Synthesis and pharmacological evaluation of novel isoquinoline N-sulphonylhydrazones designed as ROCK inhibitors

Article abstract of DOI:10.1080/14756366.2018.1490732

In this study, we synthesized a new congener series of N-sulphonylhydrazones designed as candidate ROCK inhibitors using the molecular hybridization of the clinically approved drug fasudil (1) and the IKK-β inhibitor LASSBio-1524 (2). Among the synthesized compounds, the N-methylated derivative 11 (LASSBio-2065) showed the best inhibitory profile for both ROCK isoforms, with IC₅₀ values of 3.1 and 3.8 μM for ROCK1 and ROCK2, respectively. Moreover, these compounds were also active in the scratch assay performed in human breast cancer MDA-MB 231 cells and did not display toxicity in MTT and LDH assays. Molecular modelling studies provided insights into the possible binding modes of these N-sulphonylhydrazones, which present a new molecular architecture capable of being optimized and developed as therapeutically useful ROCK inhibitors.

Full text of DOI:10.1080/14756366.2018.1490732

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and pharmacological evaluation of novel
isoquinoline N-sulphonylhydrazones designed as
ROCK inhibitors
Ramon Guerra de Oliveira, Fabiana Sélos Guerra, Cláudia dos Santos
Mermelstein, Patrícia Dias Fernandes, Isadora Tairinne de Sena Bastos,
Fanny Nascimento Costa, Regina Cely Rodrigues Barroso, Fabio Furlan
Ferreira & Carlos Alberto Manssour Fraga
To cite this article: Ramon Guerra de Oliveira, Fabiana Sélos Guerra, Cláudia dos Santos
Mermelstein, Patrícia Dias Fernandes, Isadora Tairinne de Sena Bastos, Fanny Nascimento Costa,
Regina Cely Rodrigues Barroso, Fabio Furlan Ferreira & Carlos Alberto Manssour Fraga (2018)
Synthesis and pharmacological evaluation of novel isoquinoline N-sulphonylhydrazones designed
as ROCK inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 1181-1193, DOI:
10.1080/14756366.2018.1490732
To link to this article: https://doi.org/10.1080/14756366.2018.1490732
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1181–1193
https://doi.org/10.1080/14756366.2018.1490732
RESEARCH PAPER
Synthesis and pharmacological evaluation of novel isoquinoline
N-sulphonylhydrazones designed as ROCK inhibitors
b,c
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Ramon Guerra de Oliveiraa,b , Fabiana Selos Guerra , Claudia dos Santos Mermelstein
,
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Patrıcia Dias Fernandes
, Isadora Tairinne de Sena Bastose, Fanny Nascimento Costaf
,
Regina Cely Rodrigues Barrosoe, Fabio Furlan Ferreiraf
and Carlos Alberto Manssour Fragaa,b
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Laboratorio de Avaliac¸ao e Sıntese de Substancias Bioativas (LASSBio ), Instituto de Ciencias Biomedicas, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, Brazil; Programa de Pos-Graduac¸ao em Farmacologia e Quımica Medicinal, Instituto de Ciencias Biomedicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Laboratorio de Farmacologia da Dor e da Inflamac¸ao, Instituto de Ciencias
Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Laboratorio de Diferenciac¸ao Muscular, Instituto de Ciencias
Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto de Fısica, Universidade do Estado do Rio de Janeiro, Rio
de Janeiro, Brazil; Centro de Ciencias Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), Sao Paulo, Brazil
b
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ABSTRACT
ARTICLE HISTORY
Received 7 May 2018
Revised 14 June 2018
Accepted 15 June 2018
In this study, we synthesized a new congener series of N-sulphonylhydrazones designed as candidate
ROCK inhibitors using the molecular hybridization of the clinically approved drug fasudil (1) and the IKK-b
inhibitor LASSBio-1524 (2). Among the synthesized compounds, the N-methylated derivative 11 (LASSBio-
2065) showed the best inhibitory profile for both ROCK isoforms, with IC50 values of 3.1 and 3.8 mM for
ROCK1 and ROCK2, respectively. Moreover, these compounds were also active in the scratch assay
performed in human breast cancer MDA-MB 231 cells and did not display toxicity in MTT and LDH assays.
Molecular modelling studies provided insights into the possible binding modes of these N-sulphonylhydra-
zones, which present a new molecular architecture capable of being optimized and developed as
therapeutically useful ROCK inhibitors.
KEYWORDS
Molecular hybridization;
N-sulphonylhydrazone;
Rho kinase; ROCK inhibitor;
fasudil; LASSBio-1524
Introduction
observed. ROCK1 is expressed at high levels in the thymus and
blood, whereas little or no ROCK2 expression was observed2. In
addition to the tissue distribution, these protein kinases have dif-
ferent intracellular distribution patterns: ROCK2 is predominantly
cytosolic and ROCK1 is mainly associated with centrosomes and
Rho kinases, also called ROCKs (Rho-associated protein kinases),
were the first Rho GTPases effector systems to be discovered.
ROCKs are serine/threonine kinases belonging to the AGC family
of protein kinases and were initially characterized by their roles in
the formation of stress fibres induced by RhoA and focal adhe-
sions by phosphorylating myosin light chain1.
These proteins coexist in two isoforms, ROCK1 (ROKb or
p160ROCK) and ROCK2 (ROKa), which share 65% homology at the
primary sequence level and approximately 92% homology within
the ATP binding sites2. These proteins are comprised of an N-ter-
minal lobe containing a highly conserved Ser/Thr kinase domain,
a coiled coil structure, a pleckstrin domain, and a cysteine-rich
domain at the C-terminal lobe, which is connected by a hinge
region3. These proteins typically form a self-inhibitory complex
that is disrupted upon the binding of the activated Rho-GTP pro-
tein to the Rho binding domain (RBD), leading to kinase
activation3.
the plasma membrane5,6
.
Activation of ROCKs by GTPases, mainly Rho, or by alternative
pathways involving caspases or lipid mediators leads to the phos-
phorylation of several molecular targets. One of the main sub-
strates of ROCK-mediated phosphorylation is MLC (myosin light
chain), since the activation of these proteins was initially reported
to be associated with the formation of stress fibres and changes
in the cytoskeleton4,7. In this manner, ROCKs directly phosphoryl-
ate myosin light chain, promoting the actin-myosin interaction. In
addition, ROCKs phosphorylate and inactivate MLCP (myosin light
chain phosphatase), indirectly regulating the amount of phos-
phorylated myosin4,8
.
The Rho/ROCK pathway is an important regulator of vascular
smooth muscle cell contraction and is important in controlling
migration, proliferation, differentiation, apoptosis, survival, and
transcription9. Despite the growing interest of the major pharma-
ceutical and small biotechnology companies in the Rho/ROCK
pathway and the reported pleiotropic effects of the ROCK inhibi-
Initially, ROCK1 was reported to be primarily distributed in the
lungs, liver, spleen, kidneys and testes, and ROCK2 in neuronal tis-
sues, skeletal muscles, heart, and placenta4. However, in the study
published by Linda and Olson in 2014, the distributions of ROCK
isoforms were similar, based on observations of EST markers, and
little specificity in expression between organs or tissues was tors, relatively few inhibitors that have reached clinical trials or
ꢀ
ꢀ ~ ^ ^
Laboratorio de Avaliac¸ao e Sıntese de Substancias Bioativas, Instituto de Ciencias
Biomedicas, Av. Carlos Chagas Filho 373, Cidade Universitaria, Ilha do Fundao, Rio de Janeiro 21941-902, Brazil.
CONTACT Carlos Alberto Manssour Fraga
cmfraga@ccsdecania.ufrj.br
ꢀ
ꢀ
~
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1182
R. G. OLIVEIRA ET AL.
even the market3. The most frequent indications for the clinical fasudil in the ROCK active site, and the N-sulphonylhydrazone
use of ROCK inhibitors are in the field of cardiovascular diseases10,
fragment was incorporated in the hybrids (5a–h).
since systemic exposure to ROCK inhibitors promotes a rapid and
pronounced decrease in blood pressure. A critical challenge is
therefore to differentiate the desired pharmacological activity and
adverse cardiovascular effects, since these adverse effects are not
tolerated in most cases, as well as to obtain selectivity for
ROCK isoforms11.
The first described ROCK inhibitor, i.e. the isoquinoline deriva-
tive fasudil (1), has been applied in the clinic as a treatment for
subarachnoid haemorrhage (SAH) in Japan since 199512,13. Fasudil
exerts its effects by upregulating endothelial nitric oxide synthase
(eNOS) expression and decreasing smooth muscle spasms and the
migration of inflammatory cells14. Moreover, as shown in our
recent study, the inhibition of ROCK pathway with fasudil induces
the nuclear translocation of beta-catenin to reduce MDA MB-231
tumour cell migration15.
The proposed structural design was additionally supported by
docking studies of LASSBio-1524 (2) and fasudil (1) at the active
site of ROCK, which confirmed the importance of introducing the
N-sulphonylhydrazone framework in the molecular architecture of
target compounds (5a–h) (Figure 2).
The imine substituents were chosen according to the substitu-
tion pattern present in fasudil (1) and LASSBio-1524 (2) proto-
types, exploring classical and non-classical isosteric relationships,
as well as the simplification of the homopiperazine ring to evalu-
ate its effect on molecular recognition in the active site of ROCK
isoforms and subsequent inhibition. In addition, we also proposed
the synthesis of an isoquinolinyl-N-acylhydrazone analogue to
rationalize the effects of the bioisosteric exchange between sul-
phonamide and amide functional groups, as well as an N-methy-
lated analogue to analyse the effect of N-alkylation on the kinase
inhibitory activity.
Previously, our research group proposed the use of the privi-
leged N-acylhydrazone (NAH) framework16 as a starting point for
structural modifications, leading to the discovery of novel Ser/Thr
kinase inhibitors17–19. Accordingly, the prototype LASSBio-1524 (2)
was discovered using structure-based drug design studies and
showed a moderate IKK-b inhibition profile17.
Experimental
General information
In general, protein kinases that belong to the same classes
have highly conserved domains, indicating that they possess high
structural similarity in the catalytic site. Based on the previous
study published by Avila and coworkers on the use of NAH com-
pounds as IKK-b inhibitors16 and considering that ROCKs are also
serine–threonine kinases, we propose here a series of novel
molecular hybrids between the well-known ROCK inhibitor fasudil
and LASSBio-1524, an IKK-b inhibitor. The proposed hybrids
(5a–h) presenting novel molecular architectures are expected to
participate in additional interactions with ROCK that may increase
the molecular recognition profile in the ROCK active site.
During the design of the novel hybrid analogues (5a–h) pre-
sented in Figure 1, we exploited the isosteric relationship between
the amide subunit of the N-acylhydrazone moiety present in
LASSBio-1524 (2) and the sulphonamide unit present in fasudil (1).
We have retained the isoquinoline ring substituted at the 5 pos-
ition, because it is necessary for the molecular recognition of
The melting points of the intermediates were determined using a
Quimis 340 apparatus. Hydrogen NMR spectra were determined in
deuterated chloroform or dimethylsulphoxide containing approxi-
mately 1% tetramethylsilane (TMS) as an internal standard using a
Bruker DPX-200 at 200 MHz, DRX-300 at 300 MHz, Varian 400-MR
at 400 MHz and Varian 500-MR at 500 MHz. Carbon NMR spectra
were determined using the same spectrometer at 50, 75, 100, and
125 MHz, respectively, and employing the same solvents. IR spec-
tra (cmꢀ1) were obtained using a Thermo Scientific Nicolet mod-
ule Smart ITR. The progress of all reactions was monitored
through thin-layer chromatography performed on 2.0 ꢁ 6.0 cm alu-
minium sheets precoated with silica gel 60 (HF-254, Merck) to a
thickness of 0.25 mm. The developed chromatograms were viewed
under ultraviolet light (254ꢀ366 nm) and treated with iodine
vapour. The reagents and solvents were purchased from commer-
cial suppliers and used as received. Analytical HPLC was per-
formed for compound purity determinations using a Shimadzu
LC-20AD with a Kromasil 100–5C18 column (4.6 ꢁ 250 mm) and a
Shimadzu SPD-M20A detector. The solvent system used for the
HPLC analyses was acetonitrile:water at 60:40 with or without of
Figure 2. Representation of ROCK (PDB code: 2ESM) with the co-crystallized iso-
quinoline inhibitor fasudil (1) (magenta) and best interaction mode of LASSBio-
1524 (2) (green) obtained through docking studies.
Figure 1. Design concept used to generate a novel class of N-sulphonylhydra-
zones as potential ROCK inhibitors.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1183
0.5% trifluoracetic acid. The isocratic HPLC mode was used, and (E)-N0-benzylideneisoquinoline-5-sulphonohydrazide (5b) –
the flow rate was 1.0 ml/min. The purity of the compounds was (LASSBio-2020)
higher than 95%. Ultraviolet spectroscopy was performed using a
Compound 5b (LASSBio-2020) was obtained as a white solid
through condensation of isoquinoline-5-sulphonohydrazide (4)
and benzaldehyde (227.0 mg, 73% yield); mp: 172 ꢂC. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 12.06 (s, 1H); 9.48 (s, 1H); 8.75 (d,
J ¼ 8.7 Hz, 1H); 8.66 (d, J ¼ 8.7 Hz, 1H); 8.51 (d, J ¼ 8.5 Hz, 1H); 8.47
(d, J ¼ 8.5 Hz, 1H); 7.89 (t, J ¼ 7.8 Hz, 1H); 7.89 (s, 1H); 7.45–7.47 (m,
2H); 7.32–7.34 (m, 3H). 13C NMR (100 MHz, DMSO-d6) d (ppm):
153.3; 151.4; 146.7; 144.3; 134.4; 133.9; 133.4; 130.1; 128.8; 126.7;
126.7; 117.8. IR (ATR, cmꢀ1): 3536; 1332; 1157. HRMS (ESI, m/z):
calculated for [M þ H]þ C16H13N3O2SHþ, 312.0801, found 312.0801.
Femto spectrophotometer. The wavelength used in the solubility
assay was determined as the maximum k characteristic of each
compound. The spectra were analysed using the Femto Scan soft-
ware. High resolution mass spectrometry was performed through
positive or negative ionization by Solarix XR 7Tesla, using ESI or
APCI FTICR and the data were analysed using the mMass
5.5.0 software.
Synthesis of isoquinoline-5-sulphonohydrazide (4)
In a 125-ml erlenmeyer flask, isoquinoline-5-sulphonyl chloride
hydrochloride (3) (0.5 g, 1.89 mmol) was slowly added to a satu-
rated sodium bicarbonate solution. The mixture kept its pH at a
constant value (pH 5–6), was stirred for 30 min, then extracted
with 2 ꢁ 30 ml of DCM. The organic layer was dried using magne-
sium sulphate and concentrated. The residue was re-dissolved in
2 ml of DCM and drop wise added to a solution of hydrazine
hydrate 100% (5 equiv) in DCM, maintaining the temperature
between –5 and –10 ꢂC. The reaction mixture was stirred for 5h at
room temperature (completion of the reaction was monitored by
TLC), and after the completion, the solvent was removed under
vacuum and the crude product was poured into ice flakes. The
white precipitate was filtered as a white solid (365 mg, 87% yield);
mp: 150 ꢂC. 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.47 (s, 1H);
8.82 (s, 1H); 8.67 (d, J ¼ 8.67 Hz, 1H); 8.47 (d, J ¼ 8.48 Hz, 1H); 8.45
(d, J ¼ 8.45 Hz, 1H); 8.37 (d, J ¼ 8.37 Hz, 1H); 7.85 (t, J ¼ 8.45 Hz,
1H); 4.26 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 153.1;
144.2; 134.4; 133.7; 132.7; 130.9; 128.8;126.3; 117.7. IR (ATR, cmꢀ1):
3376; 3260; 3175; 1616; 1300; 1188. LRMS (ESI, m/z): calculated for
[M þ H]þ C9H9N3O2SHþ, 224.05, found 223.85.
(E)-N0-(4-nitrobenzylidene)isoquinoline-5-sulphonohydrazide (5c)
– (LASSBio-2021)
Compound 5c (LASSBio-2021) was obtained as a yellow solid
through condensation of isoquinoline-5-sulphonohydrazide (4)
and 4-nitrobenzaldehyde as a yellow solid (267.0 mg, 75% yield);
mp: 168 ꢂC. 1H NMR (400 MHz, DMSO-d6) d (ppm): 12.70 (s, 1H);
9.71 (s, 1H); 8.86 (s, 2H); 8.65 (d, J ¼ 8.6 Hz, 1H); 8.62 (d, J ¼ 8.6 Hz,
1H); 8.20 (d, J ¼ 8.86 Hz, 2H); 8.06 (s, 1H); 8.02 (t, J ¼ 7.8 Hz, 1H);
7.75 (d, J ¼ 8.86 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm):
150.5; 147.8. 144.7; 139.5; 138.0; 136.6; 136.0; 134.0; 132.4; 128.6;
128.3; 127.8; 124.0; 120.4. IR (ATR, cmꢀ1): 2840; 1337; 1163. HRMS
(ESI, m/z): calculated for [M þ H]þ C16H12N4O4SHþ, 357.0652,
found 357.0653.
(E)-N0-(pyridin-3-ylmethylene)isoquinoline-5-sulphonohydrazide
(5d) – (LASSBio-2022)
Compound 5d (LASSBio-2022) was obtained as a white solid
through condensation of isoquinoline-5-sulphonohydrazide (4)
and nicotinaldehyde (274.5 mg, 88% yield); mp: 168 ꢂC. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 9.81 (s, 1H); 8.95 (d, J ¼ 6.5 Hz, 1H);
8.89 (d, J ¼ 6.5 Hz, 1H); 8.76 (d, J ¼ 7.8 Hz, 1H); 8.72 (t, J ¼ 7.8 Hz,
1H); 8.70 (d, J ¼ 8 Hz, 1H); 8.40 (d, J ¼ 8 Hz, 1H); 8.13 (s, 1H); 8.90
(s, 1H); 8.07 (t, J ¼ 7.8 Hz, 1H); 7.84 (dd, J1 ¼ 8 Hz, J2 ¼ 8 Hz, 1H).
13C NMR (100 MHz, DMSO-d6) d (ppm): 152.7; 148.4; 146.3; 143.1;
142.9; 135.5. 134.9; 134.6; 133.0; 131.0; 130.3; 128.5; 127.1; 124.8;
118.2. IR (ATR, cmꢀ1): 2637; 2722; 1376; 1168. HRMS (ESI, m/z): cal-
culated for [M þ H]þ C15H12N4O2SHþ, 313.0754, found 313.0754.
General procedure for the synthesis of N-
sulphonylhydrazones (5a–h)
A mixture of the required isoquinoline-5-sulphonohydrazide (4)
(1 mmol) and the appropriate aldehyde (1 mmol) were dissolved in
ethyl alcohol (15 ml) and vigorously stirred for 30–120 min at
room temperature. Once the reaction was completed, (reaction
was monitored by TLC), the solvent was vacuum removed, and
the product precipitated by the drop wise addition of cold water.
Following filtration, the precipitate was washed with H2O (10 ml)
and n-hexane (10 ml) and dried in vacuum.
(E)-N-(4-((2-(isoquinolin-5-ylsulphonyl)hydrazono)
methyl)phenyl)acetamide (5e) – (LASSBio-2023)
(E)-N0-(4-(dimethylamino)benzylidene)isoquinoline-5-
sulphonohydrazide (5a) – (LASSBio-2019)
Compound 5e (LASSBio-2023) was obtained as a yellow solid
Compound 5a (LASSBio-2019) was obtained as an orange solid through condensation of isoquinoline-5-sulphonohydrazide (4)
through condensation of isoquinoline-5-sulphonohydrazide (4) and 4-acetamidobenzaldehyde (312.8 mg, 85% yield); mp: 173 ꢂC.
and 4-(dimethylamino)benzaldehyde (318.6 mg, 90% yield); mp: 1H NMR (400 MHz, DMSO-d6) d (ppm): 11.86 (s, 1H); 10.07 (s, 1H);
165 ꢂC. 1H NMR (400 MHz, DMSO-d6) d (ppm): 11.53 (s, 1H); 9.44 9.45 (s, 1H); 8.75 (d, J ¼ 6.1 Hz, 1H); 8.63 (d, J ¼ 6,1 Hz, 1H); 8.47 (t,
(s, 1H); 8.74 (d, J ¼ 6.1 Hz, 1H); 8.67 (d, J ¼ 6.1 Hz, 1H); 8.47 (d, J ¼ 8.4 Hz, 2H); 7.88 (t, J ¼ 7.8 Hz, 1H); 7.81 (s, 1H); 7.55 (d,
J ¼ 8.4 Hz, 1H); 8.45 (d, J ¼ 8.4 Hz, 1H); 7.87 (t, J ¼ 7.8 Hz, 1H); 7.76 J ¼ 8.5 Hz, 2H); 7.38 (d, J ¼ 8.5 Hz, 2H); 2.02 (s, 3H). 13C NMR
(s, 1H); 7.27 (d, J ¼ 8.6 Hz, 2H); 6.61 (d, J ¼ 8.6 Hz, 2H); 2.89 (s, 6H). (100 MHz, DMSO-d6) d (ppm): 168.6; 153.4; 146.6; 144.6; 141.1;
13C NMR (100 MHz, DMSO-d6) d (ppm): 153.2; 151.4; 147.8; 144.3; 134.3; 133.7; 133.7; 130.7; 128.5; 128.1; 127.5; 126.6; 124.1; 118.8;
134.4; 133.9; 133.6; 130.7; 128.5; 128.0; 126.5; 120.7; 117.8; 111.6; 117.7. IR (ATR, cmꢀ1): 3898; 3216; 1663; 1321; 1158. HRMS
39.6. IR (ATR, cmꢀ1): 2889; 2722; 1359; 1163. HRMS (ESI, m/z): cal- (ESI, m/z): calculated for [M þ H]þC18H16N4O3SHþ, 369.1016,
culated for [M þ H]þ C18H18N4O2SHþ, 355.1223, found 355.1225.
found 369.1015.

1184
R. G. OLIVEIRA ET AL.
(E)-(4-((2-(isoquinolin-5-ylsulphonyl)hydrazono)methyl)
(E)-N0-benzylidene-N-methylisoquinoline-5-sulphonohydrazide
phenyl)boronic acid (5f) – (LASSBio-2024)
(11) – (LASSBio-2065)
Compound 5f (LASSBio-2024) was obtained as a white solid
through condensation of isoquinoline-5-sulphonohydrazide (4)
and 4-(dihydroxyboryl)benzaldehyde (273.0 mg, 77% yield); mp:
In
a
25-ml reaction flask, 150 mg of 5b (LASSBio-2020)
(0.48 mmol), 10 ml of acetone, 199 mg of potassium carbonate
(K2CO3) (1.44 mmol) and 0.1 ml of methyl iodide (CH3I)
(1.44 mmol) were solubilized. The flask was coupled to a conden-
ser and the reaction was kept under reflux and constant stirring
overnight. The acetone volume was reduced in a rotary evapor-
ator, and the product purified by flash column chromatography
to give a yellow solid (109.3 mg, 70% yield); mp: 108 ꢂC. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 9.42 (s, 1H); 8.71–8.75 (m, 2H);
8.47–8.51 (m, 2H); 7.90 (m, 1H); 7.79 (s, 1H); 7.55–7.57 (m, 2H);
7.33–7.41 (m, 3H); 3.30 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d
(ppm): 153.3; 144.6; 143.7; 134.9; 134.7; 133.9; 131.0; 131.0; 130.0;
128.8; 128.6; 127.0; 126.6; 118.0; 32.9. IR (ATR, cmꢀ1): 1364; 1162.
HRMS (ESI, m/z): calculated for [M þ H]þ C17H15N3O2SHþ,
326.0958, found 326.0958.
1
>250 ꢂC. H NMR (400 MHz, DMSO-d6) d (ppm): 12.06 (s, 1H); 9.48
(s, 1H); 8.77 (d, J ¼ 5.7 Hz, 1H); 8.67 (d, J ¼ 5.7 Hz, 1H); 8.51
(d, J ¼ 8.0 Hz, 1H); 8.47 (d, J ¼ 8.0 Hz, 1H); 7.90 (t, J ¼ 8.0 Hz, 1H);
7.90 (s, 1H); 7.73 (d, J ¼ 7.4 Hz, 2H); 7.42 (d, J ¼ 7.4 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6) d (ppm): 153.2; 146.8; 144.0; 134.8;
134.5; 134.4; 134.0; 133.7; 133.7; 130.8; 128.5; 126.8; 125.7; 117.9.
IR (ATR, cmꢀ1): 1325; 1161. HRMS (ESI, m/z): calculated for
[M þ H]þ C16H14BN3O4SHþ, 356.0871, found 356.0871.
(E)-N0-([1,1'-biphenyl]-4-ylmethylene)isoquinoline-5-
sulphonohydrazide (5g) – (LASSBio-2025)
Compound 5g (LASSBio-2025) was obtained as a yellow solid
through condensation of isoquinoline-5-sulphonohydrazide (4)
and [1,1'-biphenyl]-4-carbaldehyde (313.4 mg, 81% yield); mp:
166 ꢂC. 1H NMR (400 MHz, DMSO-d6) d (ppm): 12.52 (s, 1H); 9.91
(s, 1H); 9.10 (d, J ¼ 6.5 Hz, 1H); 8.92 (d, J ¼ 6.5 Hz, 1H); 8.76 (t,
J ¼ 7.8 Hz, 2H); 8.13 (t, J ¼ 7.8 Hz, 1H); 8.05 (s, 1H); 7.62–7.67 (m,
4H); 7.57 (d, J ¼ 8 Hz, 2H); 7.44 (t, J ¼ 7.5 Hz, 2H); 7.35 (t, J ¼ 7.4 Hz,
1H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 149.9; 147.0; 141.7;
139.1; 137.2; 136.5; 136.1; 134.4; 132.9; 132.4; 129.0; 128.9; 128.2;
127.9; 127.5; 127.0; 126.6; 121.2. IR (ATR, cmꢀ1): 2736; 1336; 1165.
HRMS (ESI, m/z): calculated for [M þ H]þ C22H17N3O2SHþ, 388.1114,
found 388.1114.
Methyl isoquinoline-5-carboxylate (8)
In a 125 ml flask, methanolic solutions (each 10 ml) of iodine
(2.098 g, 8.26 mmol) and KOH (926 mg, 16.5 mmol) at 0 ꢂC were
successively added to a solution of isoquinoline-5-carboxalde-
hyde (7) (1000 mg, 6.36 mmol) in 50 ml of absolute methanol
cooled to 0 ꢂC. After stirring for 10 h at 0 ꢂC, 50 ml of saturated
NaHSO3 solution was added to the reaction, resulting in the dis-
appearance of the brown colour. The methanol was then evapo-
rated under reduced pressure. The remaining content was
stirred for 30 min at 0 ꢂC, and the title compound was obtained
through filtration as a beige solid (1070 mg, 90% yield); mp:
66 ꢂC. 1H NMR (400 MHz, CDCl3): 9.33 (s, 1H); 8.82 (d, J ¼ 6 Hz,
1H); 8.61 (d, J ¼ 6 Hz, 1H); 8.46 (d, J ¼ 7 Hz, 1H); 8.19 (d, J ¼ 7 Hz,
1H); 7.65 (t, J ¼ 7 Hz, 1H); 3.99 (s, 3H). 13C NMR (100 MHz,
CDCl3): 166.6; 152.4; 143.6; 135.2; 134.5; 133.5; 128.8; 126.5;
125.8; 119.1; 52.5.
(E)-tert-butyl 4-((2-(isoquinolin-5-
ylsulphonyl)hydrazono)methyl)piperidine-1-carboxylate (6)
Compound 6 was obtained as yellow oil through condensation of
isoquinoline-5-sulphonohydrazide (4) and tert-butyl 4-formylpiperi-
dine-1-carboxylate (347.0 mg, 83% yield). 1H NMR (400 MHz,
DMSO-d6) d (ppm): 11.49 (s, 1H); 9.47 (s, 1H); 8.68 (d, J ¼ 6 Hz, 1H);
8.52 (d, J ¼ 6 Hz, 1H); 8.47 (d, J ¼ 8.0 Hz, 1H); 8.39 (d, J ¼ 8.0 Hz,
1H); 7.86 (t, J ¼ 8.0 Hz, 1H); 7.16 (d, J ¼ 4 Hz, 1H); 3.63–3.66 (m, 2H);
2.59–2.72 (m, 2H); 2.18–2.24 (m, 1H); 1.38 (s, 9H); 1.36–1.48 (m,
2H); 0.99–1.07 (m, 2H). LRMS (ESI, m/z): calculated for [M þ H]þ
C20H26N4O4SHþ, 419.17, found 419.09.
Isoquinoline-5-carbohydrazide (9)
In a 50-ml flask was added 500 mg of methyl isoquinoline-5-carb-
oxylate (8) (2.67 mmol), 15 ml ethanol and 670 mg hydrazine
hydrate (100%) (13.35 mmol). The reaction mixture was kept stir-
ring under reflux overnight. After the completion of the reaction,
ethanol was concentrated under reduced pressure, leading to the
formation of a pale yellow solid (434.3 mg, 87% yield); mp: 172 ꢂC.
1H NMR (400 MHz, DMSO-d6) d (ppm): 9.80 (s, 1H); 9.37 (s, 1H);
8.55 (d, J ¼ 6 Hz, 1H); 8.23 (d, J ¼ 7 Hz, 1H); 8.11 (d, J ¼ 6 Hz, 1H);
7.86 (d, J ¼ 7 Hz, 1H); 7.71 (t, J ¼ 7 Hz, 1H); 4.62 (s, 2H). IR (ATR,
cmꢀ1): 3216; 3002; 1643. LRMS (ESI, m/z): calculated for [M þ H]þ
C10H9N3OHþ, 188.08, found 188.02.
(E)-N0-(piperidin-4-ylmethylene)isoquinoline-5-sulphonohydrazide
hydrochloride (5h) – (LASSBio-2055)
In a 50 ml round bottom flask, 300 mg of 6 (0.7 mmol) was dis-
solved in 15 ml of dry ethanol. Then, 1.570 mg of acetyl chloride
(14 mmol) was added drop wise to a stirred solution of the N-
(tert-butoxycarbonyl)-protected sulphonylhydrazone (6) at room
temperature. The mixture was stirred overnight and evaporated in
vacuum to give the title compound as a pale yellow solid, which
was recrystallized in methanol (156 mg, 63% yield); mp: 186 ꢂC.1 H
NMR (400 MHz, DMSO-d6) d (ppm): 12.07 (s, 1H); 9.92 (s, 1H); 8.95
(d, J ¼ 6 Hz, 1H); 8.88 (d, J ¼ 6 Hz, 1H); 8.75 (d, J ¼ 8 Hz, 1H); 8.65
(d, J ¼ 8 Hz, 1H); 8.10 (t, J ¼ 8 Hz, 1H); 7.30 (d, J ¼ 4 Hz, 1H);
3.05–3.07 (m, 2H); 2.74–2.81 (m, 2H); 2.34–2.40 (m, 1H); 1.69–1.72
(m, 2H); 1.39–1.47 (m, 2H). 13C NMR (50 MHz, DMSO-d6) d (ppm):
153.2; 152.6; 149.9; 136.8; 136.0; 134.5; 132.9; 128.9; 128.2; 121.0;
42.2; 35.5; 25.0. IR (ATR, cmꢀ1): 2949; 2695; 1327; 1179. HRMS (ESI,
m/z): calculated for [M þ H]þ C15H18N4O2SHþ, 319.1223,
found 319.1223.
(E)-N0-benzylidene isoquinoline-5-carbohydrazide (10)
Compound 10 was obtained as a white solid through condensa-
tion of isoquinoline-5-carbohydrazide (9) and benzaldehyde
(223 mg, 81% yield), following the same general procedure used
1
for the synthesis of N-sulphonylhydrazones (5). H NMR (400 MHz,
DMSO-d6) d (ppm): 12.08 (s, 1H); 9.41 (s, 1H); 8.59 (d, J ¼ 6 Hz, 1H);
8.38 (s, 1H); 8.32 (d, J ¼ 7 Hz, 1H); 8.14 (d, J ¼ 6 Hz, 1H); 8.08
(d, J ¼ 7 Hz, 1H); 7.74–7.82 (m, 3H); 7.45–7.52 (m, 2H); 7.20–7.30
(m, 1H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 163.5; 152.6;
147.9; 143.7; 134.1; 133.6; 132.6; 130.5; 130.3; 130.1; 128.6; 128.4;

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1185
127.4; 126.5; 117.8. IR (ATR, cmꢀ1): 1635. HRMS (APCI, m/z): calcu- (k ¼ 1.54056 Å) and selected by a curved monochromator Ge
lated for [M þ H]þ C17H13N3OHþ, 276.1131, found 276.1129.
(111), with a tube voltage of 40 kV and a current of 40 mA. The
intensities were collected by a silicon microstrip detector, Mythen
R
1 K (DectrisV, Baden, Switzerland), in the range from 3ꢂ to 91.185ꢂ,
with steps of 0.015ꢂ and a counting time of 200 s at each 1.05ꢂ.
Water solubility assay
The solubility assay was performed considering the absorptivity of
compounds under ultraviolet spectroscopy as described by
Schneider et al.20 Assay wavelength was determined through kmax
ROCK inhibition
characteristic of each compound. Saturated aqueous solutions The inhibition of ROCK isoforms was made in EUROFINS-CEREP
were prepared (0.8–1.0 mg/mL) and kept under stirring for 4 h at France (www.cerep.fr) under the following study numbers:
37 ꢂC. The supernatant was filtered in 0.45 mm filters and trans- 100033685 and 100040481, using the protocol previously pub-
ferred to
a
quartz cuvette (10 mm) for spectra acquisition. lished by Doe et al.23 and Turner et al.24, respectively, for ROCK1
Solubility was determined by linear regression used as Excel graph and ROCK2 human recombinant enzymes. The analysis was per-
plots, solutions were prepared by dilutions of the original solution formed using software developed at Cerep (Hill software) and
R
in methanol. Data were collected in triplicate and the mean values GraphPad PrismV 5.0 (GraphPad Software, Inc., San Diego, CA) and
were used to create the graph plots. The correlation coefficient validated through comparison with results generated using the
(R2) values were between 0.9982 and 1.
SigmaPlot 4.0 software.
Chemical stability study
Cell culture
Two microliters (0.01 mmol) of a concentrated solution of the The human mammary gland/breast epithelial cell line MDA-MB
compound under examination (40 mM stock solution solubilized in 231 was obtained from the American Type Culture Collection
DMSO) and 248 lL of acid (0.2 M potassium chloride and 0.2 M (ATCCV HTB-26TM) and this use was approved by the Ethics
R
HCl; pH 2.0) or neutral (phosphate dibasic, pH 7.4) buffer were Committee for Animal Care and Use in Scientific Research from
added to a 2 ml Eppendorf microtube. After vortexing, the mixture the Federal University of Rio de Janeiro. Cells were routinely
was placed in a water bath at 37 ꢂC under vigorous stirring for 0, grown in RPMI medium containing 10% foetal bovine serum (FBS),
30, 60, 120, and 240 min. After each reaction, 248 lL of basic buf- 1% L-glutamine and 1% penicillin-streptomycin, in a humidified
fer (phosphate buffer, pH 8.4) was added to neutralize the pH of 5% CO2 atmosphere at 37 ꢂC. Cells were cultured up to 70–100%
the medium in the experiments using acidic buffer. The com- confluence and then some cultures were treated with the com-
pound was extracted using 1 ml of acetonitrile followed by vigor- pounds at different concentrations for 24 or 48 h. All cell culture
ous vortexing and freezing of the aqueous phase (ꢀ10 ꢂC). The reagents were purchased from Invitrogen (Sao Paulo, Brazil).
~
organic phase was separated, filtered, and analysed using an
HPLC-PDA (acetonitrile/water 60:4, 0.05% of TFA).
Cell viability assay
Cell viability was determined using 3–(4,5-dimethyl-2-thiazyl)-2,5-
Molecular modelling
diphenyl-2H-tetrazolium bromide (MTT) reagent (Sigma-Aldrich).
The ligands used for molecular docking studies were drawn and Briefly, cells were plated at an initial density of 2.5 ꢁ 104 cells per
their 3 D geometry optimized using the semi-empirical method well in 96-well plates and incubated for 24 h at 37 ꢂC and 5% CO2.
PM6 implemented in GAUSSIAN09W. We searched for co-crystals After 24 h cultures were treated with 5b (LASSBio-2020) and 11
containing ligands with some structural similarity to the tested (LASSBio-2065) at a final concentration of 0.1, 1, 10, 20, 30 lM and
compounds and the model used was ROCK1 structure complexed further incubated for 24 or 48 h. After treatment, the supernatant
with fasudil (PDB: 2ESM, resolution: 2.20 Å).The four fitness func- of each well was removed and cells were washed twice with
tions available in the GOLD 5.4.1 program, namely (ASP, ChemPLP, medium. Then, 10 ll of MTT solution (5 mg/ml in RPMI) and 100 ll
ChemScore and GoldScore) were evaluated for the redocking of of medium were added to each well and incubated for 4 h at
the co-crystallized ligand to identify the most adequate fitness 37 ꢂC, 5% CO2. The resultant formazan crystals were dissolved in
function for the docking studies. Crystallographic water molecules dimethylsulphoxide (100 ll) and absorbance intensities were
were removed during the docking runs, and the binding site was measured in a microplate reader (FlexStation Reader, Molecular
determined within 7 Å within the active site. The ChemPLP fitness Devices) at 570 nm. All experiments were performed in triplicate,
function was the best reproducing the binding pose as the crystal and cell viability was expressed as a percentage relative to the
structure (RMSD 0.2798 Å) and then used to perform the docking untreated control cells.
studies. Molecular graphics and analysis were made with the UCSF
Chimera package21 and 2 D representations with LigPlotþ.22
Plasma membrane integrity assay (LDH)
To measure plasma membrane integrity, we assayed serum lactate
dehydrogenase (LDH) levels and calculated percentages of LDH
X-ray diffraction
In order to get a suitable powder for the X-ray diffraction data col- release to the medium. LDH activity was measured spectrophoto-
ꢀ
lection 5f (LASSBio-2024) sample was gently hand-grinded in an metrically using a commercial kit (Doles, Goias, Brazil). After 5b
agate mortar and then packed between two 0.014-mm thick cellu- (LASSBio-2020) and 11 (LASSBio-2065) treatment, 50 ll of the
lose/acetate foils in a sample holder that was held spinning dur- supernatant were transferred to an enzymatic assay plate and
ing data collection. The data was recorded at room temperature 50 lL of LDH substrate plus 5 lL ferric albumen were added and
R
on a STADI-P powder diffractometer, from StoeV (Darmstadt, incubated for 3 min at 37 ꢂC, protected from light. Then, 10 ll of
Germany) in transmission geometry by using Cu Ka1 radiation NAD were added and absorbance intensities were measured in a

1186
R. G. OLIVEIRA ET AL.
R
microplate reader (FlexStationV 3 Reader, Molecular Devices) at Therefore, we applied the protocol described by Nudelman and
coworkers26 using acetyl chloride in ethanol to obtain the N-sul-
490 nm. The percentage of LDH release was calculated by ([LDH]
phonylhydrazone derivative 5h at a 64% yield.
sample ꢁ100)/total [LDH]. [LDH] sample was the LDH level of the
The condensation between hydrazides and aldehydes preferen-
tially produces hydrazones with a relative (E) configuration.27
However, we performed X-ray diffraction studies to further deter-
mine which diastereomer was obtained. Due to the difficulty in
obtaining all individual compounds in crystalline form, compound
5f (LASSBio-2024) was chosen as representative of the series of N-
sulphonylhydrazone derivatives. X-ray powder diffraction data
were used to index the diffraction pattern and to determine
the crystal structure of compound 5f (LASSBio-2024). Similar
detailed procedures have been reported elsewhere28–31. Briefly,
sample (released in medium) and total [LDH] was the LDH content
in the wells after addition of lysis solution (0.9% Triton X-100).
Cell-based scratch assay
Cells were cultured in 24-well culture plates for 24 h up to
90–100% confluence. Scratched wound lines were created with
the help of a 200 ll micropipette tip. Wells were washed with
RPMI for removal of non-adherent cells. Cells were incubated for
24 h with 5b (LASSBio-2020) and 11 (LASSBio-2065) at a final con-
centration of 10 or 30 lM. All cell-based scratch assays were per-
formed in the presence of the anti-mitotic reagent cytosine
arabinoside (Arac; Sigma-Aldrich) at a final concentration of 10ꢀ5
M to inhibit cell proliferation. After 5b (LASSBio-2020) and 11
(LASSBio-2065) treatment, the wound areas were observed with
an Axiovert 100 microscope (Carl Zeiss, Germany). Images were
acquired with an Olympus DP71 digital camera (Olympus, Japan)
and the wound area was quantified using Fiji software (based on
ImageJ, http://imageJ.nih.gov/ij/) from three different experiments.
compound 5f (LASSBio-2024) crystallized as
a monohydrate
(C16H14BN3O4SꢃH2O) in a monoclinic crystal system—space group
P21/c (nr. 14). The unit cell comprises four formula units (Z ¼ 4,
shown in Figure 3) and one molecule in the asymmetric unit
(Z’ ¼ 1). We assigned the relative (E) configuration to the imine
double bond (C ¼ N)32. Within the unit cell, the molecules
assumed
a
folded shape (Figure 4). Using PLATON33 and
Mercury34,35 software, we confirmed the geometry of the molecule
and the correct space group, unit cell parameters, bond distances,
angles and torsions. CCDC ID: 1816869 contains the supplemen-
tary crystallographic data for this paper.
Considering that all N-sulphonylhydrazones were obtained
using the same synthetic methodology, these experimental data
were extrapolated to the other compounds of the series.
Results and discussion
Chemistry
The synthesis of the N-sulphonylhydrazone derivatives 5a–h is
outlined in Scheme 1. The starting material was the commercially
available reagent isoquinoline-5-sulphonyl chloride hydrochloride
(3), which was neutralized and condensed with hydrazine hydrate
at 0 ꢂC, furnishing the respective sulphonylhydrazide intermediate
(4) at an 80% yield. Next, we performed the condensation of com-
pound 4 with aromatic and non-aromatic aldehydes to obtain the
corresponding N-sulphonylhydrazone derivatives 5a–h.
An additional step was performed to obtain the piperidinyl
derivative 5h, which consisted of the removal of the N-Boc group
from N-sulphonylhydrazone derivative 6 (Scheme 2). For certain
N-sulphonylhydrazones, the literature has reported the instability
of the imine bond under acidic conditions, leading to hydrolysis.25
For this reason, a mild methodology was chosen to remove the
protective group from compound 6, avoiding the possible
hydrolysis of the imine double bond, which could simultaneously
result in the formation of the corresponding hydrochloride in situ.
Figure 3. Unit cell representation of compound 5f (LASSBio-2024) along the
a-axis.
Scheme 1. Synthetic route exploited to prepare the N-sulphonylhydrazones
(5a–h). a) NaHCO3 aqueous, dichloromethane; b) N2H4.H2O, dichloromethane,
0 ꢂC, 4 h, 80%; c) EtOH, HCI (cat), r.t., 24 h, 70–90%.
Scheme 2. Synthesis of compound 5h (LASSBio-2055) following the removal of Figure 4. Crystal structure of compound 5f (LASSBio-2024) showing the labels
N-Boc from derivative 6. for all non-hydrogen atoms.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1187
Table 1. N-Sulphonylhydrazones 5a–h and their corresponding chemical yields
and purities.
Table 2. ROCK inhibition profiles and aqueous solubility of N-sulphonylhydra-
zones 5a–h and the standard compound fasudil (1).
Compound
Formula
Molecular weight Yieldsa (%) Purityc (%)
% inhibition at 3 lM
Aqueous solubility
5a (LASSBio-2019) C18H18N4O2S
5b (LASSBio-2020) C16H13N3O2S
5c (LASSBio-2021)
5d (LASSBio-2022) C15H12N4O2S
5e (LASSBio-2023) C18H16N4O3S
5f (LASSBio-2024) C16H14BN3O4S
5g (LASSBio-2025) C22H17N3O2S
5h (LASSBio-2055) C15H19ClN4O2S
354.43
311.36
356.36
312.35
368.41
355.18
387.45
354.85
92
73
75
88
85
77
81
64b
98
97
97
96
95
99
99
99
Compound
ROCK1a
ROCK2a
(mM)b
Fasudil
73.8
4.1
68.8
0.6
ND
5.4
54
26
>64
4.3
58
<0.5
>84.5
C16H12N4O4S
5a (LASSBio-2019)
5b (LASSBio-2020)
5c (LASSBio-2021)
5d (LASSBio-2022)
5e (LASSBio-2023)
5f (LASSBio-2024)
5g (LASSBio-2025)
5h (LASSBio-2055)
29
1.1
24.2
8.1
4.5
7.6
4.6
2.7
ꢀ9.1
7.3
aYields of the condensation step.
ꢀ1.1
2.4
3.9
bCumulative yield of the condensation and deprotection steps.
cDetermined by using reversed-phase HPLC analysis.
6.9
aValues are presented as averages of two experiments. Data are shown as %
inhibition of ROCK.
Moreover, the similarity of the chemical shift of the imine func- bDetermined by using the spectrophotometric method developed by Schneider
and coworkers20. ND ¼ Not determined.
1
tional group in the H NMR spectra using DMSO-d6 as solvent, as
well as the absence of additional signals in the NMR spectra,
corroborate the hypothesis that the compounds have been select-
ively obtained as (E) diastereoisomers. The chemical yields of the
condensation step and HPLC purities are described in Table 1.
Biological evaluation
ROCK inhibition assay. The N-sulphonylhydrazone derivatives
5a–h were evaluated for their ability to inhibit both ROCK1 and
ROCK2 isoforms by measuring the phosphorylation of the Ulight-
RRRSLLE substrate using human recombinant enzymes expressed
in Sf923 and Sf2124 cells, respectively. Prior to this assay, we eval-
uated the solubility of these compounds in water (buffer pH 7.4)
to ensure that the inhibition percentages were not influenced by
the precipitation of the compounds under test conditions. The
enzymatic assay was initially performed at a screening concentra-
Scheme 3. Synthetic route exploited to prepare the N-acylhydrazone derivative
tion of 3 lM, which is capable of guaranteeing the solubility of
10 (LASSBio-2064). a) KOH, I2, MeOH, 0 ꢂC, 4h, 80%; b) N2H4.H2O, EtOH, reflux,
overnight, 80%; c) EtOH, benzaldehyde, HCl (cat), overnight, 75%.
most of the compounds, and the obtained results are shown in
Table 2.
Among the N-sulphonylhydrazone derivatives that were initially
approximately 60 kcal/mol, which would be unfavourable in this
screened in the inhibition assays, only unsubstituted derivative 5b
case.31 An 1H NMR experiment with temperature variation was
(LASSBio-2020) showed a significant inhibitory profile at the
performed to completely discard the hypothesis of diastereomers.
screening concentration. Therefore, we decided to introduce add-
At 90 ꢂC, the coalescence of the adjacent signal related to the
amide nitrogen of the N-acylhydrazone group was observed
itional modifications into this derivative to better understand the
structure-activity relationships and to enhance the inhibitory pro-
(Figure 5); thus, we confirmed the presence of only one diastereo-
file towards ROCK isoforms.
isomer and attributed this effect to the mixture of conformers at
Initially, we investigated the bioisosteric replacement of the
sulphonylhydrazone group in compound 5b to an N-acylhydra-
zone group and proposed the synthesis of the N-acylhydrazone
the amide bond. This phenomenon had already been observed
for other NAH derivatives synthesized by the research group27.
As suggested above, we also aimed to investigate the impact
of the N-alkylation of the N-sulphonylhydrazone moiety on the
biological activity, as previous results from our research group
derivative 10 (LASSBio-2064). Employing the oxidative procedure
reported by Yamada36, we converted the commercially available
isoquinoline-5-carboxaldehyde (7) to the corresponding methyl
clearly indicated that this structural modification in N-acylhydra-
ester (8) after treatment with 2.6 eq. of KOH and 1.3 eq. of iodine
in methanol at 0 ꢂC and obtained an 89% yield. Next, the key N-
acylhydrazide intermediate (9) was obtained at a 70% yield by
treating an ethanolic solution of the ester (8) with hydrazine
hydrate under reflux37 (Scheme 3). The desired benzylidene-NAH
derivative 10 (LASSBio-2064) was obtained at a 75% yield after
condensing the hydrazide 9 with benzaldehyde in ethanol using
hydrochloric acid as catalyst.
Although the derivative 10 (LASSBio-2064) presented adequate
purity, as indicated by HPLC, duplicate signals in the 1H NMR
spectrum at 12.09 ppm appeared. These additional signals might
be due to a mixture of diastereoisomers or conformers. The
hypothesis of diastereoisomers was excluded because only one
singlet for the imine hydrogen was observed at 8.38 ppm. In add-
ition, if interconversion between diastereoisomers occurred, the
zones contributes to important changes in the conformational
behaviour of these derivatives and produces subsequent import-
ant modifications in their bioactivity profiles38. Compound 5b
(LASSBio-2020) was employed as precursor of the N-alkylated
derivative 11 (LASSBio-2065) and treated with potassium carbon-
ate in acetone followed by the addition of methyl iodide at room
temperature37. Under these conditions, we were able to obtain
the N-methylated derivative 11 (LASSBio-2065) at a 73% yield
(Scheme 4).
Next, we determined the IC50 values for compounds 5b
(LASSBio-2020), 10 (LASSBio-2064) and 11 (LASSBio-2065) against
both ROCK isoforms in the presence of different concentrations
ranging from 1 to 50 mM, which are within the limit of aqueous
solubility previously determined for each compound. The percent
energy barrier for the interconversion of NAH (E) to (Z) is enzyme activity was measured for each concentration to construct

1188
R. G. OLIVEIRA ET AL.
1
Figure 5. H NMR shift of the amide proton of compound 10 (LASSBio-2064). (A) Experiment performed at 25 ꢂC, where the duplication of the amide hydrogen was
observed. (B) Experiment performed at 90 ꢂC, where the coalescence of the signal was observed, indicating a conformational effect.
Scheme 4. Synthesis of the N-methyl-N-sulphonylhydrazone derivative 11
(LASSBio-2065).
Table 3. Determination of IC50 values for N-sulphonylhydrazones
5b (LASSBio-2020), 11 (LASSBio-2065) and the N-acylhydrazone
10 (LASSBio-2064) against human recombinant ROCK1 and
ROCK2 enzymes.
IC50 (mM)
Compound
ROCK1a
ROCK2a
Fasudil (1)b
5b (LASSBio-2020)
10 (LASSBio-2064)
11 (LASSBio-2065)
1.2
13
—
0.82
7.1
—
Figure 6. Predicted binding mode of compound 11 (LASSBio-2064) in complex
3.1
3.8
with ROCK. Docking studies were performed using the program GOLD 5.4.1.
aThe IC50 values displayed above are the means of two experi-
ments standard deviations presented in lM. Compounds were
examined using a five-point enzyme assay.
assay. Compound 5b (LASSBio-2020) displayed IC50 values of 13
and 7.1 lM for ROCK1 and ROCK2, respectively, whereas the N-
methylated analogue 11 (LASSBio-2065) exhibited IC50 values of
3.1 and 3.8 lM for ROCK1 and ROCK2, respectively. The N-methyla-
tion introduced in compound 11 (LASSBio-2065) resulted in a
slight increase in inhibitory potency, with a decrease in the IC50 of
approximately 4-fold for ROCK1 and 1.8-fold for ROCK2.
Furthermore, considering that 2 (LASSBio-1524) was used in the
structural design of this new family of ROCK inhibitors we also
investigated the ability of 5b (LASSBio-2020) and 11 (LASSBio-2065)
to inhibit IKK-b. However, none of these N-sulphonylhydrazone
derivatives was able to inhibit IKK-b at the screening concentration
of 10 lM, indicating their selectivity for the target kinases (data
not shown).
Among the observed binding modes for compounds 5b
(LASSBio-2020) and 11 (LASSBio-2065), all docking conformations
reproduced the key interaction with the hinge region through a
hydrogen bond between the lone pair of the isoquinoline ring and
a hydrogen of the amide backbone of MET156, which was essential
for kinase molecular recognition (Figure 7). Both compounds adopt
similar orientations in the active site, forming hydrophobic interac-
tions with PHE368, ILE80, ALA215 and VAL90. However, we also con-
sidered the differences between the binding modes of compounds
5b (LASSBio-2020) and 11 (LASSBio-2065) that could explain the
bThe IC50 values were previously determined39
.
the inhibition curves (see the Supplementary Material) and thus
determine the IC50 values shown in Table 3.
The derivative 10 (LASSBio-2064) was completely inactive in the
ROCK inhibition assays. Apparently, the conformation adopted at
the active site of target enzymes promoted by the bioisosteric
exchange of the sulphone group for the carbonyl group disfav-
oured molecular recognition at the catalytic sites of ROCK.
Additionally, as shown in Figure 6, compound 10 (LASSBio-2064)
had a different mode of interaction from the N-sulphonylhydra-
zone congener series (Figure 7). Apparently, the conformation
adopted in the active site promoted by the bioisosteric exchange
of the sulphone group for the carbonyl group disfavoured molecu-
lar recognition in the catalytic sites of ROCK isoforms. The rotation
of the carbonyl group caused the substituent bound to the imine
functional group to interact with another hydrophobic cavity of
the active site that differed from the hydrophobic cavity used by
the N-sulphonylhydrazone analogues. This mode of interaction
seems to be unfavourable and was corroborated by the study pub-
lished by Chen et al.14, who reported modifications in the structure
of fasudil (1) and revealed the pharmacophoric nature of the
sulphonyl group in relation to the respective carbonyl isostere.
In addition, we determined the IC50 values for compounds 5b
(LASSBio-2020) and 11 (LASSBio-2065), which were active in the bioactivity profiles observed in the present study. Apparently, the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1189
rotation of the N-S bond provided by the N-methyl group of com- important to address to avoid problems that require new steps of
pound 11 (LASSBio-2065) allows the compound to participate in an prototype optimization. The compounds were evaluated to deter-
mine their potential cytotoxic activities. All compounds were sub-
mitted to the MDA-MB-231 cell line viability assay using the MTT
quantitative colorimetric method15. Relative cell viability was
additional hydrophobic interaction with LEU205, which was not
observed for the analogue 5b (LASSBio-2020) and could explain the
subtle difference in the potency of these compounds.
Nevertheless, this structural modification had no effect on expressed as a percentage relative to control, untreated cells, and
ROCK1 and ROCK2 selectivity. Among the imine substituents none of the evaluated compounds showed toxicity at the tested
concentrations at 24 and 48 h. As an example, Figure 8 shows the
cell viability at concentrations of compounds 5b (LASSBio-2020)
and 11 (LASSBio-2065) ranging from 0.1 to 30 lM.
We also analysed the plasma membrane integrity by measuring
the serum lactate dehydrogenase (LDH) levels in the culture
medium to confirm the cell viability data. At concentrations of
compounds 5b (LASSBio-2020) and 11 (LASSBio-2065) ranging
chosen, the phenyl group was the most adequate for molecular
recognition in ROCK. A likely explanation may be that the chosen
4-substituted derivatives apparently did not fit well in the catalytic
cavity due to stereo-electronic effects.
Cytotoxic studies
In the development of new drug candidates, concerns regarding from 0.1 to 30 mM, LDH levels in the MDA-MB 231 cells were not
cytotoxic and pharmacokinetic properties in early stages are altered after 24 and 48 h of incubation (Figure 9).
Figure 7. (A) Predicted binding mode of compound 5b (LASSBio-2020) in complex with ROCK. (B) Predicted binding mode of compound 11 (LASSBio-2065) in complex
with ROCK. (C) Ligplot 2D representation of compound 5b (LASSBio-2020). (D) Ligplot 2D representation of compound 11 (LASBio-2065).
Figure 8. (A) Viability of MDA-MB-231 cells in the presence of compounds 5b (LASSBio-2020) and (B) 11 (LASSBio-2065).

1190
R. G. OLIVEIRA ET AL.
Figure 9. Measurements the serum lactate dehydrogenase (LDH) levels in the culture medium of MDA-MB-231 cells treated with compounds 5b (LASSBio-2020) (A)
and 11 (LASSBio- 2065) (B).
Figure 10. (A) Effects of compounds 5b (LASSBio-2020) and (B) 11 (LASSBio-2065) on the migration of MDA-MB 231 cells. Images were obtained using phase-contrast
microscopy. Filled areas represented migrating cells were calculated using ImageJ software. Scale bars represent 50lm.
Cell-based scratch assay
areas were visible in the culture dishes after 24 h of treatment
The functions of Rho-kinase include the regulation of cellular con- with all tested concentrations compared to control (Figure 10).
traction, motility, morphology, polarity, division, and gene expres- Treatment with compound 11 (LASSBio-2065) inhibited cell migra-
sion40. Therefore, we performed
a cell-based scratch assay tion in a less pronounced manner than compound 5b (LASSBio-
2020). Empty areas ranged from 21% to 43% compared to control
untreated cultures (Figure 11). Therefore, as we observed the com-
plete coverage of the scratched areas of the dish in control cells
followed by the quantification of the wounded area to analyse
whether compounds 5b (LASSBio-2020) and 11 (LASSBio-2065)
altered cell migration.
The cells were treated with compounds 5b (LASSBio-2020) or after 24 h of incubation, both compounds, 5b (LASSBio-2020) and
11 (LASSBio-2065) (10 or 30 lM) for 24 h. Interestingly, untreated 11 (LASSBio-2065), inhibited MDA-MB 231 cell migration. When
cells nearly completely covered the scratched areas of the dish in comparing with data previously obtained by our group showing
24 h, whereas in compound 5b-treated cultures, relatively empty that fasudil inhibited MDA-MB231 migration,15 50 mM fasudil

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1191
reduced cell migration by 50% in the present study. Meanwhile, imine carbon and renders it less susceptible to hydrolysis than
compound 5b (LASSBio-2020).
compounds 5b (LASSBio-2020) and 11 (LASSBio-2065) presented
similar effects at lower concentrations (10 mM). Based on our data,
these new analogues seem to be more potent than fasudil at
inhibiting tumour cell migration.
Conclusions
In the present study, we synthesized a new congener series of N-
sulphonylhydrazones as ROCK inhibitors that were designed
through molecular hybridization between the clinically approved
drug fasudil (1) and the IKK-b inhibitor LASSBio-1524 (2). After an
Chemical stability assay
Due to the possible chemical instability of the imine functional
group present in the N-sulphonylhydrazone framework, the stability initial screen of their abilities to inhibit both ROCK1 and ROCK2
profiles of compounds 5b (LASSBio-2020) and 11 (LASSBio-2065) isoforms, compound 5b (LASSBio-2020) was identified as a nonse-
lective inhibitor of ROCK1 and ROCK2 that presented moderate
potency. The corresponding NAH analogue 10 (LASSBio-2064) was
inactive in the ROCK inhibitory assay, revealing the pharmaco-
phoric nature of the sulphonyl group. On the other hand, the N-
methylated analogue 11 (LASSBio-2065) was a slightly more
potent ROCK inhibitor, but did not alter the selectivity profile
between ROCK1 and ROCK2 isoforms. In cell-based assays, deriva-
tives 5b (LASSBio-2020) and 11 (LASSBio-2065) were also effective
at inhibiting cell migration. Furthermore, none of the synthesized
compounds were cytotoxic in LDH and MTT assays. This molecular
scaffold showed similar inhibitory potency to the clinically
approved drug fasudil (1) and can be utilized for the further opti-
mization and development of ROCK inhibitors.
were determined. The study was conducted under two conditions,
pH 2.0 and pH 7.4, to mimic the acidic stomach and the neutral
plasma environments, respectively. The compounds were examined
in the presence of acid (pH 2) and neutral (pH 7.4) buffers with stir-
ring for 240 min at a controlled temperature (37 ꢂC). The results are
shown in Figure 12.
Both compounds were stable in neutral medium (pH 7.4) and
less than 20% degradation occurred, as observed from the com-
pound recovery percentage after 4 h. However, under acidic condi-
tions, both compounds underwent partial hydrolysis. After 30 min
of incubation, the recovery percentage of compound 5b (LASSBio-
2020) was 64% compared to 88% for compound 11 (LASSBio-
2065). This behaviour was progressively observed at 60, 120, and
240 min. The hydrolysis of imines are directly related to the elec-
tron density on the imine carbon. A lower electron density at this
carbon indicates that this atom is more electrophilic and available
for nucleophilic attack by water. The explanation for the better
recovery percentage, which means a better chemical stability, of
compound 11 (LASSBio-2065) is related to the presence of an
additional methyl group that increases the electron density at the
Acknowledgements
The authors thank MSc. Eduardo Miguez (IMA-UFRJ) for the vari-
able NMR experiments, and Evelyn Muguet Ferreira (NUCAT-UFRJ)
for the thermogravimetric analysis.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was co-funded by Conselho Nacional de
ꢀ
ꢀ
Desenvolvimento Cientıfico e Tecnologico [CNPq, BR, Grant No
305.991/2017–5, 432.001/2016–6, 304.872/2013–0, 307.664/2015–5
~
ꢁ
and 402.289/2013–7], Fundac¸ao Carlos Chagas Filho de Amparo a
Pesquisa do Estado do Rio de Janeiro [FAPERJ, BR, Grant No. E-26/
~
ꢁ
~
202.918/2015], Fundac¸ao de Amparo a Pesquisa do Estado de Sao
Paulo (FAPESP) [Grant No. 2015/26233–7] and Instituto Nacional
Figure 11. Effects of compounds 5b (LASSBio-2020) and 11 (LASSBio-2065) on
the migration of MDA-MB 231 cells. Filled areas representing migrating cells were
^
ꢀ
de Ciencia e Tecnologia de Farmacos e Medicamentos [INCT-
INOFAR, BR, Grant No 465.249/2014]. Thanks are also due to
calculated using ImageJ software. The results are presented as the medians
-
^
Departamento de Ciencia
e Tecnologia (DECIT) of Brazilian
standard deviations (n ¼ 3) of independent experiments. Statistical analyses were
performed using analysis of variance followed by the Newman-Keuls post-test.
Ministry of Health for the financial support [Grant No
25000.447435/2017–00].
ꢄ
p < 0.05 compared with the control group.
Figure 12. Chemical stability of compounds 5b (LASSBio-2020) and 11 (LASSBio-2065). (A) Recovery (%) of compounds at pH 2. (B) Recovery (%) of compounds at pH
7.4. The experiments were conducted in triplicate, and the values represent the averages from the experiments.

1192
R. G. OLIVEIRA ET AL.
16. Duarte CD, Barreiro EJ, Fraga CAM. Privileged structures: a
useful concept for the rational design of new lead drug can-
didates. Mini Rev Med Chem 2007;7:1108–19.
17. Avila CM, Lopes AB, Gonc¸alves AS, et al. Structure-based
design and biological profile of (E)-N-(4-Nitrobenzylidene)-2-
naphthohydrazide, a novel small molecule inhibitor of IjB
kinase-b. Eur J Med Chem 2011;46:1245–53.
18. Guedes IA, Freitas RHCN, Cordeiro NM, et al. LASSBio-1829
hydrochloride: development of a new orally active N-acylhy-
drazone IKK2 inhibitor with anti-inflammatory properties.
ChemMedChem 2016;11:234–44.
ORCID
Ramon Guerra de Oliveira
2407-5766
http://orcid.org/0000-0002-
ꢀ
Claudia dos Santos Mermelstein
8897-8526
http://orcid.org/0000-0002-
ꢀ
Patrıcia Dias Fernandes
http://orcid.org/0000-0001-6870-6745
http://orcid.org/0000-0001-9368-5640
http://orcid.org/0000-0003-1516-1221
Fanny Nascimento Costa
Fabio Furlan Ferreira
Carlos Alberto Manssour Fraga
6733-7079
http://orcid.org/0000-0001-
19. Freitas RHCN, Cordeiro NM, Carvalho PR, et al. Discovery of
naphthyl- N -acylhydrazone p38a MAPK inhibitors with in
vivo anti-inflammatory and anti-TNF-a activity. Chem Biol
Drug Des 2018;91:391–97.
References
20. Schneider P, Hosseiny SS, Szczotka M, et al. Rapid solubility
determination of the triterpenes oleanolic acid and ursolic
acid by UV-spectroscopy in different solvents. Phytochem
Lett 2009;2:85–7.
21. Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chimera-a
visualization system for exploratory research and analysis.
J Comput Chem 2004;25:1605–12.
22. Laskowski RA, Swindells MB. LigPlotþ: Multiple ligand-pro-
tein interaction diagrams for drug discovery. J Chem Inf
Model 2011;51:2778–86.
1. Riento K, Ridley AJ. Rocks: multifunctional kinases in cell
behaviour. Nat Rev Mol Cell Biol 2003;4:446–56.
2. Julian L, Olson MF. Rho-associated coiled-coil containing kin-
ases (ROCK): structure, regulation, and functions). Small
GTPases 2014;5:e29846
3. Feng Y, Lograsso PV, Defert O. Rho kinase (ROCK) inhibitors
and their therapeutic potential. J Med Chem 2016;59:
2269–300.
4. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug
target for neurological disorders. Nat Rev Drug Discov
2005;4:387–98.
23. Doe C, Bentley R, Behm DJ, et al. Novel Rho kinase inhibitors
with
anti-inflammatory
and
vasodilatory
activities.
5. Glyn M. A Rho-associated kinase mitigates reperfusion-
induced change in the shape of cardiac capillary endothelial
cells in situ. Cardiovasc Res 2003;57:195–206.
6. Stroeken PJM, Alvarez B, Rheenen J, van, et al. Integrin cyto-
plasmic domain-associated protein-1 (ICAP-1) interacts with
the ROCK-I kinase at the plasma membrane. J Cell Physiol
2006;208:620–8.
7. Leung T, Chen XQ, Manser E, et al. The p160 RhoA-binding
kinase ROK alpha is a member of a kinase family and is
involved in the reorganization of the cytoskeleton. Mol Cell
Biol 1996;16:5313–27.
8. Kimura K, Ito M, Amano M, et al. Regulation of myosin phos-
phatase by Rho and Rho-associated kinase (Rho-kinase).
Science 1996;273:245–8.
J Pharmacol Exp Ther 2007;320:89–98.
24. Turner MS, Fen-Fen-Lin Trauger JW, et al. Characterization
and purification of truncated human Rho-kinase II expressed
in Sf-21 cells. Arch Biochem Biophys 2002;405:13–20.
25. Beeren SR, Sanders JKM. Ferrocene-amino acid macrocycles
as hydrazone-based receptors for anions. Chem Sci 2011;2:
1560.
26. Nudelman A, Bechor Y, Falb E, et al. Acetyl chloride-
methanol as a convenient reagent for: A) Quantitative for-
mation of amine hydrochlorides B) carboxylate ester forma-
tion C) mild removal of N-t-Boc-protective group. Synth
Commun 1998;28:471–4.
27. Lacerda RB, da Silva LL, de Lima CKF, et al. Discovery of
novel orally active anti-inflammatory N-phenylpyrazolyl-N-
glycinyl-hydrazone derivatives that inhibit TNF-a production.
PLoS One 2012;7:e46925.
9. Dong M, Yan BP, Liao JK, et al. Rho-kinase inhibition: a novel
therapeutic target for the treatment of cardiovascular dis-
eases. Drug Discov Today 2010;15:622–9.
28. Ibiapino AL, de Figueiredo LP, Lima LM, et al. Structural and
physicochemical characterization of sulfonylhydrazone deriv-
atives designed as hypoglycemic agents. New J Chem
2017;41:6464–74.
29. Bastos ITS, Costa FN, Silva TF, et al. A combined experimen-
tal and in silico characterization to highlight additional
structural features and properties of a potentially new drug.
J Mol Struct 2017;1146:735–43.
10. Hartmann S, Ridley AJ, Lutz S. The function of rho-associ-
ated kinases ROCK1 and ROCK2 in the pathogenesis of car-
diovascular disease. Front Pharmacol 2015;6:276
11. Defert O, Boland S. Rho kinase inhibitors: a patent review
(2014 - 2016). Expert Opin Ther Pat 2017;27:507–15.
12. Asano T, Ikegaki I, Satoh S, et al. Mechanism of action of a
novel antivasospasm drug, HA1077. J Pharmacol Exp Ther
1987;241:1033–40.
13. Suzuki Y, Shibuya M, Satoh S-I, et al. A postmarketing sur- 30. de Figueiredo LP, Ibiapino AL, do Amaral DN, et al.
veillance study of fasudil treatment after aneurysmal sub-
arachnoid hemorrhage. Surg Neurol 2007;68:126–31;
discussion 131.
Structural characterization and cytotoxicity studies of differ-
ent forms of a combretastatin A4 analogue. J Mol Struct
2017;1147:226–34.
ꢀ
14. Chen M, Liu A, Ouyang Y, et al. Fasudil and its analogs: a 31. Silva TF, Bispo Junior W, Alexandre-Moreira MS, et al. Novel
new powerful weapon in the long war against central ner-
vous system disorders? Expert Opin Investig Drugs
2013;22:537–50.
orally active analgesic and anti-inflammatory cyclohexyl-N-
acylhydrazone derivatives. Molecules 2015;20:3067–88.
32. Palla G, Predieri G, Domiano P, et al. Conformational behav-
iour and E/Z isomerization of N-acyl and N-aroylhydrazones.
Tetrahedron 1986;42:3649–54.
15. Guerra FS, Oliveira RG, de Fraga CAM, et al. ROCK inhibition
with Fasudil induces beta-catenin nuclear translocation and
inhibits cell migration of MDA-MB 231 human breast cancer 33. Spek AL. Single-crystal structure validation with the program
cells. Sci Rep 2017;7:13723. PLATON. J Appl Crystallogr 2003;36:7–13.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1193
34. Macrae CF, Edgington PR, McCabe P, et al. Mercury: visual-
ization and analysis of crystal structures. J Appl Crystallogr
2006;39:453–7.
potent vasodilator arylidene N-acylhydrazone derivatives.
Eur J Med Chem 2009;44:4004–9.
€
38. Barreiro EJ, Kummerle AE, Fraga CAM. The methylation
35. Macrae CF, Bruno IJ, Chisholm JA, et al. Mercury CSD 2.0 –
new features for the visualization and investigation of crys-
tal structures. J Appl Crystallogr 2008;41:466–70.
36. Yamada S, Morizono D, Yamamoto K. Mild oxidation of alde-
hydes to the corresponding carboxylic acids and esters: alka-
effect in medicinal chemistry. Chem Rev 2011;111:
5215–46.
39. Rikitake Y, Kim H-H, Huang Z, et al. Inhibition of Rho kinase
(ROCK) leads to increased cerebral blood flow and stroke
protection. Stroke 2005;36:2251–7.
line iodine oxidation revisited. Tetrahedron Lett 40. Totsukawa G, Wu Y, Sasaki Y, et al. Distinct roles of MLCK
1992;33:4329–32.
and ROCK in the regulation of membrane protrusions and
focal adhesion dynamics during cell migration of fibroblasts.
J Cell Biol 2004;164:427–39.
€
37. Kummerle AE, Raimundo JM, Leal CM, et al. Studies towards
the identification of putative bioactive conformation of