Identification of 5H-chromeno[3,4-c]pyridine and 6H-isochromeno[3,4-c]pyridine derivatives as potent and selective dual ROCK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127474

A novel series of 5H-chromeno[3,4-c]pyridine, 6H-isochromeno[3,4-c]pyridine and 6H-isochromeno[4,3-d]pyrimidine derivatives as dual ROCK1 and ROCK2 inhibitors is described. Optimization led to compounds with sub-nanomolar inhibitory affinity for both kinases and excellent kinome selectivity. Compound 19 exhibited ROCK1 and ROCK2 IC₅₀ of 0.67 nM and 0.18 nM respectively.

Full text of DOI:10.1016/j.bmcl.2020.127474

Journal Pre-proofs
Identification of 5H-Chromeno[3,4-c]pyridine and 6H-Isochromeno[3,4-
c]pyridine Derivatives as Potent and Selective Dual ROCK Inhibitors
Zilun Hu, Cailan Wang, Doree Sitkoff, Nathan L. Cheadle, Songmei Xu, Jodi
K. Muckelbauer, Leonard P. Adam, Ruth R. Wexler, Mimi L. Quan
PII:
S0960-894X(20)30585-0
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127474
BMCL 127474
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Accepted Date:
10 July 2020
4 August 2020
Please cite this article as: Hu, Z., Wang, C., Sitkoff, D., Cheadle, N.L., Xu, S., Muckelbauer, J.K., Adam, L.P.,
Wexler, R.R., Quan, M.L., Identification of 5H-Chromeno[3,4-c]pyridine and 6H-Isochromeno[3,4-c]pyridine
Derivatives as Potent and Selective Dual ROCK Inhibitors, Bioorganic & Medicinal Chemistry Letters (2020),
doi: https://doi.org/10.1016/j.bmcl.2020.127474
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Graphical Abstract
Identification of 5H-Chromeno[3,4-c]
pyridine and 6H-Isochromeno[3,4-c]pyridine
Derivatives as Potent and Selective Dual
ROCK Inhibitors
Leave this area blank for abstract info.
Zilun Hu,* Cailan Wang, Doree Sitkoff, Nathan L. Cheadle, Songmei Xu, Jodi K. Muckelbauer, Leonard P.
Adam, Ruth R. Wexler, and Mimi L. Quan
O
Et
*
N
19
ROCK1 IC50 0.67 nM
ROCK2 IC50 0.18 nM
Kinome selectivity: FSI100 1.3
H
N
OMe
O

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Identification of 5H-Chromeno[3,4-c]pyridine and 6H-Isochromeno[3,4-
c]pyridine Derivatives as Potent and Selective Dual ROCK Inhibitors
Zilun Hu, Cailan Wang, Doree Sitkoff, Nathan L. Cheadle, Songmei Xu, Jodi K. Muckelbauer, Leonard
P. Adam, Ruth R. Wexler, and Mimi L. Quan
Research & Early Development, Bristol Myers Squibb, P.O. Box 5400, Princeton, New Jersey 08543-5400, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A novel series of 5H-chromeno[3,4-c]pyridine, 6H-isochromeno[3,4-c]pyridine and 6H-
isochromeno[4,3-d]pyrimidine derivatives as dual ROCK1 and ROCK2 inhibitors is described.
Optimization led to compounds with sub-nanomolar inhibitory affinity for both kinases and
excellent kinome selectivity. Compound 19 exhibited ROCK1 and ROCK2 IC50 of 0.67 nM and
0.18 nM respectively.
2009 Elsevier Ltd. All rights reserved.
Keywords:
Rho kinase
ROCK inhibitor
Chromenopyridine
Chromenopyrimidine
———
 Corresponding author. Tel.: +1 609 252-6125; e-mail: zilun.hu@bms.com

Rho associated protein kinases (ROCK) are members of the
serine-threonine protein kinase family. They are effector proteins
of RhoA, a small guanosine-5'-triphosphate (GTP) binding protein
that plays a key role in multiple cellular signaling pathways.1
Activated ROCK phosphorylates the myosin-binding subunit of
myosin light chain phosphatase, which inhibits activity of the
phosphatase and leads to muscle contraction by regulating the
shape and function of the actomyosin complex.2 Considerable
evidence suggests that RhoA/ROCK signaling pathways play an
important role in the pathogenesis of multiple diseases, and
inhibition of ROCK function and its downstream signaling
pathways has the potential to effect a variety of pathological
conditions.3-5 There are two known human ROCK isoforms,
ROCK1 and ROCK2, which share 65% overall sequence identity
including 92% in the kinase domain. Furthermore, there is 100%
identity within the adenosine triphosphate (ATP) binding pocket
between the two isoforms.6 The distinct physiological roles for the
individual isoforms continue to be elucidated.
Compound 1 containing the 5H-chromeno[3,4-c]pyridine
moiety was identified as a lead through our internal kinase
screening (Figure 2). It demonstrated moderate ROCK1 and
ROCK2 affinities with IC50 values of 71 nM and 54 nM,
respectively. Structure-activity relationship (SAR) optimization
on this lead is shown in Table 1. Replacement of the ether with an
amide linker, such as (D)-phenylalanine derivative 2, showed
moderate improvement in potency. (D)-Phenyl glycine homolog 3
further improved ROCK2 potency. Des-amino analog 4, however,
was less potent. To avoid a potential aniline issue, reversing the
amide resulted in benzamide 5, which was similarly potent
compared with 4. Introducing a meta-methoxy substitution on the
phenyl (6) or a benzylic methyl substitution (7) improved potency
further to single digit nanomolar ROCK2 IC50. Combination of
these two substitution groups provided 8 with ROCK1 and
ROCK2 IC50 values of 13 nM and 0.51 nM, respectively. The
corresponding pyridine analog (9) retained single digit nanomolar
Dual ROCK1/2 inhibitors, which have comparable affinity for
ROCK1 and ROCK2, have been approved to treat human diseases.
Examples of clinically approved and advanced ROCK inhibitors
are shown in Figure 1. Fasudil was marketed for cerebral
vasospasm in Japan.7-8 Ripasudil9 and Netarsudil10 (an ester which
generates active metabolite AR-13503) have been marketed for
glaucoma/ocular hypertension. Pre-clinical studies of the dual
ROCK1/2 inhibitor Y-27632 have shown hemodynamic and anti-
remodeling effects in animal models.11 ROCK2 selective inhibitor
KD025 (SLx-2119) is being clinically investigated for the
potential treatment of multiple diseases.12-14 However, the dual
ROCK1/2 inhibitors lack overall kinome selectivity, and the
affinity of KD025 for ROCK2 is modest. Given the potential
therapeutic applications, potent and kinome selective dual ROCK
O
Me
NH
Y
O
X O
N
N
O
O
S
N
Me
H
2HCl
NH2
N
Netarsudil (AR-13324)
ROCK1 Ki 1.0 nM
Fasudil
(X = H, Y = H)
ROCK1 IC50 940 nM
ROCK2 Ki 1.0 nM
AR-13503
ROCK1 Ki 0.2 nM
ROCK2 IC50 220 nM
(alcohol metabolite)
Ripasudil
(X = F, Y = Me)
ROCK1 IC50 50 nM
ROCK2 Ki 0.2 nM
ROCK2 IC50 20 nM
H
N
N
HN
Figure 1. Advanced ROCK inhibitors.
N
O
N
O
O
CH3
NH2
N
N
N
H
H
2HCl
Y-27632
ROCK1 Ki 140 nM
KD025 (SLx-2119)
ROCK1 Ki 24000 nM
ROCK2 Ki 105 nM
ROCK2 Ki 300 nM
ROCK2 potency; albeit 10-fold less potent for ROCK2.
Table 1. SAR of amide linker
N
O
R
IC50 (nM)*15
ROCK1
71
Cpd#
R
inhibitors are of interest. Herein we describe a series of 5H-
chromeno[3,4-c]pyridine, 6H-isochromeno[3,4-c]pyridine and
6H-isochromeno [4,3-d]pyrimidine dual ROCK1 and ROCK2
inhibitors.
ROCK2
54

O
1
NH2
N
O
O
NH2
1
ROCK1 IC50 71 nM
ROCK2 IC50 54 nM
Figure 2. An initial lead ROCK inhibitor.

O
(0.67 and 0.18 nM, respectively). Cyclopropyl (20) substitution
was tolerated.

N
2
19
20
H
NH2
O
Table 2. Hinge binder modifications.

3
4
5
6
7
8
16
140
81
9.4
44
N
H
NH2
O
H


N
H
R
N
OMe
H
N
O
Me
29
O
IC50 (nM)15
H
Cpd#
R

N
33
6.3
3.2
0.51
ROCK1
ROCK2
OMe
O
O
N
N
H
N
10
1.2
56
0.35

18
O
Me


O
H
N


13
OMe
OMe
NH
11
12
14
O
Me
Me
H
N
H
N
9
27
6.2
N
O
N
N
O
>2000
340
*IC50 values listed in Table 1 - 3 are Caliper assay data. See reference 15 for
details.


O
13
14
38
2.4
16
N
N
Figure 3. X-ray crystal structure of 6 in ROCK2 (2.55 Å resolution).
O
171
H2N
An X-ray crystal structure of 6 in ROCK2 was obtained (Figure
3).16 Compound 6 binds to the ROCK2 active site with DFG-in
conformation. The pyridine portion of the 5H-chromeno[3,4-
c]pyridine interacts with the hinge, and the meta-methoxyphenyl
group tucks under the p-loop towards the c-helix. The carbonyl
oxygen forms a favorable electrostatic interaction with conserved
residue Lys121. Electron density in the hinge region indicates that
both flips of the tricycle exist in roughly equal probability. In
addition, the crystal structure reveals that 2-(N-
morpholino)ethanesulfonic acid (MES) from the buffer co-
crystalized and occupied the space close to the active site near the
c-helix. The X-ray structural information provided a guide for the
possible tolerability of hinge binder modifications and extended
substitution groups towards the c-helix region (vide infra).

Me
N
O
15
15
4.7

O
O
Me
Et
N
N
16
17
0.49
0.63
2.0
0.21
0.56
0.52
0.18




O
O
Et
Et
*
*
N
N
18
(isomer 1)
19
0.67
Keeping the amide constant, SAR on the tricyclic hinge binder
was explored. It was found that the derivative of the regioisomer
of 6H-isochromeno[3,4-c]pyridine (10) was more potent for
ROCK1 and approximately equal potent for ROCK2 comparing
to 8 with ROCK1 and ROCK2 IC50 values of 1.2 nM and 0.35 nM,
respectively. However, the corresponding benzonaphthyridinones
(11, 12) were less potent than 10, significantly so for 12, in which
the lactam carbonyl may clash electrostatically with the backbone
C=O of p-loop residue Ile98 based on modeling into the active site.
Changing from pyridine to pyrimidine, 6H-isochromeno[4,3-
d]pyrimidine analog 13 retained single-digit nanomolar ROCK2
potency. Ortho-amino substitution resulted in 5 and 6-fold
reduction in ROCK1 and ROCK2 potencies respectively (14).
Modeling and the X-ray structure of 6 (Figure 3) indicated that
hydrophobic substitutions on the central ring could potentially add
affinity via hydrophobic interactions with residue Phe384 at the lip
of the hinge binding site. C5-Methyl substituted analog 15
(diastereomer mixture) maintained similar ROCK1 potency to
compound 8 as less potent for ROCK2. The corresponding
regioisomer 16 with C6-methyl substitution had excellent potency
with sub-nanomolar ROCK1 and ROCK2 IC50 values (0.49 nM
and 0.21 nM, respectively). Extending to ethyl substitution
provided similarly excellent potency (17, diastereomer mixture).
The two diastereomers in 17 were separated. Both diastereomers
showed potent ROCK activities, and diastereomer 19
demonstrated sub-nanomolar ROCK1 and ROCK2 IC50 potencies
(isomer 2)
O
N
20
4.1
0.72

The X-ray structure of 6 (Figure 3) indicated there was
available binding space near the c-helix pocket which could
accommodate extended substitution groups. Based on this insight,
a series of meta-amide analogs were evaluated (Table 3). Amides
with increasing size (21 - 23) were tolerated, and they exhibited
single digit nanomolar or sub-nanomolar IC50 values for ROCK1
and ROCK2. Analog 26 with a larger group, (1-ethylpyrrolidin-2-
yl)methylamine amide, provided a dual ROCK1 and ROCK2
inhibitor with IC50 values of 1.2 nM and 0.5 nM, respectively.
These extended groups could be combined with substitutions on
the tricyclic hinge binders and provided potent ROCK1 and
ROCK2 inhibitors (24 - 26).
Table 3. Exploration of extended c-helix binding site
R1
O
N
H
R2
N
O

IC50 (nM)15
*
Cpd#
R1
R2
ROCK1
ROCK2
Scheme 2. Reagents and conditions: (a) XPhos-Pd-PreCat-G2, K3PO4,
dioxane/H2O, microwave, 140 °C, 10 min, 58% (b) NaBH4, MeOH, 0 °C, 90%
or MeMgBr, THF, 0 °C, 30 min, 68% (c) NaH, THF, 0 - rt, 2 h, then aq. basic
treatment, 56% (R1 = Me) (d) amines, HATU, DIEA, DMF, 40 - 90%
NHMe
NHEt
21
22
23
H
H
8.0
7.0
8.9
3.2
20
1.3

O
0.8
0.85
0.8

Compounds 5 - 9 and 15 were synthesized by following the
procedures in Scheme 2. Suzuki coupling between 4-
chloronicotinaldehyde 32 and boronic acid 33 gave aldehyde 34.
Reduction of 34 by NaBH4 provided the primary alcohol, whereas
treatment with methyl Grignard reagent provided the secondary
alcohol. Cyclization of 35 under SNAr condition and basic workup
provided acid 36. Amide formation with corresponding amines
gave compounds 5 - 9 and 15.
O
H
N
H

NMe
O
NHEt
NHEt
Me
Et


24
25
O
F
3.0
O
H
N
26
Me
1.2
0.5

N
F
O
Et
F
a
b
O
O
Br
CO2Me
*Racemic/diasteromeric mixtures for 24 - 26
+
CO2Me
N
N
B
+
O
O
Kinome selectivity of selected compounds was evaluated and
the fraction selectivity indices (FSI100, obtained from a kinase
panel up to 152 kinases) are listed in Table 4. These ROCK
inhibitors exhibited good kinase selectivity. Compound 19
demonstrated excellent ROCK1 and ROCK2 potencies with
outstanding kinome selectivity, that no other kinases were
inhibited with <100x of ROCK2 IC50.
37
38
39
F
R1
R1
c
d
O
O
N
CO2Me
NHR2
O
N
N
CO2H
HO
R1
40
41
16 20
-
Scheme 3. Reagents and conditions: (a) XPhos-Pd-PreCat-G2, K3PO4,
Table 4. Kinome selectivity indices
dioxane/H2O, 2 h, 68% (b) NaBH4, MeOH, 0 °C, 30 min, 52%; or RMgBr (R
= Me, Et, cPr), THF, 0 °C - rt, 1 h, 50 - 80% (c) NaH, THF, 0 °C - rt, 16 h,
aq. workup, 30-65% (d) amines, HATU, DIEA, DMF, rt, 35 - 85%
Cpd#
8
10
19
20
22
23
25
FSI100*
2.6
2.2
1.3
2.0
2.2
5.4
2.2
* Fraction selectivity index is the ratio of active kinases with IC50 <100-fold of
ROCK2 (including ROCK1/2) out of total tested kinases (up to152 kinases).
The 6H-isochromeno[3,4-c]pyridine-8-carboxamides 16 - 20
were prepared by following the procedures shown in Scheme 3
using similar chemistry to that described in Scheme 2.
Substitutions were introduced from aldehyde 39 when treated with
different Grignard reagents before cyclization.
O
a
b
c
Br
MeO
NH2
Br
MeO
NHBoc
B
NHBoc
O
MeO
X
Y
27
28
29
X
Y
a
HN
O
N
N
CO2H
CO2H
OH
O
O
O
d
e
43
R
42
X, Y = O, C
N
NHBoc
N
NH
N
NH2
MeO
X
Y
30
31
2 4
-
b
HN
O
N
Scheme 1. Reagents and conditions: (a) Boc2O, TEA, THF, reflux, 80% (b)
B2Pin2, KOAc, PdCl2(dppf)-DCM adduct, DMF, 100 °C, 12 h, 50% (c) (i) 4-
chloronicotinaldehyde, PdCl2(dppf)-DCM adduct, K3PO4, dioxane/H2O, 100
°C, 1 h, 84% (ii) NaBH4, MeOH, 0 °C, 1 h, 98% (d) 63% aq. HBr, 100 °C, 16
h, basic workup, 83% (e) carboxylic acid, T3P, DIEA, DMF, rt, aq. workup;
TFA /DCM treatment for Boc deprotection, 70 - 95%
CONR1R2
21 - 26
Scheme 4. Reagents and conditions: (a) (i) HATU, DIEA, DMF, rt (ii) NaOH,
EtOH/H2O, rt, 2.5 h, 77% (b) amines, HATU, DIEA, DMF, rt, 45 - 90%
Scheme 4 outlines the synthesis of compounds in Table 3.
Intermediates 42 were converted to the corresponding meta-ester
benzylamide, followed by saponification to afford acids 43, which
were coupled with amines to provide 21 - 26.
Compounds 2 - 4 were synthesized according the Scheme 1. N-
Boc protected 4-bromoaniline 28 was converted to boronate 29,
which reacted with 4-chloronicotinaldehyde under Suzuki
coupling conditions, followed by aldehyde reduction, to provide
30. When 30 was treated with aqueous HBr at elevated
temperature, the Boc group was removed, and the methyl ether
was cleaved and cyclized with the benzyl alcohol, resulting in 5H-
chromeno[3,4-c]pyridin-8-amine 31. Amide coupling of 31 with
the appropriate carboxylic acids provided 2 - 4.
In summary, a novel series of potent and selective dual
ROCK1/2 inhibitors were discovered based on the
chromenopyridine and chromenopyrimidine hinge-binder
scaffolds. Representative inhibitors, such as 19, demonstrated sub-
nanomolar ROCK1/2 IC50 values of inhibitory activity with
excellent kinome selectivity.
O
O
H
Acknowledgments
a
H
(HO)2B
CO2Me
b
+
CO2Me
N
N
Cl
F
We would like to thank Dr. John S. Sack for refining the crystal
structure and preparing data for RCSB PDB deposition, and Karen
F
32
R1
33
34
R1
N
O
R1
OH
c
O
d
H
CO2Me
N
N
N
CO2H
R2
F
O
35
36
5 - 9, 15

MA). Compounds were dissolved in DMSO so that the final
concentration of DMSO was < 2%, and the reaction was initiated
with ROCK1 or ROCK2 produced at BMS. After incubation, the
reaction was terminated by the addition of EDTA and the
phosphorylated and non-phosphorylated peptides separated using a
LabChip 3000 Reader (Caliper Life Sciences). Controls consisted
of assays that did not contain compound, and backgrounds
consisted of assays that contained enzyme and substrate but had
EDTA from the beginning of the reaction to inhibit kinase activity.
Compounds were tested in dose-response format, and the inhibition
of kinase activity was calculated at each concentration of
compound. The inhibition data were fit to a four-parameter logistic
equation y = A+((B-A)/(1+((C/x)^D))), where A and B denote
minimal and maximal % inhibition, respectively, C is the IC50, D is
hill slope and x represents compound concentration equation, to
determine the IC50; i.e., the concentration of compound required to
inhibit 50% of kinase activity. Potencies of ROCK inhibitors were
also determined in homogeneous time-resolved fluorescence
(HTRF assay), a time resolved FRET-based competition binding
assay. A His-tagged form of ROCK1 or ROCK2 produced at BMS
at a concentration of 1 nM was incubated with 0.2 nM Tb-labeled
anti-His detection antibody (Cis-Bio, MA), test compound, and
fluorescein-labeled ATP competitive probe at a concentration
corresponding to the probe’s equilibrium dissociation constant for
one hour. Fluorescence at 495 nm and 520 nm was measured using
an EnVision microplate reader to quantify FRET between Tb-
labeled detection antibody and fluorescein-labeled probe.
Background subtracted FRET ratios were normalized to the
maximum signal obtained in the absence of test compound. These
values were converted to a percent inhibition. Percent inhibition
was determined for test compounds at 11 concentrations. The IC50,
defined as the concentration of competing test compound needed to
reduce specific binding of the probe by 50%, was derived by fitting
Rossi for the X-ray crystal structure pictures. The co-crystal, X-
ray diffraction data and partially refined crystal structure were
obtained from Proteros. BMS Lead Discovery and Optimization
generated kinome selectivity data. We appreciate Drs. Heather
Finlay, Peter Glunz, Ellen Kick, and Joseph Luettgen for
reviewing this manuscript.
References and notes
1.
2.
3.
Leung, T.; Manser E.; Tan, L.; Lim, L. J. Biol. Chem. 1995, 270,
29051-29054.
Schofield A. V.; Bernard O. Crit. Rev. Biochem. Mol. Biol. 2013,
48, 301-316.
Knipe, R. S.; Tager, A. M.; Liao, J. K. Pharmacol. Rev. 2015, 67,
103-117.
4.
5.
Loirand, G. Pharmacol. Rev. 2015, 67, 1074-1095.
Feng, Y.; LoGrasso, P. V.; Defert, O.; Li, R. J. Med. Chem. 2016,
59, 2269-2300.
6.
Matsui, T.; Amano, M.; Yamamoto, T.; Chihara, K.; Nakafuku, M.;
Ito, M.; Nakano, T.; Okawa, K.; Iwamatsu, A.; Kaibuchi, K. EMBO
J. 1996, 15, 2208-2216.
7.
8.
9.
Nagumo, H.; Sasaki, Y.; Ono, Y.; Okamoto, H.; Seto, M.; Takuwa,
Y. Am. J. Phys.- Cell Phys. 2000, 278, C57-65.
Naraoka, M., Munakata, A.; Matsuda, N.; Shimamura, N.; Ohkuma,
H. Transl. Stroke Res. 2013, 4, 368-374.
(a) Inoue, T.; Tanihara, H. Expert. Opin. Pharmacther. 2017, 18,
1669-1673. (b) Garnock-Jones, K. P. Drugs 2014, 74, 2211-2215.
10. Lin, C.-W.; Sherman, B.; Moore, L. A.; Laethem, C. L.; Lu, D.-
W.; Pattabiraman, P. P.; Rao, P. V.; DeLong, M. A.; Casey C.
Kopczynski, C. C. J. Ocul. Pharmacol. Ther. 2018, 34, 40-51.
11. Cantoni, S.; Pastore, F.; Cavalli, S.; Marchini, G.; Villetti,
G.; Nassini, R.; De Logu, F.; Facchinetti, F. Eur. Respiratory
J. 2016, 48, PA5100.
12. Zanin-Zhorov, A.; Weiss, J. M.; Nyuydzefe, M. S.; Chen, W.;
Scher, J. U.; Mo, R.; Depoil, D.; Rao, N.; Liu, B.; Wei, J.; Lucas,
S.; Koslow, M.; Roche, Maria; Schueller, O.; Weiss, S.;
Poyurovsky, M. V.; Tonra, J.; Hippen, K. L.; Dustin, M. L.; Blazar,
B. R.; Liu, C.-J; Waksal, S. D. Proc. Nat. Acad. Sc. USA
2014, 111, 16814-16819.
data to the 4-parameter logistic equation
y = A+((B-
A)/(1+((C/x)^D))), where A and B denotes minimal and maximal
% inhibition, respectively, C is the IC50, D is hill slope and x
represents compound concentration. ROCK1 and ROCK2
potencies were determined by both methods. The potencies of the
other kinases in the kinase panel for selectivity were determined
using either of the two methods.
16. The PDB ID is 7JNT.
13. Park, J.; Chun, K.-H. Gene, 2020, 737, 144474.
14. KD025
- Clinical Trials, ClinicalTrials.gov, NCT03919799,
December 20, 2019.
15. Caliper Assay: In vitro potencies of ROCK inhibitors were
determined in an assay containing 20 mM HEPES, pH 7.5, 20 mM
MgCl2, 0.015% Brij-35, 4 mM DTT, 5 M ATP and 1.5 M peptide
substrate (FITC-AHA-AKRRRLSSLRA-OH, Tufts University,
Click here to remove instruction text...