Inhibition of cancer cell invasion by new ((3,4-dihydroxy benzylidene)hydrazinyl)pyridine-3-sulfonamide analogs

Article abstract of DOI:10.1016/j.bmcl.2015.12.093

Rab GTPases regulate various types of intracellular membrane trafficking in all eukaryotes. Since Rab27a and its multiple effectors are involved in exocytosis of lysosome-related organelles and play a major role in malignancy, compounds targeting Rab27a could be likely used to inhibit invasive growth and tumor metastasis. Thus, we designed and synthesized several compounds based on the previously reported Rab27a-targeting synthetic compounds identified by virtual screening, and investigated their anti-metastatic effects in MDA-MB231 and A375 cells. Among the synthesized compounds, (E)-N-(3-chlorophenyl)-6-(2-(3,4-dihydroxy benzylidene)hydrazinyl)pyridine-3-sulfonamide (3d) and (E)-N-benzyl-6-(2-(3,4-dihydroxy benzylidene)hydrazinyl)-N-methylpyridine-3-sulfonamide (3f) significantly inhibited the invasiveness of both tumor cell lines. Compounds 3d and 3f also decreased the levels of signature extracellular matrix marker proteins (fibronectin, collagen, and α-smooth muscle actin) and representative mesenchymal cell markers (N-cadherin and vimentin). Taken together, our results suggest that novel sulfonamide analogs have anti-metastatic activity in breast and melanoma cancer cell lines and may be used as therapeutic agents to treat malignant cancer.

Full text of DOI:10.1016/j.bmcl.2015.12.093

Accepted Manuscript
Inhibition of cancer cell invasion by new ((3, 4-dihydroxy benzylidene)hydra-
zinyl) pyridine-3-sulfonamide analogs
Seong-Mook Kang, Ky-Youb Nam, Seung-Youn Jung, Kyung-Hee Song,
Seongho Kho, Kyoung Tai No, Hyun Kyung Choi, Jie-Young Song
PII:
S0960-894X(15)30406-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.093
BMCL 23454
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 August 2015
23 November 2015
28 December 2015
Please cite this article as: Kang, S-M., Nam, K-Y., Jung, S-Y., Song, K-H., Kho, S., No, K.T., Choi, H.K., Song, J-
Y., Inhibition of cancer cell invasion by new ((3, 4-dihydroxy benzylidene)hydrazinyl) pyridine-3-sulfonamide
analogs, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.093
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Inhibition of cancer cell invasion by new ((3, 4-dihydroxy benzylidene)hydrazinyl)
pyridine-3-sulfonamide analogs
Seong-Mook Kanga, Ky-Youb Namb, Seung-Youn Junga, Kyung-Hee Songa, Seongho
Khoa, Kyoung Tai Noc, Hyun Kyung Choid, *, Jie-Young Songa,*
aDivision of Radiation Cancer Research, Korea Institute of Radiological & Medical
Sciences, Seoul 139-706, Korea
bCenter for Development and Commercialization Anti-Cancer Therapeutics, Asan
Medical Center, Seoul 138-736, Korea
cDepartment of Biotechnology, Yonsei University, Seoul 120-749, Korea
dDepartment of Medicinal Chemistry, Jungwon University, Goesan, 367-805, Korea
*Corresponding authors. These authors contributed equally to this work. E-mails:
hkchoi45@jwu.ac.kr (H. K. Choi), immu@kirams.re.kr (J.-Y. Song)
1

Intracellular trafficking plays a key role in eukaryotic cells because it enables shuttling
proteins and lipids between cell compartments and thus facilitates constant exchange between
organelles.1, 2 It also mediates many aspects of cell transformation, invasion, and metastasis.
Small GTPases of the Rab family, the key regulators of intracellular transport, are involved in
the formation of transport carriers from donor membranes, their movement along
cytoskelecton tracks, and their fusion with target membranes.3, 4 The levels of Rab proteins
are increased in several human cancers,2, 5-8 and these GTPases play both an activating and an
inhibiting role in tumor progression.9-13 In their active GTP bound form, Rab GTPases
interact with diverse effector proteins in each subcellular location.14, 15 Among 60 members
of the Rab family, Rab27a and Rab27b are present in melanocytes and cytotoxic T
lymphocytes, and regulate intracellular transport and exosome secretion in exocrine and
17
endocrine cells.16,
In melanocytes, activated Rab27a localizes to the melanosome
membrane, where it acts as a receptor for the actin-dependent motor protein myosin Va; the
Rab27–myosin Va interaction is mediated by the melanocyte-specific Rab27a effector
melanophilin/Slac2a.18-20 A loss-of-function mutation in human RAB27A gene results in
Griscelli syndrome, which is characterized by reduced formation of the tripartite Rab27a–
melanophilin–myosin Va complex necessary for actin-dependent melanosome transport; this
in turn results in cutaneous albinism and in a loss of cytotoxic activity of T cells.21, 22 Recent
studies have shown that Rab27a is necessary for metastasis and tumor invasion due to its
essential role in exosome secretion.23-26 Wange et al. reported that Rab27a enhances the
invasive and metastatic potential of human breast cancer cells by promoting the secretion of
insulin-like growth factor-II.27
Many researchers have recently focused on targeting vesicle trafficking and exocytosis
pathways because blocking exocytosis may permit manipulation of cancer cell behavior and
give some clues for development of novel therapeutic strategies. In this regards, an
2

interesting approach was demonstrated where new blockers of the binding interface between
Rab27a and melanophilin were identified by standard pharmacophore-based virtual screening
procedures and demonstrated to significantly inhibit melanosome transport.28 Given the
previous finding of Rab27a-targeting hit compound (BMD-11) as shown in Fig. 1, we aim to
investigate whether Rab27a inhibition decreases the invasiveness of MDA-MB231 breast
cancer cells and A375 malignant melanoma cells. It was found that the di-hydroxyl groups in
BMD-11 were critical to hydrogen bond interations in the binding, therefore, we synthesized
15 novel derivatives with di-hydroxyl groups intact but replaced the diethylamine moiety of
the BMD-11 with various amine.
O
N
HO
HO
S
O
N
N
N
H
Figure 1. The structure of the BMD-11
Scheme 1 shows preparation of ((3, 4-dihydroxy benzylidene)hydrazinyl)pyridine-3-
sulfonamide analogs. Commercial 6-chloropyridine-3-sulfonyl chloride was used as the
starting material and 6-chloro sulfonamide compounds (1a–o) with various amines were
synthesized.29 Hydrazinyl sulfonamide compounds (2a–o) were obtained by nucleophilic
substitution of analogs 1a–o with hydrazine hydrate.30 The target dihydroxybenzylidene
sulfonamide analogs (3a–o) were synthesized from commercially available 3,4-
dihydroxybenzaldehyde in ethanol 31 in very high yields.
3

O
S
O
S
O
S
Cl
O
ii
R
O
R
O
i
H2N
Cl
N
Cl
N
N
N
H
1a-o
2a-o
O
S
R
O
HO
HO
iii
N
N
N
H
3a-o
OH
H
N
N
R=
3a :
3b :
3c :
3d :
3f :
3g :
3h :
3i :
3k :
N
Cl
H
N
O
N
O
3l :
N
O
O
H
N
N
H
N
3m :
OH
OH
Cl
H
N
3n :
N
N
N
H
N
N
N
3e :
3j :
3o :
Scheme 1. Reagents and conditions : (i) various amines, chloroform, diisopropylethylamine,
ethyl acetate, HCl, r.t.; (ii) hydrazine hydrate, ethanol, 60°C; (iii) 3,4-dihydroxybenzaldehyde,
ethanol
4

Table 1
Structure–activity relationship of sulfonamide analogs.
O
S
R
O
HO
N
HO
N
H
N
No.
Structure
R
Cell growth inhibition (% of control at 10 µM)
MDA-MB231
5.82
A375
0 2.78
Vehicle (DMSO)
3a
0
4.81 7.09**
1.44 3.17
8.41 4.02
N
3b
3c
14.87 3.95***
N
H
N
-25.80 4.40
3.45 7.78
Cl
H
N
3d
3e
3f
26.34 0.38***
2.10 1.34
71.66 2.22***
1.29 4.36
N
N
24.62 5.04***
0.97 6.50
49.39 2.10***
-13.68 5.94
N
O
3g
N
O
H
N
3h
3i
3j
-1.92 5.82
-15.64 11.12
-13.56 5.20
-15.12 3.14
5.06 3.68
-2.01 2.17
OH
N
N
OH
H
N
3k
7.47 0.16
-3.03 7.03
Cl
H
N
3l
-9.95 5.19
-5.98 6.11
-11.39 6.60
-9.25 7.99
O
O
3m
N
OH
3n
3o
-15.82 8.52
-20.97 6.97
-14.10 .678
-13.79 6.10
N
H
N
5

The effect of 15 synthetic compounds (3a–o) on cell viability was evaluated by MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay in MDA-MB231 and
A375 cells (Table 1). MDA-MD231 cells are a representative cell line of triple negative
breast cancer, which is clinically aggressive with limited treatment options. The cells were
treated with or without each compound (10 µM) for 24 h.
The new analogs, except 3b, 3d, and 3f, showed a tendency for low inhibition activity in
cell growth for both cells. Compound 3a, where the diethylamine moiety of BMD-11 was
modified into a dimethyl moiety, showed low inhibition value. Compound 3e with
piperazine showed somewhat higher inhibition value in both MDA-MB231 and A375 cells
when compared with other compounds (3i, 3j, 3m and 3n) containing a heterocyclic moiety.
The compound 3d with the aniline moiety inhibited cell growth more efficiently than
compounds with a aminophenol moiety (3h and 3k). Compound 3b, 3d and 3f significantly
inhibited the growth of MDA-MB231 cells, while 3b only mildly inhibited the growth of
A375 cells. However, 3d with chloroaniline and 3f with N-methyl benzyl amine suppressed
the growth of A375 cells to a greater extent as compared to that of MDA-MB231 cells.
To differentiate the cytotoxic effect of these compounds from their anti-migratory activity,
we next investigated the effect of each compound on the migration of MDA-MB231 cells.
As shown in Fig. 2a, compound 3d and 3f markedly blocked wound migration. To
quantitatively compare the activity of the synthesized compounds, we determined the density
of the cells that migrated inside the scratched areas; the results were represented in Fig. 2b.
Insonsistent with the results of cell viability test, compound 3b had not effect on cell
migration, but 3i and 3j slightly inhibited migration compared to untreated control cells.
6

Figure 2. Screening of sulfonamide derivatives (3a–3o) for their effects on breast cancer cell
migration. (a) Wound migration assay. (b) Analysis of the anti-migratory effect
To confirm the effect of the 3 compounds (3b, 3d, and 3f) on the migratory and invasive
properties of cancer cells, we analysed MDA-MB231 and A375 cells by wound scratch assay
and transwell invasion assay in the presence or absence of 3b, 3d, or 3f. Compound 3d
significantly inhibited migration and invasion of both cell lines; it also had a cytotoxic effect.
Compound 3b had no effect, whereas 3f clearly suppressed migration and invasion of both
7

cell lines compared to untreated control cells (Fig. 3).
Figure 3. Inhibitory effects of compounds 3b, 3d, and 3f on MDA-MB-231 and A375 cell
migration and invasion. (a) Wound scratch assay. (b) Transwell invasion assay
Epithelial–mesenchymal transition (EMT) signaling has been recognized as a key factor
affecting cell migration, invasion, and drug resistance in several types of cancer.32, 33
Therefore, we examined the effect of compounds 3b, 3d, and 3f on the expression of EMT-
associated proteins. The levels of representative extracellular matrix (ECM) markers,
fibronectin (FN), collagen (Col) and α-smooth muscle actin (α-SMA), were markedly
reduced by 3f. Compound 3f significantly reduced the levels of the mesenchymal marker N-
cadherin (N-Cad) and vimentin (Vm). Compound 3b had no effect except for a reduction in
Vm levels, while 3d considerably inhibited the expression of N-Cad and Vm in both cell lines
but did not alter the levels of the ECM markers. The expression of Rab27a was also
8

attenuated by treatment with 3f and 3d (Fig. 4).
Figure 4. Effects of compounds 3b, 3d, and 3f on the expression of EMT-associated markers.
(a) MDA-MB-231 cells. (b) A375 cells
Based on the results of biological evauations, a docking study of selected compounds
was performed. The X-ray structure of the Rab27a–Exophilin4/Slp-a complex was used for
modeling, which was retrieved from the PDB (PDB ID = 3BC1).34 The binding interface
between Rab27a and Exophilin4 is centered between the residues of an invariant hydrophobic
triad (Phe46, Trp73, and Phe88). An extended hydophobic cluster is formed by Ile39, Thr41,
and Val42 (Fig. 5a). Asp91 of Rab27a is a key residue for interaction with Slp-a, because an
Asp91 mutant is unable to bind Slp-a.35 The binding site for the tested compounds was
formed by four key residues (Phe46, Trp73, Phe88, and Asp91), which interacted with 3f, 3i,
and 3o (Fig. 5b). Computations were performed on a Windows platform using DS
modeling/CDOCKER (Accelrys Inc., San Diego, USA) with the CHARMM force field.36
The molecular dynamics (MD) simulated annealing process is performed using a flexible
9

ligand docking. The final minimization step is applied to ligand’s docking pose. The
minimization consists of 1,000 steps of steepest descent using an energy tolerance of 0.001
kcal/mol.
Table 2
Relation between biological activity and docking scores of 3f, 3i, and 3o
3f
3i
3o
Docking score (Ludi score37)
572
462
3.31
5.06
489
Docking score (LigScore score38)
4.01
49.39
3.77
-13.79
A375 cell growth inhibition (% of control)
These studies were performed using the LigandFit39 module as implemented in Discovery Studio 3.0
(Accelrys Inc., San Diego, CA, USA).
The relationship between cell growth inhibition and docking scores is shown in Table 2.
Given that the value of Ludi score of 600 usually implies 1.0 µM of enzyme activity,37 the
Ludi score of compound 3f (572) was high. Compound 3f also exhibited the best biological
activity. It interacted with Rab27a via hydrophobic interactions between the
dihydroxylbenzyl group and the invariant hydrophobic triad of the protein; the dihydroxyl
group also formed three strong hydrogen bonds with the side chains of Asp91 and Lys11 (red
dotted line in Fig 5c.). Dihydroxyl benzyl groups have been previously described as key
players in intermolecular interactions as hydrogen bond donors.28 Benzyl sulfonamide of 3f
also had a weak hydrogen bond (yellow dotted line in Fig. 5c.) and a hydrophobic interaction
between the terminal benzyl group and the side chain of Val42. However, 3-hydroxyl group
in compound 3i did not form a hydrogen bond with the side chain of Asp91, and did not have
a hydrophobic interaction between the piperidin-1-ylsulfonyl group and the side chain of
10

Val42 (Fig. 5d). The other compounds with terminal hydrophilic functional groups such as
ester, hydroxyl, or morpholine (3g, 3h, 3l, and 3m) showed low biological activity. Their
terminal hydrophilic functional groups did not interact with the extended hydophobic cluster
(the side chain of Val42). We suggest that the three hydrogen bonds between dihydroxyl
benzyl groups and side chains of Asp91 and Lys11, and the additional hydrophobic
interaction between the terminal sulfonamide functional group and the extened hydrophobic
cluster may play a key role in biological activity.
11

12

Figure 5. Predicted docking poses of 3,4-dihydroxybenzylidene hydrazinyl pyridine
sulfonamide analogs 3f, 3i, and 3o on Rab27a (PDB entry 3BC1). (a) The surface of Rab27a
was rendered on the basis of Kyte and Doolittle hydrophobicity and colored using a color
gradient from red (hydrophobic amino acids) to blue (hydrophilic amino acids). (b)
Superposition of the binding modes of 3f (cyan), 3i (orange), and 3o (magenta) in the binding
site of Rab27a. (c) The proposed docking conformation of compound 3f in the binding site of
Rab27a. Red and yellow dotted lines indicate hydrogen bonds between compound 3f and the
receptor. Key binding residues of the receptor are shown in stick representation (carbon, light
gray). (d) Proposed docking conformation of compound 3i in the binding site of Rab27a. The
model was generated using Pymol (http://www.pymol.org).
13

In conclusion, we have developed new compounds, characterized by a N-methyl benzyl
sulphonamide functional group, which are able to inhibit cancer cell invasion and metastasis.
Compound 3f decreased the expression of signature ECM markers (FN, Col, and α-SMA),
and mesenchymal markers (N-Cad, and Vm). Compound 3f also markedly inhibited wound
repair and tumor invasion in in vitro assays. These findings demonstrate that a new synthetic
compound targeting Rab27a could inhibit invasion and metastasis of human breast carcinoma
and melanoma cells. Our findings thus may provide a promising approach to decrease
recurrence of several solid tumors.
Acknowledgments
This work was supported by the Nuclear R&D Program of the Ministry of Science, ICT and
Future planning, Republic of Korea (Grant No. 1711021781).
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Figure Captions
17

Figure 1. The structure of the BMD-11
Figure 2. Screening of sulfonamide derivatives (3a–3o) for their effects on breast cancer cell
migration. (a) Wound migration assay. (b) Analysis of the anti-migratory effect
Figure 3. Inhibitory effects of compounds 3b, 3d, and 3f on MDA-MB-231 and A375 cell
migration and invasion. (a) Wound scratch assay. (b) Transwell invasion assay
Figure 4. Effects of compounds 3b, 3d, and 3f on the expression of EMT-associated markers.
(a) MDA-MB-231 cells. (b) A375 cells
Figure 5. Predicted docking poses of 3,4-dihydroxybenzylidene hydrazinyl pyridine
sulfonamide analogs 3f, 3i, and 3o on Rab27a (PDB entry 3BC1). (a) The surface of Rab27a
was rendered on the basis of Kyte and Doolittle hydrophobicity and colored using a color
gradient from red (hydrophobic amino acids) to blue (hydrophilic amino acids). (b)
Superposition of the binding modes of 3f (cyan), 3i (orange), and 3o (magenta) in the binding
site of Rab27a. (c) The proposed docking conformation of compound 3f in the binding site of
Rab27a. Red and yellow dotted lines indicate hydrogen bonds between compound 3f and the
receptor. Key binding residues of the receptor are shown in stick representation (carbon, light
gray). (d) Proposed docking conformation of compound 3i in the binding site of Rab27a. The
model was generated using Pymol (http://www.pymol.org).
18

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Inhibition of cancer cell invasion by new ((3,
4-dihydroxy benzylidene)hydrazinyl)
pyridine-3-sulfonamide analogs
Seong-Mook Kang, Ky-Youb Nam, Seung-Youn Jung, Kyung-Hee Song, Seongho Kho, Kyoung Tai No, Hyun
Kyung Choi, *, Jie-Young Song *
19