Optimization of 2-phenylaminoimidazo[4,5-h]isoquinolin-9-ones: Orally active inhibitors of lck kinase

Article abstract of DOI:10.1021/jm020446l

The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reaso

Full text of DOI:10.1021/jm020446l

J . Med. Chem. 2003, 46, 1337-1349
1337
Op tim iza tion of 2-P h en yla m in oim id a zo[4,5-h ]isoqu in olin -9-on es: Or a lly Active
In h ibitor s of lck Kin a se
Daniel R. Goldberg,*,† Tanja Butz,† Mario G. Cardozo,† Robert J . Eckner,§ Abdelhakim Hammach,†
J essica Huang,† Scott J akes,§ Suresh Kapadia,‡ Mohammed Kashem,† Susan Lukas,§ Tina M. Morwick,†
Maret Panzenbeck, Usha Patel,† Susan Pav,§ Gregory W. Peet,§ J effrey D. Peterson,
Anthony S. Prokopowicz, III,† Roger J . Snow,† Rosemarie Sellati, Hidenori Takahashi,† J onathan Tan,‡
Matt A. Tschantz,† Xiao-J un Wang,‡ Yong Wang,† J ohn Wolak,† Pla Xiong,† and Neil Moss†
Departments of Medicinal Chemistry, Biology, Chemical Development, and Pharmacology, Boehringer Ingelheim
Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877
Received October 8, 2002
The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have
potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors
based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did
not work in vivo upon oral administration. Our current work highlights the further use of
rational drug design and molecular modeling to produce a series of lck inhibitors that
demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in
vivo mouse model of anti-CD3-induced IL-2 production.
Tyrosine kinases play an essential role in the regula-
tion of cell signaling and cell proliferation by phos-
phorylating tyrosine residues of peptides and pro-
teins.1-3 We have been exploring a novel series of
molecules that inhibit the catalytic activity of p56lck
(lck), a member of the src family of protein tyrosine
kinases (vide infra).4
The lck (-) J urkat cell lines are unable to proliferate,
produce cytokines, and generate increases in intracel-
F igu r e 1. Initial lead 1 and optimized compound 2.
lular calcium, inositol phosphate, and tyrosine phos-
Ch em istr y
phorylation in response to T cell receptor (TCR) stimu-
lation.5,6 Therefore, an agent inhibiting lck would
Our previously reported synthetic route to the phe-
effectively block T cell function, act as an immunosup-
pressive agent, and have potential utility in autoim-
mune diseases, such as rheumatoid arthritis, multiple
sclerosis, and lupus, as well as in the area of transplant
rejection and allergic diseases.7,8
The TCR is the antigen-specific component of the T
cell, which is activated upon presentation of antigenic
peptides.9 TCR activation initiates a series of enzyme-
mediated signal transduction cascades that result in the
production of proinflammatory cytokines such as inter-
leukin-2 (IL-2).10-12
Our initial disclosure of phenylaminoimidazoisoquin-
olinones as a new class of lck inhibitors highlighted the
use of a model of inhibitors bound to lck to advance our
screening hit 1 with micromolar potency against lck, to
a nanomolar inhibitor of lck, 2 (Figure 1).4 Although
quite potent in vitro, 2 only demonstrated in vivo
efficacy when dosed at 100 mg/kg, ip. Our current work
highlights modifications to 2 that improve in vitro
potency to subnanomolar levels and also provide oral
efficacy in a mouse model of IL-2 production.
nylaminoimidazoisoquinolinone inhibitor 2 involved a
12-step linear sequence.4 We therefore looked for op-
portunities to shorten our synthesis and allow for
flexibility to introduce a variety of functionality. We
could accomplish our first goal through the use of a
selective SNAr reaction starting with 2,6-dichloro-3-
nitrobenzonitrile 3 as the key building block (Scheme
1).13 Treatment of 3 with ammonia or an alkylamine
provided benzonitrile 4 or 5, respectively. This ulti-
mately allowed access to products alkylated at the N-1
position, e.g., methylamine 12, which were not acces-
sible via our previous synthetic route. A subsequent SN-
Ar reaction with ethyl 2-methylacetoacetate to form 6
allowed ready introduction of the atoms necessary for
formation of the isoquinoline core.14 Formation of the
benzimidazole 11 was best accomplished by reacting
diamine 7, 2,6-dichlorophenyl isocyanate, and HgO in
refluxing THF. The isoquinolone was then formed by
treatment of 11 with a mixture of concentrated sulfuric
acid, water, and acetic acid to provide 2 or 12. The last
step in the reaction included the hydrolysis of the nitrile,
condensation with the ketone, and hydrolysis and
decarboxylation of the ester functionality in one step.
Reacting 5 with methyl acetoacetate to provide in-
termediate 13 accomplished introduction of a methyl
ester directly attached to C-6 (Scheme 2). Isoquinolone
formation as for 12 and 2 provided a mixture of C-6
* To whom correspondence should be addressed. Phone: 203-778-
7828. Fax: 203-791-6072. E-mail: dgoldber@rdg.boehringer-ingelhe-
im.com.
† Department of Medicinal Chemistry.
§ Department of Biology.
‡ Department of Chemical Development.
Department of Pharmacology.
10.1021/jm020446l CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/18/2003

1338 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Sch em e 1. Improved Synthesis of 2a
Goldberg et al.
a
Reagents: (a) H2NR, MeOH, 80 °C; (b) ethyl 2-methylacetoacetate, K2CO3, DMF; (c) H2, 10% Pd/C, MeOH; (d) 2,6-Cl2C6H3NCS,
EtOAc, or 2,6-Cl2C6H3NCS, HgO, THF, 80 °C; (e) concentrated H2SO4, H2O, HOAc, 100 °C.
Sch em e 2. â-Keto Ester Additionsa
a
Reagents: (a) 3-oxobutyric acid methyl ester, K2CO3, DMF; (b) 2-acetylmalonic acid diethyl ester, CsCO3, DMF; (c) H2, 10% Pd/C,
MeOH; (d) 2,6-Cl2C6H3NCS, EtOAc; (e) concentrated H2SO4; (f) (1) concentrated H2SO4 H2O, HOAc, 100 °C, (2) EtOH, H2SO4.
Sch em e 3. Amide and Amine Synthesis at C-6a
a
Reagents: (a) morpholine, TBTU, DMF; (b) BH3, SMe2, THF.
ester and decarboxylated material that could not be
separated easily. However, performing the reaction at
room temperature with concentrated sulfuric acid solved
this issue and provided 14 in good yield. The homolo-
gous ester, 15, was prepared in a similar manner with
diethylacetyl succinate. Isoquinolone formation under
the standard acidic conditions resulted in ester hydroly-
sis of the final product to provide 16. Consequently, re-
esterification with ethanol and sulfuric acid was re-
quired to obtain 17.
Ester 14 proved to be resistant to further manipula-
tion perhaps because of steric congestion around the
ester carbonyl caused by the C-7 methyl group and the
isoquinoline phenyl. The homologated analogue 16 did
not suffer from this liability and could be transformed,
via hydrolysis to the acid, into amide derivatives, e.g.,
18 (Scheme 3). The resulting amides could be reduced
to the corresponding amines, e.g., 19, with BH3-
dimethyl sulfide in THF, without affecting the isoqui-
nolone amide.
Functionalization of the C-4 position of the isoqui-
nolone was accomplished via a bromine atom as a
functional handle. Of all the potential precursors for
introduction of the bromine atom at C-4, diamine 9 was
the only intermediate that was readily brominated in
the desired manner (Scheme 4). The sequence pro-
gressed as described above to provide benzimidazole 21.
The bromine in compound 21 was further manipulated
before isoquinolone formation via palladium cross-
coupling with vinyl- or aryltin reagents to provide 22
and 23. Conversion to the corresponding isoquinolone
derivative occurred as described above. Vinyl compound
22 could be converted to aldehyde 25 that could undergo
reductive aminations, e.g., 26 and 27 (Scheme 4).
Attempts to introduce a functional handle at the C-7
position through the use of substituted â-keto esters had

Isoquinolin-9-ones as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1339
Sch em e 4. Introduction of C-4 Bromine and Cross-Coupling Reactionsa
a
Reagents: (a) Br2, CHCl3; (b) ArNCS, HgO, THF, 80 °C; (c) RSnBu3, (PPh3) 2PdCl2; (d) NaIO4, OsO4, THF; (e) H2SO4,100 °C; (f)
NaBH4, MeOH; (g) p-methoxybenzylamine, NaCNBH3, MeOH.
limited success either because of poor yields of the
SNAr reaction or difficulties in obtaining appropriately
substituted keto esters. Literature precedent demon-
strated that 3-methyl-1H-quinolin-2-one undergoes oxi-
dation with selenium dioxide to the corresponding
aldehyde.15 Reaction of 12 with SeO2 in dioxane resulted
in selective oxidation at the C-7 methyl to provide the
corresponding aldehyde 28 exclusively with no sign of
oxidation at the C6 methyl group (Scheme 5). The 2D
NMR experiments with NOESY (nuclear Overhauser
effect spectroscopy) confirmed the site of oxidation. The
predicted greater acidity of the C-7 methyl hydrogens
due to conjugation with the isoquinolone carbonyl could
explain the observed selectivity.
Secondary alcohol 31 provides another functional
handle when transformed into allylic acetate 33. Pal-
ladium-catalyzed allylic transposition of 33 worked well
with secondary amines to provide compounds such as
34.18 However, primary amines failed to react in the
allylic transposition reaction. To obtain secondary amines,
33 was treated with NaN3 as a nucleophile followed by
a Staudinger reaction to provide 35. Reductive amina-
tion with acetaldehyde provided amine 39.
Resu lts a n d Discu ssion
The two issues we felt were most important in obtain-
ing inhibitors with oral efficacy in our mouse model of
IL-2 production were to improve in vitro potency and
to improve physicochemical properties, most notably
aqueous solubility. One of the benefits of our improved
synthetic route was the ready access it allowed for pro-
viding N-1 alkylated products. We found that the corres-
ponding N-1 methyl analogue 12 shows a 5-fold increase
in potency over 2 (Table 1). The methyl appears to be
optimal because the corresponding ethyl analogue 40
is over 100-fold less potent.
This important result also helped to further vali-
date our previously developed binding model, which
predicted the 2,6-dichlorophenyl group to be point-
ing toward the N-3 nitrogen of the benzimidazole
(conformer A, Figure 2).19 Because the introduction
of a methyl group on either nitrogen would be expected
to strongly favor a conformation in which the dichlo-
rophenyl group is oriented away from the methyl, the
favorable potency of 12 strongly supports conformer
A (Figure 2) as representing the bioactive conforma-
tion of the 2,6-dichlorophenyl group. Previous SAR
studies showed that the N-3 methylated compound 41
is over 20-fold less potent than compound 2.4 How-
ever, the improved in vitro potency is likely due to a
Aldehyde 28 constitutes an intermediate for a wide
range of functionality at C-7 (Scheme 5). 28 can be
reduced directly to alcohol 29, converted to an amine
through reductive amination, e.g. 30, or converted to
secondary alcohol 31 by addition of vinylmagnesium
chloride. Although Wittig olefination procedures with
unstabilized ylides were not successful, Horner-Wad-
sworth-Emmons olefination with trimethyl phospho-
noacetate and lithium hydroxide provided R,â-unsatur-
ated ester 36.16 Alternatively, 28 could be transformed
to the simple vinyl analogue 32 via a Peterson olefina-
tion.17 R,â-unsaturated ester 36 underwent 1,4-reduc-
tion to the corresponding alkyl ester 37 upon hydroge-
nation over PtO2 in methanol. Reduction of 36 to the
corresponding allylic alcohol 38 was more troublesome
than initially expected. Hydride reducing agents such
as LiAlH4 had no effect on the ester, and returned only
un-reacted starting material. We found that if the amide
NH of the isoquinolone was deprotonated initially with
sodium bis(trimethylsilyl)amide, followed by addition of
LiAlH4, the desired alcohol could be obtained success-
fully.

1340 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Sch em e 5. C-7 Oxidation and Functionalizationa
Goldberg et al.
a
Reagents: (a) SeO2, dioxane, 100 °C; (b) LiAlH4, THF; (c) (1) benzylamine, THF, (2) NaBH4, MeOH; (d) vinylmagnesium bromide,
THF, 0 °C to room temp; (e) (1) TMSCH2MgCl, THF, -78 °C to room temp, (2) BF3(OEt)2, CH2Cl2, 0 °C to room temp; (f)
(MeO2)P(O)CH2CO2Me, LiOH, THF, H2O; (g) H2, PtO2, HOAc, EtOAH; (h) (1) NaN[Si(CH3)3]2, (2) LiALH4, THF, 0 °C to room temp; (i)
Ac2O, Et3N; (j) morpholine, Pd2(dba)3, PPh3, THF; (k) NaN3, Pd2(dba)3, PPh3, THF, (2) PPh3, THF; (l) acetaldehyde, Na(OAc)3BH, MeOH.
Ta ble 1. Methylation Studies on Benzimidazole Corec
F igu r e 2. Bioactive conformer of 2.
further modification of the N-phenyl ring. SAR at this
position tracked with our previous reported work with
no noticeable improvement in potency. Thus, we con-
centrated on functionalizing the isoquinoline core. We
had previously shown the requirement for 2,6-disubsti-
tution on the phenyl ring with either chlorine atoms or
methyl groups.
The spine of the isoquinolone core (Figure 3, C-4
through C-7) points out toward the cleft between the
N and C lobes of lck. We therefore reasoned that
substitution along this side of the molecule would be
tolerated and could provide an opportunity to improve
the potency and physicochemical properties of this
a
IC50 values for inhibition of lck. For active compounds, they
are the mean values of two or more separate determinations, in
duplicate. EC50 values for inhibition of Ca release in J urkat cells.
b
inhibitor class.
Values are the mean of two or more separate experiments in
duplicate ( SD. c NT ) not tested.
We were able to introduce a wide range of functional-
ity at both C-4 and C-6 that improved solubility;
however, most modifications provided less potent com-
pounds compared to 12 (Tables 2 and 3).
Although we were successful in introducing amine
and alcohol functionality at the C-7 position, the initial
SAR was also discouraging (Table 4). However, on
combination of locking the dichlorophenyl group into
the bioactive conformation, increasing hydrophobic
interactions with lck, and reducing desolvation factors.
On the basis of our model highlighted in Figure 3,
we had little expectation of improving potency through

Isoquinolin-9-ones as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1341
F igu r e 3. Opportunities suggested by the binding model of 12.
Ta ble 2. Effects of Substitution at C-6
Ta ble 3. Effects of Substitution at the C-4 Position
a
IC50 values for inhibition of lck. For active compounds, they
are the mean values of two or more separate determinations, in
b
duplicate. EC50 values for inhibition of Ca release in J urkat cells.
Values are the mean of two or more separate experiments in
duplicate ( SD.
The loss in activity on going from an sp2 to an sp3
group at C-7 can be rationalized on the basis of our lck
inhibitor binding model. As shown in Figure 4, the
protein is very close above and below the plane of the
molecule at the C-7 position. Therefore, planar substit-
uents at the C-7 position fit well in this narrow cleft.
Compounds that contain functionality attached to an
sp3 atom adopt a conformation that would position the
attached group out of the plane of the molecule toward
the protein, thereby lowering binding affinity. These
results parallel our initial discovery of 2 from 1 in which
removing the gem-dimethyl groups at C-6 and creating
a planar system resulted in a significant boost in
potency.4
By maintainance of the olefin substituent and intro-
duction of polar functionality, further enhancements in
cellular and molecular potency were achieved. Further-
more, compounds of this nature also resulted in an
increase in aqueous solubility. Allylic alcohol 38 showed
a small improvement in molecular potency but a sig-
nificant boost in cellular potency. Furthermore, intro-
duction of an allylic amine substituent provided an
enhancement in both molecular and cellular potency.
Although the polar allylic group does not appear to
a
IC50 values for inhibition of lck. For active compounds, they
are the mean values of two or more separate determinations, in
duplicate. EC50 values for inhibition of Ca release in J urkat cells.
Values are the mean of two or more separate experiments in
duplicate ( SD.
b
conversion of 28 to the R,â-unsaturated ester 36, we
observed our first signs, albeit modest, of improved
cellular potency. This prompted a deeper investigation
of the newly introduced ester and olefin functionality.
While the saturated ester 37 showed a 16-fold loss in
molecular potency and nearly a 2-fold loss in cellular
potency compared to 12, remarkably, the simple vinyl
group 32 maintained molecular potency and improved
cell potency nearly 10-fold when compared to 12. The
relatively poor potency of the ethyl analogue 42 further
demonstrated the importance of an olefin substituent
at C-7.

1342 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Ta ble 4. Effect of C-7 sp2 Substitutiond
Goldberg et al.
a
IC50 values for inhibition of lck. For active compounds, they are the mean values of two or more separate determinations, in duplicate.
b
EC50 values for inhibition of Ca release in J urkat cells. Values are the mean of two or more separate experiments in duplicate ( SD.
c EC50 values for SEB IL2 and human whole blood IL2 values are the mean of two or more separate experiments in duplicate ( SD. NT
) not tested.
d
F igu r e 4. Views of 12 in relationship to C-7 and the enzyme cleft.
interact with the protein directly (vide infra), overall
improvements in the physicochemical properties likely
contribute to enhanced cellular potency. Further profil-
ing this series of compounds showed them to be effica-

Isoquinolin-9-ones as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1343
F igu r e 5. Overlay of X-ray of 46 with homology model of 12.
Ta ble 5. Selectivity Profile of 43
enzyme
enzyme
kinase
IC50 (µM)a
kinase
EGFR
PDGFR
HER2
insulinRK
IKKR & â
IGFR
VEGFR
HGFR
IC50 (µM)a
lck
lyn
src
zap70
syk
PKC
PKA
p38
Btk
Itk
0.0005
0.002
0.0002
0.40
>10
0.008
>10
>100
50
>100
>100
>100
>30
>40
>300
>100
0.15
0.0004
>10
Erk
a
IC50 values for inhibition of listed kinases. For active com-
pounds, they are the mean of two or more separate determinations,
in duplicate. ATP concentrations are at or below km of the listed
kinases.
Ta ble 6. Selectivity Profile of 43
F igu r e 6. IL-2 inhibition by 43 when dosed orally.
enzyme
enzyme
kinase
CDK4
CaMK
J NK
MAPKAP-1b&2
MSK-1
PRAK
IC50 (µM)a
kinase
IC50 (µM)a
cious in both the J urkat IL-2 and human whole blood
IL-2 assay.
>100
>100
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
P70S6K
GSK3â
ROCK-II
AMPK
CHK1
CK2
PHK
CDK2
CSK
>1.0
>1.0
>1.0
X-r a y. Through the course of this work, our binding
model proved to be very useful in guiding the SAR
studies. After compound 43 was profiled, we obtained
an inhibitor-lck cocrystal structure, which confirmed
the accuracy of this model with one of our more potent
allylic amines, 46. As shown (Figure 5), our model
structure and the X-ray structure essentially overlap
identically. Although the vinyl group is clearly resolved,
the amine functionality is disordered and was therefore
modeled into the crystal structure.
>1.0
>1.0
>1.0
PDK1
PKBR
SGK
>1.0
>1.0
87% inh @ 1.0
a
IC50 values for inhibition of listed kinases. ATP concentrations
are run at 100 µM through the Dundee consortium.
30 mg/kg) might simply be due to its poor solubility
compared to 43 (0.25 vs 195 µg/mL at pH 7.4, respec-
tively).
In Vivo Activity. Compounds were tested for in vivo
efficacy in mice by measuring inhibition of IL-2 produc-
tion following stimulation with anti-CD3. In this model,
2 was only moderately active when dosed ip as reported
previously.4 However, 43 demonstrated an ED50 of 4.5
mg/kg, which was comparable with that of cyclosporin
A (ED50 ) 10 mg/kg, Figure 6). Tertiary amines 34, 44,
and 46 gave similar results in this model (data not
shown). Interestingly, allylic alcohol 38, which has in
vitro potency comparable to that of 43, proved to be
ineffective when tested in this model. The lack of
adequate oral exposure of 38 (∼0 µM at 100 and
Selectivity P r ofile. Selectivity over other kinases
will be an important consideration for developing a
chronically administered therapeutic agent. 43 demon-
strates high selectivity when compared to a variety of
both tyrosine and serine/threonine kinases (Tables 5
and 6). As expected, 43 shows no significant selectivity
over two other members of the src family. Two kinases
that demonstrated significant activity were PDGFR and
Btk, both of which have a high homology in the ATP
binding site, although less so in the whole kinase
domain.

1344 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Goldberg et al.
2-(2-Cya n o-3-m eth yla m in o-4-n itr op h en yl)-2-m eth yl-3-
oxobu tyr ic Acid Eth yl Ester (7). To a stirred solution of
potassium tert-butoxide (24.3 g, 206 mmol) in DMSO (500 mL)
was added ethyl 2-methylacetoacetate (34.3 g, 233 mmol)
dropwise over 5 min. The temperature rose to 30 °C. 6-Chloro-
2-methylamino-3-nitrobenzonitrile (43.6 g, 190 mmol) was
added in portions over 15 min. The temperature rose to 40
°C. The solution was stirred for 1 h with no external heating
or cooling. The mixture was poured into 10% aqueous NH4Cl
(500 mL) and was extracted with EtOAc. The combined
extracts were washed with water and brine and were evapo-
rated. MeOH (200 mL) was added to the residue, and the
mixture was stirred for 1.5 h. The yellow solid was filtered,
washed with cold MeOH (25 mL), and dried to provide 36.2 g
Con clu sion s
We have improved our class of phenylaminoimida-
zoisoquinolinones as potent inhibitors of lck kinase by
1000-fold over our screening hit. Our ability to ac-
complish this was aided through the use of a binding
model, whose high level of accuracy has been confirmed
by an X-ray structure of one of our inhibitors. Two key
discoveries that led to significant improvements of this
novel series were the N-1 alkylation of the benzimida-
zole and the conversion of the C-7 methyl group to a
planar, sp2 vinyl group that provided an increase in both
molecular and cellular potency. Furthermore, introduc-
tion of an allylic polar substituent at the C-7 position,
in particular an allylic amine, not only provided pico-
molar inhibitors of lck and improved aqueous solubility
but also provided in vivo oral efficacy with activity
comparable to that of cyclosporin A.
1
(60%) of the title compound. Mp 84-87 °C. H NMR (DMSO-
d6, 300 MHz): δ 1.22 (t, 3H, J ) 7 Hz), 1.81 (s, 3H), 2.36 (s,
3H), 3.55 (bs, 3H), 4.18-4.30 (bs, 2H), 6.64 (d, 1H, J ) 9 Hz),
8.06 (bs, 1H), 8.24 (d, 1H, J ) 9 Hz).
2-(3,4-Dia m in o-2-cya n op h en yl)-3-oxobu tyr ic Acid Eth -
yl Ester (8). To a solution of 2-(3-amino-2-cyano-4-nitrophe-
nyl)-3-oxobutyric acid ethyl ester (850 mg, 2.79 mmol) in
methanol (50 mL) was added 10% palladium on carbon (85
mg). The flask was pressurized with hydrogen to 50 psi and
hydrogenated until no loss of H2 could be detected. The
reaction mixture was filtered over Celite and concentrated to
Exp er im en ta l Section
Melting points were determined with an electrothermal
capillary melting point apparatus or a Fisher-J ohns apparatus
and are uncorrected. Proton NMR (1H NMR) spectra were
recorded on Bruker Avance 400, Varian Gemini 2300, and
Bruker AF270 spectrometers operating at 400, 300, and 270
MHz, respectively. Chemical shifts are reported as δ values
in ppm downfield from TMS, using the solvent peak as an
internal reference. Electron impact mass spectra (EIMS) were
run on a Finnigan SSQ7000 instrument at 70 eV. Chemical
ionization mass spectra (CIMS) were run on the same machine,
with NH3 as reagent gas. Electrospray mass spectra (ESMS)
were run on a MicroMass Platform LCZ instrument, at a cone
voltage of 30 V. Flash column chromatography was carried
out on silica gel (230-400 mesh). Organic solutions that had
been in contact with water were dried over MgSO4 prior to
concentration in a rotary evaporator.
2-Am in o-6-ch lor o-3-n itr oben zon itr ile (4). To 2,6-dichloro-
3-nitrobenzonitrile (30 g, 139 mmol) was added a solution (200
mL, 5.15 M) of freshly prepared ammonia in ethanol. The flask
was sealed and then placed in a preheated oil bath at 80 °C
for 1.5 h. After the mixture was cooled, the resulting precipi-
tate was taken up in ethyl acetate, washed with water and
brine, dried over MgSO4, and concentrated to afford 18.4 g
(68%) of the title compound. 1H NMR (CDCl3, 270 MHz): δ
6.80 (d, 1H, J ) 8.0 Hz), 8.33 (d, 1H, J ) 8.0 Hz), 8.69 (bs,
2H).
6-Ch lor o-2-m eth yla m in o-3-n itr oben zon itr ile (5). A so-
lution of 2,6-dichloro-3-nitrobenzonitrile (98.7 g, 455 mmol) in
EtOAc (910 mL) was cooled to 5 °C. A 40% aqueous methy-
lamine (79.5 mL, 1.14 mol) solution was added with vigorous
mechanical stirring, keeping the temperature at 10-15 °C.
After addition was complete, stirring was continued for 3 h at
the same temperature. More methylamine (16 mL, 230 mmol)
was added, and the mixture was stirred for a further 1.5 h at
room temperature. Water (300 mL) was added, followed by
hexane (450 mL). The mixture was stirred for 15 min and
filtered and the solid was washed with water and MeOH to
provide 80.3 g (83%) of the title compound. Mp 168-171 °C.
1H NMR (DMSO-d6, 300 MHz): δ 3.45-3.50 (m, 3H), 6.80 (d,
1H, J ) 9.0 Hz), 8.31 (d, 1H, J ) 9.0 Hz), 8.69 (bs, 1H).
2-(3-Am in o-2-cya n o-4-n itr op h en yl)-3-oxobu tyr ic Acid
Eth yl Ester (6). To a solution of 2-amino-6-chloro-3-nitroben-
zonitrile (1.97 g, 10 mmol) in DMF was added ethyl 2-methy-
lacetoacetate (3.60 g, 25 mmol) and potassium carbonate (1.50
g, 11 mmol). The reaction was stirred for 24 h at room
temperature. The reaction mixture was diluted with EtOAc,
then washed with 1 M HCl, water, and brine, and dried over
MgSO4. Column chromatography using hexanes/EtOAc (2:1)
afforded 1.78 g (58%) of the title compound. 1H NMR (DMSO-
d6, 400 MHz): δ 1.13 (t, 3H, J ) 4.0 Hz), 1.72 (s, 3H), 2.26 (s,
3H), 4.14-4.17 (m, 2H), 6.55 (d, 1H, J ) 8.0 Hz), 7.45 (bs,
2H), 8.23 (d, 1H, J ) 8.0 Hz).
1
afford the title compound. H NMR (DMSO-d6, 400 MHz): δ
1.3 (t, 3H, J ) 4.0 Hz), 1.80 (s, 3H), 2.30 (s, 3H), 4.25-4.35
(m, 2H), 6.42 (d, 1H, J ) 8.0 Hz), 6.77 (d, 1H, J ) 8.0 Hz),
8.10 (bs, 2H), 8.69 (bs, 2H).
2-(4-Am in o-2-cya n o-3-m eth yla m in op h en yl)-2-m eth yl-
3-oxobu tyr ic Acid Eth yl Ester (9). A solution of 7 (10.5 g,
32.5 mmol) in EtOAc (130 mL) was hydrogenated over 10%
palladium on carbon (0.5 g) at 50 psi for 24 h. The catalyst
was removed by filtration through diatomaceous earth, and
the filtrate was evaporated. A mixture of EtOAc/hexane (1:1,
10 mL) was added to the residue, and the resulting mixture
was stirred for 0.5 h. The crystals were filtered and washed
with hexane to provide 7.74 g (81%) of the title compound. Mp
1
130-131.5 °C. H NMR (DMSO-d6, 300 MHz): δ 1.21 (t, 3H,
J ) 7.0 Hz), 1.67 (s, 3H), 2.23 (s, 3H), 2.96-3.0 (m, 3H), 4.18-
4.21 (m, 2H), 4.89-4.91 (m, 1H), 5.17 (s, 2H), 6.35 (d, 1H, J )
8 Hz), 6.72 (d, 1H, J ) 8 Hz). CIMS m/z: 290 (MH+). Anal.
(C15H19N3O3) C, H, N.
2-(2,6-Dich lor op h en yla m in o)-6,7-d im et h yl-1,8-d ih y-
d r oim id a zo[4,5-h ]isoqu in olin -9-on e (2). To a solution of 8
(767 mg, 2.79 mmol) in ethyl acetate (30 mL) was added 2,6-
dichlorophenyl isothiocyanate (626 mg, 3.07 mmol). The reac-
tion mixture was stirred at room temperature overnight. The
reaction mixture was diluted with ethyl acetate (20 mL),
washed with water (3 × 20 mL), dried (MgSO4), and concen-
trated. The crude residue was chromatographed using hex-
anes/EtOAc (1:1) to afford the thiourea. To the intermediate
thiourea (1.30 g, 2.72 mmol) was added DCC (600 mg, 2.91
mmol) and THF. The reaction mixture was heated at 80 °C
for 3 h. After the mixture was cooled, solvent was concentrated
and crude residue was taken back up in ethyl acetate.
Insoluble material was filtered off. The filtrate was concen-
trated to afford 600 mg (50%) of the title compound. The 1H
NMR results were identical to the results reported in ref 4.
2-[4-Cya n o-2-(2,6-d ich lor op h en yla m in o)-3-m eth yl-3H-
b en zim ia zol-5-yl]-2-m et h yl-3-oxob u t yr ic Acid E t h yl
Ester (11). A solution of 9 (7.7 g, 26.6 mmol) and 2,6-
dichlorophenyl isothiocyanate (5.43 g, 26.6 mmol) in THF (150
mL) was stirred at room temperature for 5 h. Mercuric oxide
(6.34 g, 29.3 mmol) was then added in one portion, and stirring
continued overnight. The mixture was filtered through diato-
maceous earth, washing well with THF. The filtrate was
evaporated and the residue was triturated with ether to
1
provide 7.7 g (63%) of the title compound. H NMR (DMSO-
d6, 400 MHz): δ 1.17 (t, 3H, J ) 8.0 Hz), 1.82 (s, 3H), 2.30 (s,
3H), 4.08 (s, 3H), 4.16-4.25 (m, 2H), 7.16 (d, 1 H, J ) 8 Hz),
7.48 (t, 1 H, J ) 8 Hz), 7.50 (d, 1 H, J ) 8 Hz), 7.67 (d, 2 H,
J ) 8 Hz).

Isoquinolin-9-ones as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1345
2-(2,6-Dich lor op h en yla m in o)-6,7-d im et h yl-1,8-d ih y-
d r oim id a zo[4,5-h ]isoqu in olin e-9-on e (12). To a stirred
mixture of concentrated H2SO4 (40 mL), HOAc (40 mL), and
water (40 mL) at 60 °C was added 11 (7.4 g, 16 mmol) in one
portion. The solution was heated at 100 °C for 2.5 h and then
stirred overnight at room temperature. The reaction mixture
was poured onto ice and neutralized with concentrated NH4-
OH, with ice-cooling. The precipitate was filtered and washed
well with water. The solid was slurried in MeOH, stirred well,
filtered, washed with MeOH until the washings were colorless,
and dried to provide 5.5 g (88%) of the title compound. Mp
>300 °C. 1H NMR (DMSO-d6, TFA, 400 MHz): δ 2.22 (s, 3H),
2.32 (s, 3H), 4.28 (s, 3H), 7.60 (t, 1H, J ) 8 Hz), 7.70 (d, 1H,
J ) 9 Hz), 7.73 (d, 1H, J ) 9 Hz), 7.77 (d, 2H, J ) 8 Hz).
CIMS m/z: 387 (MH+). Anal. (C19H16Cl2N4O) C, H, N.
2-(2,6-Dich lor op h en yla m in o)-1-eth yl-6,7-d im eth yl-1,8-
d ih yd r oim id a zo[4,5-h ]isoqu in olin -9-on e (40). 40 was pre-
pared as described for compound 12 with ethylamine. 1H NMR
(DMSO-d6, TFA, 400 MHz): δ 2.25 (t, 3H, J ) 7.0 Hz), 2.16
(s, 3H), 2.25 (s, 3H), 5.23 (q, 2H, J ) 7, 8 Hz), 7.54 (dd, 1H, J
) 1, 8 Hz), 7.64 (dd, 2H, J ) 1, 8 Hz), 7.70 (d, 2H, J ) 8 Hz),
11.44 (s, 1H). ESMS m/z: 431 (MH+).
J ) 8 Hz), 7.67 (d, 2H, J ) 8 Hz), 7.88 (d, 1H, J ) 8 Hz), 7.98
(d, 1H, J ) 8 Hz). ESMS m/z: 458, 460 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,7-d im eth yl-6-(2-p yr r o-
lid in -1-yl-2-oxoeth yl)-1,8-d ih yd r oim id a zo[4,5-h ]isoqu in o-
lin e-9-on e (51). 51 was prepared as described above. Mp >300
1
°C. H NMR (TFA-d, 400 MHz): δ 2.12-2.20 (m, 2H), 2.25-
2.30 (m, 2H), 2.60 (s, 3H), 3.70-3.88 (m, 2H), 3.92-4.16 (m,
2H), 4.32 (s, 2H), 4.44 (s, 3H), 7.51 (t, 1H, J ) 8 Hz), 7.62 (d,
2H, J ) 8 Hz), 7.67 (d, 1H, J ) 9 Hz), 7.89 (d, 1H, J ) 9 Hz).
ESMS m/z: 484, 486 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,7-d im eth yl-6-[2-(4-m e-
th ylp ip er a zin -1-yl)-2-oxoeth yl)-1,8-d ih yd r oim id a zo[4,5-
h ]isoqu in olin e-9-on e (52). 52 was prepared as described as
above. Mp >300 °C. 1H NMR (TFA-d, 400 MHz): δ 2.66 (s,
3H), 3.30 (s, 3H), 3.41 (t, 1H, J ) 7 Hz), 3.50-3.66 (m, 2H),
3.94 (d, 1H, J ) 12 Hz), 4.07 (d, 1H, J ) 12 Hz), 4.14-4.26
(m, 1H), 4.36 (d, 1H, J ) 17 Hz), 4.46 (d, 1H, J ) 17 Hz), 4.55
(s, 3H), 4.77 (d, 1H, J ) 14 Hz), 5.08 (1H, d, J ) 14 Hz), 7.61
(1H, t, J ) 8 Hz), 7.63 (2H, d, J ) 8 Hz), 7.75 (d, 1H, J ) 8
Hz), 7.98 (d, 1H, J ) 9 Hz). ESMS m/z: 513, 515 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,7-d im et h yl-6-(2-m or -
p h olin -4-yleth yl)-1,8-d ih yd r oim id a zo[4,5-h ]isoqu in olin e-
9-on e (19). A stirred suspension of 18 (85 mg, 0.17 mmol) in
THF (9 mL) was heated to reflux, and borane methyl sulfide
(0.09 mL, 0.9 mmol) added. Stirring was continued for 3.5 h
at reflux and overnight at room temperature. Then 6 M HCl
was added and the solution was stirred for 2 h. The solution
was applied to a Varian SCX column and was washed with
MeOH/CH2Cl2, 50:50. Then the product was eluted with
MeOH/CH2Cl2/NH4OH, 50:50:1. The product was further puri-
fied on a silica column, eluting with CH2Cl2/MeOH, 98:2, to
provide 32 mg (39%) of the title compound. Mp 285-290 °C.
1H NMR (TFA-d, 400 MHz): δ 2.65 (s, 3H), 3.46-3.65 (m, 6H),
4.01 (d, 2H, J ) 12 Hz), 4.20 (t, 2H J ) 12 Hz), 4.38-4.50 (m,
2H), 4.43 (s, 3H), 7.51 (t, 1H, J ) 8 Hz), 7.63 (d, 1H, J ) 8
Hz), 7.98 (d, 1H, J ) 8 Hz), 7.97-8.0 (m, 2H). ESMS m/z: 486,
488 (MH+).
2-(2-Cya n o-3-m eth yla m in o-4-n itr op h en yl)-3-oxobu tyr -
ic Acid Meth yl Ester (13). 13 was prepared as described for
1
6 and 7. H NMR (CDCl3, 400 MHz): δ 1.95 (s, 3H), 3.48 (s,
3H), 3.91 (s, 3H), 6.52 (d, 1H, J ) 8 Hz), 8.37 (d, 1H, J ) 8
Hz), 8.61 (bs, 1H).
1,7-Dim eth yl-2-d ich lor ph en yla m in o-9-oxo-8,9-dih ydr o-
1H-im id a zo[4,5-h ]isoqu in olin e-6-ca r boxylic Acid Meth yl
Ester (14). 14 was prepared as described above for 12. 1H
NMR (TFA-d, 400 MHz): δ 2.62 (s, 3H), 4.19 (s, 3H), 4.42 (s,
3H), 7.46-7.50 (m, 1H), 7.51 (d, 2H, J ) 8 Hz), 7.87 (d, 1H, J
) 9 Hz), 7.93 (d, 1H, J ) 9 Hz). ESMS m/z: 431 (MH+).
2-Acetyl-2-(2-cya n o-3-m eth yla m in o-4-n itr op h en yl)su c-
cin ic Acid Dim et h yl E st er (15). 15 was prepared as
described for 6 and 7. 1H NMR (CDCl3, 400 MHz): δ 1.25-
1.29 (m, 5H), 1.37 (t, 3H, J ) 8 Hz), 2.08 (s, 3H), 2.43 (s, 3H),
3.41-3.45 (m, 2H), 4.15 (apt t, 2H, J ) 8 Hz), 4.39 (apt t, 2H,
J ) 8 Hz), 6.72 (d, 1H, J ) 8 Hz), 8.33 (d, 1H, J ) 8 Hz), 8.39
(bs, 1H).
2-(2,6-Dich lor op h en yla m in o)-1,7-d im et h yl-6-(3-m or -
p h olin -4-yl-3-oxop r op yl)-1,8-d ih yd r oim id a zo[4,5-h ]iso-
qu in olin e-9-on e (49). 49 was prepared as described as above.
Mp 277-282 °C. 1H NMR (TFA-d, 400 MHz): δ 2.20-2.36 (m,
2H), 2.67 (s, 3H), 3.18 (t, 2H, J ) 9 Hz), 3.43 (d, H, J ) 9 Hz),
3.54-3.62 (m, 2H), 3.80 (d, 2H, J ) 12 Hz), 4.05-4.15 (m, 2H),
4.38 (d, 2H, J ) 12 Hz), 4.45 (s, 3H), 7.54 (t, 1H, J ) 8 Hz),
7.65 (d, 2H, J ) 8 Hz), 8.00 (s, 2H). ESMS m/z: 500, 502 (MH+).
Anal. (C25H27Cl2N5O2‚0.2H2O) C, H, N.
2-(2,6-Dich lor op h en yla m in o)-1,7-d im et h yl-9-oxo-1,8-
d ih yd r oim id a zo[4,5-h ]isoqu in olin -6-yla cetic Acid (16). 16
1
was prepared as described for compounds 2 and 12. H NMR
(TFA-d, 400 MHz): δ 2.92 (s, 3H), 4.37 (s, 2H), 4.61 (s, 3H),
7.59 (t, 1H, J ) 7 Hz), 7.72 (d, 2H, J ) 7 Hz), 8.06 (s, 2H),
7.98 (d, 1H, J ) 8 Hz). ESMS m/z: 431.
2-(2,6-Dich lor op h en yla m in o)-1,7-d im et h yl-6-(3-m or -
ph olin -4-ylp r op yl)-1,8-dih yd r oim ida zo[4,5-h ]isoqu in olin -
9-on e (53). 53 was prepared as described above. Mp 277-
2-(2,6-Dich lor op h en yla m in o)-1,7-d im et h yl-9-oxo-1,8-
d ih yd r oim id a zo[4,5-h ]isoqu in olin -6-yla cetic Acid Eth yl
Ester (17). 17 was prepared from 16 by refluxing in ethanol
and H2SO4. Mp 280-285 °C. 1H NMR (TFA-d, 400 MHz): δ
1.45 (bs, 3H), 2.75 (s, 3H), 4.20 (s, 2H), 4.50 (s, 3H), 7.35-
7.40 (m, 1H), 7.75 (s, 1H), 7.95-8.0 (m, 2H), 8.37-8.39 (m,
1H), δ 8.97 (s, 1H), 11.08 (s, 1H). ESMS m/z: 500 (MH+). MSCI
m/z: 459, 461 (MH+). Anal. (C22H20Cl2N4O3‚1H2O) C, H, N.
2-(2,6-Dich lor op h en yla m in o)-1,7-d im et h yl-6-(2-m or -
ph olin -4-yl-2-oxoeth yl)-1,8-dih ydr oim idazo[4,5-h ]isoqu in -
olin e-9-on e (18). To a solution of 17 (1.0 g, 2.3 mmol) in DMF
(7 mL) was added O-benzotriazol-1-yl-N,N,N′,N′-tetramethy-
luronium tetrafluoroborate (TBTU) (0.82 g, 2.6 mmol) and
morpholine (0.24 mL, 2.8 mmol), and the mixture was stirred
for 18 h at room temperature. Ice/water was added and the
precipitate was collected, washed with water, and dried to give
0.97 g (84%) of the title compound. Mp >300 °C. 1H NMR
(TFA-d, 400 MHz): δ 2.58 (s, 3H), 3.89-3.99 (m, 2H), 4.03-
4.14 (m, 4H), 4.15-4.23 (m, 2H), 4.28 (s, 2H), 4.44 (s, 3H), 7.51
(t, 1H, J ) 8 Hz), 7.63 (d, 2H, J ) 8 Hz), 7.70 (d, 1H, J ) 9
Hz), 7.90 (d, 1H, J ) 9 Hz). ESMS m/z: 500, 502 (MH+). Anal.
(C24H23Cl2N5O3‚0.3H2O) C, H, N.
1
282 °C. H NMR (TFA-d, 400 MHz): δ 2.18 (br, 2H), 2.67 (s,
3H), 3.17 (t, 2H, J ) 8 Hz), 3.42 (t, 2H, J ) 8 Hz), 3.3-3.6 (m,
2H), 3.81 (d, 2H, J ) 12 Hz), 4.11 (t, 2H, J ) 6 Hz), 4.39 (d,
2H, J ) 12 Hz), 4.45 (s, 3H), 7.53 (t, 1H, J ) 8 Hz), 7.65 (d,
2H, J ) 8 Hz), 8.00 (s, 2 H). CIMS m/z: 500 (MH+).
2-(4-Am in o-5-br om o-2-cya n o-3-m eth yla m in op h en yl)-2-
m eth yl-3-oxobu tyr ic Acid Eth yl Ester (20). To a solution
of 9 (9.02 g, 31.2 mmol) in CHCl3 (90 mL) was added bromine
(4.98 g, 31.2 mmol) dropwise at ambient temperature. After
the addition of bromine, the reaction mixture was diluted with
EtOAc (800 mL). This solution was washed successively with
saturated NaHCO3 solution and brine and dried. The residue
after evaporation was purified by flash chromatography in
hexanes/EtOAc, 2:1, to provide 6.1 g (53%) of the title
1
compound. H NMR (CDCl3, 400 MHz): δ 1.3 (t, 3H, J ) 16
Hz), 1.89 (s, 3H), 2.45 (s, 3H), 2.95 (s, 3H), 4.30-4.40 (m, 2H),
7.01 (s, 1 H), 8.40 (bs, 2H), 8.6 (bs, 1H).
2-[8-Br om o-4-cya n o-2-(2,6-d ich lor op h en yla m in o)-3-
m eth yl-3H-ben zim idazol-5-yl]-2-m eth yl-3-oxobu tyr ic Acid
Eth yl Ester (21). To 20 (3.32 g, 9.0 mmol) in 1,4-dioxane (45
mL) was added 2,6-dichlorophenyl isothiocyanate (2.02 g, 9.9
mmol) and mercuric oxide (2.54 g, 11.7 mmol) under nitrogen
atmosphere. The resulting mixture was stirred and heated at
95 °C overnight. The reaction mixture was cooled to room
temperature and filtered though a short pad of diatomaceous
2-(2,6-Dich lor op h en yla m in o)-1,7-d im eth yl-6-(2-(eth yl-
amino)-2-oxoethyl)-1,8-dihydroimidazo[4,5-h]isoquinoline-9-
on e (50). 50 was prepared as described above. Mp >300 °C.
1H NMR (TFA-d, 400 MHz): δ 1.26 (t, 3H, J ) 8 Hz), 2.67 (s,
3H), 3.45-3.61 (m, 2H), 4.32 (s, 2H), 4.49 (s, 3H), 7.55 (t, 1H,

1346 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Goldberg et al.
earth and SiO2. The filtrate was concentrated and the residue
was purified by flash chromatography in hexanes/EtOAc, 2:1,
to provide 3.44 g (71%) of the title compound. 1H NMR (DMSO-
d6, 400 MHz): δ 1.36 (t, 3H, J ) 4.0 Hz), 1.96 (s, 3H), 2.39 (s,
3H), 4.14 (s, 3H), 4.36-4.40 (m, 2H), 7.23 (s, 1 H), 7.55 (t, 1H,
J ) 8.0 Hz), 7.77 (d, 2H, J ) 8.0 Hz), 9.62 (s, 1H).
2-[4-Cya n o-2-(2,6-d ich lor op h en yla m in o)-3-m et h yl-8-
vin yl-3H -b en zim ia zol-5-yl]-2-m et h yl-3-oxob u t yr ic Acid
Eth yl Ester (22). A mixture of 2-[8-bromo-4-cyano-2-(2,6-
dichlorophenylamino)-3-methyl-3H-benzimiazol-5-yl]-2-meth-
yl-3-oxobutyric acid ethyl ester (600 mg, 1.11 mmol), (PPh3)2-
PdCl2 (78 mg, 0.11 mmol), and tributyl(vinyl)tin (0.49 mL, 1.67
mmol) in NMP (4 mL) was degassed and heated at 100 °C for
3 days under argon. The mixture was concentrated, and the
residue was purified by flash chromatography in hexanes/
EtOAc, 3:1, to provide 530 mg (98%) of the title compound. 1H
NMR (CDCl3, 400 MHz): δ 1.38 (t, 3H, J ) 8 Hz), 1.97 (s,
3H), 2.45 (s, 3H), 4.05 (s, 3H), 4.37-4.40 (m, 2H), 5.50 (d, 1H,
J ) 8 Hz), 6.10-6.30 (m, 1H), 6.97 (s, 2H), 7.10 (d, H, J ) 8
Hz), 7.39 (d, H, J ) 8 Hz).
2-(2,6-Dich lor op h en yla m in o)-1,6,7-t r im et h yl-4-vin yl-
1,8-d ih yd r oim id a zo-[4,5-h ]isoqu in olin e-9-on e (22b). 22
(66 mg, 0.14 mmol) in a mixture of H2SO4 (0.6 mL), acetic acid
(0.6 mL), and water (0.6 mL) was heated at 100 °C for 2 h.
The resulting mixture was cooled to room temperature and
diluted with water (10 mL). The solution was adjusted to pH
8 with 10% NaOH solution. The precipitated brown solid was
filtered and purified by flash chromatography in CH2Cl2/
MeOH, 30:1, to provide 15 mg (27%) of the title compound.
Mp >250 °C (dec). 1H NMR (TFA-d, 400 MHz): δ 2.11 (s, 3H),
2.22 (s, 3H), 4.13 (s, 3H), 5.49 (d, 1H, J ) 11 Hz), 6.35 (d, 1H,
J ) 16 Hz), 7.05 (dd, 1H, J ) 11, 16 Hz), 7.31 (t, 1H, J ) 8
Hz), 7.45 (d, 1H, J ) 8 Hz), 7.68-7.71 (m, 1H), 8.86 (s, 1H),
10.9 (s, 1H). CIMS m/z: 413(MH+).
2-(2,6-Dich lor op h en yla m in o)-4-[(4-m eth oxyben zyla m i-
n o)m eth yl]-1,6,7-tr im eth yl-1,8-d ih yd r oim id a zo[4,5-h ]iso-
qu in olin -9-on e (27). A suspension of 25 (30 mg, 0.07 mmol)
in methanol (5 mL) was treated with 4-methoxybenzylamine
(94 µL, 0.72 mmol) and sodium cyanoborohydride (14 mg, 0.22
mmol) and stirred at room temperature for 16 h. The resulting
mixture was concentrated and preparative TLC (silica gel/5%
MeOH in CH2Cl2) purified the residue to give 7 mg (18%) of
1
the title compound. Mp 247-250 °C. H NMR (MeOH-d4, 400
MHz): δ 2.34 (s, 3H), 2.38 (s, 3H), 3.76 (s, 2H), 3.79 (s, 3H),
3.82 (s, 2H), 4.24 (s, 3H), 6.85 (d, 2H, J ) 8 Hz), 7.13 (d, 2H,
J ) 8 Hz), 7.24 (t, 1H, J ) 8 Hz), 7.45-7.47 (m, 3H). ESMS
m/z: 536, 538 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,6,7-tr im eth yl-4-(2,6-d i-
flu or opyr idin -3-yl)-1,8-dih ydr oim idazo[4,5-h ]isoqu in olin e-
9-on e (23b). To 2-[8-bromo-4-cyano-2-(2,6-dichlorophenylamino)-
3-methyl-3H-benzimidazol-5-yl]-2-methyl-3-oxobutyric acid ethyl
ester (100 mg, 0.186 mmol) in dry NMP (3 mL) was added
2,6-difluoro-3-tributylstannanylpyridine (113 mg, 0.28 mmol)
and dichlorobis(triphenylphosphine)palladium(II) (10 mg). The
reaction mixture was heated in a sealed tube at 100 °C
overnight. The reaction mixture was cooled to room temper-
ature and extracted with EtOAc (25 mL). The organic fraction
was washed with water, dried over anhydrous sodium sulfate,
filtered, and evaporated. Column chromatography (1:1 EtOAc/
hexanes) followed by preparative TLC (silica gel, 40% ethyl
acetate/hexanes) provided 28 mg (26%) of the title compound.
ESMS m/z: 572 (MH+).
To the coupled product (25 mg, 0.044 mmol) was added 1:1
concentrated sulfuric acid/water (3 mL). The reaction mixture
was heated at 100 °C for 2 h. The reaction mixture was cooled
to room temperature, ice/water (2 mL) was added, and the
reaction was made basic by addition of aqueous NH4OH. The
solid was filtered off and purified by preparative TLC (silica
gel/5% CH3OH/CH2Cl2) to provide 7 mg (32%) of the title
compound. Mp >300 °C. 1H NMR (DMSO-d6, TFA, 400 MHz):
δ 2.21 (s, 3H), 2.29 (s, 3H), 4.13 (s, 3H), 7.23-7.30 (m, 2H),
7.48 (s, 1H), 7.53-7.55 (m, 2H), δ 8.37-8.39 (m, 1H), 8.97 (s,
1H), 11.08 (s, 1H). ESMS m/z: 500 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,6-d im et h yl-9-oxo-8,9-
d ih y d r o -1H -im id a zo [4,5-h ]is o q u in o lin e -7-c a r b a ld e -
h yd e (28). To a suspension of 12 (521 mg, 1.3 mmol) in
dioxane (30 mL) was added selenium dioxide (430 mg, 3.9
mmol), and the mixture was heated at 100 °C for 5 h. The
reaction mixture was then cooled to room temperature, filtered
through diatomaceous earth with 10% MeOH/CH2Cl2, and then
concentrated in vacuo. The crude material was triturated with
CH2Cl2 to provide 476 mg (92%) of the title compound. Mp
>300 °C. 1H NMR (DMSO-d6, TFA, 400 MHz): δ 2.27 (s, 3H),
4.30 (s, 3H), 7.38 (t, 1H, J ) 8 Hz), 7.60 (d, 2H, J ) 8 Hz),
7.69 (s, 2H), 8.68 (bs, 1H), 10.21 (s, 1H), 10.51 (bs, 1H), 11.72
(bs, 1H). NOSEY (DMSO-d6, TFA): correlation between C5-H
and C6-CH3 and between C6-CH3 and C7-CHO. CIMS m/z:
401, 403 (MH+).
2-(2,6-Dich lor oph en ylam in o)-1,6-dim eth yl-7-(1-h ydr ox-
yp r op -2-en -1-yl)-1,8-d ih yd r oim id a zo[4,5-h ]isoqu in olin -9-
on e (31). A suspension of 28 (100 mg, 0.25 mmol) in THF (3
mL) was cooled to -78 °C. Vinylmagnesium bromide (1 M in
THF, 2.0 mmol) was added dropwise, and the brown suspen-
sion was warmed gradually to -10 °C over 2 h. The solution
was quenched with saturated NH4Cl, extracted with EtOAc,
and concentrated in vacuo to provide the title compound, which
was used in the next step without purification. Mp 235-236
°C. 1H NMR (MeOH-d4, 400 MHz): δ 2.35 (s, 3H), 4.24 (s, 3H),
5.26 (d, 1H, J ) 10 Hz), 5.40 (d, 1H, J ) 17 Hz), 5.56 (d, 1H,
J ) 5 Hz), 6.03 (ddd, 1H, J ) 5, 10, 17 Hz), 7.32 (t, 1H, J ) 8
Hz), 7.53 (d, 2H, J ) 8 Hz), 7.61 (d, 1H, J ) 9 Hz), 7.72 (d,
1H, J ) 9 Hz). ESMS m/z: 429 (MH+). Anal. (C21H18Cl2N4O3)
C, H, N.
2-[4-Cya n o-2-(2,6-d ich lor op h en yla m in o)-7-for m yl-3-
m et h yl-3H -b en zoim id a zol-5-yl]-2-m et h yl-3-oxob u t yr ic
Acid Eth yl Ester (24). To a solution of 22 (0.58 g, 1.20 mmol)
in THF (30 mL) was added osmium tetroxide (3 mL), followed
by sodium periodate (0.77 g, 3.59 mmol) and water (3.0 mL).
The mixture was stirred at room temperature for 30 min before
dilution with water and extraction of the product with ethyl
acetate. The organic layer was then diluted with an aqueous
NaHCO3 solution. The organic layer was then dried with
MgSO4 filtered, and concentrated to an oil. This was then
loaded onto a silica gel column (2:1 hexanes/ethyl acetate) to
provide 0.46 g (100% g) of the title compound. 1H NMR (CDCl3,
400 MHz): δ 1.40 (t, 3H, J ) 16 Hz), 1.60 (s, 3H), 1.97 (s, 3H),
2.45 (s, 3H), 4.01 (s, 3H), 4.05 (s, 3H), 4.31-4.45 (m, 2H), 7.01
(d, 1H, J ) 8 Hz), 7.19 (bs,1H), 7.45 (d, 1H, J ) 8 Hz), 8.95
(bs, 1H), 10.01 (bs, 1H).
2-(2,6-Dich lor op h en yla m in o)-1,6,7-tr im eth yl-9-oxo-1,8-
d ih yd r oim id a zo[4,5-h ]isoqu in olin e-4-ca r ba ld eh yd e (25).
24 (35 mg, 0.07 mmol) in a mixture of H2SO4 (0.6 mL), acetic
acid (0.6 mL), and water (0.6 mL) was heated at 100 °C for
1.5 h and cooled to room temperature. The resulting mixture
was diluted with water (10 mL), and the pH was adjusted to
7 with ammonium hydroxide solution. The precipitated orange
powder was filtered to provide 18 mg (60%) of the title
compound. 1H NMR (TFA-d, 400 MHz): δ 2.34 (s, 3H), 2.38
(s, 3H), 4.14 (s, 3H), 7.25 (t, 1H, J ) 8 Hz), 7.60 (d, 2H, J ) 8
Hz), 8.10 (s, 1H), 10.1 (s, 1H).
2-(2,6-Dich lor op h en yla m in o)-4-(2-h yd r oxyet h yla m i-
n om eth yl)-1,6,7-tr im eth yl-1,8-d ih yd r oim id a zo[4,5-h ]iso-
qu in olin -9-on e (26). A suspension of 25 (30 mg, 0.07 mmol)
in methanol (5 mL) was treated with ethanolamine (44 µL,
0.72 mmol) and sodium cyanoborohydride (14 mg, 0.22 mmol)
and stirred at room temperature for 16 h. The resulting
mixture was concentrated, and the residue was diluted with
water. The precipitated solid was filtered and dried to give 12
mg (36%) of the title compound. Mp >300 °C. 1H NMR (MeOH-
d4, 400 MHz): δ 2.36 (s, 3H), 2.38 (s, 3H), 2.71 (t, 2H, J ) 6.0
Hz), 3.62 (t, 2H, J ) 6.0 Hz), 4.14 (s, 2H), 4.20 (s, 3H), 7.25 (s,
1H), 7.49-7.52 (m, 3H). ESMS m/z: 460, 462 (MH+).
3-[2-(2,6-Dich lor op h en yla m in o)-1,6-d im eth yl-9-oxo-8,9-
d ih yd r o-1H-im id a zo[4,5-h ]isoqu in olin -7-yl]a cr ylic Acid
Meth yl Ester (36). To a suspension of 28 (329 mg, 0.82 mmol)
in THF (5 mL) was added sequentially trimethyl phospho-
noacetate (164 mg, 0.90 mmol), lithium hydroxide monohy-

Isoquinolin-9-ones as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1347
1
drate (76 mg, 1.8 mmol), and water (0.9 mL). The blood-red
solution was stirred for 2 h and quenched with water, and the
resulting solid was collected and dried in vacuo. Column chro-
matography (5% MeOH/CH2Cl2) provided 300 mg (80%) of the
title compound. Mp >300 °C. 1H NMR (DMSO-d6, TFA, 400
MHz): δ 2.45 (s, 3H), 3.76 (s, 3H), 4.20 (s, 3H), 6.89 (d, 1H, J
) 16 Hz), 7.56 (t, 1H, J ) 8 Hz), 7.73-7.77 (m, 3H), 7.81 (d,
1H, J ) 16 Hz), 7.87 (d, 1H, J ) 9 Hz), 11.29 (s, 1H). ESMS
m/z 457, 459 (MH+). Anal. (C22H18Cl2N4O3‚1.5H2O) C, H, N.
the title compound. Mp 169-171 °C. H NMR (MeOH-d4, 400
MHz): δ 2.16 (s, 3H), 2.40 (s, 3H), 4.18 (s, 3H), 5.34-5.48 (m,
2H), 6.08-6.16 (m, 1H), 6.55 (s, 1H), 7.27 (bs, 1H), 7.58 (d,
2H, J ) 8 Hz), 7.59 (d, 1H, J ) 8 Hz), 7.68 (bs, 1H). ESMS
m/z: 471 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,6-d im et h yl-7-(3-m or -
p h olin -4-yl-p r op en -1-yl)-1,8-d ih yd r oim id a zo[4,5-h ]iso-
qu in olin -9-on e (34). Tris(dibenzylideneacetone)dipalladium-
(0) (1.8 mg, 0.002 mmol) and triphenylphosphine (1.6 mg, 0.006
mmol) were stirred in THF (0.5 mL) for 20 min under inert
atmosphere until the red solution turned yellow. To this solu-
tion was added sequentially 33 (20 mg, 0.04 mmol) in THF
(0.5 mL), triethylamine (17 µL, 0.12 mmol), and morpholine
(11 µL, 0.12 mmol). The solution was stirred for 14 h and then
concentrated to an oil. Column chromatography (10% MeOH/
CH2Cl2) provided 10 mg (50%) of the title compound. Mp 175-
3-[2-(2,6-Dich lor op h en yla m in o)-1,6-d im eth yl-9-oxo-8,9-
dih ydr o-1H-im idazo[4,5-h ]isoqu in olin -7-yl]pr opion ic Acid
Meth yl Ester (37). To a solution of 36 (30 mg, 0.06 mmol) in
EtOH (3 mL) and AcOH (4 mL) in a Parr reactor was added
PtO2 (2 mg, 0.007 mmol). The Parr reactor was charged with
50 psi of H2 and shaken for 12 h. The crude reaction mixture
was filtered through diatomaceous earth with EtOH and
concentrated in vacuo. Column chromatography (2% MeOH/
CH2Cl2) provided 9 mg (30%) of the title compound. Mp 268
1
177 °C. H NMR (DMSO-d6, TFA, 400 MHz): δ 2.35 (s, 3H),
3.16 (t, 2H, J ) 12 Hz), 3.48 (d, 2H, J ) 12 Hz), 3.66 (t, 2H, J
) 12 Hz), 4.02 (d, 4H, J ) 7 Hz), 4.24 (s, 3H), 6.47 (dt, 1H, J
) 8, 16 Hz), 7.15 (d, 1H, J ) 16 Hz), 7.58 (dd, 1H, J ) 8, 9
Hz), 7.74-7.77 (m, 3H), 7.82 (d, 1H, J ) 9 Hz). ESMS m/z:
498 (MH+).
1
°C (dec). H NMR (DMSO-d6, TFA, 400 MHz): δ 2.24 (s, 3H),
2.66 (t, 2H, J ) 8 Hz), 2.90 (t, 2H, J ) 8 Hz), 3.75 (s, 3H), 4.24
(s, 3H), 7.54 (t, 1H, J ) 8 Hz), 7.70-7.76 (m, 4H), 11.37 (s,
1H). ESMS m/z: 458 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,6-d im eth yl-7-vin yl-1,8-
d ih yd r oim id a zo[4,5-h ]isoqu in olin e-9-on e (32). To a sus-
pension of 28 (100 mg, 0.25 mmol) in THF (5 mL) was added
trimethylsilylmethylmagnesium chloride (2 mL, 2 mmol) at
-78 °C. The reaction mixture was warmed to room tempera-
ture for 1 h, cooled to 0 °C, quenched with water, and extracted
with EtOAc to provide the silyl alcohol (85 mg, 70%). The crude
silyl alcohol was suspended in CH2Cl2 and cooled to 0 °C. Boron
trifluoride etherate (42 µL, 0.32 mmol) was added, and the
slurry was warmed to room temperature for 1 h. The reaction
mixture was quenched with water, and the CH2Cl2 was
removed in vacuo. Collection of the resulting solid followed by
CH2Cl2 trituration provided 17 mg (61%) of the title compound.
7-(3-Am in op r op en -1-yl)-2-(2,6-d ich lor op h en yla m in o)-
1,6-d im e t h yl-1,8-d ih yd r oim id a zo[4,5-h ]isoq u in olin -9-
on e (35). A suspension of tris(dibenzylideneacetone)dipal-
ladium(0) (185 mg, 0.25 mmol) and triphenylphosphine (320
mg, 1.2 mmol) in THF (40 mL) was stirred for 20 min under
N2. A solution of 33 (1.88 g, 4.0 mmol) in THF (5 mL) was
added, and the mixture was stirred for 20 min. Sodium azide
(280 mg, 4.4 mmol) and water (4.0 mL) were added, and the
reaction mixture was heated at 60 °C for 3 h. The solution
was cooled to room temperature, and triphenylphosphine (1.0
g, 3.8 mmol) was added. After the mixture was stirred for 45
min, ammonium hydroxide (4 mL) was added and stirring
continued overnight. The resulting solution was dried over
MgSO4 and then concentrated to an oil. Column chromatog-
raphy on silica eluting with CH2Cl2/MeOH (90:10 increasing
to 50:50) provided 1.2 g (70%) of the title compound. Mp >300
1
Mp >300 °C. H NMR (DMSO-d6, TFA, 400 MHz): δ 2.34 (s,
3H), 4.14 (s, 3H), 5.52 (1H, J ) 8 Hz), 6.13 (d, 1H, J ) 8 Hz),
7.69 (d, 1H, J ) 8 Hz), 7.9 (bs, 1H), 7.67 (bs, 4H), 11.1 (s, 1H).
ESMS m/z 399, 401 (MH+).
1
°C. H NMR (DMSO-d6, TFA, 400 MHz): δ 2.35 (s, 3H), 3.68
2-(2,6-Dich lor op h en yla m in o)-1,6-d im eth yl-7-eth yl-1,8-
d ih yd r oim id a zo[4,5-h ]isoqu in olin e-9-on e (42). 32 (50 mg,
0.13 mmol) was suspended in 3 mL of acetic acid and 3 mL of
EtOH. PtO2 (6 mg, 0.03 mmol) was added, and the reaction
mixture was stirred under 1 atm of H2(g) for 1 h. The reaction
mixture was filtered, concentrated, and extracted with EtOAc.
Column chromatography (0-10% MeOH/CH2Cl2) provided 25
mg (52%) of the title compound. Mp >300 °C. 1H NMR (DMSO-
d6, TFA, 400 MHz): δ 1.14 (t, 3H, J ) 6 Hz), 2.27 (s, 3H), 2.61
(q, 2H, J ) 6 Hz), 4.24 (s, 3H), 7.75 (t, 1H, J ) 8 Hz), 7.70 (s,
2H), 7.76 (d, 2H, J ) 8 Hz), 11.42 (s, 1H). ESMS m/z: 401
(MH+). Anal. (C20H18Cl2N4O‚1.5H2O) C, H, N.
2-(2,6-Dich lor oph en ylam in o)-1,6-dim eth yl-7-(3-h ydr ox-
yp r op en -1-yl)-1,8-d ih yd r oim id a zo[4,5-h ]isoq u in olin -9-
on e (38). A suspension of 36 (100 mg, 0.22 mmol) in THF (7
mL) was cooled to -78 °C. Sodium bis(trimethylsilyl)amide
(1 M in THF, 0.44 mmol) was added dropwise. The bright-red
solution was warmed to 0 °C for 15 min. Then lithium
aluminum hydride (1 M in THF, 2.6 mmol) was added, and
the orange solution was then warmed to room temperature
for 0.5 h. The mixture was cooled to 0 °C, quenched with
saturated ammonium chloride, and extracted with EtOAc.
Column chromatography (3-6% MeOH/CH2Cl2) provided 32
mg (34%) of the title compound. Mp 298-300 °C. 1H NMR
(DMSO-d6, TFA, 400 MHz): δ 2.32 (s, 3H), 4.16 (s, 2H), 4.19
(s, 3H), 6.63 (d, 1H, J ) 8 Hz), 6.82 (d, 1H, J ) 8 Hz), 7.42 (bs,
1H), 7.64-7.68 (m, 4H), 10.92 (s, 1H). ESMS m/z: 429, 431
(MH+).
(bs, 2H), 4.23 (s, 3H), 6.45-6.60 (m, 1H), 6.90-7.00 (m, 1H),
7.55-7.65 (m, 1H), 7.70-7.85 (m, 4H), 8.05-8.20 (m, 1H).
ESMS m/z 428 (MH+).
2-(2,6-Dich lor op h en yla m in o)-1,6-d im eth yl-7-(3-(eth yl-
am in o)pr open yl)-1,8-dih ydr oim idazo[4,5-h ]isoqu in olin -9-
on e (39). A solution of 35 (100 mg, 0.234 mmol) in acetic acid
(1 mL) was cooled to -10 °C. Sodium triacetoxyborohydride
(87 mg, 0.410 mmol) was added, and the mixture was stirred
for 20 min at -10 °C. Acetaldehyde (10 mg, 0.234 mmol) was
taken up in THF (3 mL) and added dropwise to the reaction
mixture (at -10 °C). Once all the aldehyde had been added,
the reaction mixture was warmed to room temperature and
was stirred overnight. The crude reaction mixture was diluted
with EtOAc (10 mL), washed with water (5 mL), dried
(MgSO4), and concentrated under reduced pressure. Chroma-
tography [CH2Cl2/MeOH/NH4OH (9:1:0.1)] afforded 28 mg
(26%) of the title compound. Mp 218-221 °C. 1H NMR (CDCl3,
400 MHz): δ 1.15-1.25 (m, 3H), 2.33 (s, 3H), 3.03-3.13 (m,
2H), 3.80 (bs, 2H), 4.21 (s, 3H), 6.45-6.60 (m, 1H), 7.09 (d,
1H, J ) 15 Hz), 7.55 (t, 1H, J ) 8 Hz), 7.7-7.9 (m, 4H).
2-(2,6-Dich lor op h en yla m in o)-7-(3-d ieth yla m in op r op e-
n yl)-1,6-d im eth yl-1,8-d ih yd r oim id a zo[4,5-h ]isoqu in olin -
9-on e (43). 43 was prepared as described above. 1H NMR
(DMSO-d6, TFA, 400 MHz): δ 1.27 (t, 6H, J ) 7.0 Hz), 2.38
(s, 3H), 3.04-3.40 (m, 4H), 4.01 (d, 2H, J ) 7 Hz), 4.26 (s,
3H), 6.54 (dt, 1H, J ) 7, 16 Hz), 7.26 (d, 1H, J ) 16 Hz), 7.60
(t, 1H, J ) 8 Hz), 7.78 (d, 3H, J ) 8 Hz), 7.84 (d, 1H, J ) 9
Hz).
2-(2,6-Dich lor op h en yla m in o)-7-(1-a cetoxyp r op -3-en -1-
yl)-1,6-d im eth yl-1,8-d ih yd r oim id a zo[4,5-h ]isoqu in olin -9-
on e (33). To a solution of 31 (106 mg, 0.25 mmol) in THF (1
mL) was added acetic anhydride (1 mL). Triethylamine (35
µL, 0.25 mmol) was added, and the reaction mixture was
stirred for 14 h and then concentrated in vacuo. Column
chromatography (2% MeOH/CH2Cl2) provided 85 mg (79%) of
2-(2,6-Dich lor op h en yla m in o)-1,6-d im eth yl-7-(3-p yr r o-
lid in -1-ylp r op en yl)-1,8-d ih yd r oim id a zo[4,5-h ]isoq u in o-
lin -9-on e (44). 44 was prepared as described above. 1H NMR
(DMSO-d6, TFA, 400 MHz): δ 1.91 (br, 2H), 2.06 (br, 2H), 2.38
(s, 3H), 3.11 (br, 2H), 3.59 (br, 2H), 4.04 (d, 2H, J ) 7 Hz),
4.26 (s, 3H), 6.55 (dt, 1H, J ) 7, 15 Hz), 7.16 (d, 1H, J ) 15

1348 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Goldberg et al.
Hz), 7.60 (dd, 1H, J ) 8, 8 Hz), 7.78 (d, 3H, J ) 8 Hz), 7.84 (d,
1H, J ) 8 Hz). Anal. (C25H25Cl2N5O1‚0.2H2O) C, H, N.
P-3000), and 9.8 mL of complete media. An equal volume of
7
Fluo-3 solution was added to give a 1 × 10 cells/mL solution
and 2 µM Fluo-3. The tube was wrapped in foil and incubated
at room temperature with very gentle rocking for 45 min.
Additional media to fill a 50 mL tube was added, and it was
incubated another 15 min at room temperature.
2-(2,6-Dich lor op h en yla m in o)-1,6-d im et h yl-7-[3-(b en -
zylm eth ylam in o)pr open yl]-1,8-dih ydr oim idazo[4,5-h ]iso-
qu in olin -9-on e (45). 45 was prepared as described above. 1H
NMR (DMSO-d6, TFA, 400 MHz): δ 2.38 (s, 3H), 2.76 (s, 3H),
3.81-4.10 (m, 2H), 4.27 (s, 3H), 4.31 (d, 1H, J ) 13 Hz), 4.51
(d, 1H, J ) 15 Hz), 6.62 (dt, 1H, J ) 7, 15 Hz), 7.20 (d, 1H, J
) 16 Hz), 7.40-7.67 (m, 6H), 7.71-7.92 (m, 3H), 7.85 (d, 1H,
J ) 8 Hz). Anal. (C29H27Cl2N5O1‚0.1H2O) C, H, N.
2-(2,6-Dich lor op h en yla m in o)-1,6-d im et h yl-7-(3-d im e-
th yla m in op r op en yl]-1,8-d ih yd r oim id a zo[4,5-h ]isoqu in o-
lin -9-on e (46). 46 was prepared as described above. 1H NMR
(DMSO-d6, TFA, 400 MHz): δ 2.37 (s, 3H), 2.85 (s, 6H), 3.96
(d, 2H, J ) 7 Hz), 4.26 (s, 3H), 6.52 (dt, 1H, J ) 7, 15 Hz),
7.16 (d, 1H, J ) 15 Hz), 7.60 (t, 1H, J ) 8 Hz), 7.77 (d, 3H, J
) 8 Hz), 7.84 (d, 1H, J ) 9.0 Hz). Anal. (C23H23Cl2N5O1‚0.2H2O)
C, H, N.
Cells were pelleted and washed three times with Hanks’
balanced salt solution (HBSS) (GibcoBRL no. 14175-095),
containing 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 2 mM
probenecid, and 1% fetal bovine serum (FBS), pH 7.4. Finally,
7
cells were resuspended at 2 × 10 cells/mL and kept on ice
and in the dark until ready for use.
Compounds were made up as 5 mg/mL stocks in DMSO and
diluted as appropriate with the following diluent: HBSS, 1
mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 2 mM
probenecid, 1% FBS. A 96-well V-bottom plate is used for the
compound dilutions.
2. Stim u lu s P la te. Anti-CD3 (Immunotech, clone X35,
catalog no. 0178) was prepared by taking 69 µL of a 200 µg/
mL stock into 11 mL of diluent to give a 5× stock for a final
test concentration of 0.25 µg/mL. Then 100 µL was added per
well of V-bottom 96-well plate.
3. Cell P la te P r ep a r a tion . An amount of 2 mL of loaded
cell suspension was mixed with 18 mL of diluent, and 150 µL
of this mixture was then added per well of a 96-well Black
Packard viewplate for a final cell number of 3 × 10 5/mL.
The plate was then centrifuged at 1500 rpm for 5 min at
room temperature and immediately placed in the center
position in the drawer of an FLIPR (fluorometric imaging plate
reader).
4. Assa y. The assay was performed on the FLIPR system
(Molecular Devices). All experiments were run at room tem-
perature. The cell plate was placed in the center position in
the drawer. The compound dilution plate was placed in the
left position and the stimulus plate in the right. An amount
of 50 µL of compound was added to the cells, and this was
incubated at room temperature for 15 min. An amount of 50
µL of anti-CD3 was added to the cell plate, and fluorescence
intensity was monitored for an additional 13 min.
2-(2,6-Dich lor oph en ylam in o)-1,7-dim eth yl-6-(2-h ydr oxy-
eth yl)-1,8-d ih yd r oim id a zo[4,5-h ]isoqu in olin e-9-on e (54).
To a stirred solution of 2-(2,6-dichlorophenylamino)-1,7-dim-
ethyl-9-oxo-1,8-dihydroimidazo[4,5-h]isoquinolin-6-ylacetic acid
ethyl ester (25 mg, 0.05 mmol) in THF (2 mL) was added a
solution of lithium aluminum hydride (1 M in THF, 0.25 mL,
0.25 mmol). The mixture was stirred for 30 min at room
temperature. EtOAc was added, followed by water, and then
the mixture was acidified with 1 N HCl. The crude product
was applied to a Varian SCX cartridge and washed in turn
with 1 N HCl, water, acetone, MeOH, and MeOH/CH2Cl2 (1:
1). The product was then eluted with MeOH/CH2Cl2/NH4OH
(49:49:2). Evaporation of the eluent provided 15 mg (72%) of
the title compound. Mp >300 °C. 1H NMR (TFA-d6, 400
MHz): δ 2.76 (s, 3H), 3.49 (br, 2H), 4.22 (br, 2H), 4.45 (s, 3H),
7.55 (t, 1H, J ) 8 Hz), 7.68 (d, 2H, J ) 8 Hz), 8.04 (d, 1H, J )
9 Hz), 8.19 (d, 1H, J ) 9 Hz). ESMS m/z 417, 419 (MH+). Anal.
(C20H18Cl2N4O3‚1.5H2O) C, H, N.
Tyr osin e Kin a se In h ibition Assa y. The kinase activity
is measured using DELFIA (dissociation enhanced lanthanide
fluoroimmunoassay), which utilizes europium chelate-labeled
anti-phosphotyrosine antibodies to detect phosphate transfer
to a random polymer, poly-Glu4-Tyr1 (PGTYR). The kinase
assay is performed in a neutravidin-coated 96-well white plate
(PIERCE) in kinase assay buffer (50 mM HEPES, pH 7.0, 25
mM MgCl2, 5 mM MnCl2, 50 mM KCl, 100 µM Na3VO4, 0.2%
BSA, 0.01% CHAPS). Test samples initially dissolved in DMSO
at 1 mg/mL are prediluted for dose response (10 doses with
starting final concentration of 1 µg/mL, 1-3.5 serial dilutions)
with the assay buffer. A 25 µL aliquot of this diluted sample
and a 25 µL aliquot of diluted enzyme (0.8 nM final concentra-
tion) are sequentially added to each well. The reaction is
started with a 50 µL/well of a mixture of substrates containing
2 µM ATP (final ATP concentration is 1 µM) and 7.2 ng/µL
PGTYR-biotin (CIS Biointernational) in kinase buffer. Back-
ground wells are incubated with buffer and substrates only.
Following 45 min of incubation at room temperature, the assay
plate is washed three times with 300 µL/well DELFIA wash
buffer. A 100 µL/well aliquot of europium-labeled anti-phos-
photyrosine (Eu3+-PT66, 1 nM, Wallac CR04-100) diluted in
DELFIA assay buffer is added to each well and incubated for
30 min at room temperature. Upon completion of the incuba-
tion, the plate is washed four times with 300 µL/well of wash
buffer and 100 µL/well of DELFIA wash buffer. Enhancement
solution (Wallac) is added to each well. After 15 min, time-
resolved fluorescence is measured on the LJ L’s analyst (excita-
tion at 360 nm, emission at 620 nm, EU 400 dichroic mirror)
after a delay time of 250 µs.
5. An a lysis. Calcium release was measured by fluorescence
intensity via a SAS program that measures the difference
between baseline values and peak height. These values are
plotted versus compound concentration to obtain an EC50
value. A decrease in fluorescence intensity indicated inhibition
of the release of calcium by the compound being tested.
In h ibition of IL-2 P r od u ction . The 96-well flat bottom
plates were coated with anti-CD3 and clone UCHT1 (Immu-
notech catalog no. 1304) at 4 µg/mL in phosphate-buffered
saline (PBS), 100 µL/well. The solution was prepared by taking
200 µL of 200 µg/mL anti-CD3 stock/10 mL PBS. The plate
was then incubated at 37 °C for 2 h. J urkat cells were pelleted
6
and counted. The cells were resuspended at 2.5 × 10 cells/
mL in RPMI and 10% FBS (complete media). Test compounds
were diluted from a 5 mg/mL DMSO stock directly into
complete media.
An amount of 10 µL of 20 X compound per well was added
to a separate plate, followed by 100 µL of cell suspension in
triplicate, and this plate was preincubated at 37 °C for 30 min.
The 96-well plate containing anti-CD3 was aspirated, and the
cells and compound were transferred to this plate. An amount
of 100 µL of PMA (phorbol 12-myristate 13-acetate, Sigma
catalog no. P-8139) at 20 ng/mL was added, and the plate was
incubated overnight at 37 °C. (PMA stock at 1 mg/mL in
ethanol, diluted 10 µL/mL in complete media, then 20 µL/10
mL in complete media; 100 µL/well ) 10 ng/mL, final concen-
tration). The next day, the plate was centrifuged at 1500 rpm
for 5 min at room temperature and the supernatants were
removed. The supernatants were tested using R&D Systems
Quantikine Human IL-2 Kit (catalog no. 2050). Samples were
diluted 1:5 in RPMI 1640, and 100 µL/well was used in the
ELISA. The optical density of each well was determined using
a microplate reader set to 450 nm. EC50 values were deter-
mined using Origin (nonlinear regression) or SAS by plotting
absorbance vs concentration of compound.
Ca lciu m Relea se in J u r k a t Cells. 1. Loa d in g of J u r k a t
Cells w ith F lu o-3. J urkat cells were pelleted, washed 2 times
with RPMI, 10 mM HEPES, and 10% fetal bovine serum
7
(complete media), and then resuspended at 2 × 10 cells/mL
in the above. Fluo-3 solution was prepared from 40 µL of Fluo-3
stock AM (Molecular Probes catalog no. F-1242) (4 µM), 180
µL of pluronic F127 detergent (Molecular Probes catalog no.

Isoquinolin-9-ones as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1349
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mann, K.-U.; Veillette, A.; Davidson, D.; Mak, T. W. Profound
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results did not change any of the conclusions drawn from our
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upon tyrosine phosphorylation. Nature 1996, 384, 484-489. (b)
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In h ibition of IL-2 P r od u ction in Hu m a n Wh ole Blood .
Human whole blood was obtained from in-house donors by
venipuncture collected into 25 mL heparinized Vacu-tainer
tubes.
Test compounds were dissolved in DMSO to yield 5 mg/mL
stock solutions. Stock solutions were freshly diluted in RPMI
1640 and 10% fetal bovine serum (complete media) to yield
solutions that were 10 times the final assay concentration.
Compounds were diluted serially into complete media.
SEB Solu tion . Staphylococcal enterotoxin B was dissolved
in PBS to yield a 2.5 mg/mL stock. This is further diluted
in complete media to yield a 3000 ng/mL solution (10 times
the final assay concentration). SEB stock solution is stored at
4 °C.
Assa y P r oced u r e. Add 20 µL of 10× compound dilutions,
in triplicate, to wells of a U bottom 96-well plate. For “no
compound” samples, add 20 µL of complete media. Also, dilute
DMSO to the highest concentration used in the assay and use
20 µL/well as controls. Add 160 µL of whole human blood to
each well and incubate plate for 20-30 min at 37 °C. Add 20
µL of 10x SEB solution to all test wells and to stimulated
control wells. Add 20 µL of complete media to nonstimulated
wells as controls. Incubate plate overnight at 37 °C. The next
day, centrifuge plate at 2000 rpm for 10 min at room temper-
ature and transfer supernatants to a fresh plate. Dilute
supernatants 1:5 in RPMI 1640 and then use 100 µL/well in
R&D Systems Quantikine Human IL-2 Kit.
Follow the ELISA procedure as outlined in the section
“Inhibition of IL-2 Production” in J urkat cells protocol.
1. An a lysis. EC50 values are determined using Origin
(nonlinear regression) or SAS (Secondary Screening Program,
EC50 calculation) by plotting absorbance vs actual concentra-
tion of the compound.
2. In Vivo An ti-CD3 Assa y. Female BALB/c mice (Charles
River, The J ackson Laboratories) were used for anti-CD3
studies. Animals were numbered by tail tattoo and were 6-8
weeks old at the initiation of experiments. For the induction
of IL-2 in vivo by anti-CD3, 1 µg of anti-CD3 (monoclonal
hamster-antimouse 145-2C11, lot M03283) in 200 µL of PBS
was injected intraperitoneally (ip) into experimental mice to
stimulate the polyclonal activation of T cells. Upon activation,
these T cells produced the T cell cytokine IL-2 that was
detected in the plasma.
For the measurement of plasma cytokine levels, 3 h after
anti-CD3 injection, each mouse was anesthetized with isoflu-
rane, and approximately 0.5 mL of whole blood was collected
in heparinized tubes following cardiac puncture. Plasma IL-2
levels were determined by ELISA (R&D Systems, Minneapolis
MN) of 1:10 dilutions of samples. Levels were quantitated by
linear regression analysis of samples in comparison to a
recombinant cytokine IL-2 curve, and plasma levels in vehicle-
treated groups were 1900-2800 pg/mL in each experiment.
3. Tr ea tm en t Gr ou p s. All doses of compounds and controls
are represented as milligram of compound per kilogram of body
mass and were administered orally in 100 µL of vehicle, 30%
Cremophor. The compound was administered to mice (n ) 8
per group) 1 h prior to anti-CD3 injection. Control mice
received either 100 µL of vehicle alone (negative control) or
30 mg/kg cyclosporin A (positive control).
Refer en ces
(1) Levitzki, A. Protein tyrosine kinase inhibitors as therapeutic
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kinases as therapeutic targets: the model of the MET oncogene.
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(3) Qian, D.; Weiss, A. T cell antigen receptor signal transduction.
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A. P.; Sellati, R.; Tschantz, M. A. The Discovery of 2-Pheny-
lamino-imidazo[4,5-h]isoquinolin-9-ones, a New Class of Inhibi-
tors of Lck Kinase. J . Med. Chem. 2002, 45, 3394-3405.
J M020446L