 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.5 Hz, 1H); MS (EI)
m/z 375 (M+); HRMS (EI) for C19H15F2NO3S (M+) calcd 375.0741, found 375.0743.
N-(3'-Chloro-4'-fluorobiphenyl-2-yl)-4-methoxybenzenesulfonamide (20)
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 30
The title compound was obtained in 78% overall yield from compound 27 in a manner similar to that
1
2
3
4
5
6
7
8
9
o
1
described for the preparation of 11: mp 117.0-119.0 C; H NMR (500 MHz, CDCl3) δ 3.87 (s, 3H),
6.32 (s, 1H), 6.72 (dd, J = 2.0, 7.0 Hz, 1H), 6.82-6.91 (m, 3H), 7.05 (d, J = 7.5 Hz, 1H), 7.11-7.20 (m,
2H), 7.36-7.40 (m, 1H), 7.47-7.50 (m, 2H), 7.72 (d, J = 8.0 Hz, 1H); MS (EI) m/z 391 (M+); HRMS (EI)
for C19H15ClFNO3S (M+) calcd 391.0445, found 391.0443.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(4'-Cyano-3'-fluoro-[1,1'-biphenyl]-2-yl)-4-methoxybenzenesulfonamide (21)
The title compound was obtained in 67% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 121.2-123.5 C; H NMR (500 MHz, CDCl3) δ 3.86 (s, 3H),
6.27 (s, 1H), 6.65 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.94 (dd, J = 1.0, 8.0 Hz, 1H), 7.09 (dd,
J = 1.0, 7.5 Hz, 1H), 7.23-7.26 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.59-7.63 (m,
2H); MS (EI) m/z 382 (M+); HRMS (EI) for C20H15FN2O3S (M+) calcd 382.0787, found 382.0789.
N-(3',4'-Dimethoxy-(1,1'-biphenyl)-2-yl)-4-methoxybenzenesulfonamide (22)
The title compound was obtained in 64% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 150.5-152.3 C; H NMR (500 MHz, CDCl3) δ 3.80 (s, 3H),
3.83 (s, 3H), 3.93 (s, 3H), 6.46 (d, J = 1.5 Hz, 1H), 6.51 (d, J = 8.0 Hz, 1H), 6.71 (s, 1H), 6.83-6.87 (m,
3H), 7.10-7.15 (m, 2H), 7.28-7.33 (m, 1H), 7.53 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 1H); MS (EI)
m/z 399 (M+); HRMS (EI) for C21H21NO5S (M+) calcd 399.1140, found 399.1142.
4-Methoxy-N-(3',4',5'-trimethoxybiphenyl-2-yl)benzenesulfonamide (23)
The title compound was obtained in 81% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 139.0-141.0 C; H NMR (500 MHz, CDCl3) δ 3.79 (s, 6H),
3.83 (s, 3H), 3.90 (s, 3H), 6.19 (s, 2H), 6.74 (s, 1H), 6.84 (d, J = 8.5 Hz, 2H), 7.13-7.15 (m, 2H), 7.30-
7.34 (m, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.65 (d, J = 8.5 Hz, 1H); MS (EI) m/z 429 (M+); HRMS (EI) for
C22H23NO6S (M+) calcd 429.1246, found 429.1248.
ACS Paragon Plus Environment
18

Page 19 of 30
Journal of Medicinal Chemistry
4-Methoxy-N-(2-(pyridin-4-yl)phenyl)benzenesulfonamide (24)
1
2
3
4
The title compound was obtained in 41% overall yield from compound 27 in a manner similar to that
5
6
7
8
o
1
described for the preparation of 11: mp 224.0-227.0 C; H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H),
6.75 (s, 1H), 6.86 (d, J = 8.5 Hz, 2H), 6.99 (s, 2H), 7.13 (d, J = 7.5 Hz, 1H), 7.22-7.26 (m, 1H), 7.39 (t,
J = 8.5 Hz, 1H), 7.53 (d, J = 9.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 1H); MS (EI) m/z 340 (M+); HRMS (EI)
for C18H16N2O3S (M+) calcd 340.0882, found 340.0880.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-Methoxy-N-(2-(pyridin-3-yl)phenyl)benzenesulfonamide (25)
The title compound was obtained in 73% overall yield from compound 27 in a manner similar to that
o
1
described for the preparation of 11: mp 180.0-183.0 C; H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H),
6.36 (s, 1H), 6.87 (d, J = 8.5 Hz, 2H), 7.11 (dd, J = 1.0, 7.5 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.28-7.30
(m, 2H), 7.37-7.41 (m, 1H), 7.54 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 8.5 Hz, 1H), 8.16 (s, 1H), 8.62 (t, J =
3.0 Hz, 1H); MS (EI) m/z 340 (M+); HRMS (EI) for C18H16N2O3S (M+) calcd 340.0882, found 340.0884.
N-(2-(Furan-2-yl)phenyl)-4-methoxybenzenesulfonamide (26)
The title compound was obtained in 14% overall yield from compound 27 in a manner similar to that
described for the preparation of 11: 1H NMR (500 MHz, CDCl3) δ 3.78 (s, 3H), 6.34 (d, J = 3.5 Hz, 1H),
6.43 (dd, J = 1.5, 3.5 Hz, 1H), 6.75 (d, J = 9.0 Hz, 2H), 7.12 (td, J = 1.0, 7.5 Hz, 1H), 7.25-7.29 (m, 1H),
7.35 (dd, J = 1.5, 8.0 Hz, 1H), 7.49 (dd, J = 2.0, 6.5 Hz, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H); MS
(EI) m/z 329 (M+); HRMS (EI) for C17H15NO4S (M+) calcd 329.0722, found 329.0725.
N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-4-methoxy-N-methylbenzenesulfonamide (29)
K2CO3 (0.17 g, 1.23 mmol) was added to a solution of compound 10 (0.15 g, 0.41 mmol) in DMF (3
mL), and the mixture was stirred at room temperature for 10 min, and then MeI (0.05 mL, 0.82 mmol)
was added. After stirring overnight, the reaction was extracted with H2O (10 mL) and EtOAc (3 × 10
mL). The combined organic extract was dried over anhydrous MgSO4 and evaporated to give a residue
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 30
which was purified by flash chromatography (1:1 EtOAc/n-hexane) to yield compound 29 as a white
solid (0.15 g, 99%); mp 111.0-113.0 oC; 1H NMR (500 MHz, CDCl3) δ 3.03 (s, 3H), 3.87 (s, 3H), 6.91-
6.94 (m, 3H), 7.30-7.42 (m, 3H), 7.48-7.51 (m, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.68-7.71 (m, 2H); MS
(EI) m/z 378 (M+); HRMS (EI) for C21H18N2O3S (M+) calcd 378.1038, found 378.1040.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-N-ethyl-4-methoxybenzenesulfonamide (30)
The title compound was obtained in 90% overall yield from compound 10 in a manner similar to that
o
1
described for the preparation of 29: mp 121.0-120.0 C; H NMR (500 MHz, CDCl3) δ 0.83 (t, J = 7.5
Hz, 3H), 3.25-3.40 (m, 2H), 3.88 (s, 3H), 6.90 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 7.31-7.43
(m, 3H), 7.59 (d, J = 9.0 Hz, 2H), 7.66-7.71 (m, 4H); MS (EI) m/z 392 (M+); HRMS (EI) for
C22H20N2O3S (M+) calcd 392.1195, found 392.1195.
N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-4-methoxy-N-propylbenzenesulfonamide (31)
The title compound was obtained in 78% overall yield from compound 10 in a manner similar to that
o
1
described for the preparation of 29: mp 139.0-141.0 C; H NMR (500 MHz, CDCl3) δ 0.60 (t, J = 7.5
Hz, 3H), 1.15-1.23 (m, 2H), 3.00-3.40 (m, 2H), 3.90 (s, 3H), 6.92 (dd, J = 1.0, 8.0 Hz, 1H), 6.96 (d, J =
9.0 Hz, 2H), 7.31-7.42 (m, 3H), 7.62 (d, J = 9.0 Hz, 2H), 7.67-7.72 (m, 4H); MS (EI) m/z 406 (M+);
HRMS (EI) for C23H22N2O3S (M+) calcd 406.1351, found 406.1349.
N-(2-Chloroethyl)-N-(4'-cyano-(1,1'-biphenyl)-2-yl)-4-methoxybenzene sulfonamide (32)
The title compound was obtained in 86% overall yield from compound 10 in a manner similar to that
o
1
described for the preparation of 29: mp 150.0-152.0 C; H NMR (500 MHz, CDCl3) δ 3.00-3.40 (m,
2H), 3.50 (t, J = 7.5 Hz, 2H), 3.90 (s, 3H), 6.96-7.00 (m, 3H), 7.34-7.37 (m, 2H), 7.42-7.46 (m, 1H),
7.63-7.72 m, 7H); MS (EI) m/z 426 (M+); HRMS (EI) for C22H19ClN2O3S (M+) calcd 426.0805, found
426.0802.
N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide (33)
ACS Paragon Plus Environment
20

Page 21 of 30
Journal of Medicinal Chemistry
The title compound was obtained in 81% overall yield from compound 10 in a manner similar to that
1
2
3
4
5
6
7
8
9
o
1
described for the preparation of 29: mp 130.0-133.0 C; H NMR (500 MHz, CDCl3) δ 2.98-3.17 (m,
4H), 3.54 (s, 3H), 4.23 (s, 2H), 6.58 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 9.0 Hz, 2H), 6.99-7.12 (m, 3H),
7.13 (d, J = 9.0 Hz, 2H), 7.36-7.42 (m, 4H); MS (EI) m/z 408 (M+); HRMS (EI) for C22H20N2O4S (M+)
calcd 408.1144, found 408.1144.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methyl 2-(N-(4'-cyano-(1,1'-biphenyl)-2-yl)-4-methoxyphenylsulfonamido) acetate (34)
The title compound was obtained in 99% overall yield from compound 10 in a manner similar to that
described for the preparation of 29: 1H NMR (500 MHz, CDCl3) δ 3.55 (s, 3H), 3.88 (s, 3H), 6.93 (d, J
= 8.5 Hz, 2H), 7.22-7.26 (m, 1H), 7.29-7.33 (m, 2H), 7.38-7.42 (m, 1H), 7.60-7.70 (m, 6H); MS (EI)
m/z 436 (M+); HRMS (EI) for C23H20N2O5S (M+) calcd 436.1093, found 436.1094.
N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-N-((4-methoxyphenyl)sulfonyl)acetamide (35)
The title compound was obtained in 31% overall yield from compound 10 in a manner similar to that
o
1
described for the preparation of 29: mp 206.0-208.0 C; H NMR (500 MHz, CDCl3) δ 1.90 (s, 3H),
3.88 (s, 3H), 6.91(d, J = 9.0 Hz, 2H), 7.25 (d, J = 7.5 Hz, 1H), 7.45-7.61 (m, 5H), 7.70-7.75 (m, 4H);
MS (EI) m/z 406 (M+); HRMS (EI) for C22H18N2O4S (M+) calcd 406.0987, found 406.0988.
2-(N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-4-methoxyphenylsulfonamido)acetic acid (36)
The title compound was obtained in 99% overall yield from compound 34 in a manner similar to that
o
1
described for the preparation of 14: mp 175.0-178.0 C; H NMR (500 MHz, CDCl3) δ 3.88 (s, 3H),
6.91 (d, J = 9.0 Hz, 2H), 7.24-7.26 (m, 1H), 7.30-7.36 (m, 2H), 7.40-7.43 (m, 1H), 7.55-7.60 (m, 4H),
7.65 (d, J = 8.0 Hz, 2H); MS (EI) m/z 422 (M+); HRMS (EI) for C22H18N2O5S (M+) calcd 422.0936,
found 422.0938.
2'-((4-Methoxybenzyl)amino)-(1,1'-biphenyl)-4-carbonitrile (37)
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 30
A mixture of compound 28d (0.4 g, 1.78 mmol), iron powder (0.3 g, 5.35 mmol), NH4Cl (0.19 g, 3.57
mmol), H2O (4 mL), and i-PrOH (18 mL) was heated to reflux for 3 h. Then the reaction was cooled and
filtrated through a pad of Celite. The filtrate was concentrated and extracted with H2O (10 mL) and
EtOAc (3 × 10 mL). The organic layer was purified by flash chromatography (1:2 EtOAc/n-hexane) to
yield the corresponding amine product. Anisaldehyde (0.08 mL, 0.65 mmol), AcOH (0.1 mL), and
MeOH (10 mL) were added to the resulting product (0.2 g, 1.03 mmol). After stirring for 10 min,
sodium cyanoborohydride (0.08 g, 1.27 mmol) was added and stirred at room temperature overnight.
The reaction was filtered and the filtrate was concentrated to afford compound 37 as a white solid (0.21
g, 100%): mp 159.0-161.0 oC; 1H NMR (500 MHz, CDCl3) δ 3.79 (s, 3H), 4.24 (s, 2H), 6.72 (d, J = 8.5
Hz, 1H), 6.80 (t, J = 7.5 Hz, 1H), 6.84-6.88 (m, 2H), 7.06 (dd, J = 1.0, 8.0 Hz, 1H), 7.20-7.27 (m, 4H),
7.58 (dd, J = 1.5, 6.5 Hz, 2H), 7.71 (d, J = 1.5, 6.5 Hz, 2H); MS (EI) m/z 314 (M+); HRMS (EI) for
C21H18N2O2 (M+) calcd 314.1419, found 314.1421.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(4'-Cyano-(1,1'-biphenyl)-2-yl)-4-methoxybenzamide (38)
A mixture of compound 28d (0.4 g, 1.78 mmol), iron powder (0.3 g, 5.35 mmol), NH4Cl (0.19 g, 3.57
mmol), H2O (4 mL), and i-PrOH (18 mL) was heated to reflux for 3 h. The reaction was cooled and
filtrated through a pad of Celite. The filtrate was concentrated and extracted with H2O (10 mL) and
EtOAc (3 × 10 mL). The organic layer was purified by flash chromatography (1:2 EtOAc/n-hexane) to
yield the corresponding amine product. A mixture of the resulting product (0.2 g, 1.03 mmol), p-anisoyl
chloride (0.14 mL, 1.03 mmol), and pyridine (3 mL) was stirred at 90 °C overnight. The mixture was
concentrated in vacuo, and then the residue was purified by flash chromatography (1:2 EtOAc/n-hexane)
to afford compound 38 as a white solid (0.22 g, 65%): mp 136.0-138.0 oC; 1H NMR (500 MHz, CDCl3)
δ 3.84 (s, 3H), 6.89-6.93 (m, 2H), 7.24-7.29 (m, 2H), 7.45-7.49 (m, 1H), 7.57 (d, J = 8.5 Hz, 4H), 7.62
(s, 1H), 7.77 (d, J = 8.5 Hz, 2H), 8.31 (d, J = 8.0 Hz, 1H); MS (EI) m/z 328 (M+); HRMS (EI) for
C21H16N2O2 (M+) calcd 328.1212, found 328.1212.
N-(2-Bromobenzyl)-4-methoxybenzenesulfonamide (40)
ACS Paragon Plus Environment
22

Page 23 of 30
Journal of Medicinal Chemistry
A mixture of compound 39 (0.5 g, 2.69 mmol), 4-methoxybenzenesulfonyl chloride (0.4 g, 1.92 mmol),
and pyridine (5 mL) was stirred at 90 °C overnight. The mixture was concentrated in vacuo, and then
the residue was purified by flash chromatography (1:1 EtOAc/n-hexane) to afford compound 40 as a
yellow solid (0.94 g, 99%): 1H NMR (500 MHz, CDCl3) δ 3.85 (s, 3H), 4.21 (s, 1H), 4.22 (s, 1H), 4.99
(s, 1H), 6.91 (d, J = 9.0 Hz, 2H), 7.10 (dt, J = 1.5, 8.0 Hz, 1H), 7.21 (dt, J = 1.0, 7.5 Hz, 1H), 7.26-7.31
(m, 3H), 7.45 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 8.60 (d, J = 4.0 Hz, 1H).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-((4'-Cyano-(1,1'-biphenyl)-2-yl)methyl)-4-methoxybenzenesulfonamide (41)
A mixture of compound 40 (0.2 g, 0.56 mmol), Pd(PPh3)4 (0.05 g, 0.17 mmol), 4-cyanophenylboronic
acid (0.09 g, 0.62 mmol), 2M K2CO3 (0.7 mL), and in DMF (5 mL) was heated to reflux for 18 h. The
reaction mixture was filtrated through a pad of Celite and the filtrate was extracted with H2O (10 mL)
and EtOAc (3 × 10 mL). The organic layer was purified by flash chromatography (1:3 EtOAc/n-hexane)
o
1
to yield compound 41 as a light yellow solid (0.09 g, 42%): mp 163.0-166.0 C; H NMR (500 MHz,
CDCl3) δ 3.90 (s, 3H), 4.00-4.02 (d, J = 6.0 Hz, 2H), 4.40-4.43 (m, 1H), 6.92 (d, J = 8.5 Hz, 2H), 7.16-
7.19 (m, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.34-7.41 (m, 3H), 7.59 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 9.0 Hz,
2H); MS (EI) m/z 378 (M+); HRMS (EI) for C21H18N2O3S (M+) calcd 378.1038, found 378.1040.
(B) Biology.
(a) Materials
Sulforhodamine B (SRB) (Sigma-Aldrich, 230162, St. Louis, MO), 4',6-diamidino-2-phenylindole
(DAPI) was purchased from Roche Molecular Biochemicals (Cat. 10236276001, Mannheim, Germany).
Antibodies to STAT3 (Cat. 610189) were purchased from BD Biosciences (San Jose, CA). The
antibody to STAT3 (Tyr705) (Cat. 2236-1) was obtained from Epitomics (Burlingame, CA), and the
antibody to actin (Cat. MAB1501) was purchased from Millipore (Temecula, CA). Stat3 inhibitor S3I-
201 (Cat. 14336) was purchased from Cayman Chemical (Ann Arbor, MI).
(b) Cell culture
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 30
Human NSCLC cell line A549, human pancreatic adenocarcinoma cell line AsPC-1, human hepatoma
cell line Hep3B and human prostate cancer cell line PC-3 were obtained from American Type Culture
Collection (ATCC) (Manassas, VA). Cells were maintained in RPMI 1640 medium or DMEM with
10% fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 g/mL)/amphotericin (0.25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
g/mL) at 37 °C in a humidified incubator with 5% CO2.
(c) Sulforhodamine B (SRB) assay
All cells were seeded in 96-well culture plates at a density of 3-5 x 103 cells/well. After attachment,
cells were fixed with 10% trichloroacetic acid (TCA) to provide a measurement of the cell population at
the time of drug addition. The following day, cells were treated with vehicle (0.1% DMSO) or an
increasing gradient concentration of the indicated compounds for 48 h, after which cells were fixed with
10% TCA and stained with 0.4% (w/v) SRB dissolved in 1% AcOH. The protein-bound dye was
subsequently extracted with 10 mM trizma base to determine the absorbance at a wavelength of 515 nm.
The inhibition rate on cell proliferation (GI50) as a function of test drug concentration was calculated for
each well as 100-[(A515treated cells - A515time)].
(d) In vitro tubulin polymerization assay
The effect of identified compounds on tubulin polymerization was determined kinetically using the
CytoDYNAMIX Screen kit (BK006P, Cytoskeleton Inc., Denver, CO). Cold porcine tubulin protein (>
99% purity) was added to G-PEM buffer (80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP,
pH 6.9) containing 15% glycerol with or without the identified compounds. The sample mixture was
dotted onto a prewarmed 96-well plate, which was immediately transferred to a 37 °C plate reader
(SpectraMax Plus, Molecular Devices Inc., Sunnyvale, CA). The absorbance was read every minute for
30 min at 340 nm.
(e) Immunofluorescence confocal microscopy
A549 cells were seeded sparsely in eight-well chamber slides and treated with or without identified
compounds for 24 h. Following treatment, cells were fixed with cold MeOH at -20 °C for 15 min,
washed three times with PBS and blocked with 1% PBS plus 0.1% Triton X-100 for 30 min at 37 °C.
ACS Paragon Plus Environment
24

Page 25 of 30
Journal of Medicinal Chemistry
Microtubules were detected by incubation with a monoclonal anti--tubulin at 37 °C for 1 h. Then, the
cells were washed with PBS and incubated with a FITC-conjugated anti-mouse IgG antibody. Nuclei
were stained with DAPI, and microtubule distribution images were acquired with a Leica TCS SP2
Confocal Spectral Microscope.
1
2
3
4
5
6
7
8
9
(f) [3H]-Colchicine competitive binding assay
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 21 was submitted to Eurofins Panlabs Pharma Services (Taiwan Ltd) for [3H]-colchicine
competitive binding assay. In briefly, tubulin was preincubated with compound 21 or colchicine in
incubation buffer (10 mM Sodium Phosphate, pH 6.7, 10 mM MgCl2, 1 mM EGTA, 0.1 mM GTP) for 1
h followed by the addition of [3H]-colchicine. After 3 h of incubation at 37 C, bound [3H]-colchicine
were analyzed by scintillation counter.
(g) STAT3 kit
The level of STAT3 phosphorylation in response to test compounds treatment was determined using the
PathScan® Phospho-Stat3 (Try705) Sandwich ELISA kit (Cell Signaling Technology) according to the
manufacturer's instructions. The absorbance was read within 30 min at 405 nm using a microplate
ELISA reader.
(h) Western blot analysis
For Western blot analysis, cell lysates were prepared, and proteins were separated by 7.5-15% SDS-
PAGE, transferred onto PVDF membrane, and then immunoblotted with specific antibodies. Proteins
were visualized with an ECL detection system.22
(i) Flow Cytometry
Cells were treated with indicated concentration of S3I-201 for 24 h and then fixed in ethanol (75%, v/v)
overnight at -20°C. After centrifugation, fixed cells were washed with ice-cold PBS once and incubated
in 0.1M of phosphate–citric acid buffer (0.2 M NaHPO4, 0.1 M citric acid, pH 7.8) for 30 min at room
temperature, and then stained with propidium iodide staining buffer containing Triton X-100 (0.1%, v/v),
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 30
RNase A (100 mg/ml) and propidium iodide (80 mg/ml). Cell cycle distribution was performed using a
FACScan flow cytometry with CellQuest software (Becton Dickinson, Mountain View, CA, USA).
(j) Statistical Analysis
1
2
3
4
5
6
7
8
9
Results are expressed as the mean ± SD for the indicated number of separate experiments. Means were
assessed for significant differences using t-test and P-values < 0.05 were considered significant.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Author Information.
Corresponding Author
*Phone: 886-2-2736-1661 ext 6130. E-mail: jpl@tmu.edu.tw.
Acknowledgment. This research were supported by the Ministry of Science and Technology of the
Republic of China (grant no. MOST 103-2113-M-038-001-MY3).
Abbreviations Used
STAT3, Signal transducer and activator of transcription 3; LiOH, lithium hydroxide; Pd(PPh3)4,
tetrakis(triphenylphosphine)palladium(0);
DMF,
dimethylformamide;
NaBH3CN,
sodium
cyanoborohydride.
Supporting Information Available: The HPLC results and 1H-NMR spectrum of target compounds 7-
26, 29-38, and 41. This material is available free of charge via the Internet at http://pubs.acs.org.
ACS Paragon Plus Environment
26

Page 27 of 30
Journal of Medicinal Chemistry
References
1
2
3
4
5
6
1. Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. High-resolution model of the
microtubule. Cell 1999, 96, 79–88.
7
8
9
2. Liu, Y. M.; Chen, H. L.; Lee, H. Y.; Liou, J. P. Tubulin inhibitors: a patent review. Expert Opin.
Ther. Pat. 2014, 24, 69–88.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. Westermann, S.; Weber, K. Post-translational modifications regulate microtubule function. Nat.
Rev. Mol. Cell Biol. 2003, 4, 938–947.
4. Darnell, J. E. Jr. STATs and gene regulation. Science 1997, 277, 1630–1635.
5. Yu, H.; Jove, R. The STATs of cancer-new molecular targets come of age. Nat. Rev. Cancer. 2004,
4, 97–105.
6. Bromberg, J.; Chen, X. STAT proteins: signal transducers and activators of transcription. Methods
Enzymol. 2001, 333, 138–151.
7. Bromberg, J. F.; Wrzeszczynska, M. H.; Devgan, G.; Zhao, Y.; Pestell, R. G.; Albanese, C.;
Darnell, J. E. Jr. Stat3 as an oncogene. Cell. 1999, 98, 295–303.
8. Bowman, T.; Garcia, R.; Turkson, J.; Jove, R. STATs in oncogenesis. Oncogene 2000, 19, 2474–
2488.
9. Bromberg, J. Stat proteins and oncogenesis. J. Clin. Invest. 2002, 109, 1139–1142.
10. Walker, S. R.; Chundhury, M.; Nelson, E. A.; Frank, D. A. Microtubule-targeted chemotherapeutic
agents inhibit signal transducer and activator of transcription 3 (STAT3) signaling. Mol.
Pharmacol. 2010, 78, 903–908.
11. Walker, S. R.; Chaudhury, M.; Frank, D. A. STAT3 inhibition by microtubule-targeted drugs: dual
molecular effects of chemotherapeutic agents. Mol. Cell Pharmacol. 2011, 3, 13–19.
12. http://clinicaltrials.gov/
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 30
13. Chang, J. Y.; Hsieh, H. P.; Chang, C. Y.; Hsu, K. S.; Chiang, Y. F.; Chen, C. M.; Kuo, C. C.; Liou,
J. P. 7-Aroyl-aminoindoline-1-sulfonamides as a novel class of potent antitubulin agents. J. Med.
Chem. 2006, 49, 6656–6659.
1
2
3
4
5
6
7
8
9
14. Chang, J. Y.; Lai, M. J.; Chang, Y. T.; Lee, H. Y.; Cheng, Y. C.; Kuo, C. C.; Su, M. C.; Chang, C.
Y.; Liou, J. P. Synthesis and biological evaluation of 7-arylindoline-1-benzenesulfonamides as a
novel class of potent anticancer agents. Med. Chem. Commun. 2010, 1, 152–155.
15. Lee, H. Y.; Pan, S. L.; Su, M. C.; Liu, Y. M.; Kuo, C. C.; Chang, Y. T.; Wu, J. S.; Nien, C. Y.;
Mehndiratta, S.; Chang, C. Y.; Wu, S. Y.; Lai, M. J.; Chang, J. Y.; Liou, J. P. Furanylazaindoles:
potent anticancer agents in vitro and in vivo. J. Med. Chem. 2013, 56, 8008–8018.
16. Xie, F.; Zhao, H.; Li, D.; Chen, H.; Quan, H.; Shi, X.; Lou, L.; Hu, Y. Synthesis and biological
evaluation of 2,4,5-substituted pyrimidines as a new class of tubulin polymerization inhibitors. J.
Med. Chem. 2011, 54, 3200–3205.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17. Marzaro, G.; Coluccia, A.; Ferrarese, A.; Brun, P.; Castagliuolo, I.; Conconi, M. T.; La Regina, G.;
Bai, R.; Silvestri, R.; Hamel, E.; Chilin, A. Discovery of biarylaminoquinazolines as novel tubulin
polymerization inhibitors. J. Med. Chem. 2014, 57, 4598–4605.
18. Youn, S. W.; Bihn, J. H.; Kim, B. S. Pd-catalyzed intramolecular oxidative C-H amination:
synthesis of carbazoles. Org. Lett. 2011, 13, 3738–3741.
19. Rajeshkumar, V.; Lee, T. H.; Chuang, S. C. Palladium-catalyzed oxidative insertion of carbon
monoxide to N-sulfonyl-2-aminobiaryls through C-H bond activation: access to bioactive
phenanthridinone derivatives in one pot. Org. Lett. 2013, 15, 1468–1471.
20. Liang, Z.; Zhang, J.; Liu, Z.; Wang, K.; Zhang, Y. Pd (II)-catalyzed C (sp2)-H carbonylation of
biaryl-2-amine: synthesis of phenanthridinones. Tetrahedron 2013, 69, 6519–6526.
21. Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 2004, 4,
253-265.
ACS Paragon Plus Environment
28

Page 29 of 30
Journal of Medicinal Chemistry
22. Yang, C. R.; Hsieh, S. L.; Teng, C. M.; Ho, F. M.; Su, W. L.; Lin, W. W. Soluble decoy receptor 3
induces angiogenesis by neutralization of TL1A, a cytokine belonging to tumor necrosis factor
superfamily and exhibiting angiostatic action. Cancer Res. 2004, 64, 1122-1129.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
29

Journal of Medicinal Chemistry
Page 30 of 30
1
2
3
4
Graphic for manuscript
5
6
7
8
O
S
2
N
H
9
OMe
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
CN
21
AsPC-1 cells GI = 0.02 μM
Hep3B cells GI = 0.03 μM
STAT3 IC = 0.2 μM
50
50
50
ACS Paragon Plus Environment