Amino acid derived quinazolines as Rock/PKA inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.01.109

SAR and lead optimization studies for Rock inhibitors based on amino acid-derived quinazolines are described. Studies demonstrated that these amino acid derived quinazolinones were mainly pan-Rock (I & II) inhibitors. While selectivity against other kinas

Full text of DOI:10.1016/j.bmcl.2013.01.109

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1592–1599
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Amino acid derived quinazolines as Rock/PKA inhibitors
Sarwat Chowdhury a, Yen Ting Chen a, Xingang Fang a, Wayne Grant b, Jennifer Pocas b,
Michael D. Cameron c,e, Claudia Ruiz c, Li Lin c, HaJeung Park d, Thomas Schröter b, Thomas D. Bannister a,
Philip V. LoGrasso b,e, Yangbo Feng a,
⇑
a Medicinal Chemistry, Translational Research Institute, The Scripps Research Institute, 130 Scripps Way, 2A1, Jupiter, FL 33458, USA
b Discovery Biology, Translational Research Institute, The Scripps Research Institute, 130 Scripps Way, 2A1, Jupiter, FL 33458, USA
c Drug Metabolism and Pharmacokinetics, Translational Research Institute, The Scripps Research Institute, 130 Scripps Way, 2A1, Jupiter, FL 33458, USA
d Crystallography/Modeling Facility, Translational Research Institute, The Scripps Research Institute, 130 Scripps Way, 2A1, Jupiter, FL 33458, USA
e Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, 2A1, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
SAR and lead optimization studies for Rock inhibitors based on amino acid-derived quinazolines are
described. Studies demonstrated that these amino acid derived quinazolinones were mainly pan-Rock
(I & II) inhibitors. While selectivity against other kinases could be achieved, selectivity for most of these
compounds against PKA was not achieved. This is distinct from Rock inhibitors based on non-amino acid
derived quinazolinones, where high selectivity against PKA could be obtained.22 The inhibitors presented
here in some cases possessed sub-nanomolar inhibition of Rock, nanomolar potency in ppMLC cell based
assays, low to fair cytochrome P-450 inhibition, and good human microsomal stability.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 30 November 2012
Revised 14 January 2013
Accepted 22 January 2013
Available online 31 January 2013
Keywords:
Rock
PKA
Quinazoline
Kinase inhibitor
Rho-associated protein kinase (Rock), a serine-threonine kinase
of the AGC family, is a major downstream effector of the Rho-med-
iated signaling cascade.1 Two isoforms of Rock (I & II) have been
isolated that share 65% overall sequence homology and 92% in
their ATP-binding domain.2 Upon activation following binding of
Rho, Rock phosphorylates targets such as myosin light chain,3
myosin light chain phosphatase,4 LIM kinase5 and zipper-interact-
ing kinase (ZIPK)6 which lead to cell signaling that primarily pro-
motes changes in cell motility, adhesion,7 stress fiber formation,7
and force generation through smooth muscle contraction.8 Inhibi-
tion of Rock is thus considered to have potential therapeutic value
for conditions such as hypertension,9,10 glaucoma,11 cancer metas-
tasis12 and multiple sclerosis13 among others.
Fasudil, the only clinically approved Rock inhibitor, has been
used for the treatment of cerebral vasospasm in Japan since
1995.14 The proven safety and efficacy of Fasudil have engendered
considerable interest in the development of Rock inhibitors for
therapeutic purposes and consequently, several reports have dis-
closed the development of Rock inhibitors of various classes.15
Over the course of several years our group has reported Rock inhib-
itors of various scaffolds including benzadioxane, chroman, and
tetrahydroisoquinoline amides,16 benzimidazoles,17 benzothiaz-
oles,18 ureas,19 indazoles,20 and indole and azaindole-based21 com-
pounds. Recently we also reported a series of highly potent and
selective quinazoline-based Rock inhibitors.22 Herein, we report
our efforts towards development of quinazoline-based Rock inhib-
itors derived from amino acids. The reason for the introduction of
an amino group (from amino acids) was mainly to enhance the
aqueous solubility of Rock-II inhibitors, which is a highly desirable
property for topical anti-glaucoma applications.
The amino acid-quinazoline-based Rock inhibitors were de-
signed to maximize three key interactions (Fig. 1): (i) hydrogen
bond donor/acceptor interactions with the backbone (Glu-170
and Met-172) in the ATP hinge-binding site, (ii) electrostatic inter-
action with the conserved lysine side chain group (Lys-121), and
(iii) hydrophobic interaction with the glycine-rich flexible P-loop
as depicted in Figure 1a. We elected to probe three amino acid
sub-structures (Fig. 1b): phenylglycine analogs 1, phenylalanine
derivatives 2, and pyrrolidine-2-carboxylic acid based compounds
(3). The later substructure, a b-amino acid, was derived from a
Rock-II inhibitor discovered in our HTS campaign and showed
promises in our earlier SAR studies.17b
Synthesis of phenylalanine and phenylglycine-based Rock
inhibitors began with HATU-mediated coupling of a desired amino
acid 5 and 2-amino-5-bromobenzamide 4 (Scheme 1). The result-
ing amide 6 was then subjected to Suzuki heteroarylation under
microwave irradiation to give the Boc-protected quinazolinone 8.
The coupling reaction and the ring-closure reaction (to form the
quinazolinone) were carried out in one-pot as described in
⇑
Corresponding author. Tel.: +1 561 228 2201.
E-mail address: yfeng@scripps.edu (Y. Feng).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.109

S. Chowdhury et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1592–1599
1593
Scheme 1. Deprotection of Boc by treatment with TFA in DCM at
room temperature furnished the final compounds 1 and 2.
The two-step synthesis consisting of an amide coupling fol-
lowed by palladium-catalyzed Suzuki coupling (Scheme 1) was
also adapted to access N-protected pyrrolidine-based compound
11 (Scheme 2). Boc-protected amino acids (9) were obtained from
commercial sources while N–Bn or N–Me compounds were syn-
thesized following established routes.23 Rock inhibitor 12 with
the basic pyrrolidine ring was obtained from appropriate starting
materials by Boc-deprotection using TFA in DCM at room temper-
ature or debenzylation by hydrogenation using Pd(OH)2 in AcOH.
Scheme 3 illustrates a representative procedure for the synthe-
sis of N-substituted phenylalanine-based inhibitors. Briefly, EDC
mediated coupling of compound 13 with a desired carboxylic acid
followed by reverse phase preparative HPLC purification gave the
desired product 14.
(a)
(b)
Lys-121
Glu-170
Met-172
O
R1
p-loop
Het
NH
N
N
R2
O
O
R1
R1
Het
Het
NH
NH
( )n
N
N
NH2
NH
1, n= 0
2, n= 1
3
In the cyclic b-aminoacid series, pyrrolidine N-functionalized
Rock inhibitors were accessed first by treatment of 15 using TFA
in DCM to remove the Boc protecting group as shown in Scheme 4.
The free pyrrolidine NH group was then acylated (2i) by reaction
with a desired acid chloride or treated with CDI followed by an
Figure 1. (a) Design rationale for amino acid derived quinazoline Rock inhibitors.
(b) -Amino acid derived inhibitors (1 & 2) and cyclic b-amino acid derived Rock
inhibitors (3).
a
CONH2
O
Br
O
R1
NHBoc
n = 0,1
Br
HATU, NMM,
DMF, rt
+
N
H
NH2
NH2
n = 0,1
COOH
BocHN
R1
5
4
6
O
O
O
Het
Het
NH
N
Het B
7
NH
TFA:DCM (1/1)
rt
NH2
O
NHBoc
n = 0,1
N
Pd(PPh3)4, K2CO3 aq.,
EtOH:tol (3/2), 1 h,
microwave,140 oC,
n = 0,1
R1
R1
1 n= 0,
2 n= 1,
8
Scheme 1. Synthesis of Rock inhibitors 1 and 2.
R1
R1
O
CONH2
O
HOOC
Br
Br
NH2
HATU, TEA,
DMF, r.t
+
N
H
NH2
N
9
R2
N
10
R2
4
R2 = Boc, Bn or Me
R2 = Boc, Bn or Me
O
R1
O
TFA:DCM (1/1),
Het
r.t (when R2 = Boc)
Het B
7
NH
O
N
H2, Pd(OH)2, AcOH,
r.t (when R2 = Bn)
Pd(PPh3)4, K2CO3 aq.,
EtOH:tol (3/2), 1 h,
microwave, 140 °C,
N
11
R2 = Boc, Bn or Me
R2
O
R1
Het
NH
N
12
NH
Scheme 2. Synthesis of cyclic b-amino acid derived Rock inhibitors.

1594
S. Chowdhury et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1592–1599
N
N
HN
O
N
O
N
HN
N
NH
NH
O
H
N
NH2
HO
N
O
DMF, NMM, HOBt,
EDC, r.t, overnight
Cl
Cl
13
14
Scheme 3. Synthesis of N-substituted quinazolinone Rock inhibitors.
N
O
N
1) TFA:DCM (1/1), r.t
2i) DMF, TEA, R3OCl, r.t
or
HN
Br
O
NH
2ii) CDI, THF, TEA,
N
H
R3OH or R3NH2
N
N
16
R = C(O)R3 (2i)
O
NH2
R
Boc
3) 7, Pd(PPh3)4, K2CO3 aq.,
EtOH:tol (3/2), 1 h,
microwave, 140°C
15
R = C(O) OR3 or C(O)NR3 (2ii)
Scheme 4. Synthesis of N-substituted b-amino acid based inhibitors.
N
R4
O
N
N
HN
N
N
HN
NH
N
NHBoc
NHBoc
BOP, R4NHCH3,TEA,
DMF, r.t ,1 h
F
F
17
18
R4
N
N
HN
N
NH2
DCM/TFA (1:1),
r.t, 1 h
N
F
19
Scheme 5. Synthesis of 4-amino quinazolines as Rock inhibitors.
alcohol or an amine to furnish target carbamate or urea com-
pounds (2ii) (following procedures in Scheme 2). Finally, a one-
pot reaction of palladium mediated Suzuki heteroarylation and
ring-closure was used to access the desired final product 16.
Scheme 5 depicts a representative synthetic strategy employed
to obtain 4-amino substituted quinazoline Rock inhibitors from
their corresponding quinazolinones. Briefly, a solution of Boc-pro-
tected compound 17 in DMF was treated with BOP and a desired
amine under basic conditions to smoothly furnish compound 18.
Boc-deprotection by TFA as the final step followed by HPLC purifi-
cation gave the target Rock inhibitor 19.
Taking phenylalanine (2), phenylglycine (20), and pyrrolidine 3-
carboxylic acid (3) based compounds as model systems, our SAR
began with the exploration of the hinge-binding groups in these
three subclasses. As shown in Table 1, the unsubstituted pyrazole
(in 22, 24, and 28) was a better hinge binding moiety in terms of
Rock-II inhibition24 compared to the 3-methyl pyrazole (23, 27),
the pyridine (21, 25), and the amino-pyrimidine (26) groups. Inter-
estingly, compounds in sub-class 3 always gave higher Rock po-
tency than those in sub-classes 2 and 20, probably due to a more
rigid pyrrolidine structure in 3. It is important to point out that,
in all three classes, these inhibitors also exhibited high PKA inhibi-
tion which was comparable to the Rock-II potency (Table 1). PKA is
always selected as the control to monitor the general kinase selec-
tivity in our development of Rock inhibitors.16–22 Various SAR stud-
ies were thus carried out for all three sub-classes (2, 3, and 20) in
order to increase selectivity against PKA.
Since most amino acids contain at least one chiral center, deter-
mination of the preferred chirality (absolute configuration) is of
high importance. Thus, pairs of stereoisomers in the phenylalanine
series were prepared and evaluated. Data in Table 2 for three pairs
of inhibitors (ca. 32 vs 33, 34 vs 35, and 36 vs 37) demonstrated
that there were no significant differences between stereoisomers
for both Rock and PKA activities. Chiral inhibitors for series 30
and 31 were not prepared but similar results are expected based
on our docking studies. Therefore, the chirality of applied amino
acids will not be stressed in future optimizations.
Using pyrazole as the hinge-binder we also explored the P-loop
binding region (see Fig. 1) by installing various substituents on the
phenyl ring. As shown in Table 2, these substitutions increased the
Rock potency for phenylglycine series 30 (compared to unsubsti-
tuted 24), slightly improved the potency for the phenylalanine ser-

S. Chowdhury et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1592–1599
1595
Table 1
Enzyme inhibition data for three sub-classes of Rock inhibitors
O
O
N
O
N
Het
Het
OMe
Het
NH
NH
NH
N
NH2
NH2
20
NH
2
3
a
ID
Structure
Het
IC50 (nM)
PKA/Rock-II
Rock-II
PKA
49
21
22
(S)-2
(S)-2
N
52
12
0.9
0.6
NH
N
7
NH
N
23
( )-20
98
nd
—
NH
N
24
25
( )-20
( )-3
43
23
20
29
0.5
1.3
N
NH2
N
26
( )-3
6
10
1.6
N
NH
N
27
28
( )-3
( )-3
7
1
7
4
1.0
4.0
NH
N
a
Values are means of two or more experiments with errors within 35% of the mean. nd = not determined.
ies 29 (compared to compound 22), but had almost no effects on
the Rock activity for pyrrolidine series 31 (compounds 49–51, com-
pared to unsubstituted 28). Interestingly, substitutions in series 29
at 3- or 4- position by both electron-donating and electron-with-
drawing groups all yielded potent Rock inhibitors and simulta-
neously reversed selectivity (Rock vs PKA) slightly favoring Rock-
II as compared to unsubstituted compound 22. Similar selectivity
pattern was also observed for the phenylglycine series 30 (45–48
vs 24). However, it should be pointed out that these modified com-
pounds, including Rock inhibitors based on the pyrrolidines (49–
51), are still highly potent PKA inhibitors.
In our recent report on quinazoline-based Rock inhibitors that
were not derived from amino acids,22 we disclosed that substitu-
tion at the 8-position in the bicyclic quinazoline ring significantly
increased selectivity against PKA. In our efforts to improve selectiv-
ity in this aminoacid-derived series, 8-substituted quinazoline
compounds 36/37 and 47/48 were also prepared. As demonstrated
by data in Table 2, this substitution was not able to enhance selec-
tivity and still produced very potent PKA inhibitors in these amino
acid derived quinazoline Rock inhibitors. In addition, benzyl sub-
stitution to the NH group of the pyrrolidine ring (52–54) produced
inhibitors with lower potency against both Rock and PKA as com-
pared to the free NH analogs (Table 2). Again, similar activities for
Rock and PKA were observed, indicating that this substitution
could not increase selectivity against PKA either.
(58, 63, and 64). Remarkably, the small and charged quaternary
ammonium salt N,N-dimethyl pyrrolidine 63 also exhibited high
Rock-II inhibition although with only a slight improvement in PKA
selectivity. In contrast, potent Rock-II compounds (as compared to
the parent unsubstituted structure 24) were obtained in the phenyl-
glycine series 56. Among all candidates 59–62, compound 59
showed impressive picomolar Rock inhibitory activity in enzyme as-
says with fair selectivity against PKA. Therefore, phenylglycine 56 is
more tolerant to N-substitutions than phenylalanine 55.
Further exploration by modification of the quinazoline 4-posi-
tion (group Y in Table 4) was also undertaken. In phenylalanine
series 66, substitutions with amines in general reduced the Rock-
II potency. Amine substitutions terminating in an N,N-dimethyl-
amino group (68) was proved to be most detrimental for binding
affinity while substitutions with smaller amino groups gave
slightly improved selectivity at a cost of lower Rock-II inhibition
(69). Interestingly, stereoisomers 69 and 71 showed significant dif-
ference in PKA selectivity while Rock-II inhibition remained un-
changed. In the b-aminoacid series 67, as a general trend, both
Rock and PKA potency fell off drastically as the size of amine group
increased. A small dimethylamino substitution (73) showed high
Rock-II potency as well as a remarkably high selectivity over PKA
(ca. 110-fold). For Rock inhibitors based on amino acid derived
quinazolines (the three sub-classes developed in our labs), com-
pound 73 was the best overall in terms of both Rock-II affinity
and PKA selectivity. Data in Table 5 seem to suggest that large sub-
stitutions on the 4-position of the quinazoline ring hurt the affinity
to both Rock and PKA, but more significantly to Rock inhibitions.
To help understand the observed SAR and to guide further opti-
mizations, representative inhibitors from each sub-scaffold were
More N1-substitutions were evaluated for inhibitors from all
three sub-classes (Table 3). Similar to compounds 52–54 (Table 2),
a relatively large substitution in series 55 (14) and series 57 (65)
reduced kinase activities. On the other hand, small un-branched
substitutions in general were well tolerated by both Rock and PKA

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S. Chowdhury et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1592–1599
Table 2
Enzyme inhibition data for optimized compounds
N
O
N
N
O
R1 HN
O
N
R1
HN
HN
R1
NH
NH
NH
N
N
R2
NH2
R2
NH2
N
R3
30
31
29
R1
R2
R3
IC50 (nM)
PKA/Rock-II
a
ID
Structure
Rock-II
PKA
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
(S)-29
(R)-29
(S)-29
(R)-29
(S)-29
(R)-29
(S)-29
(S)-29
(S)-29
(R)-29
(R)-29
(R)-29
(R)-29
( )-30
( )-30
( )-30
( )-30
( )-31
( )-31
( )-31
( )-31
( )-31
( )-31
4-Cl
4-Cl
4-F
4-F
3-F
3-F
4-OMe
4-OEt
3,4-di-OMe
3,4-di-F
3-Cl
3-F
3-CF3
4-Cl
3-Cl
4-Cl
H
H
H
H
OMe
OMe
H
H
H
H
H
H
H
H
H
OMe
OMe
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
H
6
4
9
8
8
7
4
19
39
7
13
10
16
10
13
9
7
5
1.2
1.2
1.8
2.1
3.3
3.4
2.0
3.2
1.0
1.7
0.5
0.9
1.7
0.9
nd
1.6
1.4
3.5
>2.0
3.0
1.3
1.7
4.8
16
17
26
24
8
60
38
12
6
9
28
9
nd
14
11
7
3-Cl
4-F
4-Cl
4-CF3
4-OMe
3-F
8
2
H
H
H
H
H
H
Bn
Bn
Bn
<1
2
88
225
49
2
6
117
423
233
2-F
H
a
Values are means of two or more experiments with errors within 35% of the mean. nd = not determined.
Table 3
Enzyme inhibition data for N-substituted Rock inhibitors
N
HN
N
N
HN
O
R1
O
O
N
R1
R1
HN
NH
NH
NH
N
N
X
X
X
57
56
55
R1
X
IC50 (nM)
PKA/Rock-II
a
ID
Structure
Rock-II
239
PKA
nd
O
N
14
58
59
(R)-55
(R)-55
( )-56
4-Cl
4-Cl
4-Cl
—
N
H
N
H
14
<1
17
7
1.2
>7
O
N
N
H
O
NH2
60
61
( )-56
( )-56
4-Cl
4-Cl
6
21
96
3.5
5.3
N
H
O
18
NH2
N
H

S. Chowdhury et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1592–1599
1597
Table 3 (continued)
a
ID
Structure
R1
X
IC50 (nM)
PKA/Rock-II
Rock-II
PKA
28
O
62
63
( )-56
( )-57
4-Cl
H
20
1.4
5.7
N
H
NH2
4
23
N
64
65
( )-57
( )-57
4-OMe
4-OMe
NMe
NBn
5
88
26
117
5.2
1.3
a
Values are means of two or more experiments with errors within 35% of the mean. nd = not determined.
Table 4
Enzyme inhibition data for 4-substituted quinazoline Rock inhibitors
N
N
Y
Y
N
R1
R1
HN
HN
N
N
N
NH2
NH
66
67
a
ID
Structure
R1
Y
IC50 (nM)
PKA/Rock-II
1.8
Rock-II
627
PKA
N
68
(R)-66
3-F
1160
N
OH
69
70
71
(R)-66
(R)-66
(S)-66
3-F
3-F
3-F
N
N
27
44
1.6
-
OH
OH
159
27
nd
N
284
10.5
72
(S)-66
3-F
N
67
259
3.4
73
74
( )-67
( )-67
H
N
N
2.5
71
279
443
110
6.2
4-F
N
75
( )-67
4-F
371
884
2.4
N
H
a
Values are means of two or more experiments with errors within 35% of the mean. nd = not determined.
docked into a homology model of human Rock-II.16–22 Shown in
Figure 2 are the docking modes of the phenylalanine based inhib-
itor 32 (Fig. 2A) and the pyrrolidine based compound 50 (Fig. 2B).
Similar modes were also observed for the phenylglycine based
Rock inhibitors. Several identical interactions were observed for
both compounds 32 and 50, and these interactions were also ob-
tained in the binding modes of non-amino acid based quinazoline
Rock inhibitors.22 For example, the pyrazole moiety formed two
hydrogen bonds with residues Glu170 and Met172 in the hinge re-
gion. The quinazolinone amide carbonyl group H-bound to the side
chain amino group of Lys121, and the amide NH group formed an
H-bond to residue Asp232. An important hydrophobic interaction
was formed between the phenyl moiety and the hydrophobic
binding pocket under the P-loop. Unlike the binding modes of
non-amino acid based quinazoline Rock inhibitors, additional
H-bonds were observed in these amino acid based quinazoline
Rock inhibitors. The NH on the pyrrolidine ring of compound 50
formed an H-bond to residue Asn219, while the NH2 group of
inhibitor 32 H-bound to both residue Asn219 and residue Asp232.
The docking modes in Figure 2 explain very well the observed
SAR. For example, a large Y substitution on series 66 and 67 reduced
binding affinity because there was no space in the ATP pocket to
accommodate such a large group. Functionalization to the NH2
group of series 56 and the pyrrolidine NH of series 57 did not affect
much of the binding potency since these amino groups were orien-
tated toward outside of the ATP pocket. On the other hand, function-
alization to the NH2 of phenylalanine series 55 by a large group
reduced the Rock potency significantly (14) because the NH2 group
in this sub-scaffold bound toward the inside of the ATP pocket and
there was not enough space to accommodate a large group.
In addition to Rock-II inhibition, the Rock-I inhibitory activity
was also assessed for amino acid derived quinazoline compounds.
As shown in Table 5, all tested compounds were found to be pan-
Rock inhibitors with very similar potency for both Rock isoforms.

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S. Chowdhury et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1592–1599
tude higher than their enzyme inhibition IC50 values, indicating a
necessity to further improve the cell permeability of these Rock
inhibitors. Interestingly, the two compounds having the highest
cell potency (50, 64) are both from the cyclic b-amino acid pyrrol-
idine-quinazoline scaffold. Considering that compounds with the
highest PKA selectivity (71, 73) are also from the same sub-class,
this pyrrolidine-quinazoline scaffold will therefore be the focus
for future optimizations.
In vitro DMPK were assayed for most of these amino acid de-
rived Rock inhibitors (Table 5). Basically, most inhibitors exhibited
high stability in human microsomes. The low stability for 38, 59,
and 64 is probably due to methoxy substitutions on the phenyl
ring, or the demethylation on the pyrrolidine ring, or a combina-
tion. The rat microsomal stability was generally lower than that
in human microsomes with the exception of compound 50, which
exhibited an extremely high stability (t1/2 = 105 min) in rats. It is
important to note that high stability is normally associated with
free NH2 or NH groups present in the compounds. Remarkably,
the stability of inhibitor 61 was extremely high in both human
and rat microsomes (Table 5), which was probably due to its high
CYP-450 inhibitions. As shown in Table 5, at 10
strongly inhibited three of the four selected CYP-450 enzymes 1A2,
2C9, and 2D6, (P90% at 10 M) and also moderately inhibited 3A4
(63% at 10 M). Except for 48 and 61, the general CYP-450 inhibi-
lM compound 61
l
l
tion profiles of inhibitors in Table 5 were fair to good. Indeed, most
compounds had low inhibition of 3A4, the most important CYP
enzyme.
In summary, aminoacid-quinazoline chimeric scaffold-based ki-
nase inhibitors with phenylglycine, phenylalanine, and pyrroli-
dine-2-carboxylic acid structures were synthesized and their
Rock-II and PKA inhibitory properties were evaluated. These com-
pounds generally exhibited pan-Rock inhibition as well as high
PKA inhibitory properties although good selectivity against PKA
could be achieved in a few cases. The overall best inhibitors (po-
tency, stability, and CYP-450 inhibition profiles) developed for this
scaffold so far were compounds 28, 32, 50, 59, and 64. These com-
pounds might find applications that do not require high PKA selec-
tivity, such as use as topical anti-glaucoma therapeutics.
Figure 2. Docking to a homology model of the Rock-II kinase for a phenylalanine
based Rock inhibitor 32 (A), and a pyrrolidine based Rock inhibitor 50 (B).
While selectivity against PKA could be achieved in only a few cases,
these Rock inhibitors were found to be indeed selective against
References and notes
many other kinases such as JNK1/2/3, p38a, MRCKa etc. (IC50
>1 M, data not shown). The cell activities for those inhibitors
l
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which showed high enzyme potency were also evaluated in ppMLC
target modulation cell assays.25 As shown in Table 5, compounds
50 and 64 exhibited high cell potency with IC50 values of
<50 nM. Other Rock inhibitors had cell IC50 values of between 90
and 800 nM. These cell IC50 values were several orders of magni-
Table 5
Rock-I and II enzyme inhibition data, cell activity data, and in vitro DMPK properties for some selected Rock inhibitors
a
a
ID
IC50 (nM)
Cell ppMLC IC50 (nM)
Mic. stability T1/2 (min)
Cyp-450 % inh. @ 10 lM 1A2/2C9/2D6/3A4
Rock-II
Rock-I
Hum.
rat
24
28
32
34
35
37
38
46
48
50
59
61
64
73
43
<1
6
9
8
48
2
nd
17
8
nd
64
35
47
42
65
36
21
46
51
75
15
420
21
86
6
À1/37/36/40
19/5/72/19
312
300
300
nd
470
nd
91
nd
23
500
nd
29
19
10
20
5
51/54/78/42
12/38/89/31
19/36/89/37
34/10/57/63
10/52/76/53
18/84/89/75
54/79/63/80
65/34/75/30
10/83/55/23
90/94/96/63
0/55/66/À3
49/47/88/77
7
4
7
16
15
8
5
13
8
<1
<1
18
5
13
11
105
7
104
3
1
<1
22
13
nd
30
800
3
9
a
Values are means of two or more experiments with errors within 35% of the mean. nd = not determined.

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