Synthesis and biological evaluation of 4-quinazolinones as Rho kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.039

Rho kinase (ROCK) is an attractive therapeutic target for various diseases including glaucoma, hypertension, and spinal cord injury. Herein, we report the development of a series of ROCK-II inhibitors based on 4-quinazolinone and quinazoline scaffolds. SAR studies at three positions of the quinazoline core led to the identification of analogs with high potency against ROCK-II and good selectivity over protein kinase A (PKA).

Full text of DOI:10.1016/j.bmcl.2011.01.039

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1844–1848
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 4-quinazolinones as Rho kinase inhibitors
⇑
Xingang Fang, Yen Ting Chen , E. Hampton Sessions, Sarwat Chowdhury, Tomas Vojkovsky, Yan Yin,
Jennifer R. Pocas, Wayne Grant, Thomas Schröter, Li Lin, Claudia Ruiz, Michael D. Cameron, Philip LoGrasso,
⇑
Thomas D. Bannister, Yangbo Feng
Translational Research Institute and Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 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:
Rho kinase (ROCK) is an attractive therapeutic target for various diseases including glaucoma, hyperten-
sion, and spinal cord injury. Herein, we report the development of a series of ROCK-II inhibitors based on
4-quinazolinone and quinazoline scaffolds. SAR studies at three positions of the quinazoline core led to
the identification of analogs with high potency against ROCK-II and good selectivity over protein kinase
A (PKA).
Received 24 November 2010
Revised 9 January 2011
Accepted 11 January 2011
Available online 14 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Rho kinase
Quinazolinone
Pyrazole
ROCK inhibitor
Protein kinase A
The Rho-associated coiled-coil containing protein kinase, also
known as ROCK, is an integral component of the Rho signaling
pathway. Two isoforms of ROCK, ROCK-I and ROCK-II, have been
identified to date. These isoforms share 65% overall sequence iden-
tity with 92% homology in the kinase domain.1 By phosphorylating
various substrates including myosin light chain (MLC), MLC phos-
phatase (MLCP), ezrin/radixin/moexin (ERM), and LIM kinases,
ROCK controls actin cytoskeleton assembly and cell contraction.2
Inhibition of ROCK was demonstrated to contribute to effects such
as smooth muscle relaxation3 and alteration of intercellular junc-
tions in the trabecular meshwork of the eye.4 Therefore, inhibitors
of ROCK may have beneficial implications for the treatment of dis-
eases including hypertension5 and glaucoma.6
Figure 1. Structure and biological properties of quinazolinone 1.
homologous protein kinase A (PKA), chosen to gauge the potential
for broad selectivity over other kinases. Hence, we aimed to
improve the PKA selectivity of this class while maintaining potent
inhibition of ROCK-II and favorable PK properties.
Our strategy to optimize 4-quinazolinone 1 was set in two
stages. The first stage involved the modification of the benzodiox-
ane portion of inhibitor 1. Based on our experience in ROCK inhib-
itor design, we observed that various alterations on the
benzodioxane group were well tolerated for ROCK inhibition. In
general, such substitutions include an aromatic group to generate
hydrophobic interactions with the glycine-rich phosphate-binding
loop (P-loop) region of ROCK.18 The second stage was aimed to
introduce functionalities at either the 4- or 8-position of the qui-
nazoline core to identify functionalities that could confer favorable
potency and selectivity.
The synthesis of quinazolines and related C-4-substituted quin-
azolines with no substitution at the 8-position (Scheme 1, R2 = H)
began with the coupling of aniline 319 with acid 2 via either acid
chloride formation or HATU-mediated conditions. Subsequent
Suzuki heteroarylation and dehydration were achieved in one step
Over the past 5 years, several groups have reported the develop-
ment of potent ROCK inhibitors.7–9 We previously reported the
identification of chroman-containing inhibitors with high affinity
and selectivity for ROCK10,11 and disclosed the effects of several
analogs in reducing intraocular pressure (IOP).12–14 In our contin-
ued efforts in this area, we identified benzodioxane-substituted
quinazolinone 1 (Fig. 1), a compound with high affinity for
ROCK-II (IC50 = 1 nM).15 This compound was potent in our cell-
based myosin light chain bis-phosphorylation (ppMLC) assay16
(IC50 = 10 nM), and displayed an excellent pharmacokinetic (PK)
profile in Sprague–Dawley rats, including high oral bioavailability
(F = 79%).17 No selectivity was observed against the highly
⇑
Corresponding authors. Tel.: +1 561 228 2204; fax: +1 561 228 3089 (Y.T.C.);
tel.: +1 561 228 2201; fax: +1 561 228 3089 (Y.F).
E-mail addresses: ytchen@scripps.edu (Y.T. Chen), yfeng@scripps.edu (Y. Feng).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.039

X. Fang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1844–1848
1845
Scheme 1. Reagents and conditions: (a) (COCl)2, pyridine, CH2Cl2, 2 h; (b) HATU, N-
methylmorpholine, DMF, 36 h; (c) 1H-pyrazole-4-boronic acid pinacol ester,
Pd(PPh3)4, K2CO3, toluene, ethanol, H2O, 1 h, 135 °C,
l
W; (d) R3XH, BOP, DBU,
DMF, 2 h.
under microwave conditions from amide 4 to obtain quinazolinone
5. Further substitution at C-4 was achieved by BOP-mediated cou-
pling20 with an amine or thiol nucleophile (R3XH, X = N or S) to af-
ford quinazoline 6.
The construction of inhibitors with an amide or ketone at the 60-
position of the chroman ring is described in Scheme 2. Ester 7,
which was synthesized according to Scheme 1 with the appropri-
ate chroman-3-carboxylic acid, was saponified and coupled with
an amine to obtain amide 8. Ketone 9 was prepared by hydrolysis
of ester 7, followed by formation of a Weinreb amide,21 and treat-
ment of cyclopropylmagnesium bromide. For the preparation of
quinazoline 10, coupling at the quinazoline C-4 was performed
first, followed by ester hydrolysis and amide formation.
To access quinazolinones with substituent R2 at C-8, aniline 3
with either a fluoro or methoxy group at the appropriate location
was prepared by carboxamide formation, followed by bromination
(Scheme 3). Compound 15, which contains a dimethylaminoethyl-
methyl amine group, was obtained in three steps involving con-
densation, nucleophilic substitution, and finally Suzuki coupling
under microwave conditions. The dimethylaminoethoxy analog
(19) was synthesized from bromide 16 in four steps. Demethyla-
tion with BBr3, amide formation via an acid chloride intermediate,
and nucleophilic substitution of 2-dimethylaminoethyl chloride
Scheme 3. Reagents and conditions: (a) SOCl2, DMF, 2 h, 75 °C; (b) NH4OH, CH2Cl2,
1 h; (c) N-bromosuccinimide, DMF, 8 h; (d) K2CO3, dioxane, H2O, 1 h, 140 °C,
(e) N,N,N0-trimethylethylenediamine, 1 h, 165 °C,
W; (f) 1H-pyrazole-4-boronic
acid pinacol ester, Pd(PPh3)4, K2CO3, dioxane, H2O, 1 h, 140 °C, W; (g) BBr3, CH2Cl2,
lW;
l
l
1 h, ꢀ78 °C; (h) 6-methoxychroman-3-carboxylic acid, (COCl)2, DIEA, CH2Cl2, cat.
DMF, 1 h; (i) 2-dimethylaminoethyl chloride, NaHCO3, DMF, 6 h, 60 °C.
gave bromide 18. Finally, Suzuki heteroarylation gave the desired
product, 19.22
In agreement with earlier studies, a chroman functional group
could readily replace the benzodioxane moiety of quinazolinone
1 (Table 1).10,11 The inhibition of ROCK-II was assessed by HTRF
methods.12,23 With the exception of chromene 20 and chroman
24, these compounds were highly effective inhibitors of ROCK,
with IC50 values less than 50 nM. Chromans 21 and 22 have similar
affinity for ROCK-II as benzodioxane 1, with at least 10-fold
improvement in selectivity over PKA and higher stability to human
liver microsomes. Selectivity was further enhanced with 60-meth-
oxychroman 25, which displayed an IC50 value of 2 nM for ROCK-
II and 380-fold selectivity against PKA. Consistent with our previ-
ous studies, chroman regioisomers with 50- or 80-methoxy groups
(23 and 24, respectively) were less effective.24 The asymmetric
synthesis of chroman-3-amide-based inhibitors was recently dis-
closed and we observed that the chirality of these compounds con-
tributes to inhibition of ROCK-II.23 Thus, the enantiomers of
quinazoline 25 were prepared with slightly modified conditions25
and the enantiomeric purity was verified by chiral reverse-phase
HPLC (Chiralpak AD-RH). The (R)-enantiomer of chroman 25 was
the eutomer, with IC50 values of <1 nM against ROCK-II and at
the lowest limits of detection (4 nM) in the cell-based ppMLC as-
say.16 In comparison, the (S)-enantiomer was a modest inhibitor
Scheme 2. Reagents and conditions: (a) LiOH, dioxane, H2O, 3 h; (b) R4NH2, HATU,
Et3N, DMF, 1 h; (c) N,O-dimethylhydroxylamine, HATU, Et3N, DMF, 1 h; (d)
cyclopropylmagnesium bromide, THF, 16 h, 0 °C; (e) R3XH, BOP, DBU, DMF, 2 h.

1846
X. Fang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1844–1848
Table 1
Table 1 (continued)
Kinase inhibition and stability of 2-substituted quinazolinones
a
b
Compd R1
IC50 (nM)
t1/2 (min)
HLM
ROCK-II PKA
33
34
35
876
17
a
b
Compd R1
IC50 (nM)
t1/2 (min)
123
2830 330
HLM
ROCK-II PKA
a
b
c
Average of two or more measurements with error within 30% of the mean.
1 mg/mL human liver microsomes were used in stability studies.
Not determined.
20
21
22
>5000
2
>20,000 438
47
34
62
66
of ROCK-II (IC50 = 87 nM) with no detectable activity in the ppMLC
assay even at 2.7 M. This difference in ROCK-II affinity was likely
l
attributed to more favorable interactions between the (R)-chroman
group and the glycine-rich P-loop. Interestingly, chroman (R)-25
was also more stable than its stereoisomer in the microsome assay.
Since substituents at the 60-position of the chroman ring were
best tolerated, other small functional groups were introduced at
this position (chromans 26, 27, and 9). These analogs were simi-
larly potent ROCK-II inhibitors with high stability to human liver
microsomes. In particular, cyclopropylketone 9 showed the highest
level of stability (>3.5 h) and selectivity over PKA (>1000-fold) in
this series. In our earlier work on benzimidazole-based ROCK
inhibitors, we observed that the installation of amides usually con-
taining a polar group on the chroman C-60 could enhance potency
and selectivity.24 Inspired by these results, several analogs were
synthesized and evaluated (28–32). These inhibitors were indeed
highly potent inhibitors of ROCK-II, with most IC50 values in the
subnanomolar range. Compounds 30 and 31 possessed high micro-
somal stability and moderate levels of PKA selectivity, and mor-
pholine 32 demonstrated the best PKA selectivity in this
carboxamide series.
<1
23
24
31
591
51
423
>5000 ndc
25
2
<1
87
6
760
144
17
62
2
(R)-25
(S)-25
26
>20,000
702
31
We also investigated the effect of using non-chroman groups,
including phenoxymethyl analogs 33 and 34, and the cyclopropyl
analog, 35. These compounds were moderate inhibitors of ROCK-
II with up to 25-fold selectivity over PKA. The modest ROCK affinity
of cyclopropyl 35 suggested that while a chroman or a similar aro-
matic group may assist in improving affinity to the enzyme, it was
less important for binding than the quinazolinone scaffold.
Based on our experience in the development of Rho kinase
inhibitors using other scaffolds, we believed that the addition of
a side chain at an appropriate location could enhance favorable
properties such as kinase selectivity.11,13,14 To this end, we first
introduced a side chain at the C-4 position of the quinazoline scaf-
fold, which was synthetically accessible as depicted in Scheme 1.
Compared to chroman 25, mercapto analog 36 showed a slight de-
crease in inhibition of ROCK-II while retaining the same level of
PKA selectivity and having higher microsomal stability (Table 2).
Since the other C-4 substituents studied conferred less favorable
properties (37–39), we turned our attention to analogs of thioether
36. Inhibitors 40–42 had similar affinity for ROCK-II as quinazoline
36 and improved selectivity over PKA compared to their 4-hydroxy
counterparts. In particular, more than 1000-fold selectivity was ob-
served for morpholine 42 and methoxy ether 41 was as potent as
benzodioxane 1 in the cell-based ppMLC assay.
27
3
326 143
9
18
<1
<1
1
>20,000 218
28
29
30
31
32
29
31
42
67 107
<1
102 102
32
33
<1
269
18
We next examined the effects of substituents on the C-8 site of
the quinazolinone core (Table 3), accessed as shown in Scheme 3.
Incorporation of a methoxy group (43) maintained a similar level
of affinity for ROCK-II inhibition and vastly improved PKA selectiv-
ity to 7000-fold. In contrast, fluoro substitution was not tolerated
at this position. Installation of basic chains (15 and 19) slightly
188
257 ndc

X. Fang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1844–1848
1847
Table 2
Effects of quinazoline C-4 substitution on kinase inhibition, cell-based potency, and half-life in human liver microsomes
R3
R4
IC50 (nM)
IC50 (nM)
t1/2 (min)
a
a
b
Compound
ppMLC
HLM
ROCK-II
PKA
36
37
38
39
S(CH2)2NMe2
OMe
OMe
OMe
OMe
6
22
358
>5000
2600
1870
ndc
ndc
55
37
N(Me)(CH2)2OH
N(Me)(CH2)2NMe2
NHCH2(2-pyridine)
14
ndc
ndc
ndc
ndc
>5000
40
41
S(CH2)2NMe2
S(CH2)2NMe2
6
3
1680
1850
50
10
19
13
42
S(CH2)2NMe2
3
4450
81
9
a
b
c
Average of two or more measurements with error within 30% of the mean.
1 mg/mL human liver microsomes were used in stability studies.
Not determined.
Table 3
Effects of quinazolinone C-8 substitution on kinase inhibition, cell-based potency, and stability
R1
R2
IC50 (nM)
IC50 (nM)
t1/2 (min)
a
a
b
Compound
ppMLC
HLM
ROCK-II
2
PKA
43
44
15
19
45
OMe
F
14,200
4120
12
77
104
21
16
1
ndc
1700
370
11
ndc
ndc
64
805
>20,000
1020
OMe
124
46
47
48
49
OMe
OMe
OMe
OMe
5
11
2
12,500
>20,000
111
28
2700
39
94
73
22
39
1
197
33
a
b
c
Average of two or more measurements with error within 30% of the mean.
1 mg/mL human liver microsomes were used in stability studies.
Not determined.

1848
X. Fang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1844–1848
Table 4
References and notes
Pharmacokinetic profile for ROCK-II inhibitors in ratsa
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min/kg)
Vss (L/
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Oral F
(%)
(l
(l
25
1.4
0.27
0.14
0.49
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2.7
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43.4
63.2
2.36
87.9
0.15
9.63
66
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4. Rao, V. P.; Epstein, D. L. Biodrugs 2007, 21, 167.
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19
49
1.4
0.22
15
a
Data was generated from three determinations. Formulated and dosed as
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A. Q.; Xu, W. W.; Ye, G. S.; Zhang, D. H.; Zhao, Y. D.; Jolivette, L. J.; Head, M. S.;
Semus, S. F.; Elkins, P. A.; Kirkpatrick, R. B.; Dul, E.; Khandekar, S. S.; Yi, T.; Jung,
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9. Wu, F.; Büttner, F. H.; Chen, R.; Hickey, E.; Jakes, S.; Kaplita, P.; Kashem, M. A.;
Kerr, S.; Kugler, S.; Paw, Z.; Prokopowicz, A.; Shih, C.-K.; Snow, R.; Young, E.;
Cywin, C. L. Bioorg. Med. Chem. Lett. 2010, 20, 3235.
10. Sessions, E. H.; Yin, Y.; Bannister, T. D.; Weiser, A.; Griffin, E.; Pocas, J.;
Cameron, M. D.; Ruiz, C.; Lin, L.; Schürer, S. C.; Schröter, T.; LoGrasso, P.; Feng,
Y. B. Bioorg. Med. Chem. Lett. 2008, 18, 6390.
11. Chen, Y. T.; Bannister, T. D.; Weiser, A.; Griffin, E.; Lin, L.; Ruiz, C.; Cameron, M.
D.; Schürer, S.; Duckett, D.; Schröter, T.; LoGrasso, P.; Feng, Y. Bioorg. Med.
Chem. Lett. 2008, 18, 6406.
12. Feng, Y.; Yin, Y.; Weiser, A.; Griffin, E.; Cameron, M. D.; Lin, L.; Ruiz, C.; Schürer,
S. C.; Inoue, T.; Rao, P. V.; Schröter, T.; LoGrasso, P. J. Med. Chem. 2008, 51, 6642.
13. Yin, Y.; Cameron, M. D.; Lin, L.; Khan, S.; Schröter, T.; Grant, W.; Pocas, J.; Chen,
Y. T.; Schürer, S.; Pachori, A.; Lo Grasso, P.; Feng, Y. ACS Med. Chem. Lett. 2010, 1,
175.
14. Fang, X.; Yin, Y.; Chen, Y. T.; Yao, L.; Wang, B.; Cameron, M. D.; Lin, L.; Khan, S.;
Ruiz, C.; Schröter, T.; Grant, W.; Weiser, A.; Pocas, J.; Pachori, A.; Schürer, S.;
Lograsso, P.; Feng, Y. J. Med. Chem. 2010, 53, 5727.
15. In this communication, only the biological data for ROCK-II is presented. ROCK-
I inhibition was also assessed, however isoform selectivity of compounds in
this series did not exceed 10-fold.
described in Ref. 17.
reduced inhibition of ROCK-II, and more than 1200-fold selectivity
over PKA was observed for ether 19. Thus, other compounds con-
taining the C-8 methoxy group were synthesized and evaluated.
In general, these compounds displayed 10- to 50-fold improve-
ment in selectivity for ROCK-II compared to their counterparts
with no C-8 substituent. Chromans 45 and 46 demonstrated
1000- and 2500-fold greater affinity for ROCK-II over PKA, respec-
tively. The selectivity of benzodioxane 48 was significantly en-
hanced compared to quinazolinone 1, and compound 49 had a
30-fold increase from quinazolinone 34 in ROCK-II inhibition.
ppMLC assays were performed on these compounds and chromans
43 and 45 were equipotent in cells as benzodioxane 1. Several
other inhibitors also had IC50 values less than 50 nM in the cell-
based assay. A notable exception is tetrahydronaphthalene 47,
which showed no inhibition at 2.7 lM despite good potency in
the enzyme assay. We concluded that both the 60-substituted chro-
man moiety and the 8-methoxyquinazolinone group contributed
to improved PKA selectivity with high ROCK affinity, and the com-
bination of these two factors (chroman 43) led to a particularly
desirable profile. Based upon the higher ROCK affinity of (R)-25
versus its (S)-enantiomer, the corresponding (R)-enantiomer of
quinazolinone 43 is expected to be preferred.
16. Schröter, T.; Griffin, E.; Weiser, A.; Feng, Y.; LoGrasso, P. Biochem. Biophys. Res.
Commun. 2008, 374, 356.
The PK data of selected compounds in Sprague–Dawley rats was
next determined (Table 4). Quinazolinones 25 and 43 showed low
17. Compounds were formulated at 1 mg/mL in a 1:1:8 DMSO:Tween 80:water
solution and dosed at 1 mg/kg intravenously into the femoral vein or 2 mg/kg
by oral gavage.
18. Gohda, K.; Hakoshima, T. P-Loop Pliability of Rho-Kinase for Inhibitor Binding.
In Drug Design Research Perspectives; Kaplan, S. P., Ed.; Nova Science Publishers:
Hauppauge, 2007; pp 39–55.
19. Dolzhenko-Podchezertseva, A. V.; Korkodinova, L. M.; Vasilyuk, M. V.; Kotegov,
V. P. Pharm. Chem. J. 2002, 36, 647.
20. Wan, Z.-K.; Wacharasindhu, S.; Levins, C. G.; Lin, M.; Tabei, K.; Mansour, T. S. J.
Org. Chem. 2007, 72, 10194.
21. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.
22. Feng, Y.; Lograsso, P.; Bannister, T.; Schroeter, T.; Fang, X.; Yin, Y.; Chen, Y. T.;
Sessions, H.; Chowdhury, S.; Luo, J.; Vojkovsky, T. WO 2010056758, 2010.
23. Chen, Y. T.; Vojkovsky, T.; Fang, X.; Pocas, J. R.; Grant, W.; Schröter, T.;
LoGrasso, P.; Bannister, T. D.; Feng, Y. Med. Chem. Commun. 2011, 2, 73.
24. Sessions, E. H.; Smolinski, M.; Wang, B.; Frackowiak, B.; Chowdhury, S.; Yin, Y.;
Chen, Y. T.; Ruiz, C.; Lin, L.; Pocas, J.; Schröter, T.; Cameron, M. D.; LoGrasso, P.;
Feng, Y.; Bannister, T. D. Bioorg. Med. Chem. Lett. 2010, 20, 1939.
25. Synthesis of quinazolines (S)-25 and (R)-25 involved modifications of the
reactions described in Scheme 1. In brief, the carboxylic acid (prepared in Ref.
20) and aniline were coupled with EDC and HOAt in dichloromethane without
the presence of base. The following Suzuki heteroarylation was then performed
with sodium bicarbonate in place of potassium carbonate as the base.
clearance, low volume of distribution, high AUC, and high oral Cmax
.
Compound 49 also displayed low clearance and volume of distribu-
tion, but with lower oral absorption. Comparing the pharmacoki-
netic properties of the enantiomer of inhibitor 25, the eutomer,
(R)-25 was more favorable for systemic applications. Less desirable
systemic PK properties were observed for quinazolinone 19, which
was likely ascribed to its dimethylaminoethoxy group.
In conclusion, we have described a series of quinazoline and 4-
quinazolinone pyrazoles as potent ROCK inhibitors. SAR studies at
the C-2, C-4 and C-8 positions of the quinazoline scaffold led to the
identification of several chroman analogs with excellent affinity for
ROCK and selectivity over PKA. In addition, compounds 25 and 43
possessed oral exposure levels favorable for systemic applications.
Future studies to be reported in due course include more extensive
target selectivity profiling of these potent ROCK inhibitors26 as well
as in vivo pharmacological studies to assess their efficacy as ther-
apeutic agents for hypertension or glaucoma.
26. Compounds 9 and 25 were also screened against three other kinases, MRCK
a,
JNK3, and p38 and no inhibition was observed at 20 M concentration.
l
Acknowledgments
We thank Prof. William Roush and Prof. Patrick Griffin for their
support.