Design, Synthesis, and Structure-Activity Relationships of Pyridine-Based Rho Kinase (ROCK) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b00424

The Rho kinases (ROCK1 and ROCK2) are highly homologous serine/threonine kinases that act on substrates associated with cellular motility, morphology, and contraction and are of therapeutic interest in diseases associated with cellular migration and contraction, such as hypertension, glaucoma, and erectile dysfunction. Beginning with compound 4, an inhibitor of ROCK1 identified through high-throughput screening, systematic exploration of SAR, and application of structure-based design, led to potent and selective ROCK inhibitors. Compound 37 represents significant improvements in inhibition potency, kinase selectivity, and CYP inhibition and possesses pharmacokinetics suitable for in vivo experimentation.

Full text of DOI:10.1021/acs.jmedchem.5b00424

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Article
Design, Synthesis and Structure-Activity Relationships of Pyridine-Based Rho Kinase (ROCK) Inhibitors
Jeremy Green, Jingrong Cao, Upul Bandarage, Huai Gao, John J. Court, Craig Marhefka, Marc Jacobs, Paul Taslimi, David Newsome, Tomoko Nakayama, Sundeep Shah, and Steve Rodems
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 03 Jun 2015
Downloaded from http://pubs.acs.org on June 3, 2015
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Design, Synthesis and Structure-Activity Relationships of Pyridine-
Based Rho Kinase (ROCK) Inhibitors.
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Jeremy Greena*, Jingrong Caoa, Upul K. Bandaragea, Huai Gaoa, John Courta, Craig
Marhefkaa, Marc Jacobsa, Paul Taslimia,√, David Newsomea, Tomoko Nakayamaa,†,
Sundeep Shahb, Steve Rodemsb,∫.
aVertex Pharmaceuticals, Inc., 50 Northern Avenue, Boston, MA 02210, USA
bVertex Pharmaceuticals, Inc., 11010 Torreyana Road, San Diego, CA 92121, USA
* Corresponding author. Telephone: 617ꢀ341ꢀ6315; email: jeremy_green@vrtx.com
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√ Present address: Ariad Pharmaceuticals, 26 Landsdowne Street, Cambridge, MA 02139
† Present address: Digilab, Inc., 100 Locke Drive, Marlborough, MA 01752
∫ Present address: Retrophin, Inc., 12255 El Camino Real, Suite 250, San Diego, CA
92130
Abstract
The Rho kinases (ROCK1 and ROCK2) are highly homologous serine/threonine kinases
that act on substrates associated with cellular motility, morphology and contraction, and
are of therapeutic interest in diseases associated with cellular migration and contraction,
such as hypertension, glaucoma and erectile dysfunction. Beginning with compound 4, an
inhibitor of ROCK1 identified through highꢀthroughput screening, systematic exploration
of SAR and application of structureꢀbased design, led to potent and selective ROCK
inhibitors. Compound 37 represents significant improvements in inhibition potency,
kinase selectivity and CYP inhibition and possesses pharmacokinetics suitable for in vivo
experimentation.
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Introduction
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The Rho kinases (ROCK1 and ROCK2) are 160 kDa serine/threonine kinases consisting
of an Nꢀterminal kinase domain, a central coiledꢀcoil region containing the Rho binding
domain and a Cꢀterminal pleckstrin homology (PH) domain, containing an internal
cysteineꢀrich region.1 The Cꢀterminal region negatively regulates ROCK activity,2
however upon binding of activated RhoAꢀGTP, the interaction between the Cꢀterminal
inhibitory region and the kinase domain is disrupted and kinase function is activated.
ROCK1 and ROCK2 are highly homologous in their kinase domains (92% sequence
identity) with 65% overall sequence identity3 and both are ubiquitously expressed in rat
and mouse tissues. ROCK phosphorylates a variety of cellular substrates, mainly
associated with actinꢀcytoskeletal reorganization affecting cell adhesion, cell
morphology, cell motility and smooth muscle contraction.4 Notable substrates include
myosin light chain (MLC) and the myosin binding subunit of myosin phosphatase
(MYPT). Other substrates include proteins acting on actin, such as the LIM kinases,
adducin and the ERM proteins.5 The action of ROCK on these substrates implicates
ROCK as a potential therapeutic target for indications wherein intervention in smooth
muscle contraction is desired, such as hypertension and cardiovascular disease,6
glaucoma,7 Raynaud’s disease8 or erectile dysfunction.9
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A number of molecules have been reported that inhibit ROCK, the most widely
studied of which are compound 1 (Fasudil)10 and compound 3 (Yꢀ27632).11 Fasudil, is
approved for clinical use in Japan for the treatment of subꢀarachnoid hemorrhage.12
Interestingly, compound 1 is metabolized in vivo to the quinolone, 2 (widely referred to
as hydroxyfasudil),13 which is also a ROCK inhibitor, even though the mode of binding
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to ROCK is changed.14 Compound 311 has also been widely utilized as a ROCK inhibitor
standard in a large number of studies.15 Compounds 1ꢀ3 are modest inhibitors of ROCK,
with reported comparable Ki values of 530, 150 and 150 nM respectively.14 More
recently, numerous publications have reported potent ROCK inhibitors based on
derivatives of indazoles, isoquinolines, phthalimides, aminofurazans, pyrazoles,
aminopyrimidines, 7ꢀazaindoles and pyridines. Much of this work has been reviewed.16
In particular, a number of pyridineꢀcontaining inhibitors of ROCK have been published,17
prompting us to report our findings in this area.
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Figure 1. Structures of ROCK inhibitors
High throughput screening of our compound collection identified compound 4 as an
interesting inhibitor of ROCK1, with Ki = 1.1 µM. This compound had not been
previously identified in our other kinase screens and thus we felt that it may offer
opportunities to develop both potent and selective inhibitors. It was also less active
against PKAα (Ki > 5 µM), a closely related member of the AGC kinase subfamily,18
suggesting that selectivity within this subfamily might also be achieved. An Xꢀray
crystallographic structure of 4 bound to ROCKꢀ1 (PDB ID: 4YVC) showed several
notable features, illustrated in Figure 2. The pyridine group is hydrogen bonded to the
backbone NH of hinge residue Met156. In contrast to most other kinases, the hinge
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region constitutes a deep, distinct binding pocket as the lower region is blocked by the
sidechains of residues Tyr155 and Phe368, blocking access to solvent from the pyridine
2ꢀposition. The amide carbonyl of 4 is within Hꢀbonding distance of catalytic Lys105.
The 2ꢀfluorophenyl ring lies unproductively beneath the glycineꢀrich loop and suggests
an obvious area for the design of improved inhibitors. We reasoned that insertion of one
or more* sp3ꢀhybridized atoms next to the carbonyl (for example, an Nꢀphenylacetyl
group) would allow this ring (or any other group) to occupy space beneath the glycineꢀ
rich loop and for substituents to consummate further enzymeꢀinhibitor interactions with
loop residues.
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Figure 2. Xꢀray crystallographic structure of 4 bound to ROCK1 (PDB ID: 4YVC)
* Extending the chain length between aryl group and carbonyl was also investigated and results
are summarized in Supporting Material Table S2. Although the parent phenylpropionyl
compound offered potency and selectivity, the crystal structure of 10 suggested that less space is
available for substituents to make productive interactions.
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Results and Discussion
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Exploration of acyl substituents showed that extending the substituent into the pocket
defined by the glycineꢀrich loop enhanced ROCK inhibition potency relative to 4, and
established a baseline for selectivity against PKA (Table 1). The Xꢀray crystal structure
of compound 10 (Ki = 100 nM) (Figure 3, PDB ID: 4YVE) indicated that aryl
substituents, such as the 3ꢀmethoxy of 10, are capable of additional, productive
interactions with the glycineꢀrich loop. Using this structure as a guide, substitutions
capable of forming hydrogenꢀbonds with the main chain amide of Phe87 were explored.
The 3ꢀhydroxyꢀ and 3,4ꢀmethylenedioxyꢀcompounds 13 and 15 were similarly potent at
28 nM and 100 nM respectively, while combination of a 2ꢀchloro substituent, with the
4,5ꢀmethylenedioxy in compound 16 further enhances potency into the single digit
nanomolar range, presumably as a result of conformational constraint and reduced
entropic contribution. Other 3ꢀsubstituents capable of Hꢀbonding interaction with the
Phe87 NH were also investigated. Among these, compound 18 bearing a 3ꢀ
methanesulfonylamino group, provided the best overall combination of potency (Ki = 26
nM) and selectivity (127ꢀfold vs PKAα).
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Figure 3. Xꢀray crystallographic structure of 10 bound to ROCK1 (PDB ID: 4YVE)
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Table 1. SAR of substituted phenylacetyl series
Compound
R
ROCK Ki, nMa PKA Ki, nM
Selectivity
5
H
170
130
120
81
3500
2090
21
16
6
2ꢀF
7
3ꢀF
2000
17
8
4ꢀF
1580
20
9
2ꢀOMe
3ꢀOMe
4ꢀOMe
2ꢀOH
>2500
100
540
900
28
>5000
2500
NC
25
10
11
12
13
14
15
16
17
18
>5000
>5000
1700
9
>6
3ꢀOH
61
4ꢀOH
70
>5000
>5000
310
>71
>50
23
3,4ꢀOCH2Oꢀ
2ꢀClꢀ3,4ꢀOCH2Oꢀ
3ꢀNH2
100
7
350
>5000
>14
3ꢀNHSO2CH3
26
>3300
127
a Minimum Significant Ratio (MSR)19 = 3.6: i.e. compounds that have a difference in Ki of at
least a factor of 3.6 are considered significantly different.
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Substitution at the methylene of the phenylacetyl series was next investigated to improve
PKA selectivity over ROCK. In most cases, neither ROCK potency nor PKA selectivity
was improved significantly, though lactam 19, showed improved potency for ROCK and
135ꢀfold selectivity against PKA (Figure 4). Other attempts to capitalize on Nꢀ or
Cꢀsubstitution did not lead to significant improvements in potency or selectivity (see
Supporting Information Table S3).
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Figure 4.
Replacement of the thiazole ring with other 5ꢀmembered rings was investigated and the
results are summarized in Table 2. Nonꢀsulfurꢀcontaining heterocycles such as pyrazole,
triazole and oxadiazole derivatives showed relatively weak ROCK inhibition (data not
shown), while thiophenes, regioisomeric thiazoles and thiadiazoles showed good ROCK
potency. For these sulfurꢀcontaining heterocycles, those with NH adjacent to S atom (10,
22, 25) showed better activity than the corresponding isomers in which the 4ꢀpyridyl
system was adjacent to sulfur (20, 23), while systems in which sulfur bridged the two
substituted carbons was least effective (21, 24, 26). Thus, thiazole 10 and thiophene 22
were identified as the best leads in this series, with the latter showing some
improvements in potency and selectivity.
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Table 2. SAR of central heterocycle replacements
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Cpd
HET
ROCK Ki, nM PKA Ki, nM
Selectivity
>25
10
100
530
490
10
>2500
>5000
520
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25*
26
>10
1
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6
>2500
1700
<1
* 4ꢀfluorophenylacetyl derivative
Despite the pyridine ring providing a simple template for binding to the hinge pocket of
ROCK, compounds such as 10 and 22 also possessed less desirable qualities, notably
panꢀcytochrome P450 inhibition. These compounds inhibited CYP 3A4 with IC50 = 1.9
and 0.80 µM respectively. Applying lessons from p38 inhibitors, in which a pyridine
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hinge binding ring was successfully replaced with an unsubstituted pyrimidine to
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maintain target affinity and reduce CYP inhibition,20 we investigated the analogous
modification (Table 3). However this tactic did not succeed. Compound 27 containing the
pyrimidinylꢀthiazole system, lessened CYP 3A4 inhibition (CYP 3A4 IC50 = 10 µM), but
ROCK potency was lost (Ki > 2.5 µM).
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Table 3. SAR and CYP 3A4 inhibition of the 4ꢀPyridyl and 4ꢀPyrimidinyl Substituted
Thiazole and Thiophenes
ROCK Ki,
nM
CYP 3A4
IC50, ꢁM
Cpd
X
Y
PKA Ki, nM Selectivity
10
22
27
28
CH
CH
N
N
CH
N
100
10
>2500
600
>25
60
ꢀ
0.75
0.48
10
>2500
120
>5000
2900
N
CH
24
2.0
Our interpretation for the loss of ROCK inhibition is that the low energy conformation
orients the two aromatic rings such that the nitrogen atoms are separated by 180° as
shown in Figure 5A. In the case of 27 this places the pyrimidine nitrogen in an
unfavourable hydrophobic location in the binding site. This energy barrier is remedied by
replacing thiazole with thiophene, since the rotational difference is minimal, and ROCK
inhibition is restored. Unfortunately the thiophene compound 28 retained significant CYP
inhibitory activity (CYP 3A4 IC50 = 3.7 µM).
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Figure 5. Rationale for observed pyrimidine activities.
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A. Unfavourable orientation of low energy
B. Favourable orientation of thiophene 28
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conformation of 27
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C. Comparison of the relative rotational energies of 26 and 27 about the biaryl bond.
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2
0
0
50
100
150
Torsion Angle (degrees)
blue = 27
red = 28
One strategy to circumvent CYP inhibition is to interfere with the interaction of the heme
group of the CYP enzyme with the pyridine.21 Therefore, we next investigated
substitution of the pyridine ring, focusing on position 2. The results are summarized in
Table 4. In general, pyridine 2ꢀsubstitution had a desirable effect on reducing CYP
inhibition to acceptable levels. The 2ꢀaminopyridine compound 30 demonstrated 35ꢀfold
less CYP 3A4 inhibition liability compared with unsubstituted compound 10 (IC50 =
26 µM and 0.75 µM respectively) while maintaining the ROCK potency (Ki = 170 nM).
However, most of the pyridine 2ꢀsubstitutions had little significant effect on ROCK
inhibitory activity, with 2ꢀmethylꢀ (29), 2ꢀfluoroꢀ (34) and 2ꢀchloroꢀpyridines (35) within
2ꢀfold of the unsubstituted system, 10. Larger substituents such as 2ꢀmethylaminoꢀ (31),
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2ꢀdimethylaminoꢀ (32) and 2ꢀmethoxyꢀpyridine (33) significantly decreased ROCK
potency. In the case of compound 31, modeling suggested that even the relatively small
methylamino group requires sufficient space to result in steric overlap with residues
Tyr155 and Phe368 at the base of the pyridineꢀbinding pocket. In compound 33, potential
unfavourable interactions between the methoxy oxygen and carbonyls of hinge residues
Gln 154 and Met156 explains the poor ROCK potency.
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Table 4. SAR of 2ꢀSubstituted pyridines
ROCK Ki,
nM
CYP 3A4,
IC50, ꢁM
Cpd
R
PKA Ki, nM Selectivity
10
29
30
31
32
33
34
35
2ꢀH
2ꢀCH3
2ꢀNH2
2ꢀNHMe
2ꢀNMe2
2ꢀOMe
2ꢀF
100
210
>2500
>3300
1850
>25
>15
11
>4
ꢀ
0.75
7.4
170
26
770
>3300
>3300
>3300
>3300
>3300
11.4
3.5
>2500
>2500
110
ꢀ
8.3
>30
>27
1.5
2ꢀCl
120
3.6
The above described scaffold modifications, taken in toto, led to a set of molecules that
encapsulate the key findings, and that offered the best opportunities to secure ROCK
potency and PKA selectivity while minimizing CYP inhibition. Compounds containing
the optimal phenylacetyl, thiophene and 2ꢀpyridine substitutions are summarized in Table
5. In addition Table 5 includes cellular activity of the top compounds, representing the
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effects of ROCK inhibitors on cellular migration of THPꢀ1 cells towards the chemokine
attractant MCPꢀ1.
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Table 5. Activity profiles of combinations of preferred substituents
THPꢀ1
migration,
IC50
ROCK
Ki, nM
PKA Ki,
nM
Selectivity,
fold
CYP 3A4,
IC50, ꢁM
Cpd
R1
R2
36
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F
F
3ꢀOMe
3ꢀNHSO2Me
3ꢀOMe
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10
44
45
22
30
780
3000
2200
>3300
140
43
300
50
840
240
3.8
>30
5.0
Cl
1600
650
Cl
3ꢀNHSO2Me
3ꢀOMe
>73
6
7.5
NH2
NH2
590
5.6
3ꢀNHSO2Me
850
28
450
>15
The 2ꢀfluoroꢀ and 2ꢀaminoꢀsubstituted pyridines 37 and 41 showed reduced CYP
inhibition while maintaining good ROCK potency and PKA selectivity. The 2ꢀ
chloropyridines 38 and 39 were less potent than the analogous 2ꢀfluoropyridines 36 and
37. Compound 37 represented the best overall combination of ROCK potency, PKA
selectivity, cellular efficacy and CYP inhibition for further investigation. The crystal
structure of 37 bound to the active ste of ROCK is illustrated in Figure 6 and shows
additional contact between the methylsulfonamide and the glycineꢀrich loop, contacting
both Phe87, as previously seen with compound 10, as well as a new interaction with the
NH of Ala86. Kinase selectivity screening of 37 at 2 µM against a panel of 46 kinases,
revealed that only three kinases, all from the AGC family kinases, were inhibited at
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greater than 25%, namely MSK1 (54%), PRK2 (90%) and ROCK2 (99%). Further CYP
profiling showed that potent inhibition had been abrogated by the combination of the
thiophene and 2ꢀfluoropyridine (Table 5). In vivo rat pharmacokinetics of compound 37
showed excellent properties and is suitable for oral administration (Table 6).
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Figure 6. Xꢀray crystallographic structure of 37 bound to ROCK1 (PDB ID: 5BML)
Table 5. CYP inhibition profile of 37
CYP isozyme
1A2
IC50, µM
>100
12
2C9
2C19
7.3
2D6
8.7
3A4
>30
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Table 6. Rat pharmacokinetic profile of compound 37
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6
7
Pharmacokinetics (i.v.)
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9
CL (L/min/kg)
T1/2 (h)
3.45
2.6
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60
Vdss (L/kg)
Cmax (µg/mL)
AUC (µg h/mL)
0.675
2.92
4.31
Pharmacokinetics (p.o.)
Formulation
0.5% aqueous
methyl cellulose
Cmax (µg/mL)
AUC (µg h/mL)
Tmax (h)
T1/2 (h)
%F
3.2
32.6
3
3.4
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Chemistry
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8
Acylated aminothiazoles were synthesized as shown in Scheme 1. The synthesis began
with αꢀbromination of 4ꢀacetylpyridine with bromine, followed by reaction of αꢀ
bromoketone 42 with thiourea to obtain 4ꢀ(pyridinꢀ4ꢀyl)thiazolꢀ2ꢀamine 43. The weak
nucleophilicity of 43 required a convenient and consistent method for acylation for which
we identified the coupling agent Nꢀ(1ꢀmethanesulfonyl)benzotriazole (MeSO2Bt)
reported by Katritzky et al.22 Microwave heating of a mixture of 43 with carboxylic acids
in the presence of MeSO2Bt and Et3N in THF at 160°C reliably afforded compounds 5-18
in good yields.
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60
Scheme 1. Preparation of NꢀAcylꢀ2ꢀAminothiazole Derivatives
(a) Br2, 48% HBr, 70°C; (b) thiourea, EtOH, reflux; (c) carboxylic acid, MeSO2Bt, Et3N,
THF, µW, 160°C, 10 min
Compound 19 was synthesized by Mitsunobu reaction with 3ꢀhydroxybromopropane and
compound 10 and followed by sodium hydrideꢀmediated cyclization as shown in Scheme
2.
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Scheme 2. Aminothiazole Alkylation via Mitsunobu Reaction and Subsequent
Cyclization.
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(a) Br(CH2)3OH, Ph3P, DIAD, THF, 0°C; (b) NaH, THF, 0°C.
2ꢀAminoꢀ4ꢀ(pyridinꢀ4ꢀyl)thiophene 46 was synthesized in 3 steps as shown in Scheme 3.
Knoevenagel condensation of 4ꢀpyridylacetophenone with ethyl cyanoacetate, followed
by Gewald synthesis of thiophene using elemental sulfur in morpholine afforded
compound 45 in good yield.23 Hydrolysis and decarboxylation of compound 45 afforded
amino thiophene 46.
Scheme 3. Preparation of 2ꢀAminoꢀ4ꢀ(pyridinꢀ4ꢀyl)thiophene.
(a) NH4OAc, AcOH, benzene, reflux; (b) S8, morpholine, EtOH, RT; (c) KOH (20% aq),
EtOH, reflux; (d) 3ꢀmethoxyphenylacetic acid, MeSO2Bt, Et3N, THF, µW, 160°C, 10
min
4ꢀ(Pyrimidinꢀ4ꢀyl)thiophenꢀ2ꢀamine 50 was synthesized in 4 steps as illustrated in
Scheme 4. 3ꢀAcetylthiophene was treated with DMFꢀDMA, followed by formamidine to
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give the 3ꢀ(pyrimidineꢀ4ꢀyl)ꢀthiophene 48, which was subjected to nitration and followed
by reduction to give the desired aminothiophene 50.
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Scheme 4. Preparation of 4ꢀ(pyrimidinꢀ4ꢀyl)thiophenꢀ2ꢀamine.
(a) DMFꢀDMA, reflux; (b) CH(NH)NH2.HCl, K2CO3, DMF, 80°C; (c) KNO3, H2SO4,
0°C; (d) H2, PdꢀC, RT; (e) 3ꢀmethoxyphenylacetic acid, MeSO2Bt, Et3N, THF, µW,
160°C, 10 min
2ꢀSubstituted 4ꢀ(pyridinꢀ4ꢀyl)ꢀ2ꢀaminothiazoles were generally prepared by the method
previously reported for 2ꢀaminopyridines (Scheme 5).24 Starting from either 2ꢀ
substitutedꢀ4ꢀacetylpyridines (54-58), or the precursor 4ꢀnitriles (51-53) (converted to 4ꢀ
acetyl by the action of methylmagnesium bromide), treatment with bromine in
hydrobromic acid affords the 4ꢀbromomethylketones 59, which were converted directly
to the acyl aminothiazoles 29-35 (X = F, Cl, Me, NH2, NH2Me) by the modified
Hantzsch reaction with the phenylacetylthioureas. In the case of the 2ꢀNH2 system, in situ
cleavage of the pꢀmethoxybenzyl group under brominating conditions occurs, allowing
direct access to the desired products on condensation of the crude product with the
phenylacetylthioureas. For the 2ꢀmethoxy and 2ꢀdimethylamino substitutions, the 2ꢀ
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chloropyridinꢀ4ꢀylꢀ2ꢀaminothiazole 60 was treated with sodium methoxide or
dimethylamine respectively to afford compounds 61 and 62 respectively then by
subsequent acylation gave final acylated products.
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Scheme 5. Synthesis of 2ꢀMethylꢀ, 2ꢀFluoroꢀ, 2ꢀChloroꢀ, 2ꢀMethoxy, 2ꢀAmino, 2ꢀ
Methylamino and 2ꢀDimethylamino Substituted Pyridines
(a) X = pMBNH2: DMA, 130°C, 1 hr. X = NHMe: 40% CH3NH2, DMA, 130°C, 1 hr; (b)
(i) 3M MeMgBr, (ii) 6N HCl; (c) Br2, 48% HBr, AcOH, 70°C; (d) 3ꢀ(MeO)ꢀ
PhCH2CONHCSNH2, EtOH, 70°C; (e) NH2CSNH2, EtOH, 70°C; (f) X=NMe2:
Me2NH.HCl, EtOH, Et3N, µW, 160°C. X = OMe: 25% NaOMe, MeOH, toluene, 110°C;
(g) 3ꢀmethoxyphenylacetic acid, MeSO2Bt, Et3N, THF, µW, 160°C, 10 min
Compound 37 was synthesized in four steps as outlined in Scheme 6. Curtius
rearrangement of 4ꢀbromothiopheneꢀ2ꢀcarboxylic acid afforded compound 63, which
was coupled with 2ꢀfluoropyridineꢀ4ꢀboronic acid to give compound 64. NꢀBoc
deprotection afforded amino thiophene 66. Coupling of 65 with 2ꢀ(3ꢀmethylꢀ
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sulfonylaminophenyl)acetic acid by means of of MeSO2Bt afforded compound 37 in
5
6
good yield.
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Scheme 6. Synthesis of Compound 37
(a) DPPA, tꢀBuOH, Et3N, 100°C, 5 h; (b)(i) 2ꢀfluoroꢀpyridineꢀ4ꢀboronic acid, Pd(PPh3)4,
K2CO3, dioxane, water, 100°C, 4 h; (c) TFA, DCM, RT, 4 h; (d) 3ꢀ(methylsulfonylꢀ
amino)ꢀphenylacetic acid, MeSO2Bt, Et3N, THF, 85°C, 18 h.
Conclusion
We have synthesized a series of Nꢀacylated aminothiazoles and evaluated their biological
activity against the Rho kinase. These compounds possess high ROCK potency and
moderateꢀtoꢀexcellent PKA selectivity. Xꢀray crystallography was used to help guide
productive interaction with the target enzyme, as well as understand and forecast
unproductive interactions in the closely related kinase PKA. The critical liability of the
pyridine system, CYP inhibition, was addressed through pyridine replacement and
substitution, leading to small 2ꢀsubstituents that reduced CYP inhibition, yet did not
affect ROCK inhibition. Compound 37 was identified as an optimal development of this
series, with excellent potency, selectivity against a panel of kinases, good cellular activity
and excellent pharmacokinetics. Thus, compound 37 is an excellent compound for further
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study of the pharmacologic effects of ROCK inhibition via oral dosing experiments,
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6
results of which will be reported elsewhere.
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Acknowledgements
The authors thank their colleagues Cameron Moody and Christine Memmott for the high
throughput screen and assay development, Mike Arnost and John Cochran for synthesis
support, and Barry Davis, Frank Holland and Leonard Kalkevijc for analytical chemistry
support.
Experimental Section
Reagents and anhydrous solvents were obtained from commercial sources and were used
without further purification. Reactions heated by microwave were performed in sealed
tubes on a Biotage Initiator microwave synthesis reactor (Biotage, Uppsala, Sweden).
LCMS data was acquired by the following methods:
Method A: Samples were analyzed on a Micro Mass ZQ mass spectrometer operated in a
single MS mode with electrospray ionization. Samples were introduced into the mass
spectrometer by direct (flow) injection (FIAꢀMS) or following HPLC chromatography on
an Agilent 1100 system (LCꢀMS). Separation was effected on a YMC ODSꢀAq C18
column (3.0 × 150 mm) eluted with a linear gradient from 10% to 90% CH3CN in H2O
(each containing v/v 0.2% formic acid) over 7 min at a flow rate of 1.5 mL/min.
Method B: Separations were performed on a tandem Waters Acquity Classic UPLCꢀMS
system using a single quadrupole mass spectrometer (model SQ2, Waters) and a 50x 2.1
mm BEH C18, 1.7 mm column from Waters Corporation. The column was heated at
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50°C and eluted with a gradient of 1% to 99% CH3CN in H2O (each containing 0.05%
v/v TFA) over 2.9 minutes at 1.2 mL/min.
7
8
9
High resolution mass spectra were acquired on a Thermo QExactive Orbitrap mass
spectrometer following chromatography on a Waters Acquity UPLC system. Preparative
HPLC was performed on a Gilson HPLC system using a gradient of 10ꢀ90% MeCN in
H2O (0.1% TFA) at a flow rate of 15 mL/min over 10 min on either YMC ODSꢀAQ C18
or Kromasil C8 columns (10 micron particle size; 21 x 150 mm). 1H NMR spectra (δ,
ppm) were recorded using a Bruker DRXꢀ500 (500 MHz), Bruker Avance 300 (300
MHz) or Bruker AvanceꢀIII (400 MHz) instrument. Exemplary methods are described for
key compounds. The purity of compounds was determined by LC absorbance at two
wavelengths, typically 220 nm and 254 nm, and all tested compounds were >95% pure
by peak area. Additional compound characterization data is available in Table S1.
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60
ROCK Enzyme Assay
Compounds were screened for their ability to inhibit ROCK I (AA 6ꢀ553) activity using a
standard coupled enzyme system.25 Reactions were carried out in a solution containing
100 mM HEPES (pH 7.5), 10 mM MgCl2, 25 mM NaCl, 2 mM DTT and 1.5% DMSO.
Final substrate concentrations in the assay were 45 µM ATP (Sigma Chemicals, St Louis,
MO) and 200 µM peptide (LysꢀLysꢀArgꢀAsnꢀArgꢀThrꢀLeuꢀSerꢀVal) (American Peptide,
Sunnyvale, CA). Reactions were carried out at 30 °C and 45 nM ROCK1. Final
concentrations of the components of the coupled enzyme system were 2.5 mM
phosphoenolpyruvate, 350 µM NADH, 30 µg/ml pyruvate kinase and 10 µg/ml lactate
dehydrogenase. The minimum significant ratio (MSR)19 for this assay is 3.6, determined
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using a retrospective analysis of 150 compounds whose Ki’s were determined at least
twice. Compounds that have a difference in Ki of at least a factor of 3.6 are considered
significantly different.
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PKA Enzyme Assay
The assay for PKAα was run according to the method described for ROCK, but using 10
µM ATP, 100 µM substrate (LeuꢀArgꢀArgꢀAlaꢀSerꢀLeuꢀGly) and 20 nM PKA (AA 1ꢀ
351).
THP-1 Cellular Migration Assay
Compounds were tested for the ability to block THPꢀ1 cell migration using a filter plate
assay. Briefly, 150,000 cells in Iscoves media with 1% FBS are placed in the top filter
well of a 96 well filter plate (5 micron filter, Millipore MAMIC5S10) and incubated with
a bottom plate containing a 10 ng/mL MCPꢀ1 as a chemoattractant in the same media.
Compound was preꢀadded to both the top and bottom chambers of the plate in the same
media (100 µl and 150 µl final volumes respectively). After a 1 hr incubation period at
37°C, 100 µl from the bottom chamber was transferred to a fresh 96 well plate, and cells
were quantified using an ATP luciferase kit (Promega G7571). Luminescence was read
using an LJL instrument with standard luminescence settings. The outer edge wells are
not used for data collection due to inconsistent preliminary data. Instead the outer wells
are filled with media to prevent evaporation in assay wells.
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General procedure for acylation of aminothiazoles: 2-(2-Fluorophenyl)-N-(4-
pyridin-4-yl-thiazol-2-yl)-acetamide (6)
7
8
9
4ꢀ(4ꢀPyridyl)ꢀ2ꢀaminothiazole (43) (329 mg, 1.86 mmol), 2ꢀfluorophenylacetic acid (377
mg, 2.25 mmol) and Nꢀ(1ꢀmethanesulfonyl)benzotriazole (MeSO2Bt) (440 mg, 2.23
mmol) were placed in a microwave reaction vessel. THF (2 mL) was added followed by
Et3N (0.52 mL, 3.73 mmol) and the mixture heated in the sealed tube at 160 °C for 10
minutes. The product precipitated upon cooling to room temperature. The precipitate was
filtered, washed with acetonitrile and dried to afford compound 6 (462 mg, 76%).
1H NMR (DMSOꢀd6) δ 12.68 (1H, s), 8.63 (2H, d), 8.00 (1H, s), 7.84 (2H, d), 7.43ꢀ7.17
(4H, m), 3.90 (2H, s).
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LCꢀMS (Method B) Rt = 2.09 min, [M+H]+ = 314, [MꢀH]ꢀ = 312.
HRMS: m/z calcd. for C16H12FN3OS [M+H]+ 314.07579; found, 314.07581.
3-(3-Methoxyphenyl)-1-(4-pyridin-4-yl-thiazol-2-yl)-piperidin-2-one (19)
2ꢀ(3ꢀMethoxyꢀphenyl)ꢀNꢀ(4ꢀpyridinꢀ4ꢀylꢀthiazolꢀ2ꢀyl)ꢀacetamide (360 mg, 1 mmol:
prepared from 4ꢀ(4ꢀpyridyl)ꢀ2ꢀaminothiazole and 3ꢀmethoxyphenylacetic acid as
described above), triphenylphosphine (315 mg, 1.2 mmol), diisopropyl azodicarboxylate
(239 mg, 1.2 mmol) and THF (10 mL) were stirred overnight at room temperature. The
mixture was then cooled to 0°C and NaH (60% dispersion: 48 mg, 1.2 mmol) added and
the reaction mixture was stirred at 0 °C for 30 minutes. MeOH was added to quench the
reaction. The solvent was evaporated and the residue purified by chromatography on
silica gel (EtOAc/hexane gradient) to afford compound 19 in 60% yield.
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1H NMR (DMSOꢀd6) δ 8.67 (d, 2H), 7.80 (d, 2H), 7.46 (s, 1H), 7.30 (m, 1H), 6.85 (m,
3H), 4.57 (m, 1H), 4.32 (m, 1H), 3.93 (m, 1H), 3.82 (s, 3H), 2.36 (m, 1H), 2.25 (m, 1H),
2.15 (m, 2H).
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LCꢀMS (Method B) Rt = 2.43 min, [M+H]+ = 366, [MꢀH]ꢀ = 364.
HRMS: m/z calcd. for C20H19N3O2S [M+H]+ 366.12708; found, 366.12704.
2-Amino-4-pyridin-4-yl-thiophene-3-carboxylic acid ethyl ester (45)
Ethyl cyanoacetate (6.78 g, 60 mmol) and 4ꢀacetylpyridine (7.26 g, 60 mmol) were
dissolved in dry benzene (35 mL) to which NH4OAc (0.54 g, 7 mmol) and glacial acetic
acid (1.5 mL) were added. The mixture was refluxed under a DeanꢀStark trap until the
formation of H2O ceased. The mixture was cooled, diluted with benzene, and washed
with water. The organic layer was dried with Na2SO4, filtered, and concentrated in vacuo
to give compound 44 in quantitative yield. To a stirred solution of 44 in EtOH (100 mL)
was added sulfur (1.9 g, 60 mmol) and morpholine (1 mL). The mixture was stirred at
room temperature overnight. The precipitate was filtered, washed with cold EtOH and
hexane to give compound 45 as a light yellow solid. The filtrate was concentrated and
redissolved in cold ethanol, filtered and washed with hexane to give more product 45 in
overall yield of 60% (2 steps).
1H NMR (CDCl3) δ 8.57 (2H, m), 7.31 (2H, m), 6.25 (2H, br), 6.17 (1H, s), 4.10 (2H,
q), 0.99 (3H, t).
LCꢀMS (Method A) Rt = 2.26 min, [M+H]+ = 248.
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4-(Pyridin-4-yl)-thiophen-2-ylamine 46
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A solution of compound 45 (2.49g, 10 mmol) in 20% KOH and EtOH (10 mL) was
refluxed for 18 hours. The mixture was cooled to room temperature and acidified with 2N
H2SO4. The mixture was stirred at RT until no further evolution of gas was observed and
the precipitate was filtered, washed with water, and dried to afford compound 46 as a
light yellow solid (60% yield).
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1H NMR (400 MHz, DMSOꢀd6) δ 8.81 (2H, d), 8.22 (2H, d), 7.81 (1H, s), 6.71 (1H, s)
LCꢀMS (Method B) Rt = 0.28 min, [M+H]+ = 177.
2-(3-Methoxyphenyl)-N-(4-(pyridin-4-yl)thiophen-2-yl)acetamide (22).
Compound 46 (1.75 g, 9.9 mmol), 3ꢀmethoxyphenylacetic acid (1.98 g, 11.9 mmol), Et3N
(25 mmol) and MeSO2Bt (2.34 g, 11.9 mmol) were placed in a microwave reaction vessel
in THF (20 mL) and the mixture heated at 160 °C for 10 minutes. Upon cooling to room
temperature the solvent was evaporated and the residue was taken in EtOAc and washed
with 2 N NaOH. The organic layer was dried (Na2SO4) and evaporated to afford 22 as an
oil. The product was converted to the HCl salt by redissolving in EtOAc and addition of
4N HCl in dioxane. (3.25 g, 90%)
1H NMR (CD3OD) δ 8.82 (2H, s), 8.27 (2H, d), 7.99 (1H, s), 7.32 (1H, s), 7.25 (1H, m),
6.92 (2H, m), 6.83 (1H, m), 3.80 (3H, s), 3.72 (2H, s).
LCꢀMS (Method B) Rt = 2.07 min, [M+H]+ = 325
HRMS: m/z calcd. for C18H16N2O2S [M+H]+ 325.10053; found, 325.10041.
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3-Dimethylamino-1-thiophen-3-yl-propenone 47
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3ꢀAcetylthiophene (42.8 g, 339 mmol) was mixed with DMFꢀDMA (250 mL) and heated
to reflux 18 hr. The red solution was evaporated in vacuo. The resulting oil was dissolved
in CH2Cl2 and washed with water. The organic solution was dried (MgSO4), and
evaporated in vacuo to afford compound 47 (61.04 g, 99%) as a brown oil that
crystallized upon standing.
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1H NMR (400 MHz, CDCl3) δ 7.93 (1H, dd), 7.79 (1H, d), 7.55 (1H, dd), 7.28 (1H, m),
5.60 (1H, d), 3.15 (3H, brs), 2.93 (3H, brs).
LCꢀMS (Method B) Rt = 0.63 min, [M+H]+ = 182.
4-Thiophen-3-yl-pyrimidine (48)
A solution of compound 47 (7 g, 38.62 mmol), formamidine hydrochloride (11 g, 136.6
mmol) and DIPEA (13.5 mL, 77 mmol) in DMF (50 mL) were heated at 80°C for 4 days.
The brown solution was cooled to room temperature and poured into water (200 mL).
The aqueous layer was extracted with EtOAc (3x 100 mL) and the combined organic
layers were dried (Na2SO4). The solvent was evaporated under reduced pressure and the
crude product was purified by silca gel chromatography (80g, 0ꢀ80% EtOAc in hexanes)
to afford compound 48 (4.6 g, 73%) as white solid.
1H NMR (400 MHz, CDCl3) δ 9.21 (1H, d), 8.74 (1H, d), 8.17 (1H, dd), 7.72 (1H, dd),
7.58 (1H, dd), 7.47 (1H, dd).
LCꢀMS (Method B) Rt = 0.67 min, [M+H]+ = 163.
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4-(5-Nitrothiophen-3-yl)-pyrimidine (49)
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Potassium nitrate (686 mg, 6.78 mmol) was added to a stirred solution of compound 48
(1 g, 6.2 mmol) in conc. H2SO4 (15 mL) at 0°C. The pale brown solution was stirred at
room temperature for 2 hr and poured into water (50 mL). The aqueous layer was
extracted with EtOAc (3x 25 mL), dried (Na2SO4) and evaporated under reduced
pressure. The crude product was purified by silica gel chromatography (80g, 0ꢀ80%
gradient EtOAC in hexanes) to afford compound 49 (0.50 g, 40%) as off white solid.
1H NMR (400 MHz, CDCl3) δ 9.27 (1H, d), 8.85 (1H, d), 8.52 (1H, d), 8.37 (1H, d), 7.60
(1H, dd).
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LCꢀMS (Method B) Rt = 0.80 min, [M+H]+ = 208.
4-Pyrimidin-4-yl-thiophen-2-ylamine hydrochloride (50)
10% Palladium on carbon (50 mg) was added to N2 purged solution of compound 49
(0.20 g, 0.97 mmol) in EtOAc:MeOH (4 mL, 3:1). The reaction was stirred at ambient
temperature under 1 atmosphere of H2 for 4 hours. The catalyst was filtered through a
celite plug and the plug was washed with EtOAc. The combined filtrate was cooled to
0°C and the filtrate volume was doubled with Et2O, and then to the solution was added a
solution of 4N HCl in dioxane (0.5 L, 2 mmol). A light yellow solid precipitated
immediately, which was stirred for 5 minutes at 0°C, and then filtered. The solids were
washed with copious amounts of Et2O with care taken not to expose the compound to air,
and quickly transferred to high vacuum for drying. The solid was dried in vacuo to give
4ꢀpyrimidinꢀ4ꢀylꢀthiophenꢀ2ꢀylamine hydrochloride 50 (160 mg, 77%) as a pale yellow
solid.
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