Discovery of (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide as potent and isoform selective ROCK2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.02.047

ROCK1 and ROCK2 are highly homologous isoforms. Accumulated studies indicate that they have distinct different functions, and the development of isoform selective ROCK inhibitors will pave new roads for the treatment of various diseases. In this work, a series of amide-chroman derivatives were synthesized and biologically evaluated in order to develop potent and isoform selective ROCK2 inhibitors. Remarkably, (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide ((S)-7c) possessed ROCK2 inhibitory activity with an IC₅₀ value of 3 nM and 22.7-fold isoform selectivity (vs. ROCK1). Molecular docking indicated that hydrophobic interactions were the key element for the high potency and isoform selectivity of (S)-7c. The binding free energies predicted by MM/GBSA were in good agreement with the experimental bioactivities, and the analysis of individual energy terms suggested that residue Lys105 in ROCK1 or Lys121 in ROCK2 was the key residue for the isoform selectivity of (S)-7c.

Full text of DOI:10.1016/j.bmc.2019.02.047

Accepted Manuscript
Discovery of (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phe-
nyl)-amide as potent and isoform selective ROCK2 inhibitors
Jinpeng Pan, Yan Yin, Lianhua Zhao, Yangbo Feng
PII:
S0968-0896(19)30055-0
https://doi.org/10.1016/j.bmc.2019.02.047
BMC 14783
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
10 January 2019
15 February 2019
20 February 2019
Please cite this article as: Pan, J., Yin, Y., Zhao, L., Feng, Y., Discovery of (S)-6-methoxy-chroman-3-carboxylic
acid (4-pyridin-4-yl-phenyl)-amide as potent and isoform selective ROCK2 inhibitors, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.047
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Discovery of (S)-6-methoxy-chroman-3-
carboxylic acid (4-pyridin-4-yl-phenyl)-
amide as potent and isoform selective
ROCK2 inhibitors
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Jinpeng Pana, Yan Yina,*, Lianhua Zhaoa,Yangbo Fengb,*
aSchool of Chemical and Environment Engineering, Shanghai Institute of Technology, 100 Hai Quan Rd.,
Shanghai, 201418, P. R. China
bMedicinal Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States

Bioorganic & Medicinal Chemistry
Discovery of (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-
amide as potent and isoform selective ROCK2 inhibitors
Jinpeng Pana, Yan Yina,*, Lianhua Zhaoa, Yangbo Fengb,*
a School of Chemical and Environment Engineering, Shanghai Institute of Technology, 100 Hai Quan Rd., Shanghai, 201418, P. R. China
b Medicinal Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States
ARTICLE INFO
ABSTRACT
Keywords:
ROCK
Isoform selectivity
ROCK inhibitors
Chromans
ROCK1 and ROCK2 are highly homologous isoforms. Accumulated studies indicate that they
have distinct different functions, and the development of isoform selective ROCK inhibitors will
pave new roads for the treatment of various diseases. In this work, a series of amide-chroman
derivatives were synthesized and biologically evaluated in order to develop potent and isoform
selective ROCK2 inhibitors. Remarkably, (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-
4-yl-phenyl)-amide ((S)-7c) possessed ROCK2 inhibitory activity with an IC50 value of 3 nM
and 22.7-fold isoform selectivity (vs. ROCK1). Molecular docking indicated that hydrophobic
interactions were the key element for the high potency and isoform selectivity of (S)-7c. The
binding free energies predicted by MM/GBSA were in good agreement with the experimental
bioactivities, and the analysis of individual energy terms suggested that residue Lys105 in
ROCK1 or Lys121 in ROCK2 was the key residue for the isoform selectivity of (S)-7c.
Computational theoretical studies
2009 Elsevier Ltd. All rights reserved.
decade.18-21 Therefore, the discovery of isoform-selective ROCK
inhibitors remains a great challenge due to the high conservation
and similarity between the two homologous kinase isoforms.
1. Introduction
Rho-associated kinases (ROCKs), originally identified as the
effectors of small GTPase Rho, play a major role in mediating
rearrangement of the actomyosin cytoskeleton.1-5 Two ROCK
isoforms, ROCK1 and ROCK2, have been identified in
SR3677, one of the best known ATP-competitive ROCK
inhibitors, demonstrated an approximately 17.5-fold isoform
selectivity of ROCK 2 over ROCK1 (IC50 = 3.2 nM against
ROCK2, and 56 nM against ROCK1, Figure 1).22 Investigation
into Alzheimer's disease (AD) mouse models with SR3677
suggested ROCK inhibition reduced Aβ levels through
mammalian system. They have been shown to function
independently of each other and play non-overlapping roles in
cellular processes,6-8 so selective inhibition of the two kinase
isoforms can result in different biological effects on
independent mechanisms, and the potential for isoform-selective
ROCK inhibitors to combat Aβ production for the treatment of
AD raised considerable enthusiasm.23 Molecular docking study
indicated the hydrophobic interaction of the benzodioxane phenyl
ring in SR3677 with the hydrophobic surface of hydrophobic
pocket in ROCKs is the dominating factor that contributes to the
high inhibitory potency of SR3677 against ROCK2. 22 To our
delight, modifications on the benzodioxane group as well as the
bottom pyrazol group of SR3677 (Figure 1) led to (S)-6-
methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-
amide ((S)-7c) which has ROCK2 inhibitory potency of IC50= 3
nM and up to 22.7-fold isoform selectivity against ROCK1.
Modeling studies including molecular docking, molecular
dynamics simulations demonstrated that the difference of hydro-
physiological and pathological processes. Recently, isoform
selective inhibition of ROCK2 has been suggested to have wide
applications in treating Alzheimer disease (AD),9-11 Parkinson’s
disease (PD),12 vascular diseases,13 focal cerebral ischemia,14 and
neuroblastoma.15 In addition, the hypertensive effects of systemic
ROCK inhibition are speculated to be mainly due to ROCK1
inhibition.16 Therefore, isoform selective ROCK2 inhibitors
might provide medications with less safety concerns. However
the two isoforms share an overall 65% homology in amino acid
sequence and 92% homology in kinase domain,17 and most small-
molecule ROCK inhibitors cannot distinguish between the two
kinases, resulting in non-isoform-selective pharmacological
inhibition. Although significant efforts have been applied to
develop isoform selective ROCK inhibitors, few high-subtype
selective ROCK inhibitors were publicly reported over the past
*Corresponding authors. (86)-21-6087-7220. E-mail: yinyan@sit.edu.cn (To Yan Yin)
*Corresponding authors. 561-909-9820. E-mail: yangbof@gmail.com (To Yangbo Feng)

that the difference of hydrophobic contacts formed in ROCK1
and ROCK2 could be the main reason for the generation of
isoform selectivity and residue Lys105 in ROCK1 or Lys121 in
ROCK2 played a key role in isoform selectivity. Herein, the
discovery of (S)-7c as a potent and isoform selective ROCK2
inhibitor including synthesis, in vitro activity evaluation, and
computational studies will be reported in detail.
(R)-7c with HATU as the coupling reagent and DIEA as the
organic base (Scheme 2).
Inhibitory activities against ROCK1 and ROCK2 of all
synthesized compounds (7a-7g, (S)-7c, and (R)-7c) were
evaluated and presented in Table 1 as IC50 values. Effects of
bottom aromatic groups on the inhibition and isoform selectivity
were initially investigated. Compound 7a (SR3850) with a
pyrazole group displayed strong inhibition toward both ROCK
isoforms (IC50 = 3.4nM for ROCK1, and 0.7nM for ROCK2) and
poor isoform selectivity (4.8 fold).28 Compound 7b with the 3-
methyl-1H-pyrazole group had single digital nM potency against
ROCK2 (IC50 = 1.2nM) and IC50 = 16.3nM value against ROCK1
so as to improve isoform selectivity compared with 7a (13.6 fold
for 7b vs 4.8 fold for 7a). Most encouraging was the finding that
inhibitor 7c with a pyridine group as the hinge binding moiety
had a significant drop in ROCK1 potency (IC50 = 170nM against
ROCK1 and 9nM against ROCK2), thus resulting in an 18.9 fold
isoform selectivity. The SAR from the hinge binding region
indicated that replacement of pyrazole with pyridine might
significantly weaken ROCK1 inhibitory activity while still
retaining excellent ROCK2 potency thus yielding higher isoform
selectivity.
Figure 1. New ROCK inhibitors.
2. Results and discussion
Cyclization of 3-substituted salicaldehydes 1 with acrylonitrile
in the presence of equivalent DABCO, followed cyano
hydrolysis gave chromene acids 2. Then hydrogenation of 2
provided chroman-3-carboxylic acids 3 in high yields (Scheme
1).
Scheme 1. Reagents and conditions: (a) (i) Acrylonitrile, DABCO,
110°C; (ii) NaOH, H2O, reflux; (b) H2, 10% Pd/C, methanol, rt.
Although optical 3-methoxyl chroman-3-carboxylic acids can
be obtained by chiral auxiliaries or metal-catalyzed asymmetric
hydrogenation,24 the chiral auxiliary method can only provide
sub-gram quantities of each enantiomer as well as the need for
expensive selectride. In asymmetric hydrogenation environment-
tally unfriendly metal catalysts and expensive chiral ligands were
used. To overcome the above shortcomings, a cheap and green
synthesis method was developed in the preparation of optical 3a
from racemic 3a. Both (R)-3a and (S)-3a were obtained with
more than 95% enantiomeric excess (ee) via salification with
(1R,2R) or (1S,2S) 2-amino-1-(4-nitrophenyl)-propane-1,3-diol,
recrystalli-zation in CH3CN/CH3OH, dissociation with HCl, and
recrystalli-zation in Hexane/CH3CN. Both (1R, 2R) and (1S, 2S)
chiral resolving agents can be recovered after a series of
treatment of the crystal or mother liquor including acidification,
extraction, alkalization of aqueous phase, and extraction, etc.
(Figure 2). 25-27
Scheme 2. Reagents and conditions: (a) 2-Dimethylamino-ethanol, NaH,
THF, rt; (b) Ar-boronic acid pinacol ester or Ar-boronic acid, Pd(PPh3)4,
Na2CO3, dioxane, H2O, 100°C; (c) SnCl2, EtOAc, rt;(d) 3, (S)-3a,or (R)-3a,
HATU, DIEA, DMF, rt.
The SAR information of substitutions at the C6 position of the
chroman ring was then studied. Compared with 7c, compounds
7d (with a 6-CH3) and 7f (with a 6-Cl) showed similar ROCK1
inhibition and around 4 times weaker ROCK2 inhibition (IC50
=
169 nM against ROCK1, 24 nM against ROCK2 for 7d; 135 nM
against ROCK1, 22 nM against ROCK2 for 7f). 7e (with no 6-
substitution) had a similar ROCK2 inhibitory activity and better
ROCK1 inhibition (IC50 = 30 nM against ROCK1, 7 nM against
ROCK2), and 7g (with a 6-F group) exhibited a little higher
ROCK1 inhibition (IC50 = 104 nM) while retaining the same
ROCK2 potency (IC50 = 7 nM). SAR information of substitutions
at the C6 position of the chroman ring suggested that a 6-
methoxyl group would be less tolerated for ROCK1 inhibition
and consequently produced better isoform selectivity.
Molecules with a chiral center have two enantiomers.29
Although the physical and chemical properties of the two isomers
are the same, the pharmacological activities needs rewording
because of stereoselective reactions at the binding sites. Due to
the high ROCK2 inhibitory activity and impressive isoform
selectivity of 7c, it was chosen to probe possible stereochemical
preferences and the influence of chirality on isoform selectivity.
As shown in entries 8 and 9 of Table 1, (S)-7c showed 22.7-fold
isoform selectivity with IC50 = 68 nM against ROCK1 and 3 nM
against ROCK2 inhibitory activities, while (R)-7c had only 3-
fold isoform selectivity and weaker inhibitory activities toward
Figure 2. Preparation of (R)-3a and (S)-3a from racemic 3a.
Nucleophilic substitution reaction between commercially
available 4-bromo-2-fluoro-nitrobenzene and 2-dimethylamino-
ethanol, followed by Suzuki coupling gave nitro intermediates 5.
Then 5 were reduced to aniline derivatives 6 with stannous
chloride. Amide formation reaction between acids 3, (S)-3, or
(R)-3 and 6 produced the desired compounds 7a-7g, (S)-7c, and

Table 1. The chemical structures and inhibitory activities
ROCK1
IC50 (nM)a
ROCK2
IC50 (nM)a
Entry
1
Compd.
R
Ar
Selectivity
4.8
7a (SR3850)
OCH3
3.4
0.7
1.2
2
7b
OCH3
16.3
13.6
3
4
5
6
7
8
9
7c
OCH3
CH3
H
170
169
30
9
18.9
7.0
7d
24
7
7e
4.3
7f
Cl
135
104
68
22
7
6.1
7g
F
14.8
22.7
3.0
(S)-7c
(R)-7c
OCH3
OCH3
3
523
172
a Values are means of three or more experiments. The error in these values is within ±30% of mean.
both ROCK1 and ROCK2 kinases (IC50 = 523 nM against
ROCK1, 172 nM against ROCK2). These results suggested that
the S configuration was stereochemically preferred for both
ROCK inhibition and isoform-selectivity among this series of
compounds.
clearly explains why both (S)-7c and (R)-7c were potent against
both ROCK1 and ROCK2.
Hydrophobic interactions can be subdivided into pi-
hydrophobic interaction, alkyl hydrophobic interaction, and
mixed pi/alkyl hydrophobic interaction, which were shown as
deep pink, pink, and faded pink, respectively. Electronic
interactions and hydrophobic interactions in these systems were
quite different. In the (S)-7c-ROCK1 complex, there was one
electronic interaction between chrome and Lys105, and nine
hydrophobic interactions (Ala103-C···pyridine ring, Tyr155-
phenyl ring···pyridine ring, Met156-C···pyridine ring, Leu205-
C···middle phenyl ring, Lys105-C···phenyl ring in chroman,
Val90-C···middle phenyl ring, Ala215-C···middle phenyl ring,
Phe87-phenyl ring···pyran ring, and Phe87-phenyl ring···phenyl
ring in chroman). In the (S)-7c-ROCK2 complex, there was one
electronic interaction between chroman and Lys121, and thirteen
hydrophobic interactions (Ala119-C···pyridine ring, Met169-
C···pyridine ring, Tyr171- phenyl ring···pyridine ring, Met172-
S···pyridine ring, Leu221-C···pyridine ring, Leu221-C···middle
phenyl ring, Ala231-C···middle phenyl ring, Val106-
Molecular docking is one of the most frequently used methods
in drug discovery due to its ability to predict the binding-
interactions between ligand and protein.30,31 To explain the
inhibitory activity and isoform selectivity variation between the
two enantiomers of 7c, (S)-7c and (R)-7c were docked into the
active sites of ROCK1 (PDB: 2ETR) and ROCK2 (PDB: 4L6Q)
using the Surflex-Dock program and their binding behaviors in
the ligand-protein complexes were predicted from the amino acid
residue level (Figure 3). Comparison of the (S)-7c-ROCK1
complex, the (S)-7c-ROCK2 complex, the (R)-7c-ROCK1
complex, and the (R)-7c-ROCK2 complex, showed that the
binding poses of one compound at the binding site of either
ROCK1 or ROCK2 was quite similar, while binding poses of
(S)-7c-protein complex and (R)-7c-protein complex were
obviously different.
C···pyridine ring, Val106-C···middle phenyl ring, Lys121-
C···pyran ring, Lys121-C···phenyl ring in chroman, Phe103-
phenyl ring···pyran ring, Phe103-phenyl ring···phenyl ring in
chroman, Leu123-C···phenyl ring in chroman, and Phe136-
phenyl ring···phenyl ring in chroman). In the (R)-7c-ROCK1
complex, there were five hydrophobic interactions (Ala103-
The hydrogen bond interactions in the above four enzyme-
ligand complexes were the same: the first one is between the
amide carbonyl and Lys105 in ROCK1 or Lys 121 in ROCK2;
the second is between the protonated (CH3)2N and Asp160 in
ROCK1 or Asp 176 in ROCK2, and the third is between the
pyridine N and Met156 in ROCK1 or Met 172 in ROCK2, this

C···pyridine ring, Leu205-C···middle phenyl ring, Leu205-
C···pyridine ring, Val90-C···middle phenyl ring, and Ala215-
C···middle phenyl ring). In the (R)-7c-ROCK2 complex, there
were five hydrophobic interactions (Ala119-C···pyridine ring,
Leu221-C···phenyl ring, Leu221-C···pyridine ring, Ala231-
C···middle phenyl ring, and Val106-C···middle phenyl ring).
studies with the structures shown in Figure 3 as the initial
structures. As shown in Figure 4, both complexes reached a
stabilization at 10 ns with subtle rmsd values (around 1.5Å,
Figure 4a). The initial and final pharmacophore structures and
other structures were basically similar (Figure 4b and 4c). As a
result it could be inferred that the docking results were reliable.
We believed that the number of amino acids involved in the
binding, electronic interactions and hydrophobic interactions
between the ligand and protein contributed to the higher
inhibitory activities of (S)-7c against ROCK isoforms than (R)-
7c. At the same time hydrophobic interactions in the (S)-7c-
ROCK2 complex (Met169-C···pyridine ring, Leu221-
C···pyridine ring, Val106-C···pyridine ring, Lys121-C···pyran
ring, Leu123-C···phenyl ring in chroman, Phe136-
phenyl···phenyl ring in chroman) may contribute the isoform
selectivity of (S)-configuration of 7c.
Figure 4. Molecular dynamics simulation. (a) Plot of RMSD versus time.
(b) (S)-7c-ROCK1 complex. (c) (S)-7c-ROCK2 complex. The initial and
final structures are represented in green and magenta, respectively.
The predicted binding free energy (∆Gbind) can give a good
explanation of the activity difference between the studied
inhibitors.32 The binding free energies of both the ROCK1-(S)-7c
and ROCK2-(S)-7c complexes were predicted by the Molecular
Mechanics/Generalized Born Surface Area (MM/GBSA) method
in Amber using 50 snapshots extracted from 5 to 10 ns. At the
same time, individual energy components of the binding free
energies including van der Waals contribution (∆Gvdw),
electrostatic contribution (∆Gele), the polar contribution to
solvation free energy (∆GGB), and the non-polar contribution to
solvation free energy (∆GSA) were also calculated (Table 2).
∆Gvdw, ∆Gele, ∆GGB, ∆GSA, and ∆Gbind were -52.22 kcal mol-1, -
55.02 kcal mol-1, 64.19 kcal mol-1, -7.25 kcal mol-1, and -50.30
kcal mol-1 in ROCK1-(S)-7c, and -60.01 kcal mol-1, -55.68 kcal
mol-1, 65.54 kcal mol-1, -7.66 kcal mol-1, and -57.81 kcal mol-1 in
ROCK2-(S)-7c, respectively.
According to the predicted energies, the following conclusions
can be drawn: (a) In both systems, ∆Gvdw, ∆Gele and ∆GSA were
favorable, and ∆GGB was unfavorable for the binding and
inhibitory activities. (b) The differences of ∆Gele, ∆GGB, and
∆GSA between the two systems were subtle. (c) The calculated
∆Gbind value of the ROCK2-(S)-7c complex was lower than the
ROCK1-(S)-7c complex, indicating that (S)-7c had a higher
potency to ROCK2 than to ROCK1, which was in good
agreement with the experimental activities (IC50 = 3 nM against
ROCK2 and 68 nM against ROCK1). (d) Usually, the van der
Waals energy is closely correlated to the hydrophobic
interactions, ∆Gvdw value of the ROCK2-(S)-7c complexes was
significantly lower compared with the ROCK1-(S)-7c
complexes. So it was hypothesized that the hydrophobic
interactions formed in the protein-(S)-7c complexes played a key
role in the isoform selectivity of ROCK kinases, which was
consistent with the docking results.
Figure 3. Docking results. (a) 3D schematic view of (S)-7c-ROCK1
complex; (b) 2D schematic view of (S)-7c-ROCK1 complex; (c) 3D
schematic view of (S)-7c-ROCK2 complex; (d) 2D schematic view of (S)-7c-
ROCK2 complex; (e) 3D schematic view of (R)-7c-ROCK1 complex; (f) 2D
schematic view of (R)-7c-ROCK1 complex; (g) 3D schematic view of (R)-
7c-ROCK2 complex; (h) 2D schematic view of (R)-7c-ROCK2 complex.
Hydrogen bonds, hydrophobic interactions and electrostatic interactions were
shown as green, pink and orange dotted lines, respectively.
Table 2. The predicted binding free energies and individual
energy componentsa
System
ROCK1-(S)-7c
-52.22
1.30
-55.02
4.26
ROCK2-(S)-7c
-60.01
1.84
-55.68
4.18
Ave.
Std.Dev.
Ave.
Std.Dev.
Ave.
∆Gvdw
In order to evaluate the reliability of the docking results, the
(S)-7c-ROCK1 complexes and (S)-7c-ROCK2 complexes were
submitted to the studies for molecular dynamics (MD) simulation
∆Gele
∆GGB
64.19
65.54

Std.Dev.
Ave.
Std.Dev.
Ave.
3.63
-7.25
0.14
-50.30
2.04
2.99
-7.66
0.09
-57.81
2.09
column (5μm, 4.6mm*250mm) and UV detection in 292 nm. The
solvents used on the mobile phase were hexane and isopropanol.
NMR spectra were recorded with a Bruker 500 MHz
∆GSA
∆Gbind
spectrometer. The high-resolution mass spectra (HRMS
Std.Dev.
a All energies are in kcal mol-1.
electrospray ionization) were performed with Solaril X70 FT-MS
apparatus. The IR spectra were recorded on a Nicolet 6700 FT-IR
spectrometer in the range of 400-4000 cm-1. The melting points
were measured on a SGW X-4 melting point instrument. Optical
rotations were measured using a SGW®-1 automatic polarimeter.
All compounds were determined to be >95% pure by analytical
HPLC on Agilent technologies 1200 series with CH3CN (solvent
B)/H2O+0.9%CH3CN+0.1%TFA(solvent A) as eluents.
Docking studies indicated that Val106, Lys121, Leu123,
Phe136, Met169, and Leu221 had different interactions in the
two ROCK isoforms. To further investigate the protein-ligand
interactions, the energy contributions of these residues33 were
calculated and listed in Figure 5. In the ROCK2-(S)-7c
complexes, the energy differences were 1.140, 4.201, 0.873,
0.820, 0.474, 1.131kcal/mol (Energy contributions of Val106,
Lys121, Leu123, Phe136, Met169, and Leu221 were -3.396, -
5.697, -0.939, -0.828, -1.164, and -2.382 kcal/mol in the
ROCK2-(S)-7c complexes, respectively, and -2.256, -1.496, -
0.066, -0.007, -0.689, and -1.251 kcal/mol in the ROCK1-(S)-7c
complexes, respectively). Energy decomposition demonstrated
residue Lys105 in ROCK1 or Lys121 in ROCK2 was the largest
contribution to the isoform selectivity.
4.1. Synthesis procedure of compounds 7a-7g, (S)-7c and (R)-7c
1,4-Diazabicyclo[2,2,2]octane (DABCO, 17.5 mmol) was
added to a solution of 2-hydroxy-benzaldehyde derivative (1,
17.5 mmol) in acrylonitrile (20 mL) . The mixture was refluxed
under argon until 1 was completely conversed. The reaction
mixture was cooled, and washed with 10% aqueous NaOH and
ether. The organic phase was collected and concentrated under
reduced pressure to give the intermediate nitrile.
The nitrile was added to 10% NaOH solution and the reaction
mixture was heated at 60 OC until the complete disappearance of
nitrile. Then the mixture was extracted with ether and the water
phase was acidified with concentrated HCl, and extracted with
ether (20 mL X 3). The combined organic phases were collected,
washed with saturated NaCl, dried over anhydrous Na2SO4, and
concentrated under vacuum to produce chromene -3-carbozylic
acid 2.
10% palladium on carbon (20 mg) was added to a mixture of
chromene-3-carbozylic acid 2 (0.6 mmol) in methanol (5 mL).
The mixture was stirred under a hydrogen atmosphere for 20 h at
room temperature. After removal of the catalyst by filtration, the
filtrate was concentrated in vacuo and purified by flash column
chromatography to produce chroman-3-carboxylic acid 3a-3e.
Figure 5. Contribution of key residues
3. Conclusion
In summary, a series of amide-chromans were synthesized and
biologically evaluated in order to develop potent and isoform
selective ROCK2 inhibitors. Remarkably, compound (S)-7c,
which had a pyridyl group as the hinge-binding group exhibited
high potency at ROCK2 with 22.7-fold isoform selectivity.
Docking (S)-7c into the active site of ROCK1 and ROCK2
kinases provided insights in the binding features and
demonstrated that the differences of hydrophobic interactions
formed in ROCK1 and ROCK2 kinases were the main reason for
the generation of isoform selectivity. MD simulations further
verified the docking results. The inhibitory activities of (S)-7c
against ROCK1 and ROCK2 were further backed by binding free
energy analysis. The free energy deposition studies highlighted
that residue Lys 105 in ROCK1 or Lys121 in ROCK2 play a key
role in the isoform selectivity. These results deepen our
understanding of the differences of hydrophobic region within
ROCK1 and ROCK2, and facilitate the development of more
isoform selective ROCK2 inhibitors. More biological data, such
cellular activity, and pharmacokinetics in vitro and in vivo, will
be reported in due course.
Racemic 6-methoxychroman-3-carboxylic acid (3a, 15 mmol)
was dissolved in acetonitrile (300 mL) and the solution was
warmed to reflux. (1S, 2S)-2-amino-1-(4-nitrophenyl)propane-
1,3-diol (15 mmol) was added in one portion and the mixture was
stirred at gentle reflux for 30 min. Methanol was then added
dropwise until the solution became completely clear. The mixture
was then allowed to crystallize at ambient temperature overnight.
The precipitate was collected by filtration, washed with MeCN,
and dried to afford a salt. Then this salt was treated with ethyl
acetate (100 mL) and water (100 mL), acidified with 6 M
aqueous HCl (10 mL), and stirred for 10 min. The organic layer
was separated and washed with water (100 mL), dried over
Na2SO4, and concentrated to dryness to obtain (S)-3a. Finally, the
obtained (S)-3a was then recrystallized with hexane/ethyl acetate
to produce (S)-3a in 36% yield and 97.1% ee (mobile phase:
isopropanol/hexane/TFA = 50 mL: 50 mL: 0.5mL).
(R)-6-methoxychroman-3-carboxylic acid ((R)-3a) was
prepared in 37% yield and 95.6% ee with the synthesis procedure
of (S)-3a from racemic 6-methoxychroman-3-carboxylic acid and
(1R, 2R)-2-amino-1-(4-nitrophenyl)propane-1,3-diol. (mobile
phase: isopropanol/hexane/TFA = 50 mL:50 mL:0.5 mL).
4. Experimental Section
Commercially available reagents and anhydrous solvents were
used without further purification unless otherwise specified. Thin
layer chromatography (TLC) analysis was performed on 1 mm
thick precoated silica plates with ethyl acetate/hexane or
methanol/dichloromethane as eluent. Column chromatography
was performed on silica gel (200-300 Mesh) with ethyl
4-Bromo-2-fluoro-1-nitrobenzene (4) (1.00 mmol) was added
to a mixture of NaH (60% oil dispersion, 1.2 mmol) and 2-
dimethylamino-ethanol (1.04 mmol) in THF (15 mL) at 0℃.The
resulting mixture was allowed to warm to room temperature and
stirred overnight. The solvent was removed by rotary evaporation
and the obtained residue was partitioned between saturated
aqueous NaHCO3 (20 mL) and ethyl acetate (20 mL). The
acetate/hexane or methanol/dichloromethane as eluent. Chiral
analytical HPLC analyses were performed with a Chiralpak® IF

4.1.3. N-(2-(2-(Dimethylamino)ethoxy)-4-(pyridine-
4-yl)phenyl)-6-methoxychromane-3-carboxamide
(7c).
aqueous layer was further extracted with ethyl acetate (3x20 mL)
and the combined organic layers were washed with brine (30
mL), dried over Na2SO4, filtered, and concentrated to dryness.
Without further purification, the bromide residue was dissolved
in a mixture of dioxane and water (4:1, 10 mL). Then boronic
acid pinacol ester or boronic acid (1.5 mmol), 2M K2CO3 (3.00
mmol), and tetrakis (triphenyphosphine) palladium (0.05mmol)
were added to the mixture. Under argon protection, the reaction
mixture was refluxed until the complete conversion of the
bromide. After removal of the solvent under reduced pressure,
the obtained residue was extracted with ethyl acetate (3 x 10
mL). The combined organic layers were dried, concentrated
under reduced pressure, and purified by flash column
Yellow solid: 44% yield over four steps. Mp: 68-70°C. 1H
NMR (500 MHz, DMSO-d6) δ 9.73 (NH, br , 1H), 8.61 (s, 2H),
8.14 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 6.0 Hz, 2H), 7.55 (s, 1H),
7.44 (d, J = 8.5 Hz, 1H), 6.74-6.68 (m, 3H), 4.39 (d, J = 10.5 Hz,
1H), 4.26 (t, J = 5.0 Hz, 2H), 3.98 (t, J = 10.0 Hz, 1H), 3.69 (s,
3H), 3.21-3.15 (m, 1H), 3.08-3.03 (m, 1H), 2.97-2.92 (m, 1H),
2.68 (t, J = 5.0 Hz, 2H), 2.27 (s, 6H)； 13C NMR (125 MHz,
DMSO-d6) δ 171.06, 153.55, 150.58, 149.64, 148.13, 146.88,
133.58, 129.88, 122.43, 122.21, 121.39, 120.07, 117.24, 114.59,
113.80, 112.95, 68.26, 67.55, 66.82, 58.02, 55.81, 45.83, 28.28.
IR (KBr, cm-1): 2926, 1683, 1595, 1527, 1497, 1211, 1041, 811.
HRMS calcd for C26H30N3O4: 448.2231 [M+H+], found
448.2246.
chromatography to give intermediate 5.
SnCl2 (3 mmol) was added to a mixture of 5 (0.7 mmol) in
ethyl acetate (5 mL) at room temperature. After the complete
conversion of 5 detected by TLC, the reaction mixture was cool
to 0 oC. Then Na2SO4·12H2O was added slowly with strong
magnetic stirring. Upper organic phase was filtrated, washed with
saturated aqueous NaHCO3 and saturated aqueous NaCl, dried
with anhydrous Na2SO4, filtered, concentrated under reduced
pressure, and purified by flash column chromatography to give
aniline 6.
4.1.4. (S)-N-(2-(2-(Dimethylamino)ethoxy)-4-
(pyridin-4-yl)phenyl)-6-methoxychromane-3-
carboxamide((S)-7c).
[α]D20 = +10.42 (c = 0.45, CHCl3).
4.1.5. (R)-N-(2-(2-(Dimethylamino)ethoxy)-4-
(pyridin-4-yl)phenyl)-6-methoxychromane-3-
carboxamide((R)-7c).
[α]D20 = -6.83 (c = 0.34, CHCl3).
Racemate or optically pure acid 3 (0.12 mmol), HATU (0.
1mmol), and DIEA (0.1 mmol) were added to the mixture of
aniline 6 (0.1mmol) in DMF (2 mL). The resulting mixture was
stirred at room temperature until 6 was completed conversed.
Then the reaction mixture was concentrated under reduced
pressure, extracted with ethyl acetate (2 X 20 mL). The combined
organic phases were washed with brine (5 mL), dried over
Na2SO4, filtered, concentrated to dryness, and purified by flash
column chromatography to provide targeted compounds 7a-7g,
(S)-7c and (R)-7c.
4.1.6. N-(2-(2-(Dimethylamino)ethoxy)-4-(pyridine-
4-yl)phenyl)-6-methylchromane-3-carboxamide(7d).
White solid: 40% yield over four steps. Mp: 187-189°C. 1H
NMR (500 MHz, DMSO-d6) δ 9.69 (NH, br, 1H), 8.62 (d, J = 4.5
Hz, 2H), 8.12 (d, J = 8.0 Hz, 1H) 7.75 (d, J = 5 Hz, 2H), 7.56 (s,
1H), 7.44 (d, J = 8.0 Hz, 1H), 6.95 (s, 1H), 6.90 (d, J = 8.0 Hz,
1H), 6.69 (d, J = 8.0 Hz, 1H), 4.41 (d, J = 10.5 Hz, 1H), 4.27 (t, J
= 5.5 Hz, 2H), 3.99 (t, J = 10.0 Hz, 1H), 3.19-3.13 (s, 1H), 3.04-
2.99 (m, 1H), 2.94-2.90 (m, 1H), 2.68 (t, J = 5.5 Hz, 2H), 2.27 (s,
6H), 2.21 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 171.21,
152.01, 150.55, 149.61, 146.94, 133.52, 130.49, 129.75, 129.45,
128.19, 122.39, 121.40, 121.22, 119.95, 116.38, 112.48, 67.72,
67.50, 57.94, 45.62, 31.73, 28.05, 20.57. IR (KBr, cm-1): 2943,
1685, 1595, 1528, 1500, 1219, 1031, 811. HRMS calcd for
C26H30N3O3: 432.2282 [M+H+], found 432.2281.
4.1.1. N-(2-(2-(Dimethylamino)ethoxy)-4-(pyrazol-
4-yl)phenyl)-6-methoxychromane-3-carboxamide
(7a).
Yellow solid: 46% yield over four steps. Mp: 63-65°C. 1H
NMR (500 MHz, DMSO-d6) δ 9.58 (NH, br, 1H), 8.06 (s, 2H),
7.88 (d, J = 8.5 Hz, 1H), 7.35 (s, 1H), 7.20 (d, J = 8.0 Hz, 1H),
6.73-6.68 (m, 3H), 4.38 (d, J = 10.0 Hz, 1H), 4.19 (t, J = 5.0 Hz,
2H), 3.96 (t, J = 10 Hz, 1H), 3.69 (s, 3H), 3.11-3.02 (m, 2H),
2.94-2.91 (m, 1H), 2.66 (t, J = 5.0 Hz, 2H), 2.27 (s, 6H); 13C
NMR (125 MHz, DMSO-d6) δ 170.62, 153.54, 149.91, 148.15,
130.26, 126.58, 122.95, 122.27, 121.41, 118.19, 117.22, 116.88,
114.61, 113.78, 113.29, 111.66, 68.16, 67.63, 58.07, 55.82,
45.85, 28.33, 25.43. IR (KBr, cm-1): 2919, 1674, 1596, 1530,
1498, 1211, 1040, 843. HRMS calcd for C24H29N4O4: 437.2183
[M+H+], found 437.2184.
4.1.7. N-(2-(2-(Dimethylamino)ethoxy)-4-(pyridine-
4-yl)phenyl)chromane-3-carboxamide(7e).
White solid: 45% yield over four steps. Mp: 137-139°C. 1H
NMR (500 MHz, DMSO-d6) δ 9.78 (NH, br, 1H), 8.61 (d, J = 4.5
Hz, 2H), 8.14 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 5.5 Hz, 2H), 7.56
(s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H), 7.10 (t.
J = 7.5 Hz, 1H), 6.87 (t, J = 7.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H),
4.45 (d, J = 10.5 Hz, 1H), 4.262 (t, J = 5.5 Hz, 2H), 4.03 (t, J =
10 Hz, 1H), 3.24-3.18 (m, 1H), 3.09-3.03 (m, 1H), 2.99-2.94 (m,
1H), 2.68 (t, J = 6.0 Hz, 2H), 2.26 (s, 6H); 13C NMR (125 MHz,
DMSO-d6) δ 171.01, 154.19, 150.58, 149.72, 146.89, 133.62,
130.30, 129.85, 127.66, 122.52, 121.60, 121.39, 120.88, 120.04,
116.63, 112.87, 68.21, 67.54, 58.03, 45.82, 29.47, 28.13. IR
(KBr, cm-1): 2926, 1684, 1595, 1527, 1490, 1209, 1041, 811.
HRMS calcd for C25H28N3O3: 418.2125 [M+H+], found
418.2128.
4.1.2. N-(2-(2-(Dimethylamino)ethoxy)-4-(3-methyl-
1H-pyrazol-4-yl)phenyl)-6-methoxychromane-3-
carboxamide (7b).
Yellow solid: 42% yield over four steps. Mp: 110-112°C. 1H
NMR (500 MHz, DMSO-d6) δ 9.65 (NH, br, 1H), 7.92 (d, J = 8.0
Hz, 1H), 7.79 (s, 1H), 7.14 (s, 1H), 7.03 (d, J = 8.5 Hz, 1H),
6.74-6.68 (m, 3H), 4.38 (d, J = 10.0 Hz, 1H), 4.17 (t, J = 5.0 Hz,
2H), 3.96 (t, J = 10.0 Hz, 1H), 3.69 (s, 3H), 3.13-3.10 (m, 1H),
3.07-3.02 (m, 1H), 2.96-2.90 (m, 1H), 2.66 (t, J = 5.0 Hz, 2H),
2.38 (s, 3H), 2.26 (s, 6H); 13C NMR (125MHz, DMSO-d6) δ
170.71, 153.53, 149.68, 148.14, 131.05, 126.39, 122.87, 122.27,
119.69, 118.65, 117.22, 116.59, 114.59, 113.77, 113.05, 109.73,
74.02, 67.99, 67.64, 58.01, 55.80, 45.77, 28.33, 25.42. IR (KBr,
cm-1): 2928, 1679, 1595, 1532, 1498, 1211, 1041, 843. HRMS
calcd for C25H31N4O4: 451.2340 [M+H+], found 451.2332.
4.1.8. N-(2-(2-(Dimethylamino)ethoxy)-4-(pyridine-
4-yl)phenyl)-6-chlorochromane-3-carboxamide(7f).
Yellow solid: 38% yield over four steps. Mp: 105-107°C. 1H
NMR (500 MHz, DMSO-d6) δ 9.77 (NH, br, 1H), 8.61 (s, 2H),
8.11 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 5.5 Hz, 2H), 7.55 (s, 1H),
7.44 (d, J = 10.0 Hz, 1H), 7.24 (s, 1H), 7.13 (d, J = 6.0 Hz, 1H),
6.83 (d, J = 8.5 Hz, 1H), 4.45 (d, J = 11 Hz, 1H), 4.27 (t, J = 5.5
Hz, 2H), 4.07 (t, J = 10 Hz, 1H), 3.08-2.95 (m, 3H), 2.69 (t, J =

5.5 Hz, 2H), 2.27 (s, 6H); 13C NMR (125 MHz, DMSO-d6) δ
170.69, 153.10, 150.58, 149.73, 146.88, 133.68, 129.75, 129.63,
127.45, 124.39, 123.96, 122.55, 121.40, 120.03, 118.34, 112.86,
68.16, 67.78, 58.00, 45.80, 29.47, 27.80. IR (KBr, cm-1): 2919,
1685, 1596, 1527, 1495, 1206, 1029, 811. HRMS calcd for
C25H27ClN3O3: 452.1735 [M+H+], found 452.1744.
solute species was restrained by the force of 100 kcal·mol-1·Ǻ-2.
Second step, the entire systems were optimized energy by 5000
steps of steepest descent method followed by 5000 steps of
conjugated gradient method. Then the system was heated from 0
to 300K, followed by equilibration at 300K over 50 ps. Finally, a
10 ns production run was carried out by NPT ensemble at 300K
with 1.013x105 kPa. All energy components were calculated
using 50 snapshots extracted from 5 to 10 ns based on the RMSD
results mentioned above.
4.1.9. N-(2-(2-(Dimethylamino)ethoxy)-4-(pyridine-
4-yl)phenyl)-6-fluorochromane-3-carboxamide(7g).
Yellow solid: 40% yield over four steps. Mp: 101-103°C. 1H
NMR (500 MHz, DMSO-d6) δ 9.73 (NH, br, 1H), 8.62 (s, 2H),
8.11 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 5.0 Hz, 2H), 7.56 (s, 1H),
7.44 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 1H), 6.96-6.92 (m,
1H), 6.83-6.80 (m, 1H), 4.44 (d, J = 10.5 Hz, 1H), 4.27 (t, J = 5.5
Hz, 2H), 4.04 (t, J = 10 Hz, 1H), 3.23-3.16 (m, 1H), 3.09-3.04
(m, 1H), 3.00-2.95 (m, 1H), 2.68 (t, J = 5.5 Hz, 2H), 2.27 (s, 6H);
13C NMR (125 MHz, DMSO-d6) δ 170.77, 157.54, 155.67,
150.59, 150.46, 149.74, 146.87, 133.69, 129.81, 123.32, 123.26,
122.56, 121.40, 120.07, 117.78, 117.71, 116.14, 115.96, 114.41,
114.22, 112.99, 68.32, 67.71, 58.04, 45.88, 29.48, 28.02. IR
(KBr, cm-1): 2919, 1685, 1596, 1527, 1495, 1206, 1029, 811.
HRMS calcd for C25H27FN3O3: 436.2031 [M+H+], found
436.2030.
At length, a production run for 10 ns under conditions of
constant pressure and temperature was carried out. MD
trajectories of all systems were taken to calculate the binding free
energy with Molecular Mechanics/Generalized Born Surface
Area (MM/GBSA) method reported by Onufiev (igb = 2).41 In
these calculations, the dielectric constants were set to 1.0 for the
interior solute and 80.0 for the exterior solvent.
The contribution of residue to the total free energy was
calculated by sander in MMPBSA.py.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
4.2. ROCK-1/2 Assays.30,34
Assays were performed using the STK2 kinase system from
Cisbio. A 5 μL mixture of a 1μM STK2 substrate and ATP
(ROCK1: 4 μM, ROCK2: 20μM) in STK-buffer was added to
the wells of the 384-well plate using a BioRAPTR FRD
Workstation (Aurora Discovery, Carlsbad, CA). Twenty
nanoliters of test compound was then dispensed using a 384-head
offline Pintool system (GNF System, San Diego, CA). The
reaction was started by adding either 5μL of 2.5 nM ROCK1
(Upstate #14-601) or 5μL of 0.5 nM ROCK2 in STK-buffer.
After 4h at RT the reaction was stopped by adding 10 μL of 1X
antibody and 62.5 nM Sa-XL in Detection Buffer. After 1h
incubation at RT, the plates were read on the Viewlux in HTRF
mode.
Financial supports from National Natural Science Foundation
of China (Grant No. 21502117), and the collaboration Innovation
Foundation of Shanghai Institute of Technology, China
(No.XTCX2016-3). We are also grateful to Prof. Gang Zhao and
Prof. Guanjun Wang for technical assistance.
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