Discovery of lipoic acid-4-phenyl-1: H -pyrazole hybrids as novel bifunctional ROCK inhibitors with antioxidant activity

Article abstract of DOI:10.1039/c6ra12081d

A series of lipoic acid (LA) and 4-phenyl-1H-pyrazole hybrids as bifunctional Rho-associated kinase (ROCK) inhibitors were designed, synthesized and evaluated. Compound 15 is identified to be a novel potent bifunctional ROCK inhibitor with antioxidant activity and neuroprotection.

Full text of DOI:10.1039/c6ra12081d

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DOI: 10.1039/C6RA12081D
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Discovery of Lipoic acid-4-Phenyl-1H-pyrazole Hybrids as Novel
Bifunctional ROCK Inhibitors with Antioxidant Activity†
Received 00th January 20xx,
Accepted 00th January 20xx
Ya-lin Tu,abc Qiu-he Chen,abc Sheng-nan Wang,abc Asko Uri,d Xiao-hong Yang,abc Jia-qi Chu, abc Jing-
be
abcg
kao Chen, abc Bing-ling Luo,be Xiao-hong Chen,f Shi-jun Wen,* Rong-biao Pi*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of lipoic acid (LA) and 4-phenyl-1H-pyrazole hybrids as ‘‘one-drug one-target’’ strategy is not good to hit the multiple
bifunctional Rho-associated kinases (ROCK) inhibitors were targets implicated in CNS complex diseases, combining LA with
designed, synthesized and evaluated. Compound 15 is identified other agents or using LA as a pharmacophore in designing new
to be a novel potent bifunctional ROCK inhibitor with antioxidant multi-target ligands (MTLs) may be more effective and safety
activity and neuroprotection.
although more difficult to fulfill.8, 9
Rho-associated kinases (ROCK1 and ROCK2), downstream
Central nervous system (CNS) progressive neurodegenerative
diseases, such as Alzheimer’s disease (AD), Parkinson’s disease
(PD) and amyotrophic lateral sclerosis (ALS), are identified as
multifactorial diseases, bringing not only physical and mental
suffering to the patients, but also heavy financial burden to the
society and family.1 However, due to the particularity of brain,
eg, blood-brain-barrier (BBB), and especially the complex
network of interconnected pathological processes, the
therapeutic drugs to effectively fighting against these diseases
remain a longstanding challenge.
eff
ectors of the small GTPase Rho A, play remarkable roles in
numerous cellular processes, such as cell motility, contraction,
survival, di
erentiation, invasion and migration.10 Mounting
ff
evidences show that there is an abnormal activation of the
Rho/ROCK pathway in several CNS diseases, and inhibiting the
Rho/ROCK pathway is considered as a promising approach for
the treatment of these diseases.11, 12 For example, it is believed
that ROCK inhibitors show significant beneficial effects on AD
by reducing the production of amyloid-beta protein (Aβ),
decreasing inflammation response, and regulating synaptic
function and plasticity, etc.13, 14
Given of the failures in fighting against CNS diseases in
the past decades, now researchers realize that single-target
drugs are limited in the treatment of complex diseases such as
CNS diseases, which have multiple pathogenic mechanisms
and are found to overlap and influence one another.15
Therefore, it is of high interest to seek molecules that are able
to influence on two or more complementary targets involving
complex diseases. To date, the MTLs strategy have brought us
lights of success on the treatment of CNS diseases.15, 16
The pathological mechanisms of CNS diseases are
complex, involving known and unknown signaling cascades,
oxidative stress, protein misfolding and so on.2 One of the
culprits, reactive oxygen species (ROS) is considered as a major
determinant of pathogenesis and progression of CNS diseases.
3, 4
Consequently, compounds with antioxidative activity have
been proposed either for the prevention or the treatment of
CNS diseases. Alpha lipoic acid (LA, Fig. 1) possesses vigoroso
capability to scavenge free radicals and neuroprotection,
which is beneficial for patients with CNS diseases.5-7 However,
Fig. 1 The strategy to develop MTL compound 15 from Lipoic acid and SR3677.
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SR3677 (Fig. 1) is one of potently selective ROCK2
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DOI: 10.1039/C6RA12081D
Scheme 1. Synthesis of compounds 1-8
inhibitors, and has been shown to dramatically reduce Aβ
production as well as the levels in vitro and in vivo.17, 18 SR3677
and its derivatives contain a hydrophobic moiety that interacts
with the hydrophobic pocket under the P-loop of ROCK,
contributing to their high potency.17 However, according to the
reported results, we found the hydrophobic moiety permits
more flexibility.19-23 Basing MTLs strategy, in this work, we
combined LA as an antioxidative hydrophobic moiety with
ROCK inhibition fragments in the hope of discovering novel
bifunctional ROCK inhibitors, which exhibit
a balanced
bifunctional profile covering antioxidant and ROCK inhibition.
Herein, the synthesis and biological evaluation of a series of
designed novel hybrids for CNS diseases were described.
Compounds 1-8 were prepared as the scheme 1. The side
chain groups(R) was introduced by nucleophilic substitution
following the reported procedure to provide the nitrobenzene
derivatives 1817 (or obtained from commercial sources).
Coupling of 18 with pyridine-4-boronic acid pinacol ester using
Pd[P(Ph)3]4 as the catalyst in the presence of K2CO3 were
applied to provide 19. The intermediates were then reduced
Reagents and conditions: (a) NaH, THF, R-OH, rt. (b) Pyridine-4-boronic acid pinacol
cyclic ester, Pd[P(Ph)3]4, K2CO3, toluene/H2O/EtOH, 100 ℃. (c) Raney-nickel, Hydrazine,
MeOH, rt. (d) lipoic acid，HATU, DIEA, DMF, rt.
Scheme 2. Synthesis of compounds 9-16
with Raney-nickel and hydrazine to arylamine 20. 1-8 were
obtained after an amide formation on the newly formed amino
group with LA, in which HATU was used as the coupling
reagent with the presence of DIEA in DMF.
Similar to the synthetic process of 1-8, 9-16 were
prepared as the scheme 2. The intermediates contained side
chain (18) prepared in the scheme 1 were used directly or
synthesized as mentioned. N-Boc-protected pyrazole-4-
boronic acid pinacol ester was used as another material, and a
Suzuki coupling reaction using PdCl2(dppf) as the catalyst in
the presence of Cs2CO3 was likewise applied to provide 21. The
intermediates were then reduced with Pd/H2 to arylamine 22
.
Amide reaction were taken place as mentioned above to give
23, and 9-16 were obtained after the N-Boc group of 23 were
removed using TFA in DCM.
Glutamate (Glu) induced neurotoxicity in HT22 cells, is
considered to be one of excellent models for studying the
consequences of oxidative stress.24 And neuroprotective drugs
are able to protect cells against Glu-induced damage.25
Therefore, firstly, we examined neuroprotective effects of
compounds against Glu-induced cell death by MTT assay.
Reagents and conditions: (a) Cs2CO3, PdCl2(dppf), N-Boc-4-pyrazoleboronic acid pinacol
ester, dioxane/H2O, 70 ℃. (b) Pd/C, H2, MeOH, rt. (c) lipoic acid, HATU, DIEA, DMF, rt.
(d) TFA, DCM, rt.
Pretreatment with test compounds, except
4 and 8, showed
better protection against Glu-induced HT22 cell death than
that with LA, while fasudil, a clinical used ROCK inhibitor, did
not exert protective effect under similar concentrations.
Moreover, several compounds (2, 6, 7, 10, 11
showed satisfactory effects even at low concentration, at
which LA had no effects. Compounds 11 14 and 15 could
, 14 and 15)
,
completely reverse Glu-induced cell death at 10 μM.
Obviously, as mentioned at the beginning, our goal is to
discover bifunctional compounds that not only exhibit potent
selective ROCK inhibition but also possess moderate
antioxidative effects. So that, we detected the kinases
inhibition of some of our compounds that have good to
moderate antioxidative effect in vitro. As shown in Table 1,
Fig. 2 The neuroprotective effects of compounds on glutamate-induced cytotoxicity in
HT22 cells. Cells were pretreated with/without compounds for 0.5
h and then
incubated with glutamate (2mM) for 24 h. Cells viability was determined using MTT
assay. Data are presented as means ± S.D. One-way ANOVA followed by Tukey’s test.
###
**
P < 0.001 vs. control group. *** P < 0.001, P < 0.01,* P <0.05 vs. glutamate treated
pyrazoles (
1
,
2
,
3
,
6
and
7
) were more potent inhibitary against
10 11 14 and 15) and compounds
ROCK than pyridines (
9
,
,
,
group.
that possess an alkaline side chain have better inhibitary
2 | J. Name., 2012, 00, 1-3
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Fig. 3 Docking pose of compound 15 (green) in the catalytic domain of ROCK2 (PDB ID:
against ROCK2. These results were consistent with the
previous report.22 The IC50 of the most potent compound 15
for ROCK2 was 0.8 μM, which was close to the value of fasudil
(0.46 μM in this assay), and had a better selectivity against PKA
(~453-fold) and PKG (~30-fold).
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2F2U). Interactions are displayed by light blue dotted lines. Key residues that could
DOI: 10.1039/C6RA12081D
form interactions with 15 are shown. Direct hydrogen bond interactions are formed
between the pyrazole ring of 15 and Glu 170, Met 172 on the bottom of the pocket as
well as the protonated tertiary amine of the 1-methyl-4-(oxymethyl)piperidine group of
15 and the Asp-176 carboxylate side chain.
Table 1 Kinases inhibition activity of tested compounds against ROCK2, PKA and
PKG
Better BBB penetration capacity plays a crucial role in the
effect of CNS drugs. To explore whether our compounds could
Compound
ROCK2 IC50(μM)
PKA IC50(μM)
＞5000
＞5000
＞5000
＞5000
252
PKG IC50(μM)
penetrate into the brain, we used
a parallel artificial
1
2
3
6
7
156
70
92
49
5
76
60
105
14
membrane permeation assay for the BBB (PAMPA-BBB),
similar to the procedure described by Di et al.27 The results
(Table S1†, S2†) showed that the selected compounds except
1
and
9 were able to cross the porcine brain lipid, which
6
indicated these compounds could penetrate the BBB.
9
64
46
37
10
0.8
0.46
＞5000
＞5000
＞5000
256
363
5.3
81
From the neuroprotective experiment, we were pleased
to find that most of targeted compounds displayed a better
neuroprotective capacity compared to LA. Furthermore, those
compounds were predicted to be able to penetrate the BBB.
Considering the inhibiatry activity against ROCK, we selected
compound 15 for further study. LA exhibits its antioxidant
activity not only by directly scavenging free radicals, but also
by regenerating other endogenic antioxidants, including
glutathione (GSH).28 Thus, to examine the effects of 15 on ROS,
we measured the free radical and the intracellular GSH levels
by radical fluorescent probe dihydroethidium (DHE) and a GSH
assay kit respectively. The results indicated that Glu caused an
significant increase of ROS, and 15 potently scavenged free
radical compared with LA (Fig. 4 A, B). Furthermore, Glu
dramatically decreased the intracellular level of GSH, while 15
remarkably reversed the process at 3 and 10 μM but LA did not
show any effect even at 10 μM (Fig. 4 C) and merely slight
increase the level of GSH at 30μM in our previous report.29
10
11
14
15
fasudil
168
147
33
24
2.5
There are sufficient evidences indicating that the
beneficial effects of ROCK inhibitors for CNS diseases,
especially AD, are mainly attributed to the ROCK2 inhibition
while ROCK1 inhibition may be more closely associated with
their side effects.18, 26 Therefore, we determined the subtype
selectivity of 15. The result (Fig. S1†) indicated that 15
displayed modulate ROCK2 selectivity against ROCK1 (IC50
=
2.110 μM to ROCK1, IC50 = 0.437 μM to ROCK2 in this assay,
there were some subtle differences between two methods†).
In order to understand the interactions between 15 and
ROCK2 in the active site in detail, a Molecular Operating
Environment (MOE) program was used for molecular docking.
Through the best scoring pose (Fig. 3 and Fig. S2†), we found
that the interaction model between 15 and ROCK2 was very
similar to the reported SR3677.17 The two H-bonds from
pyrazole ring and two amino acid residues of ROCK2 are
proposed necessary for the potency of 15. And the H-bond
between the protonated tertiary amine of the 1-methyl-4-
(oxymethyl)piperidine group of 15 and the Asp-176
carboxylate side chain is crucial to the selectivity of 15 against
other kinases. Docking score of
is lower than 15, and there is only one hydrogen bond
between and ROCK2 on the bottom of the pocket (data not
7, the pyridine analogue of 15,
7
shown). This may explain the higher potency of pyrazoles than
pyridines. The longish alkyl chain and the weaker
hydrophobicity of LA-group, compared to aromatic ring or Fig.
heterocyclic aromatics, may explain the reason for the milder
activity of 15. However, dramatically, it accomplished the
balance between the antioxidative effect and ROCK inhibition.
4
Effects of compound 15 on intracellular ROS and GSH levels. Cells were
pretreated with/without 15 and LA for 30 min and then exposed to 2 mM glutamate for
10 h. (A) Cells were followed by incubation with 10 µM DHE for 30 min, then cells were
photographed and fluorescence was recorded using a high content screening system.
(a) CT; (b) Glu; (c) 1 µM 15 + Glu; (d) 3 µM 15 + Glu; (e) 10 µM 15 + Glu; (f) 10 µM LA +
Glu; scale bar = 100 μm. (B) Quantitative analysis for relative DHE flurosence. (C) The
levels of GSH were measured using commercial assay kits. The basal contents of GSH in
untreated control cells were taken as 100%. The data were represented as mean ± SD
(n=3). * P < 0.05, *** P < 0.001 vs. Glu treated cells. ### P < 0.001 vs. control cells.
The significant blood pressure (BP) reduction associated
with ROCK inhibitions is one of the main challenges in the
development of ROCK inhibitors for systemic applications and
it is speculated that the side effect of BP reduction is mainly
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associated with ROCK1 inhibition.26 However, on the flip side,
increasing research results indicate that appropriate
vasodilatation of brain is beneficial for some CNS diseases, e.g.
AD.30, 31 The IC50 value of 15 for ROCK1 was 2.11 μM, while for
ROCK2 was 0.437 μM as mentioned above. It indicates that 15
may possess a moderate vasodilatation activity. To confirm the
ROCK inhibit activity and the effects of compound 15 on blood
vessels, we evaluated the vasorelaxant effect of 15 by a rat
aorta assay. Similar to our previous report,29 pro-contracting of
aorta was induced by KCl (60 mM) and fasudil was used as a
positive control. As shown in Fig. 5, compared with the vehicle
control group, compound 15 did not exert a significant
relaxation at 10 μM, and only had a mild relaxation (23%) even
at 30 μM compared to that of fasudil (53%).
7
D. P. De Araujo, F. Lobato Rde, J. R. Cavalcanti, L. R. Sampaio,
P. V. Araujo, M. C. Silva, K. R. Neves,DMOI.: 1M0..10F3Vo9ien/wCteA6lrRetiAcsl1,e2OF0n.8l1iCnDe.
Sousa and S. M. Vasconcelos, Int. J. Neurosci., 2011, 121, 51-
57.
O. Prezzavento, E. Arena, C. Parenti, L. Pasquinucci, G. Arico,
G. M. Scoto, S. Grancara, A. Toninello and S. Ronsisvalle, J.
Med. Chem., 2013, 56, 2447-2455.
A. Di Stefano, P. Sozio, A. Cocco, A. Iannitelli, E. Santucci, M.
Costa, L. Pecci, C. Nasuti, F. Cantalamessa and F. Pinnen, J.
Med. Chem., 2006, 49, 1486-1493.
8
9
10 F. Amigoni, E. Legnaghi and P. Pevarello, Pharm. Pat. Anal.,
2012, , 177-192.
11 B. K. Mueller, H. Mack and N. Teusch, Nat. Rev. Drug Discov.,
2005, , 387-398.
12 M. Chen, A. Liu, Y. Ouyang, Y. Huang, X. Chao and R. Pi,
Expert Opin. Investig. Drugs, 2013, 22, 537-550.
13 N. Hensel, S. Rademacher and P. Claus, Front. Neurosci.,
1
4
2015, 9, 198.
14 R. Lefort, Neurotherapeutics, 2015, 12, 19-28.
15 H. Zheng, M. Fridkin and M. Youdim, Pharmaceuticals (Basel),
2014, 7, 113-135.
16 C. Berk, G. Paul and M. Sabbagh, Expert Opin. Investig. Drugs,
2014, 23, 837-846.
17 Y. Feng, Y. Yin, A. Weiser, E. Griffin, M. D. Cameron, L. Lin, C.
Ruiz, S. C. Schurer, T. Inoue, P. V. Rao, T. Schroter and P.
Lograsso, J. Med. Chem., 2008, 51, 6642-6645.
18 J. H. Herskowitz, Y. Feng, A. L. Mattheyses, C. M. Hales, L. A.
Higginbotham, D. M. Duong, T. J. Montine, J. C. Troncoso, M.
Thambisetty, N. T. Seyfried, A. I. Levey and J. J. Lah, J.
Neurosci., 2013, 33, 19086-19098.
Fig. 5 Effects of compound 15 on aortic rings contracted by KCl. *** P < 0.001, ** P < 0.01
vs. vehicle control group (n=6).
19 X. Fang, Y. Yin, Y. T. Chen, L. Yao, B. Wang, M. D. Cameron, L.
Lin, S. Khan, C. Ruiz, T. Schroter, W. Grant, A. Weiser, J. Pocas,
A. Pachori, S. Schurer, P. Lograsso and Y. Feng, J. Med. Chem.,
2010, 53, 5727-5737.
20 R. Li, M. P. Martin, Y. Liu, B. Wang, R. A. Patel, J. Y. Zhu, N.
Sun, R. Pireddu, N. J. Lawrence, J. Li, E. B. Haura, S. S. Sung,
W. C. Guida, E. Schonbrunn and S. M. Sebti, J. Med. Chem.,
2012, 55, 2474-2478.
In summary, we synthesized a series of LA based
bifunctional compounds and evaluated their biological activity
from antioxidant to ROCK inhibition. Compounds 7, 14 and 15
exhibited a balanced bifunctional profile covering antioxidant
and ROCK inhibition with good BBB penertration property.
Notably,
compound
15
displayed
a
remarkable
21 Y. T. Chen, T. D. Bannister, A. Weiser, E. Griffin, L. Lin, C. Ruiz,
M. D. Cameron, S. Schurer, D. Duckett, T. Schroter, P.
neuroprotective activity by prominently scavenging free
radicals and increasing the level of intracellular GSH.
Furthermore, 15 was identified as a potent ROCK2 inhibitor
with good selectivity against ROCK1, so that exhibited milder
vasorelaxant effect compared to fasudil. The satisfactory
inchoate biological evaluation results strongly encourage
further pharmacological studies and optimization of
compound 15 in the development of bifunctional ROCK
inhibitors for specified CNS disease. Such studies have been
undertaken, and the progress will be reported in short future.
LoGrasso and Y. Feng, Bioorg. Med. Chem. Lett., 2008, 18
6406-6409.
,
22 Y. Yin, M. D. Cameron, L. Lin, S. Khan, T. Schroter, W. Grant, J.
Pocas, Y. T. Chen, S. Schurer, A. Pachori, P. LoGrasso and Y.
Feng, ACS Med. Chem. Lett., 2010, 1, 175-179.
23 Y. Feng, P. V. LoGrasso, O. Defert and R. Li, J. Med. Chem.,
2016, 59, 2269-2300.
24 A. Pfeiffer, M. Jaeckel, J. Lewerenz, R. Noack, A. Pouya, T.
Schacht, C. Hoffmann, J. Winter, S. Schweiger, M. K. Schafer
and A. Methner, Br. J. Pharmacol., 2014, 171, 2147-2158.
25 M. Koufaki, E. Theodorou, D. Galaris, L. Nousis, E. S.
Katsanou and M. N. Alexis, J. Med. Chem., 2006, 49, 300-306.
26 Y. Feng and P. V. LoGrasso, Expert Opin. Ther. Pat., 2014, 24
,
Notes and references
295-307.
27 L. Di, E. H. Kerns, K. Fan, O. J. McConnell and G. T. Carter, Eur.
J. Med. Chem., 2003, 38, 223-232.
1
X. Ning, Y. Guo, X. Wang, X. Ma, C. Tian, X. Shi, R. Zhu, C.
Cheng, Y. Du, Z. Ma, Z. Zhang and J. Liu, J. Med. Chem., 2014,
57, 4302-4312.
28 M. Jalali-Nadoushan and M. Roghani, Brain Res., 2013, 1505
,
68-74.
2
3
4
5
6
M. P. Mattson and T. Magnus, Nat. Rev. Neurosci., 2006,
7
,
29 M. Chen, Q. Liu, A. Liu, M. Tan, Z. Xie, A. Uri, Z. Chen, G.
278-294.
Huang, Y. Sun, H. Ge, P. Liu, M. Li, X. Li, S. Wen and R. Pi, RSC
P. J. Vernon and D. Tang, Antioxid. Redox Signal., 2013, 18
,
Advances, 2014, 4, 37266-37269.
677-691.
30 Y. Zhao, J. H. Gu, C. L. Dai, Q. Liu, K. Iqbal, F. Liu and C. X.
Gong, Front. Aging Neurosci., 2014, , 10.
31 A. ElAli, P. Theriault, P. Prefontaine and S. Rivest, Acta
Neuropathol. Commun., 2013, , 75.
M. Valko, D. Leibfritz, J. Moncol, M. T. Cronin, M. Mazur and
J. Telser, Int. J. Biochem. Cell Biol., 2007, 39, 44-84.
K. Hager, A. Marahrens, M. Kenklies, P. Riederer and G.
Munch, Arch. Gerontol. Geriatr., 2001, 32, 275-282.
F. Di Domenico, E. Barone, M. Perluigi and D. A. Butterfield,
Expert Rev. Neurother., 2015, 15, 19-40.
6
1
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DOI: 10.1039/C6RA12081D
A potently selective ROCK2 inhibitor with antioxidative property