4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones as potent and selective inhibitors of the cyclin-dependent kinase 4 (CDK4)

Article abstract of DOI:10.1021/jm800072z

The cyclin-dependent kinases (CDKs), as complexes with their respective partners, the cyclins, are critical regulators of cell cycle progression. Because aberrant regulations of CDK4/cyclin D1 lead to uncontrolled cell proliferation, a hallmark of cancer, small-molecule inhibitors of CDK4/cyclin D1 are attractive as prospective antitumor agents. The series of 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione derivatives reported here represents a novel class of potent inhibitors that selectively inhibit CDK4 over CDK2 and CDK1 activities. In the headpiece of the 4-(phenylaminomethylene) isoquinoline-1,3(2H,4H)-dione, a basic amine substitutent is required on the aniline ring for the CDK4 inhibitory activity. The inhibitory activity is further enhanced when an aryl or heteroaryl substituent is introduced at the C-6 position of the isoquinoline-1,3(2H,4H)-dione core. We present here SAR data and a CDK4 mimic model that explains the binding, potency, and selectivity of our CDK4 selective inhibitors.

Full text of DOI:10.1021/jm800072z

J. Med. Chem. 2008, 51, 3507–3525
3507
4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones as Potent and Selective Inhibitors of
the Cyclin-Dependent Kinase 4 (CDK4)
Hwei-Ru Tsou,*,† Mercy Otteng,† Tritin Tran,†,§ M. Brawner Floyd, Jr.,† Marvin Reich,† Gary Birnberg,† Kristina Kutterer,†
Semiramis Ayral-Kaloustian,† Malini Ravi,† Ramaswamy Nilakantan,† Mary Grillo,‡ John P. McGinnis,‡ and
Sridhar K. Rabindran‡,|
Wyeth Research, 401 N. Middletown Road, Pearl RiVer, New York 10965
ReceiVed January 24, 2008
The cyclin-dependent kinases (CDKs), as complexes with their respective partners, the cyclins, are critical
regulators of cell cycle progression. Because aberrant regulations of CDK4/cyclin D1 lead to uncontrolled
cell proliferation, a hallmark of cancer, small-molecule inhibitors of CDK4/cyclin D1 are attractive as
prospective antitumor agents. The series of 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione
derivatives reported here represents a novel class of potent inhibitors that selectively inhibit CDK4 over
CDK2 and CDK1 activities. In the headpiece of the 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-
dione, a basic amine substitutent is required on the aniline ring for the CDK4 inhibitory activity. The inhibitory
activity is further enhanced when an aryl or heteroaryl substituent is introduced at the C-6 position of the
isoquinoline-1,3(2H,4H)-dione core. We present here SAR data and a CDK4 mimic model that explains the
binding, potency, and selectivity of our CDK4 selective inhibitors.
Introduction
pharmacological inhibition of CDK4/cyclin D1 complexes is a
useful strategy to inhibit the growth of tumors. Malumbres6
provided additional evidence that knockin mice expressing a
mutant form of cyclin D1 that binds to CDK4/6, but cannot
activate their catalytic activity, are resistant to c-neu/erbB-2
tumorigenesis in spite of undergoing normal epithelial cell
expansion during pregnancy. Moreover, knockdown of CDK4
in mammary tumor cells prevents tumor formation.6 These
results strongly suggest that inhibition of CDK4 kinase activity
might be beneficial for cancer therapy. In contrast, several
experimental results suggest that highly selective inhibition of
CDK2 may not be useful therapeutically. For example, a variety
of cancer cell lines were able to proliferate after specific and
acute depletion of CDK2 by siRNA or antisense oligonucle-
otides.7 Moreover, CDK2 knockout mice are viable.8
Cyclin-dependent kinases (CDKs), a family of serine/threo-
nine kinases, are critical regulators of cell cycle progression
that form complexes with their activating partners, the cyclins,
in order to be active.1 Different CDK/cyclin complexes serve
as switches that regulate each of the cell cycle transitions through
four distinct phases: G1, S, G2, and M. In the G1-S transition,
the retinoblastoma susceptibility protein (pRB) plays a central
role. In its hypophosphorylated state, pRB blocks cell progres-
sion from G1 to S by binding with E2F transcription family
members. In response to mitogenic stimulation, cells synthesized
D-type cyclins that complex with CDK4 and CDK6. In turn,
pRB is phosphorylated by CDK4- or CDK6-cyclin complexes
in early G1 and by CDK2/cyclin E in late G1.2 The hyperphos-
phorylation of RB causes the release of the E2F proteins, leading
to transcriptional activation of a set of genes required for entry
into S-phase of the cell cycle.3 In normal cells, CDK4/6 activities
are negatively regulated by cyclin-dependent kinase inhibitors
of the INK4 family, such as p16, a tumor suppressor, and that
of CDK2 is negatively regulated by CKIs of the Cip/Kip family,
such as p21. The CDK4/6-cyclin D-INK4-Rb pathway is
universally disrupted in human cancer.2 For example, p16 is
inactivated due to mutation, deletion, or genetic silencing, the
catalytic subunit of CDK4 is overexpressed, and the activating
partner, cyclin D1, is amplified in many human cancers. These
changes lead to uncontrolled cell proliferation, a hallmark of
cancer.4
In the past few years, several small-molecule CDK inhibitors
have been advanced to clinical trials.9 Among them, PD-
0332991 (pyridopyrimidine)10 is a selective CDK4 inhibitor,
whereas flavopiridol is a pan-CDK inhibitor, R-Roscovitine
(CYC202, Seliciclib)11 and BMS-387032 (aminothiazole)12 are
inhibitors selective for CDK2 and CDK1. When we started our
work, there were few examples of inhibitors selective for CDK4.
Among those reported, 3-(R-heteroarylaminobenzylidene)-2-
indolinones13 were noteworthy as inhibitors selective for CDK4
over CDK1.
Recently, our laboratory5 reported that inhibition of endog-
enous cyclin D1 or CDK4 expression by RNA interference
resulted in hypophosphorylation of RB and accumulation of cells
in G1 phase. This result supports the prevailing view that
Our initial chemical effort involved preparation of a few
prelimiary analogues of ring-expanded isoquinoline-1,3-dione,
a novel scaffold. To our delight, they showed selectivity for
CDK4 over CDK1 and CDK2. We then began our medicinal
chemistry effort to find analogues with enhanced potency which
maintained a favorable selectivity profile. This paper provides
details on our optimization efforts and the structure-activity
relationships (SAR) relevant to the substituents at the C-4, C-6,
and C-7 positions of the isoquinoline-1,3-dione core. In this
paper, we also present one of our inhibitors docked in the ATP
binding domain of the CDK4 mimic model to show the critical
* To whom correspondence should be addressed. Phone: (845) 602-4712.
Fax: (845) 602-5561. E-mail: tsouh@wyeth.com.
†
Chemical and Screening Sciences, Wyeth Research.
Oncology Research, Wyeth Research.
Current address: GlaxoSmithKline, 709 Swedeland Road, King of
‡
§
Prussia, Pennsylvania 19406.
|
Current address: GlaxoSmithKline, 1250 S. Collegeville Road, UP1450,
Collegeville, Pennsylvania 19426.
10.1021/jm800072z CCC: $40.75
2008 American Chemical Society
Published on Web 05/22/2008

3508 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
Scheme 1a
a (i) Urea, reflux; (ii) HC(OMe)3, Ac2O, DMF, reflux; (iii) DMF, reflux; (iv) amine, EtOH; (v) Fe, NH4Cl, MeOH, H2O, reflux.
Scheme 2a
H-binding interactions that may explain the CDK4 selectivity
as well as potency.
Chemistry
As shown in Scheme 1, compounds 5a-d were prepared
starting from the commercially available homophthalic acid 1.
Urea was mixed well with 1 and heated to yield isoquinoline-
1,3-dione 2.14 Condensation of 2 with methyl orthoformate in
the presence of acetic anhydride gave the enol ether 3, which
was further reacted with anilines 4a-d to afford the phenyle-
namines 5a-d.15 The phenylenamines could be prepared from
2 without isolation of the intermediate 3. Substituted anilines
4a-d were readily synthesized by treatment of the amines with
p-fluoronitrobenzene 6 or p-nitrobenzyl bromide 8, followed by
reduction of the nitro group.
Substituted homophthalic acids 11a-e were prepared by two
different methods, as shown in Scheme 2. Substituted methyl-
benzolic acids 9 were first treated with lithium diisopropylamide,
and the resulting dianions were then quenched with dimethyl-
carbonate16 to afford 11a-d. Alternatively, 2-chloro-4-nitroben-
zoic acid was condensed with ethyl acetoacetate in the presence
of sodium ethoxide and cuprous bromide, in refluxing ethanol,17
to produce 11e. The homophthalic acids 11a-e were converted
into the corresponding isoquinoline-1,3-diones 12a-e, using the
same method as for the preparation of 2. Additionally, refluxing
11e with acetyl chloride afforded anhydride 13, which upon
treatment with concentrated ammonium hydroxide yielded the
a (i) (for 11a-d) Lithium diisopropylamide, (MeO)2CO, then H2O; (ii)
(for 11e) ethyl acetoacetate, NaOEt, CuBr, EtOH, then NaOH; (iii) urea,
with or without 1,2- dichlorobenzene, reflux; (iv) MeCOCl, dioxane; (v)
NH4OH; (vi) 1,2-di-Cl-benzene or carbonyldiimidazole, DMF, reflux; (vii)
TBDMSCI, imidazole, DMF, room temperature; (viii) t-BuLi, THF, -78
°C; then for 12f: I2, -78 °C; for 12g: Me2NCOCl, THF, -78 °C; (ix) Pd/
C, H2; (x) for 12h: 2,4-(MeO)2-tetrahydrofuran, 4-chloropyridine HCl salt;
for 12i: Ac2O, DMF.
amide 14. Refluxing 14 in 1,2-dichlorobenzene produced the
desired isoquinoline-1,3-dione 12e.
Isoquinoline-1,3-diones bearing other substituents at C-6 such
as iodo (12f) and N,N-dimethylacetamido (12g) were prepared
from the corresponding bromo derivative 12b. Theoretically,
metal-halogen exchange of 12b would provide compounds with
other functional groups. However, because of the presence of
acidic methylene protons, the bromide 12b was converted into
1,3-disiloxyisoquinoline 15 before treatment with tert-butyl
lithium. The resulting lithium intermediate was reacted with
iodine or N,N-dimethylcarbamoyl chloride to yield 12f and 12g,
respectively. Other 6-substituted isoquinoline-1,3-diones 12h and
12i were prepared from the corresponding nitro derivative 12e.
The nitro group of 12e was reduced to amine 16, which was
either condensed with 2,4-dimethoxytetrahydrofuran catalyzed
by 4-chloropyridine hydrochloride, to yield the pyrrole derivative
12h, or reacted with acetic anhydride to produce the acetamide
derivative 12i.
As depicted in Scheme 3, various C-6 substituted isoquino-
line-1,3-diones 12a-i were converted into the enol ethers 17a-i
using the same conditions described in Scheme 1 for the
unsubstituted isoquinoline-1,3-dione (from 2 to 3). The enol

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3509
Scheme 3a
a (i) HC(OMe)3, Ac2O, DMF, reflux; (ii) 4-(N-Me-piperazinyl)aniline, DMF, reflux; (iii) 4-(N-piperidinylmethy)aniline, DMF, reflux.
Scheme 4a
Scheme 5a
a (i) Pd2(dba)3, KOt-Bu, 1,3-bis(2,6-di-isopropylphenyl)imidazonium
chloride, DMF, 100 °C, (for 20a, 21a) piperidine, (for 21b) morpholine,
(for 21c) 4-Me-piperazine, (for 21d) PhNH2.
a (i) (for 22a,b) XB(OH)2, Pd(PPh3)4, Cs2CO3, DMF, microwave, 150
°C; (for 23a-d,f,k) XB(OH)2, Pd(PPh3)4, Na2CO3, DMF, 120 °C; (for
22c-e, 23i,j) XB(OH)2, Pd2(dba)3, Ph-PhP(t-Bu)2, Na2CO3, DMF, 120 °C;
(for 23e,g,h) XB(OH)2, Pd2(dba)3, Ph-PhP(t-Bu)2, Cs2CO3, DMF, 120 °C;
(for 22f) styrene, n-Bu4NBr, (o-tolyl)3P, Cs2CO3, Pd(OAc)2, DMF, micro-
wave, 200 °C, 2 min; (for 22g) phenylacetylene, PdCl2(PPh3)2, Cul, Et3N,
DMF, 70 °C; (for 23l) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C.
ethers 17a-i were then reacted with 4-(N-methylpiperazinyl)-
aniline to form 18a,b,e-i or with 4-(piperidinylmethyl)aniline
to yield 19b-i.
As described above, the 6-bromo group of isoquinoline-1,3-
dione 12b was first converted to other functional groups
(Scheme 2), followed by introduction of the aniline headgroup
(Scheme 3). An alternate method is shown in Scheme 4, where
the aniline headgroups were already attached to the 6-bromoiso-
quinoline-1,3-dione 18b and 19b, and the 6-bromo substitutent
was subsequently converted to various cyclic amines 20a,
21a-c, and aniline 21d via Buchwald chemistry, using
Pd2(dba)3, imidazolium ionic liquid, and potassium tert-butoxide
in DMF. The 6-aryl (22a, 22e, 23a, 23e-k) and 6-heteroaryl
(22b-d, 23b-d) were also prepared from the correponding
6-bromo derivatives 18b and 19g via Suzuki cross coupling
reactions, as shown in Scheme 5. The 6-alkene 22f and 6-alkyne
22g were prepared from 18b via Heck coupling and Sonogashira
coupling, respectively. Treatment of 19b with zinc cyanide and
Pd(PPh3)4 yielded the nitrile 23l.
carrying a 6-I group to generate the desired 6-iodo-isoquinoline-
1,3-dione derivatives 34-38, respectively, as shown in Scheme
6.
Other headgroups 41a-e (Scheme 7), where the pheny ring
of aniline 4a was replaced with a pyridine, pyridazine, flurophe-
nyl, and pyrimidine, respectively, were prepared in the same
manner as aniline 4a. Whereas the pyrazine headgroup 41f was
prepared from N-methylpiperazine and aminobromopyrazine 42
protected as the N,N-diacetyl derivative. The resulting bis-amide
43 was then deprotected by acid to yield the desired headgroup
41f. These headgroups 41a-f were then coupled with the enol
ethers 17b and 17f to afford the desired final compounds 44a-f.
The halogens at C-6 were further converted into 3-furyl via
Suzuki coupling reactions to afford the 6-(3-furyl)isoquinoline-
1.3-dione derivatives 45a-f.
Headgroups carrying bulky piperizines were prepared as
shown in Scheme 6. Amination of 4-fluoronitrobenzene 6 with
commerically available N-Boc-2,5-diaza-bicyclo[2.2.1]heptane
gave 24, which upon treatment with trifluoroacetic acid afforded
25. Further methylation of the nitrogen of 25, followed by
reduction of the nitro group yielded the aniline 27. Other anilines
carrying bulky piperazines 30 and 31 were prepared in a similar
way. Anilines 27, 30-33 were reacted with enol ether 17f,
Results and Discussion
CDK4 Inhibitory Activity, Selectivity and Inhibition of
Cell Proliferation. Our initial efforts focused on modifying the
substituent on the aniline ring of 4-(phenylaminomethylene)iso-
quinoline-1,3(2H,4H)-dione. As shown in Table 1, 5a, which
has a 4-methylpiperazine group on the aniline ring, showed

3510 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
Scheme 6a
a (i) 2-Boc-(1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane, i-Pr2NEt, CH3CN, 80°C; (ii) TFA, MeOH, room temperature; (iii) HCOOH, (CH2O)n, 80 °C;
(iv) Pd/C, H2; (v) DMF, 90-120 °C, 2,6-Me2-piperazine; (vi) DMF, reflux.
moderate CDK4 activity and selectivity over CDK1 and CDK2.
In contrast, the morpholino derivative, 5b, showed IC50 values
greater than 29 µM for all three CDKs. Interestingly, addition
of a methylene bridge between the morpholine and the phenyl
ring, to provide 5c, resulted in improved CDK4 inhibitory
activity. The methylene-linked morpholine in 5c is basic,
whereas the same group in 5b linked directly to a phenyl ring
lacks basicity. This suggests that a basic amino substituent in
the 4-phenylaminomethylene headpiece is needed for CDK4
activity. As expected, the methylene-linked piperidine, 5d, is
also a CDK4 inhibitor.
Table 2 shows the effect of varying substituents at C-6 or
C-7 of 5a, which has a 4-(methylpiperazinyl)phenylami-
nomethylene headpiece at C-4 of the isoquinoline-1,3(2H,4H)-
dione core. The 6-Br derivative 18b was almost a log order
more potent in CDK4 inhibition than the 7-Br derivative 18a.
This result led us to focus solely on 6-substituted analogues.
The larger 6-iodo analogue 18f showed a 3-fold increase in
CDK4 inhibition and more than 100-fold selectivity over CDK1
and CDK2. The 6-phenyl derivative 22a is as potent and
selective as the 6-iodo derivative 18f. The 6-(4-formylphenyl)
derivative 22e was about 3-fold more potent than 22a. However,
addition of a double bond or a triple bond between the phenyl
group and the core, yielded the respective analogues 22f and
22g that showed much reduced activity. Replacement of the
phenyl group at C-6 of 22a by heteroaryls, such as 2-furyl and
3-furyl, generated the corresponding compounds 22b and 22c
that are as potent and selective as 22a. Other 6-heteroaryl
derivatives such as 6-(pyrrolyl) (18h) and 6-(3-thienyl) (22d)
analogues are 3- to 4-fold more potent than the 6-phenyl
analogue 22a, with excellent selectivity (more than 357- and
500-fold for 19h and 22d, respectively) over CDK1 and CDK2.
However, replacement of the pyrrolyl group by a piperidinyl
group to provide 20a resulted in a log order decrease in potency
for CDK4 inhibition. In addition, dramatic decreases in CDK4
activity and selectively were seen with analogues bearing
electron-withdrawing substituents at C-6, such as nitro (18e),
acetamido (18i), and N,N-dimethylcarboxamido (18g).
Effects of C-6 substituents on analogue 5d, which has a
4-(piperidinylmethyl)phenylaminomethylene headpiece of the
core at C-4, were also evaluated, as shown in Table 3. It is
interesting to see that SAR for substitution at C-6 is quite similar
for 5a and 5d. For example, comparable activity and selectivity
were seen for the pairs of 5a and 5d subseries that carry bromo
(18b vs 19b), iodo (18f vs 19f), phenyl (22a vs 23a), 3-thienyl
(22d vs 23d), pyrrolyl (18h vs 19h), piperidinyl (20a vs 21a),
nitro (18e vs 19e), acetamido (18i vs 19i), or N,N-dimethyl-
carboxamido (18g vs 19g) at C-6. The only exception is the
pair of 6-(3-furyl) derivatives, where 23b was 6-fold more potent
and selective than 22c. Among the three halogen derivatives at
C-6, the chloro analogue (19c) is the least potent, whereas the
iodo compound (19f) is most the potent, as expected. The

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3511
Scheme 7a
a (i) N-Me-piperazine, CH2Cl2; (ii) Pd/C, H2, or Fe, HOAc, MeOH; (iii) 5-Cl-pyridine HCl, N-Me- piperazine; (iv) NaH, DMF, AcCl, 0 °C; then N-Me-
piperazine, Pd2(dba)3, (t-Bu)3P, KO-t-Bu, DMF, microwave, 120°C; (v) HCl; (vi) DMF, 110°C; for 44a-d: R ) Br; for 44e,f: R ) I; (vii) 3-furanboronic
acid, Pd2(dba)3, (t-Bu)3P or Ph2t-BuP, Na2CO3 or Cs2CO3, DMF, 100-120 °C.
Table 1. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities
Table 2. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities
kinase assays (IC50)a
kinase assays (IC50)a
compd
R
CDK4
CDK1
CDK2
R
CDK4
CDK1
CDK2
5a
H
4.1
12.1
1.40
11
0.48
41
0.14
11.0
1.62
0.39
0.33
0.22
0.10
0.13
14.3
2.30
>28
>50
>50
7.2
>28
47
>50
>50
>50
47
>50
>50
>50
>50
>50
>50
>50
7.7
a
b
c
4-Me-piperazinyl
4-morpholinyl
CH2-(4-morpholinyl)
CH2-(1-piperidinyl)
4.1
>29
10
>28
>29
>27
>27
>28
>29
21
18a
18b
18e
18f
18g
18h
18i
20a
22a
22b
22c
22d
22e
22f
22g
7-Br
6-Br
6-NO2
6-I
d
3.3
19.6
>50
>50
>50
13.8
>50
>50
>50
>50
>50
8.2
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%,
as determined from the dose-response curve. Determinations were done
in duplicate and repeat values agreed, on average, with a mean 2-fold
difference.
6-C(O)NMe2
6-pyrrolyl
6-NHAc
6-piperidinyl
6-phenyl
6-(2-furyl)
6-nitrile derivative 23l was not only a poor inhibitor for CDK4,
but also lost selectivity. Introducing a methoxy or an aniline
group at C-6 on 5d to yield 19d and 21d did not affect the
potency. Replacement of the 6-phenyl group of 23a with
3-pyridyl, as in derivative 23c, resulted in enhanced potency.
Further investigation was carried out by introducing substituents
on the 6-phenyl ring of 23a. The 6-(4-fluorophenyl) derivative
23e was as potent as 23a in inhibiting CDK4 activity. Addition
of a hydroxyl substituent on the meta or para position of the
6-phenyl derivative 23a yielded 23g and 23h, respectively,
which interestingly showed a log order higher potency than the
unsubsituted 6-phenyl derivative 23a. This result suggests that
the hydroxyl group may form a hydrogen bond interaction at
the binding site of the CDK4 enzyme. Compared to the
6-(hydroxyphenyl) derivatives 23g and 23h, the corresponding
6-(3-furyl)
6-(3-thienyl)
6-[4-HC(O)-phenyl]
6-(CHdCH-phenyl)
6-(Ct sphenyl)
33.2
>50
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%,
as determined from the dose-response curve. Determinations were done
in duplicate and repeat values agreed, on average, with a mean 2-fold
difference.
6-(methoxyphenyl) derivatives 23i and 23j were less potent.
The para-methoxyphenyl derivative 23j showed a 4-fold
decrease in CDK4 potency compared to the corresponding para-
hydroxyphenyl derivative 23h. However, the meta-methoxyphe-
nyl derivative 23j lost its potency by 1000-fold with respect to
the corresponding meta-hydroxyphenyl derivative 23g. The latter

3512 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Table 3. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities
Tsou et al.
Table 4. Inhibition (IC50) of CDK4 Activity and Cell Proliferation
kinase assays (IC50)a
kinase assaya
CDK4
cell-based assaysb
compd
R
CDK4
CDK1
CDK2
compd
R
HCT116
MCF-7
5d
H
Br
Cl
3.3
1.1
2.5
2
15.1
0.3
>27
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
15.8
19.6
>50
>50
>50
>50
20.4
39.8
>50
8.1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
26.5
18b
18f
I
I
I
I
I
I
II
II
II
II
II
II
II
II
II
II
Br
I
1.4
3.1
2.8
1.5
2.8
1.2
1.4
5
2.7
2.7
1.5
2.4
1.9
1.0
1.0
1.3
1.6
3.0
2.5
2.3
7.3
1.1
2.3
3.1
1.0
2.1
2.3
2.9
1.1
3.4
0.55
2.0
3.0
19b
19c
19d
19e
19f
19g
19h
19i
0.48
0.14
0.39
0.22
0.10
1.1
0.3
0.14
1.8
0.32
0.037
0.05
0.31
0.027
0.041
18h
22a
22c
22d
19b
19f
19h
21d
23a
23b
23d
23e
23g
23h
pyrrolyl
phenyl
3-furyl
3-thienyl
Br
OMe
NO2
I
C(O)NMe2
pyrrolyl
21.2
0.14
3.5
I
NHAc
pyrrolyl
NH-Ph
phenyl
21a
21b
21c
21d
23a
23b
23c
23d
23e
23f
23g
23h
23i
piperidinyl
morpholinyl
4-Me-piperazinyl
NH-Ph
phenyl
3-furyl
1.0
0.92
34.7
1.8
3-furyl
3-thienyl
4-F-phenyl
3-OH-phenyl
4-OH-phenyl
0.32
0.037
0.05
0.13
0.31
19.4
0.027
0.041
27.8
0.13
3-pyridyl
3-thienyl
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%,
as determined from the dose-response curve. Determinations were done
in duplicate and repeat values agreed, on average, with a mean 2-fold
difference. b Dose-response curves were determined at five concentrations.
The IC50 (µM) values are the concentrations needed to inhibit cell growth
by 50%, as determined from these curves.
4-F-phenyl
4-Cl-phenyl
3-OH-phenyl
4-OH-phenyl
3-MeO-phenyl
4-MeO-phenyl
4-PhO-phenyl
CN
23j
23k
23l
>50
15.8
of the headpiece to prevent stacking. Five piperazinyl derivatives
with different degrees of steric bulk were prepared and evalu-
ated, as shown in Table 5. Compared to 18f, all five new
analogues, 34-38, showed 4- to 18-fold enhancement in
aqueous solubility. However, four of these analogues, 35-38,
lost CDK4 activity by 3- to 10-fold, although they maintained
good selectivity for CDK4 over CDK1 and CDK2. Only the
bridged piperazinyl derivative 34 showed enzyme activity
comparable to that of 18f. It is interesting to note that, although
35 was 3-fold less potent than 18f in inhibiting CDK4 activity,
it showed a 3-fold enhancement in its ability to inhibit the
proliferation of HCT116 cells.
Replacements of the p-phenylene moiety in the N-methylpip-
erazinylphenyl headpiece of 22c are shown in Table 6.
Compared to 22c, the fluorophenyl analogue 45d was almost
3-fold more potent in inhibiting CDK4 activity. Replacement
of the phenyl ring in the headgroup of 22c with a pyridine, to
provide 45a and 45b, resulted in a slight enhancement of
the CDK4 inhibitory activity. Compared to 22c, the pyrimidine
derivative 45e showed comparable CDK4 activity, but the
pyrazine isomer 45f was 6-fold less potent and the pyridazine
isomer 45c was 4-fold less potent in CDK4 inhibitory activity.
These new derivatives 45a-c,e,f bearing one or two nitrogens
in the aryl ring did not display enhanced aqueous solubility at
pH 7.4. Furthermore, no major improvement in their ability to
inhibit cell proliferation was observed.
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%,
as determined from the dose-response curve. Determinations were done
in duplicate and repeat values agreed, on average, with a mean 2-fold
difference.
result further supports the significance of the hydroxyl group
as a participant in favorable electrostatic interaction with binding
site of the CDK4 enzyme. This speculation was subsequently
corroborated by a binding model for 23g in the ATP binding
site of a CDK4 mimic, which will be discussed in detail in the
molecular modeling section. Although the 4-methoxy substi-
tutent is somewhat tolerated on the 6-phenyl, as shown in 23j,
the corresponding 6-(4-phenoxyphenyl) derivative 23k lost
inhibitory activity for all three CDKs, suggesting a space
limitation between the inhibitor and the enzyme.
The compounds were also evaluated for their ability to inhibit
the growth of selected cell lines, as shown in Table 4. Two
human carcinoma cell lines were used: HCT116 (colon), which
has deregulated cyclin D1 due to a mutation in the APC/ꢀ-
catenin pathway that regulates the cyclin D protmoter,18 and
MCF-7 (breast), which overexpresses cyclin D1 due to gene
amplification.19 Compared to the 6-halogen derivatives, the
6-aryl and 6-heteroaryl derivatives are among the slighly more
effective inhibitors of cell growth. The magnitude of the en-
hancement of CDK4 inhibitory activity does not always correlate
with the improvement of cellular activity. For example, com-
pared to the 6-phenyl derivative 23a, the 6-(hydroxyphenyl)
derivatives 23g and 23h were a log order more potent CDK4
inhibitors, however, there was no significant improvement in
cellular activity. On the other hand, the 6-(4-fluorophenyl)
derivative 23e was as potent as the 6-phenyl 23a vs CDK4, yet
the former showed 2- to 5-fold better potency than 23a in
inhibiting the proliferation of cells.
Molecular Modeling. A binding model for 23g in the ATP
binding site is presented in Figure 1. The protein coordinates
used in this study are those reported in the X-ray crystal structure
of the catalytic domain of CDK2, with three residues of the
binding site mutated to mimic CDK4.20 The CDK4 mimic
structure of Ikuta et al., includes three mutations at residues
F82H, L83V, and K89T. The resulting protein is a mutant of
the CDK2 kinase domain that possesses the CDK2 amino acid
sequence in all other regions except the ATP binding pocket,
Attention was then focused on enhancing the aqueous
solubility of 18f by adding steric bulk on the piperazinyl ring

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3513
Table 5. Inhibition (IC50) of CDKs Activities, Cell Proliferation, and Aqueous Solubility
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%, as determined from the dose-response curve. Determinations were done in
duplicate and repeat values agreed, on average, with a mean 2-fold difference. b Dose-response curves were determined at five concentrations. The IC50
(µM) values are the concentrations needed to inhibit cell growth by 50%, as determined from these curves.
Table 6. Inhibition (IC50) of CDKs Activities and Cell Proliferation
kinase assaysa
CDK1
cell-based assaysb
compd
X
Y
Z
W
CDK4
CDK2
HCT116
MCF-7
22c
45a
45b
45c
45d
45e
45f
CH
N
CH
N
CH
CH
CH
CH
CH
N
N
CF
N
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
0.22
0.13
0.11
0.82
0.08
0.25
1.25
>50
>50
>50
24.7
>50
31
>50
>50
>50
>50
>50
>50
>50
1.2
0.76
2.1
1.9
2.2
1.1
1.6
1.7
4.5
0.9
3.2
1.3
1.1
0.95
N
CH
2.2
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%, as determined from the dose-response curve. Determinations were done in
duplicate and repeat values agreed, on average, with a mean 2-fold difference. b Dose-response curves were determined at five concentrations. The IC50
(µM) values are the concentrations needed to inhibit cell growth by 50%, as determined from these curves.
where the three amino acid mutations confer a sequence that
more closely resembles that of CDK4. The amino acid number-
ing of the CDK4 mimic structure will therefore be that of CDK2,
which is the parent structure. This CDK4 mimic structure was
used by Ikuta et al.20 to study CDK4 specific inhibitors. The
inhibitor was docked using the GLIDE docking algorithm in
the XP (extra precision) mode.21 The resulting model success-
fully identifies key hydrogen bond interactions between the
ligands and residues of the protein’s ATP binding pocket. The
specific orientation of the ligand in the binding model exploits
the range of residues unique to CDK4, namely 82, 83, and 84.
We do not see any interactions between the 4-(phenylaminom-
ethylene)isoquinoline-1,3(2H,4H)-dione inhibitors and Thr 89
in our binding models. A more detailed discussion on Thr 89
follows. Although the public domain CDK4 mimic structure
used in this study does not include a CDK4 specific mutation
at residue 84, a brief discussion of our in silico structure with
residue 84 mutated to ASP follows.
In the most favored bound conformation, the NH of the
isoquinoline-1,3-dione core is found 2.0 Å from the backbone
carbonyl of Glu 81. The binding model places a carbonyl oxygen
of the isoquinoline-1,3-dione core at 2.0 Å from the backbone
NH of Val 83, a position in the binding cavity where the L83V
mutation differentiates CDK4 from CDK2. Additionally, the

3514 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
Figure 1. Proposed Binding Model for 23g in the ATP binding site of the CDK4 mimic model.
amino NH of the enamine headpiece is found within 2.6 Å from
the backbone carbonyl oxygen of Val 83. As the sequence of
amino acids confers specific spatial features to the protein cavity,
we postulate that the close proximity of these interactions lends
support to the selectivity of the isoquinoline-1,3-dione core for
the CDK4 binding pocket. A second carbonyl oxygen of the
isoquinoline-1,3-dione core is found within 4.2-4.8 Å of the
center of the phenyl ring of Phe 80. It is postulated that
interactions between at least two of the aromatic hydrogens of
the phenyl ring and the carbonyl oxygen of the isoquinoline-
1,3-dione core play a significant role in supporting inhibitor
binding. Two more residues were mutated in our computational
studies, and this is evident in the structures of the protein
complexed with 23g (Figure 1). These were carried out to
evaluate resulting models and possible protein-ligand interac-
tions. The CDK4 mimic structure of Ikuta et al. contains a His
84, which is unique for CDK2. However, we mutated this
residue to the Asp that is found at this position in CDK4.
Similarly, residue 131 in the CDK4 mimic structure is Gln,
which is a unique residue for CDK2, was mutated in our models
to Glu, which is found in CDK4. In our models, Glu 131 is
found to be approximately 5.0 Å from the hydroxy oxygen at
the meta position of the 6-phenyl substitutent in 23g and the
ring nitrogen of the piperidine headpiece of 23g is found ∼5 Å
from the His 82 side chain and approximately 6.4 Å from the
Asp 84 side chain. Both of these groups are found in solvent
exposed regions, involving very flexible side chains. Short
dynamics runs were carried out and they indicate a closer
approach of these interacting pairs, however, the distances do
not vary dramatically in our simulation runs. It is expected that
the flexibility and the solvent exposed nature of these interac-
tions will result in closer distances of approach between the
respective interacting partners, and possibly water-mediated
interactions.
numbering of these homology models differs from those of the
CDK4 mimic models used by our group, Ikuta20 and Pratt,24
by a value of 13 for the residues of the ATP binding site.
Docking studies using CDK4 homology models identified
interactions of inhibitors with the backbone NH and carbonyl
group of Val 96 (Val 83 in our model) and the carbonyl of Glu
94 (Glu 81 in our model) along with the side chain of His 95
(His 82 in our model). As well, Aubry et al.22c observed π-π
interactions with the side chain of Phe 93 (Phe 80 in our model).
A key finding of the study by McInnes et al.22a is the presence
of a positively charged moiety proximal to the Asp and Glu
residues that correspond to residues numbered 86 and 131 in
the CDK2 structure used in our study. Although the 4-(pheny-
laminomethylene)isoquinoline-1,3(2H,4H)-dione analogues of
our study are not found to interact with Asp 86, our binding
models place a piperidine moiety proximal to the His 82 and
Asp 84 side chains and a hydroxy moiety proximal to the Glu
131 side chain; all these are residues that are unique to CDK4
and found in regions of the binding pocket where protein
flexibility and the presence of solvent molecules are expected.
While the Gln 131 of the CDK4 mimic structure was mutated
to Glu 131 and short molecular dynamics simulation runs were
carried out, we believe this is insufficient to realize any
significant rearrangement resulting from protein-ligand binding.
Park et al.23 have shown that protein flexibility is crucial to
inhibitor binding and that CDK4 undergoes significant confor-
mational changes resulting from solvent and inhibitor interac-
tions. We postulate that detailed molecular dynamics studies
will identify the key role that Glu 131 plays in stabilizing
protein-4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-di-
one binding.
The K89T mutation, which places a threonine at the entrance
to the ATP binding cleft of CDK4, is singled out as a significant
source of CDK2/CDK4 selectivity by several investigators.18,24
Our binding studies, however, do not indicate any involvement
of the threonine residue in ligand binding. Pratt et al.24 have
pointed to the K89T mutation as being the primary source of
CDK4 selectivity for the bis-anilino-pyrimidine analogues. We
Other researchers such as McInnes,22a Honma,22b and
Aubry22c carried out computational studies using homology
models of CDK4, produced using the known crystal structures
of CDK2 and CDK6 as templates. As such, the amino acid

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3515
Figure 2. Effect of 18b and 19b on pRB phosphorylation in MCF-7 cells. MCF-7 cells were incubated in the presence of 18b or 19b at the
indicated concentrations for 24 h at 37 °C. Protein extracts were analyzed by SDS-PAGE, followed by immunoblotting using Rb antibody.
point to intrinsic differences in the shapes of the 4-(phenylami-
nomethylene)isoquinoline-1,3(2H,4H)-dione analogues, which
result in optimal orientations in the CDK4 binding cavity that
do not involve the threonine residue in question. Consequently,
the 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione
inhibitors of our study represent a class of molecules that form
interactions with several CDK4 specific amino acids that are
described in earlier literature, as well as with the side chains of
His 82 and Asp 84, both of which are CDK4 specific amino
acid residues proximal to the hinge region of the CDK4 catalytic
domain.
We believe that the protein-ligand interactions detailed in
our binding models characterize the selectivity and potency of
the 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione
analogues of this study with corroboration of several trends in
the SAR. First, the interactions of the inhibitor core with the
backbone of residues 81-83, and the presence of a basic
nitrogen proximal to the side chains of His 82 and Asp 84, as
shown in Figure 1, are fundamental to inhibitor potency. The
R groups, listed in Table 3, that facilitate such an orientation
(i.e., compounds 19f, 23a, 23c, 23d, 23g, 23h) result in increased
potency. However, bulky R groups (i.e., compounds 23f, 23k)
and R groups with undesirable moieties (i.e., compounds 19g,
21c) where steric hindrance or electrostatic incompatibility with
residues such as Glu 131 and Lys 129, preclude the optimal
alignment of the basic piperidinyl nitrogen. A similar trend is
seen in Table 2, where R groups that facilitate the basic nitrogen
of the piperizinyl moiety to orient proximally to His 82 and
Asp 84 (i.e., compounds 18f, 22a, 22d) render enhanced potency
with excellent selectivity. In contrast, the best binding models
obtained for compound 5b of Table 1 place a morpholinyl
oxygen proximal to the side chains of His 82 and Asp 84, where
we see a significant drop in potency.
Inhibition of pRB Phosphorylation and Cell Cycle
Analysis. The biological activity of 18b and 19b was evaluated
in MCF-7 cells, which overexpress cyclin D1 due to gene
amplification. We first examined the phosphorylation state of
pRB, the physiological substrate for CDK4/cyclin D complexes,
after overnight incubation with 18b or 19b. As shown in Figure
2, treatment of MCF-7 cells with either compound, resulted in
a reduction of phosphorylation of pRB, as indicated by the
decrease of the band corresponding to ppRB in polyacrylamide-
SDS gels. Inhibition was clearly detected at the lowest
concentration tested (0.5 µM) and increased in a concentration-
dependent manner. This effect is observed at a concentration
which is consistent with the effect of these compounds in the
cellular proliferation assays (IC50 3 µM). Inhibiton of pRB
phosphorylation was also confirmed with a specific antibody
against the phosphorylated Ser 807/811 in pRB (data not
shown). Consistent with the effect of these compounds on
inhibition of pRB phosphorylation, overnight treatment of
MCF-7 cells with these inhibitors (18b and 19b) caused a G1-
phase cell cycle arrest, as indicated by the decrease in the
S-phase fraction in flow cytometry (Figure 3). Compared with
untreated cells, the percentage of cells in S-phase decreased with
increasing concentration of the inhibitors. Almost complete
disappearence of S-phase cells (2-3% of total cells) was
observed with 3 µM of each compound, corroborating the data
from the pRB phosphorylation studies and the cell proliferation
assays. No significant sub-G1 fraction was observed in these
experiments, indicating that the primary effect of these com-
pounds is cell cycle arrest rather than apoptosis. We next
examined the effects of inhibiting CDK4 on other cell cycle
regulatory proteins. Overnight treatment of MCF-7 cells with
19b results in substantial decrease in cyclin D1 levels (85% at
7.5-10 µM). Smaller decreases were observed for CDK4 (60%
at 7.5-10 µM), CDK2 (55% at 7.5 µM), cyclin B (60% at 4
µM), and CDK1 (40% at 7.5 µM), as shown in Figure 4. There
appeared to be a slight overall increase (5-20%) in cyclin E
levels at all concentrations >2 µM. This could result from
compensatory effects of cyclin D1 loss. Alternatively, it may
reflect arrest of the cells in late G1, when cyclin E levels begin
to rise. Surprisingly, although the flow cytometric analyses
indicate a cell cycle arrest at G1, a decrease in the cell cycle
kinase inhibitor p27 was also observed (45% decrease at 7.5
µM). Taken together, the biological activities of these com-
pounds are consistent with the expected effects of CDK4
inhibition.
Conclusions
In summary, we reported the discovery of 4-(phenylaminom-
ethylene)isoquinoline-1,3(2H,4H)-dione derivatives as a novel
class of potent inhibitors that selectively inhibit CDK4 over
CDK2 and CDK1. We have shown that attaching a basic
functional group onto the 4-(phenylaminomethylene) headpiece
is essential for CDK4 inhibition. Systematic addition of a variety
of substituents on the C-6 and C-7 positions of the isoquinoline-
1,3(2H,4H)-dione led us to identify 23g, carrying a 3-hydrox-
yphenyl substituent at C-6, that is 122-fold more potent than
the corresponding C-6 unsubstituted compound 5d. We also
presented a proposed binding model for 23g at the ATP binding
site of a CDK4 mimic structure that contains three mutations
at residues F82H, L83V, and K89T with two additional in silico
mutations at H84D and Q131E. On the basis of this binding
model, several hydrogen bond interactions were postulated
between the NH and carbonyl oxygen of the core, the NH of
the enamine headpiece, the basic amine on the aniline ring of
the 4-(phenylaminomethylene) headpiece and the amino acid
residues that are unique to the CDK4 enzyme. These interactions
could explain the high selectivity of this series of compounds
for CDK4 over CDK2 and CDK1. In cells, selected compounds
18b and 19b from this series have demonstrated biological
activity: they inhibit phosphorylation of pRB, the physiological
substrate for CDK4/cyclin D1 and cause cell cycle arrest.

3516 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
Figure 3. Effect of 18b and 19b on cell cycle progression in MCF-7 cells. MCF-7 cells were treated with the indicated concentrations of 18b or
19b for 24 h at 37 °C. Untreated and treated cells were pulse-labeled for 30 min with BrDU. Cells were stained with BrDU antibodies/FITC
conjugates, counterstained with propidium iodide and analyzed by flow cytometry. The percentage of cells in S-phase of the cell cycle is shown as
determined from the histogram of propidium iodide-stained cells.
Figure 4. Effect of 19b on cell cycle regulatory proteins. MCF-7 cells were incubated in the absence or presence of 19b at the indicated doses for
24 h at 37 °C. Protein extracts were analyzed by SDS-PAGE, followed by immunoblotting using the indicated antibodies. Equivalent protein
loading was confirmed using actin antibodies.
purifications were by flash chromatography using Baker 40 µ silica
gel. Melting points were determined in open capillary tubes on a
Meltemp melting point apparatus and are uncorrected. Semiprepara-
tive reverse-phase high-pressure liquid chromtography (RP-HPLC)
was performed using a Gilson Preparatory HPLC. The sample was
dissolved in DMSO, applied on a Phenomenex C18 Luna column
(21.2 mm × 60 mm, 5 µ), and eluted at 22.5 mL/min with a 19
min of gradient: 5% B; 2.5 min: 5% B; 18 min: 95% B; 19 min:
95% B, where solvent A ) water (0.02% TFA buffer) and solvent
B ) acetonitrile (0.02% TFA buffer). Analytical high-pressure
liquid chromatograpy (HPLC) and LC-MS analyses were conducted
using the following two instruments and conditions. LCMS1:
analytical HPLC was conducted on an HP 1100 liquid chromatog-
raphy system over a 2.1 mm × 30 mm xterra C18 Luna column (5
µ) at 50 °C using multiple wavelength UV detection (typically 215,
Consistent with these effects, the compounds inhibit the
proliferation of cells in vitro. Further refinement of these series
of compounds is required to demonstrate antitumor activity in
animal tumor models.
Experimental Section
Chemistry. 1H NMR spectra were determined with a Bruker
DRX400 spectrometer at 400 MHZ or a NT-300 WB spectrometer
at 300 MHz. Chemical shifts (δ) are expressed in parts per million
relative to the internal standard tetramethylsilane. Electrospray mass
spectra were recorded in positive mode on a Micromass Platform
spectrometer. Electron impact (EI) and high-resolution mass spectra
(HRMS) were obtained on a Finnigan MAT-90 spectrometer. Some
high-resolution electrospray mass spectra with higher precision were
obtained on a Brucker 9.4T FTMS spectrometer. Chromatographic

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3517
254 nm) and MS detection (API-ES scanning mode positive/
negative 100-1000, fragmentor: positive 140 mV, negative 170
mV). A gradient elution of increasing concentrations of acetonitrile
in water containing 0.02% formic acid (10-90% over 3.5 min, and
was held at 90% for additional 1.5 min) and a flow rate of 1 mL/
min were employed. LCMS2: Analytical HPLC was conducted on
a Agilent LC-1100-MSD liquid chromatography system over a 50
mm × 2.1 mm Aquasil C18 column (Thermo Electron Corporation,
5 µ) using multiple wavelength UV detection (typically 215, 254
nm) and MS detection (single quadrupole mass filter scanning from
100-1000 Da). A gradient elution of increasing concentrations of
acetonitrile in water containing 0.1% formic acid (0-100% over
2.5 min, and was held at 100% for additional 1.5 min) and a flow
rate of 1 mL/min were employed.
Molecular Modeling. The inhibitor structures were minimized
using the MMFF94 force field of SYBYL.25 A public domain
structure of CDK420 was used as the 3-D representation for
molecular docking studies. This is a CDK4 mimic structure with
the mutations in the ATP binding cleft at residues F82H, L83V,
and K89T. The inhibitor structures were docked using the GLIDE21
docking algorithm in the XP (extra precision) mode. Details of the
algorithm are found in GLIDE documentation. Briefly, GLIDE’s
proprietary conformational expansion and exhaustive search of the
binding site produces a multitude of ligand poses that undergo an
initial refinement, energy minimization on a precomputed grid, and
a final scoring and ranking. GLIDE uses proprietary scoring
functions that are variations of the ChemScore26 empirical scoring
function and the OPLS-AA27 force field to compute van der Waals
and electrostatic grids for the receptor. The final ligand binding
poses are ranked according to a computed E model score that
encompasses the grid score, the proprietary Glide Score, and the
internal energy strain. The HIS to ASP mutation at position 84
and the GLN to GLU mutation at position 131 were carried out
using the GLIDE software package. The molecular dynamics
simulations mentioned above were carried out using the SYBYL
software package and the MMFF94 force field.
Preparation of the Substituted Cores. 4-Bromo-2-(carbo-
xymethyl)benzoic acid (11b). In a 500 mL 3-neck round-bottom
flask, an amount of diisopropylamine (28.0 mL, 200 mmol) in 65
mL of tetrahydrofuran was cooled to -78 °C and slowly added
80.0 mL (200 mmol) of n-butyllithium (2.5 M in hexane) with
vigorous stirring. Allow to warming up to 0 °C and keeping at this
temperature for 5 min, the reaction was cooled back to -78 °C.
To this mixture was slowly added a solution of 10.8 g (50.0 mmol)
of 4-bromo-2-methylbenzoic acid (9b) and 8.42 mL (100 mmol)
of dimethylcarbonate in 65 mL of tetrahydrofuran keeping the
internal temperature of the reaction mixture below -50 °C. After
addition, the dry ice bath was removed and the reaction mixture
was stirred at room temperature for 4 h. Precipitate was observed
as the internal temperature raising to room temperature. The reaction
was quenched with 80 mL of water and stirred overnight to give a
homogeneous solution. The organic layer was separated, and the
aqueous solution was acidified with concentrated HCl to pH ∼ 2
and extracted with 3 × 100 mL of ethyl acetate. The combined
organic solution was washed twice with water. After drying over
MgSO4, the organic solution was filtered and concentrated to give
a white solid. Recrystallization from EtOAc (hot)/hexane yielded
8.86 g (68.2%) of white solid. 1H NMR (DMSO-d6) δ ppm: 12.62
(bs, 1H); 7.82 (d, J ) 6.3 Hz, 1H), 6.18 (m, 2H), 3.95 (s, 2H). MS
(ESI) m/z 257.1 and 259.1 (M - H)-1. Anal. (C9H7BrO4 ·0.2
EtOAc) C, H.
(d, J ) 8.1 Hz, 1H), 7.66 (s, 1H), 7.92 (d, J ) 8.1 Hz, 1H) 11.37
(s, 1H). Anal. (C9H6BrNO2) C, H, N.
(4E)-6-Bromo-4-(methoxymethylene)isoquinoline-1,3(2H,4H)-
dione (17b). A solution of 12b (4.15 g, 7.29 mmol), 3.78 mL (34.55
mmol) of trimethyl orthoformate, and 30 mL of acetic anhydride
in 10 mL of N,N′-dimethylformamide was heated at 120 °C under
N2 for 2 h. After cooling, ethyl ether was added, and the precipitate
was collected and washed successively with MeOH, Et2O, and
hexane. After drying in oven (60 °C) overnight, 3.4 g (70% yield)
of 17b was obtained as a solid. MS (ESI) m/z 281.9 (M + H)+1
.
1H NMR (400 MHz, DMSO-d6) δ ppm: 4.26 (s, 3H), 7.62 (dd, J
) 8.1, 2.2 Hz, 1H), 7.98 (d, J ) 8.1 Hz, 1H), 8.08 (s, 1H), 8.38 (d,
J ) 2.2 Hz, 1H), 11.40 (s, 1H). Anal. (C11H8BrNO3) C, H, N.
6-Bromo-1,3-bis{[tert-butyl(dimethyl)silyl]oxy}isoquinoline
(15). A solution of 12b (1.0 g, 4.17 mmol), 1.875 g (12.51 mmol)
of tert-butyldimethylsilyl chloride, and 1.13 g (16.68 mmol) of
imidazole in N,N′-dimethylformamide was stirred at room temper-
ature overnight. After evaporating to dryness, the brown oil was
extracted with 4 × 100 mL of 25% diethyl ether/hexane. The
organic solution was washed with 3 × 100 mL of water, dried over
MgSO4, filtered, and concentrated to give a brown oil. This brown
oil was dissolved in 50 mL of 20% CH2Cl2/hexane and passed
through a pad of magnesol, followed by rinsing with 500 mL of
the same solvent mixture. The organic solution was concentrated
to give 1.184 g (60.6%) of 15 as a colorless solid. MS (ESI) m/z
468.2 and 470.2 (M + H)+1. 1H NMR (400 MHz, CDCl3) δ ppm:
0.31 (s, 6H), 0.42 (s, 6H), 1.00 (s, 9H), 1.07 (s, 9H), 6.46 (s, 1H),
7.33 (dd, J ) 8.81, 1.76 Hz, 1H), 7.71 (d, J ) 1.76 Hz, 1H), 7.94
(d, J ) 8.81 Hz, 1H). Anal. (C21H34BrNO2Si2) C, H, N.
6-Iodoisoquinoline-1,3(2H,4H)-dione (12f). An amount of 15
(15.0 g, 32.0 mmol) in 100 mL of anhydrous tetrahydrofuran was
cooled to -78 °C and then 47 mL (80.0 mmol) of tert-butyllithium
(1.7 M in pentane) was added slowly with stirring. After stirring
at this temperature for 2 h, 12.0 g (48 mmol) of fresh iodine crystal
was quickly added into the mixture and stirred at this temperature
for additional 5 h. The dry ice bath was removed, and the reaction
mixture was allowed to warm up to room temperature and stirred
over a weekend. Evaporating the brown solution yielded a brown
oil. The reaction mixture was acidified with 48 mL of 2 M HCl
solution and stirred at room temperature for 1 h. The mixture was
filtered to give a light-tan solid. The solid was dissolved in hot
DMSO, and then 20% MeOH/H2O solution was added to give a
precipitate. The precipitate was collected and washed successively
with water, methanol, ether, and hexane to afford 5.1 g (55.6) of
1
12f as an off-white solid. MS (ESI) m/z 286.08 (M - H)-1. H
NMR (400 MHz, DMSO-d6) δ ppm: 4.01 (s, 2H), 7.73 (d, J ) 8.2
Hz, 1H), 7.83 (d, J ) 8.2 Hz, 1H), 7.84 (s, 1H), 11.35 (s, 1H).
6-(N,N-Dimethylcarbamyl)isoquinoline-1,3(2H,4H)-dione (12g).
An amount of 15 (0.5 g, 1.07 mmol) in 10 mL of anhydrous
tetrahydrofuran was cooled to -78 °C and then 1.42 mL (2.41
mmol) of tert-butyllithium (1.7 M in pentane) was added slowly
with stirring. After stirring at this temperature for 30 min, 0.118
mL (1.28 mmol) of N,N-dimethylcarbamyl chloride was added and
stirred at this temperature for 45 min. After it was warmed up to
room temperature, 1.6 mL (3.2 mmol) of 2 M aqueous HCl solution
was added and stirred for 3 h. The solvents were removed, and the
resulting gum was purified by column chromatography eluted with
5% methanol in chloroform. The desired fractions were pooled and
dried to give 42 mg (57%) of 12g as a yellow solid. MS (ESI) m/z
1
233.2 (M + H)-1. H NMR (400 MHz, DMSO-d6) δ ppm: 2.87
(s, 3H), 2.98 (s, 3H), 4.06 (s, 2H), 7.40 (s, 1H), 7.45 (d, J ) 6.9
Hz, 1H), 8.05 (d, J ) 6.9 Hz, 1H), 8.05 (s, 1H), 11.38 (s, 1H).
2-Carboxy-5-nitrobenzeneacetamide (14). A stirred mixture of
2-carboxy-5-nitrobenzeneacetic acid, 11e (2.25 g, 10 mmol)
(prepared from 2-chloro-4-nitrobenzoic acid, 10 according to
literature procedure28), 2.5 mL (35 mmol) of acetyl chloride, and
8 mL of acetone was refluxed for 60 min. The resulting solution
was evaporated to dryness. The resulting tan solid was shown to
6-Bromoisoquinoline-1,3(2H,4H)-dione (12b). A suspension of
11b (343 g, 1.324 mol) and urea (171.5 g, 2.858 mol) in
1,2-dichlorobenzene (3 L) was stirred and heated. The starting
material went into solution at 115 °C and urea melted at 120 °C.
The product started to precipitate as a yellow solid at 135 °C. The
reaction mixture was heated at 150 °C for 2.5 h. After it was cooled
to room temperature, the yellow solid was collected by filtration,
washed with ethyl acetate, water, and methanol and air-dried to
give 271.6 g (85.5%) of 12b as a solid. (ESI) m/z 237.9 (M -
1
be the corresponding cyclic anhydride, 13, by H NMR (DMSO-
d6) (a singlet of two protons at δ 4.40). The anhydride 13 was mixed
at 0 °C with 16 mL of conc NH4OH and 16 mL of H2O. The
1
H)-1. H NMR (400 MHz, DMSO-d6) δ ppm: 4.04 (s, 2H), 7.65

3518 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
resulting mixture was warmed to 25 °C, stirred 15 min, and
evaporated to dryness at <30 °C. The residue was stirred in 25
mL of H2O, acidified at 10 °C with 4 mL of 4 N HCl, and stirred
10 min. The resulting tan solid was filtered, washed with H2O, and
dried to give 2.01 g (90%) of 14 as a solid: mp 185-190 °C (dec).
1H NMR (DMSO-d6) δ ppm: 13.50 (s, 1H), 8.20 (d, J ) 2.4, 1H),
8.16 (dd, J ) 2.4, 8.4, 1H), 8.02 (d, J ) 8.4, 1H), 7.47 (s, 1H),
6.96 (s, 1H), 3.96 (s, 2H). MS (ES-) m/z 223.1 (M - H)-1. Anal.
(C9H8N2O5) C, H, N.
carbonate, and extracted three times with ethyl acetate. The organic
solution was dried with magnesium sulfate and evaporated to afford
6 g (99%) of 4c as an orange solid.
3,5-Dimethyl-1-(4-nitrophenyl)piperazine (28). An amount
4-nitrophenylfluoride, 6 (2.0 mL, 18.85 mmol), in 20 mL of
acetonitrile was added to 2,6-dimethylpiperazine (2.58 g, 22.62
mmol). The reaction mixture was reflux under N2 overnight. Mass
spectroscopy suggested the completion of reaction. Solvent was
subsequently evaporated under vacuum. The collected yellow solid
was dissolved in chloroform and washed twice with 200 mL of
saturated NaHCO3 solution and once with 100 mL of brine. The
organic portion was dried over MgSO4, filtered, evaporated to give
yellow solid, which was recrystallized from EtOAc/hexane to give
3.98 g (89.8%) of 28 as bright-yellow crystals: mp 124-125 °C.
MS (ESI) m/z 236.1 (M + H)+1. 1H NMR (400 MHz, DMSO-d6)
δ ppm: 1.03 (d, J ) 6.04 Hz, 6H) 2.30 (s, 1H) 2.34-2.45 (m, 2H)
2.75 (d, J ) 3.02 Hz, 2H) 3.89 (dd, J ) 12.34, 2.01 Hz, 2H) 7.02
(d, J ) 9.57 Hz, 2H) 8.03 (d, J ) 9.32 Hz, 2H). Anal. (C12H17N3O2)
C, H, N.
6-Nitroisoquinoline-1,3(2H,4H)-dione (12e). A stirred suspen-
sion of 14 (11.1 g, 49.3 mmol) in 99 mL of 1,2-dichlorobenzene
was refluxed for 3 h. The residue obtained after evaporation of the
solvent under vacuum was washed with ether and dried to give
7.34 g (72%) of 12e as a tan solid: mp 255-260 °C (dec). 1H
NMR (DMSO-d6) δ ppm: 11.6 (s, 1H), 8.2-8.3 (m, 3H), 4.17 (s,
2H). MS (ES-) m/z 205.2 (M - H)-1
.
(4E)-4-(Methoxymethylene)-6-nitroisoquinoline-1,3(2H,4H)-
dione (17e). To a stirred mixture of 12e (0.41 g, 2.0 mmol), 3.2
mL (34 mmol) of acetic anhydride, and 0.80 mL of DMF was added
trimethyl orthoformate (0.44 mL, 4.0 mmol). The mixture was
heated to 125 °C and maintained for 30 min, cooled, diluted with
ether, and stirred for 10 min. The resulting brown solid was filtered,
washed with ether, and dried to give 372 mg (74%) of 17e as a
[4-(3,5-Dimethylpiperazin-1-yl)phenyl]amine (30). An amount
of 28 (1.86 g, 7.90 mmol) in EtOH was hydrogenated with Pd/C
catalyst for 4 h. The solid was removed by filtration, and solvent
was evaporated under vacuum to give a pinkish residue. Recrys-
tallization of this residue from MeOH/ether gives 1.30 g (80.2%)
of 30 as pinkish crystals: mp 124-125 °C. MS (ESI) m/z 206.1
1
solid. H NMR (DMSO-d6) δ ppm: 11.55 (s, 1H), 8.99 (d, J )
2.0, 1H), 8.30 (d, J ) 8.6, 1H), 8.19 (dd J ) 2.0, 8.6, 1H), 4.33 (s,
3H).
1
(M + H)+1. H NMR (400 MHz, CDCl3) δ ppm: 1.12 (d, J )
6-Aminoisoquinoline-1,3(2H,4H)-dione (16). A solution of 12e
(6.19 g, 30 mmol) in 15 mL of MeOH and 150 mL of DMF was
hydrogenated at 1 atm of H2 at 25 °C in the presence of 1.5 g of
10%Pd/C for 7 h. The catalyst was removed by filtration through
a pad of Celite. The filtrate was evaporated to give 5.4 g (100%)
6.55 Hz, 6H) 2.21 (t, J ) 10.83 Hz, 3H) 2.86-3.20 (m, 3H) 3.32
(dd, J ) 11.83, 2.27 Hz, 3H) 6.56-6.69 (m, 2H) 6.75-6.91 (m, 2
H). Anal. (C12H19N3) C, H, N.
1-Methyl-4-(5-nitro-pyridin-2-yl)-piperazine (40b). An amount
of 2 g (9.85 mmol) of 2-bromo-5-nitro-pyridine (39b) was stirred
in dichloromethane (50 mL), followed by addition of 1-methylpip-
erazine (10.9 mL, 98.5 mmol). The reaction mixture was refluxed
for 1 h. After cooling, the mixture was washed with sodium
bicarbonate, followed by additional washing with brine, dried over
sodium sulfate, filtered, and evaporated to afford 2 g (91%) of 40b
as yellow crystals: mp 75-76 °C. MS (ESI) m/z 223.1 (M + 1)+.
6-(4-Methylpiperazin-1-yl)pyridin-3-amine (41b). An amount
of 1 g (4.48 mmol) of 40b was dissolved in methanol (50 mL),
followed by catalytic amount of 10% Pd/C. The mixture was
hydrogenated at 35-40 psi for 3 h. After filtration through a pad
of Celite, the solution was evaporated to give 900 mg (100%) of
41b as a purple solid: mp 97-98 °C. MS (ESI) m/z 193.1 (M +
1)+.
1
of 16 as a tan solid: mp 200-220 °C (dec). H NMR (400 MHz,
DMSO-d6) δ ppm: 10.75 (s, 1H), 7.66 (d, J ) 8.8, 1H), 6.55 (dd,
J ) 2.1, 8.8, 1H), 6.36 (d, J ) 2.1, 1H), 6.09 (s, 2H), 3.82 (s, 2H).
MS (ES+) m/z 177.2 (M + H)+1. Anal. (C9H8N2O2) C, H, N.
6-Pyrrol-1-yl-isoquinoline-1,3(2H,4H)-dione (12h). A solution
of 16 (1 g, 5.676 mmol), 2,4-dimethoxytetrahydrofuran (0.736 mL,
5.676 mmol), 4-chloropyridine hydrochloride (0.417 g, 2.78 mmol)
in 20 mL of dioxane was heated at 70 °C for 2 h. After filtration,
the filtrate was evaporated to dryness. The residue was washed with
ether, filtered, and washed with water, ether, and hexane to give
1
0.86 g (67%) of 12h as a light-brown solid. H NMR (400 MHz,
DMSO-d6) δ ppm: 4.06 (s, 2H), 6.33 (m, 2H), 7.51 (m, 2H), 7.66
(d, s, 1H), 7.71 (dd, J ) 8.3, 2.1 Hz, 1H), 8.05 (d, J ) 8.3 Hz,
1H), 11.28 (s, 1H). MS (ES+) m/z 227.1 (M + H)+1. Anal.
(C13H10N2O2 ·0.4 H2O) C, H, N.
[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amine (41a). Using the
procedure described for the preparation of 41b, 2.6 g (90%) of 41a
was obtained as a purple solid from 3.4g (15.3 mmol) of 1-methyl-
4-(2-nitro-pyridin-2-yl)-piperazine (40a). MS (ESI) m/z 193.1 (M
+ 1)+
N-(1,3-Dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl)acetamide
(12i). A solution of 500 mg (2.8 mmol) of 16 in 2 mL of N,N-
dimethylacetamide at 0 °C was dropwise added to 1.1 mL (14.2
mmol) of acetyl chloride and 1.1 mL (14.2 mmol). After 0.5 h, it
was warm up to room temperature. After the solvents were removed,
the solid was isolated and washed with ether to give 450 mg (73%)
of 12i as a green solid. MS (ESI) m/z 217.1 (M-1). 1H NMR (400
MHz, DMSO-d6) δ ppm: 3.65 (s, 3 H), 3.81 (s, 3H), 4.66 (d, J )
6.30 Hz, 2H), 7.15-7.43 (m, 3H), 7.70 (d, J ) 8.56 Hz, 1H), 7.87
(d, J ) 8.31 Hz, 1H), 8.10 (d, J ) 1.51 Hz, 1H), 8.71 (d, J )
13.35 Hz, 1H), 9.37 (s, 1H), 10.53-10.76 (m, 1H), 11.10 (s, 1H).
Preparation of the Headgroups. 4-(N-Nitro-benzyl)-morpho-
line (7c). An amount of 10 g (46.30 mmol) of 1-bromomethyl-4-
nito-benzene (8) was stirred in dichloromethane (125 mL), followed
by addition of triethylamine (12.90 mL, 92.6 mmol) and morpholine
(4.03 g, 46.30 mmol). The reaction mixture was refluxed for 1 h,
subsequently washed three times with aqueous sodium bicarbonate,
dried over sodium sulfate, followed by evaporation to dryness, to
give 7.5 g (73%) of 7c as white crystals.
.
[6-(4-Methylpiperazin-1-yl)pyridazin-3-yl]amine (41c). An
amount of 1.0 g (7.72 mmol) of 3-amino-6-chloropyridazine (39c),
5-chloro pyridine hydrogen chloride (4.46 g, 38.6 mmol), and
N-Me-piperazine (5.1 mL, 72 mmol) were placed in a preheated
oil bath at 165-170 °C for 4 h. After cooling, the mixture was
basified with saturated potassium carbonate, extracted with ethyl
acetate, and dried over magnesium sulfate. The oily residue was
purified by column chromatography to give 800 mg (53%) of 41c
as a brown solid. MS (ESI) m/z 194.3 (M + 1)+.
1-(2-Fluoro-4-nitrophenyl)-4-methylpiperazine (40d). Using
the procedure described for the preparation of 40b, 4.89 g (45%)
of 40d as a light-brown solid was obtained from 5 mL (45.16 mmol)
of 3,4-difluoronitrobenzene (39d) and 5 mL (45.16 mmol) of
N-methyl-piperazine in 20 mL of dichloromethane and 3 equivalent
of triethylamine: mp 69-70 °C. MS (ESI) m/z 240.1 (M + H)+1
.
1H NMR (400 MHz, CDCl3) δ ppm: 2.37 (s, 3H) 2.48-2.81 (m,
4H) 3.20-3.44 (m, 4H) 6.92 (t, J ) 8.81 Hz, 1H) 7.90 (dd, J )
13.22, 2.64 Hz, 1H) 7.98 (dd, J ) 9.06, 1.01 Hz, 1H).
4-Mopholin-4-yl-methyl-phenylamine (4c). Seven g (31.52
mmol) of 7c, ammonium chloride (15.14 g, 283.68 mmol), and
iron (10.56 g, 189.12 mmol) were added to 266 mL of methanol/
water (4.75:1) and refluxed until there was no appearance of starting
materials. After filtering through celite, the solvent was evaporated.
The residue was dissolved in water, basified with potassium
[3-Fluoro-4-(4-methylpiperazin-1-yl)phenyl]amine (41d). Us-
ing the procedure described for the preparation of 41b, 3.8 g
(90.5%) of 41d was obtained as a colorless solid from 4.8 g (20.06
mmol) of 40d: mp 89-90 °C. MS (ESI) m/z 210.1 (M + H)+1. 1H

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3519
NMR (400 MHz, CDCl3) δ ppm: 2.35 (s, 3H) 2.59 (s, 4H) 3.01 (s,
4H) 3.54 (s, 2H) 6.19-6.53 (m, 2H) 6.81 (t, J ) 9.06 Hz, 1H).
[2-(4-Methylpiperazin-1-yl)pyrimidin-5-yl]amine (41e). By the
procedure described above for 41b, 2-(4-methyl-piperazin-1-yl)-
5-nitro-pyrimidine29 (40e) (0.446 g, 2 mmol) was reduced in the
presence of iron (0.432 g, 7.7 mmol), acetic acid (0.884 mL), and
methanol (8 mL) to give 0.3 g (78%) of 41e. MS (ESI) m/z 194.1
(M + 1)+.
1H NMR (400 MHz, DMSO-d6) δ ppm: 1.39 (m, 2H), 1.45-1.54
(m, 4H), 2.32 (s, 4H), 3.41 (s, 2H), 7.21-7.39 (m, 3H), 7.50 (d, J
) 8.3 Hz, 2H), 7.55-7.72 (m, 1H), 8.03 (dd, J ) 7.93, 1.4 Hz,
1H), 8.16 (d, J ) 8.3 Hz, 1H), 8.88 (d, J ) 12.6 Hz, 1H), 11.32 (s,
1H), 12.41 (d, J ) 12.6 Hz, 1H). Anal. (C22H23N3O2 ·0.6H2O) C,
H, N.
(4Z)-7-Bromo-4-({[4-(4-methylpiperazin-1-yl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (18a). By the procedure
described above for 5a, 300 mg (1.06 mmol) of (4E)-7-bromo-4-
(methoxymethylene)isoquinoline-1,3(2H,4H)-dione (17a) and (211.10
mg, 1.06 mmol) of 4-(4-methylpiperazin-1-yl)-phenylamine (4a)
were reacted to give 380 mg (81%) of 18a as a green-yellow solid:
MS (ESI) m/z 441.33 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 2.23 (s, 3H), 2.39-2.48 (m, 4H), 3.07-3.19 (m, 4H), 6.99
(d, J ) 8.9 Hz, 2H), 7.44 (d, J ) 8.9 Hz, 2H), 7.73 (dd, J ) 8.4,
2.2 Hz, 1H), 8.12 (dd, J ) 8.4, 2.2 Hz, 2H), 8.84 (d, J ) 12.8 Hz,
1H), 11.40 (s, 1H), 12.50 (d, J ) 12.8 Hz, 1H). Anal. (C21H21-
BrN4O2 ·0.5H2O) C, H, N.
[5-(4-Methylpiperazin-1-yl)pyrazin-2-yl]amine (41f). An amount
of 3.0 g (17.24 mmol) of 2-amino-5-bromopyrazine (42) was
dissolved in N,N-dimethyformamide (20 mL) and cooled to 0 °C.
Sodium hydride (1.05 g, 43.09 mmol) was added and stirred at 0
°C for 10 min. Acetyl chloride (6.2 mL, 86.2 mmol) was added
and stirred at room temperature overnight. The mixture was
quenched with water and extracted with ether. The organic layer
was dried over magnesium sulfate, dried, and purified by column
chromatography to give 1.35 g (31%) of N-acetyl-N-(5-bromo-
pyrazin-2-yl)-acetamide as a yellow oil. MS (ESI) m/z 259.0 (M
+ 1)+. A mixture of N-acetyl-N-(5-bromo-pyrazin-2-yl)-acetamide
(1.33 g, 5.14 mmol), N-methylpiperazine (2.9 mL, 25.7 mmol),
Pd2(dba)3 (470 mg, 0.1 mmol), (t-Bu)3P (1 mL, 10.28 mmol), KO-
t-Bu (1.15 g, 10.28 mmol) in 10 mL of DMF was heated at 120 °C
in a microwave for 20 min. After it was filtered, it was dried and
washed with ether to give 500 mg (35%) of N-acetyl-N-(4-methyl-
3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-acetamide (43). Then
43 (500 mg, 1.8 mmol) was stirred with 6 mL of conc hydrochloric
acid at room temperature overnight. It was basified with potassium
carbonate and extracted with methylene chloride. It was dried to
give 200 mg (57%) of 41f as a dark oil. MS (ESI) m/z 195.1 (M +
1)+.
Preparation of the Final Compounds. (4Z)-4-({[4-(4-Meth-
ylpiperazin-1-yl)phenyl]amino}methylene)isoquinoline 1,3(2H,4H)-
dione (5a). A mixture of 4-methoxymethylene-4H-isoquinoline-
1,3-dione (3) (101.5 mg, 0.5 mmol) and 4-(4-methyl-piperazin-1-
yl)-phenylamine(4a)(95.6mg,0.5mmol)in1mLofdimethylformamide
was heated at 110 °C for 1 h. After cooling in the refrigerator, the
precipitate was collected, and washed with ether to give 163 mg
(90%) of 5a as a yellow solid: mp 245-246 °C. MS (ESI) m/z
363.19 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.22 (s,
3H), 2.39-2.49 (m, 4H), 3.02-3.18 (m, 4H), 6.99 (d, J ) 9.1 Hz,
2H), 7.24 (m, 1H), 7.43 (d, J ) 9.1 Hz, 2H), 7.55-7.66 (m, 1H),
8.02 (dd, J ) 8.1, 1.26 Hz, 1H), 8.13 (d, J ) 8.31 Hz, 1H), 8.81
(d, J ) 12.8 Hz, 1H), 11.25 (s, 1H), 12.47 (d, J ) 12.8 Hz, 1H).
Anal. (C21H22N4O2) C, H, N.
(4Z)-6-Bromo-4-({[4-(4-methylpiperazin-1-yl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (18b). A mixture of 17b
(3.49 g, 12.4 mmol) and 4a (2.36 g, 12.4 mmol) in 12 mL of DMF
was heated in a preheated oil bath at 110 °C for 25 min. After the
solvent was evaported, the solid was collected and washed with
cold DMF, ether, and hexane to give 3.0 g (55%) of 18b as an
amber solid: mp 220-223 °C. MS (ES+) m/z 441.0, 443.0 (M +
H)+1 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.23 (s, 3H),
.
2.44-2.49 (m, 4H), 3.07-3.21 (m, 4H), 7.00 (d, J ) 9.1 Hz, 2H),
7.37 (dd, J ) 8.4, 1.6 Hz, 1H), 7.48 (d, J ) 9.1 Hz, 2H), 7.90 (d,
J ) 8.4 Hz, 1H), 8.43 (d, J ) 1.6 Hz, 1H), 8.88 (d, J ) 12.8 Hz,
1H), 11.34 (s, 1H), 12.59 (d, J ) 12.8 Hz, 1H). Anal. (C21H21-
BrN4O2 ·0.4H2O) C, H. N: calcd, 12.49; found, 12.06.
(4Z)-6-Nitro-4-({[4-(4-methylpiperazin-1-yl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (18e). By the procedure
described above for 18b, 0.116 g (0.469 mmol) of 17e and 0.09 g
(0.469 mmol)of 4a were reacted to give 0.13 g (68%) of 18e as an
amber solid, mp 250-260 °C (dec). MS (ES+) m/z 408.2 (M +
1
H)+1. H NMR (400 MHz, DMSO-d6) δ ppm: 2.27 (s, 3H), 2.54
(m, 4H), 3.01-3.26 (m, 4H), 7.02 (d, J ) 9.1 Hz, 2H), 7.51 (d, J
) 9.1 Hz, 2H), 7.92 (dd, J ) 8.6, 2.0 Hz, 1H), 8.22 (d, J ) 8.6
Hz, 1H), 8.94 (d, J ) 2.0 Hz, 1H), 9.02 (d, J ) 12.8 Hz, 1H),
11.58 (s, 1H), 12.67 (d, J ) 12.8 Hz, 1H). Anal. (C21H21-
N5O4 ·0.5H2O) C, H. N: calcd, 16.82; found, 16.06.
(4Z)-6-Iodo-4-({[4-(4-methylpiperazin-1-yl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (18f). To a solution of
0.15 g (0.61 mmol) of (4E)-6-iodo-4-(methoxymethylene)isoquino-
line-1,3(2H,4H)-dione (17f) in 2 mL of N,N′-dimethylformamide
was added 4a (0.096 g, 0.67 mmol). The reaction mixture was
heated at 120 °C for 2 h. The reaction mixture was concentrated
under high-pressure vacuum, then treated with MeOH to give a
tan precipitate. It was filtered and washed successivly with MeOH,
Et2O, and hexane to afford 0.202 g (91%) of 18f as a brown solid:
mp 220-221 °C. MS (ESI) m/z 489.1 (M + H)+1. 1H NMR (400
MHz, DMSO-d6) δ ppm: 2.23 (s, 3H), 2.42-2.48 (m, 4H),
3.09-3.20 (m, 4H), 7.00 (d, J ) 8.8 Hz, 2H), 7.47 (d, J ) 8.8 Hz,
2H), 7.56 (d, J ) 8.5 Hz, 1H), 7.72 (d, J ) 8.5 Hz, 1H), 8.56 (s,
1H), 8.86 (d, J ) 12.8 Hz, 1H), 11.31 (s, 1H), 12.59 (d, J ) 12.8
Hz, 1H). Anal. (C21H21IN4O2 ·0.33H2O) C, H, N.
(4Z)-4-{[(4-Morpholin-4-ylphenyl)amino]methylene}isoquin-
oline-1,3(2H,4H)-dione (5b). By the procedure described above
for 5a, 3 (101.5 mg, 0.5 mmol) and 4-morpholin-4-yl-phenylamine
(4b) (89.12 mg, 0.5 mmol) were reacted to give 139 mg (80%) of
5b as a greenish-yellow solid: mp 257-258 °C. MS (ESI) m/z
350.17 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.97-3.21
(m, 4H), 3.66-3.86 (m, 4H), 7.01 (d, J ) 9.0 Hz, 2H), 7.25 (m,
1H), 7.46 (d, J ) 9.0 Hz, 2H), 7.49-7.79 (m, 1H), 8.02 (dd, J )
7.9, 1.1 Hz, 1H), 8.13 (d, J ) 8.3 Hz, 1H), 8.82 (d, J ) 12.8 Hz,
1H) 11.24 (s, 1H), 12.46 (d, J ) 12.8 Hz, 1H). Anal. (C20H19N3O3)
C, H, N.
(4Z)-4-({[4-(Morpholin-4-ylmethyl)phenyl]amino}methylene)-
isoquinoline-1,3(2H,4H)-dione (5c). By the procedure described
above for 5a, 3 (300 mg, 1.47 mmol) and 4c (283.5 mg, 1.47 mmol)
were reacted to give 283.5 mg (1.47 mmol) of 5c as a yellow solid:
(4Z)-N,N-Dimethyl-4-({[4-(4-methylpiperazin-1-yl)phenyl]-
amino}methylene)-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-6-
carboxamide (18g). A slurry of 81.7 mg (0.298 mmol) of 17g and
56.9 mg (0.298 mmol) of 4a in 1.5 mL of DMF was heated at 100
°C under N2 for 0.5 h. After the reaction was chilled in ice, the
solid product was collected, washed with DMF and Et2O and dried
to give 111 mg (86%) of 18g as yellow crystals: mp >300 °C.
HRMS (ESI) m/e calcd for C24H27N5O3 432.20411, found 432.20337
1
mp 221-222 °C. MS (ESI) m/z 463.1 (M - 1). H NMR (400
MHz, DMSO-d6) δ ppm: 2.35 (m, 4H), 3.36 (s, 2H), 3.57 (m, 4H),
7.28 (m, 1H), 7.35 (d, J ) 8.3 Hz, 2H), 7.52 (d, J ) 8.3 Hz, 2H),
7.62 (m, 1H), 8.06 (d, J ) 7.1 Hz, 1H), 8.16 (d, J ) 8.3 Hz, 1H),
8.89 (d, J ) 12.5 Hz, 1H), 11.32 (s, 1H), 12.41 (d, J ) 12.5 Hz,
1H). Anal. (C21H21N3O3 ·0.3H2O) C, H, N.
(4Z)-4-({[4-(Piperidin-1-ylmethyl)phenyl]amino}methylene)-
isoquinoline-1,3(2H,4H)-dione (5d). By the procedure described
above for 5a, 3 (101.5 mg, 0.5 mmol) and 4-piperidin-1-ylmethyl-
phenylamine (4d) (95.14 mg, 0.5 mmol) were reacted to give 92
mg (51%) of 5d as a yellow solid: mp 185-186 °C. HRMS (ESI)
(M+H)+1 1H NMR (DMSO-d6) δ ppm: 2.22 (s, 3H), 2.46 (m,
.
4H), 2.90 (s, 3H), 3.04 (s, 3H), 3.14 (m, 4H), 6.98 (d, J ) 8.9 Hz,
2H), 7.16 (d, J ) 8.1 Hz, 1H), 7.46 (d, J ) 8.9, 2H), 8.03 (d, J )
8.1 Hz, 1H), 8.14 (s, 1H), 8.88 (d, J ) 12.8 Hz, 1H), 11.25 (s,
1H), 12.55 (d, J ) 12.8 Hz, 1H); Anal. (C24H27N5O3) C, H, N.
m/z calcd for C22H23N3O2 362.18546, found 362.18631 (M+H)+1
.

3520 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
(4Z)-4-({[4-(4-Methylpiperazin-1-yl)phenyl]amino}methylene)-
6-(1H-pyrrol-1- yl)isoquinoline-1,3(2H,4H)-dione (18h). A mix-
ture of 17h (100 mg, 0.3727 mmol) and 4a (71.3 mg, 0.3727 mmol)
in 1 mL of N,N-dimethylformamide was heated at 100 °C for 0.5 h.
After the solvent was evaporated, ether was added to the residue.
The precipitate was collected and washed with ether to give 122
mg (77%) of 18h as a light-brown solid: mp 239-240 °C. HRMS
(ESI) m/z calcd for C25H25N5O2 428.20811, found 428.20865 (M
7.59 (d, J ) 8.3 Hz, 2H), 7.96 (dd, J ) 8.8, 2.0 Hz, 1H), 8.23 (d,
J ) 8.8 Hz, 1H), 8.98 (d, J ) 2.0 Hz, 1H), 9.10 (d, J ) 12.6 Hz,
1H), 11.65 (s, 1H), 12.62 (d, J ) 12.6 Hz, 1H). Anal. (C22H22N4O4)
C, H, N.
(4Z)-6-Iodo-4-({[4-(piperidin-1-ylmethyl)phenyl]amino}meth-
ylene)isoquinoline-1,3(2H,4H)-dione (19f). By the procedure
described above for 5a, 0.2 g (0.60 mmol) of (4E)-6-iodo-4-
(methoxymethylene)isoquinoline-1,3(2H,4H)-dione (17f) was re-
acted with 4d (0.127 mL, 0.67 mmol) to give 0.143 g (48.3%) of
19f as a tan solid: mp 202-203 °C. MS (ESI) m/z 488.1 (M +
+ H)+1 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.23 (s, 3H),
.
2.40-2.48 (m, 4H), 3.02-3.23 (m, 4H), 6.18-6.45 (m, 2H), 7.02
(d, J ) 9.1 Hz, 2H), 7.33-7.58 (m, 3H), 7.59-7.76 (m, 2H), 8.06
(d, J ) 8.6 Hz, 1H), 8.11 (d, J ) 1.8 Hz, 1H), 8.95 (d, J ) 12.8
Hz, 1H), 11.22 (s, 1H), 12.59 (d, J ) 12.8 Hz, 1H). Anal.
(C25H25N5O2 ·0.33H2O) C, H, N.
H)+1 1H NMR (400 MHz, DMSO-d6) δ ppm: 1.39 (m, 2H)
.
1.43-1.65 (m, 4H) 2.32 (s, 4H) 3.42 (m, 2H) 7.33 (d, J ) 8.31
Hz, 2H) 7.54 (d, J ) 8.5 Hz, 2H) 7.60 (dd, J ) 8.1, 1.4 Hz, 1H)
7.73 (d, J ) 8.3 Hz, 1H) 8.59 (s, 1H) 8.92 (d, J ) 12.6 Hz, 1H)
11.38 (s, 1H) 12.53 (d, J ) 12.6 Hz, 1H). Anal. (C22H22IN3O2) C,
H, N.
N-[(4Z)-4-({[4-(4-Methylpiperazin-1-yl)phenyl]amino}meth-
ylene)-1,3-dioxo-1,2,3,4-tetrahydroisoquinolin-6-yl]aceta-
mide (18i). By the procedure described above for 5a, 410 mg (1.57
mmol) of (4E)-6-acetamido-4-(methoxymethylene)isoquinoline-
1,3(2H,4H)-dione (17i) and 300 mg (1.57 mmol) of 4a were reacted
to give 450 mg (68%) of 18i as a yellow solid: mp 292-293 °C.
MS (ESI) m/z 420.2 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ
ppm: 2.10 (s, 3H), 2.24 (s, 3H), 2.45-2.49 (m, 4H), 2.96-3.22
(m, 4H), 7.02 (d, J ) 9.2 Hz, 2H), 7.34 (d, J ) 9.2 Hz, 2H), 7.52
(dd, J ) 8.8, 0.8 Hz, 1H), 7.94 (d, J ) 8.8 Hz, 1H), 8.01 (d, J )
0.8 Hz, 1H), 8.42 (d, J ) 12.8 Hz, 1H), 10.21 (s, 1H), 11.13 (s,
(4Z)-N,N-Dimethyl-1,3-dioxo-4-({[4-(piperidinylmethyl)phenyl]-
amino}methylene)-1,2,3,4-tetrahydroisoquinoline-6-carboxam-
ide (19g). By the procedure described above for 5a, 70 mg (0.255
mmol) of 17g and 48.5 mg (0.255 mmol) of 4d were reacted to
give 82.4 mg (74%) of 19 g as a bright-yellow crystals: mp
266-267 °C (dec). HRMS (ESI) m/e calcd for C25H28N4O3
431.20886, found 431.20820 (M - H)-1. 1H NMR (DMSO-d6) δ
ppm: 1.38 (m, 2H), 1.48 (m, 4H), 2.32 (m, 4H), 2.90 (s, 3H), 3.04
(s, 3H), 3.42 (s, 2H), 7.20 (d, J ) 6 Hz, 1H), 7.32 (d, J ) 9 Hz,
2H), 7.53 (d, J ) 9 Hz, 2H), 8.06 (d, J ) 6 Hz, 1H), 8.17 (s, 1H),
8.95 (d, J ) 9 Hz, 1H), 11.40 (s, 1H), 12.45 (d, J ) 9 Hz, 1H).
Anal. (C25H28N4O3 ·0.25H2O) C, H, N.
(4Z)-4-({[4 Piperidin-1-ylmethyl]phenyl}amino)methylene)}-
6-(1H-pyrrol-1-yl)isoquinoline-1,3(2H,4H)-dione (19h). By the
procedure described above for 5a, 100 mg (0.38 mmol) of (4E)-
4-(methoxymethylene)-6-(1H-pyrrol-1-yl)isoquinoline-1,3(2H,4H)-
dione (17h) and 4d (70.70 mg, 0.38 mmol) were reacted to give
50 mg (31%) of 19h as a yellow solid: mp 207-208 °C. MS (ESI)
m/z 426.21 (M + 1).; 1H NMR (400 MHz, DMSO-d6) δ ppm: 1.39
(m, 2H), 1.50 (m, 4H), 2.34 (m, 4H), 3.45 (s, 2H), 6.35 (m, 2H),
7.36 (d, J ) 8.4 Hz, 2H), 7.46-7.57 (m, 3H), 7.66 (m, 2H), 8.07
(d, J ) 8.8 Hz, 1H), 8.13 (d, J ) 2.0 Hz, 1H), 9.01 (d, J ) 12.59
Hz, 1H), 11.30 (s, 1H), 12.6 (d, J ) 12.6 Hz, 1H). Anal.
(C26H26N4O2 ·0.1H2O·0.1TFA) C, H, N.
N-[(4Z)-1,3-Dioxo-4-({[4-piperidin-1-ylmethyl]phenyl}amino)-
methylene]-1,2,3,4-tetrahydroisoquinolin-6-yl]acetamide (19i).
By the procedure described above for 5a, 94 mg (0.36 mmol) of
N-[(4E)-1,3-dioxo-4-(methoxy)methylene-1,2,3,4-tetrahydroiso-
quinolin-6-yl]acetamide 17i and 4d (76 mg, 0.40 mmol) were
reacted to give 89 mg (59%) of 19i as a brown solid, mp >320 °C.
1H NMR (400 MHz, DMSO-d6) δ ppm: 1.41 (m, 2H), 1.54 (m,
4H), 2.10 (s, 3H), 2.13 (m, 4H), 3.56 (m, 2H), 7.42 (m, 2H), 7.57
(m, 2H), 7.98 (m, 1H), 8.10 (m, 1H), 8.60 (m, 1H), 9.00 (d, J )
12.7, 1H), 10.24 (m, 1H), 11.31 (m, 1H), 12.38 (m, 1H). MS (ES+)
m/z 419.3 (M + H)+1. Anal. (C24H26N4O3 ·2.6H2O) C, N. H: calcd,
6.76; found, 4.47.
1H),
12.26
(d,
J
)
12.8
Hz,
1H).
Anal.
(C23H25N5O3 ·0.2H2O·0.5TFA) C, H, N.
(4Z)-6-Bromo-4-{[(4-piperidin-1-ylmethyl)phenyl]amino}-
methyleneisoquinoline-1,3(2H,4H)-dione (19b). (way-186084). By
the procedure described above for 5a, 141 mg (0.50 mmol) of 17b
and 100 mg (0.525 mmol) of 4d were reacted to give 138 mg (63%)
of 19b as an off-white solid, mp 222-225 °C. MS (ES+) m/z 440.1,
1
442.1 (M + H)+1. H NMR (400 MHz, DMSO-d6) δ ppm: 1.39
(m, 2H), 1.42-1.62 (m, 4H), 2.32 (m, 4H), 3.42 (s, 2H), 7.33 (d,
J ) 8.3 Hz, 2H), 7.41 (d, J ) 8.0 Hz, 1H), 7.55 (d, J ) 8.6 Hz,
2H), 7.92 (d, J ) 8.3 Hz, 1H), 8.46 (s, 1H), 8.94 (d, J ) 12.7 Hz,
1H), 11.41 (s, 1H), 12.53 (d, J ) 12.7 Hz, 1H). Anal.
(C22H22BrN3O2 ·0.3H2O) C, H, N.
(4Z)-6-Chloro-4-({[4-(piperidin-1-ylmethyl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (19c). By the procedure
described above for 5a, 0.15 g (0.63 mmol) of (4E)-6-chloro-4-
(methoxymethylene)isoquinoline-1,3(2H,4H)-dione (17c) and 0.145
g (0.756 mmol) of 4d were reacted to give 0.165 g (66%) of 19c
as a yellow solid: mp 225-226 °C. MS (ESI) m/z 396.1 (M +
H)+1. HRMS (ESI) m/e calcd for C22H22ClN3O2396.14733, found
1
396.14670 (M + H)+1. H NMR (400 MHz, DMSO-d6) δ ppm:
1.39 (m, 2H), 1.49 (m, 4H), 2.33 (s, 4H), 3.44 (m, 2H), 7.28 (dd,
J ) 8.5, 1.7 Hz, 1H), 7.34 (d, J ) 8.1 Hz, 2H), 7.55 (d, J ) 8.1
Hz, 2H), 8.00 (d, J ) 8.5 Hz, 1H), 8.32 (d, J ) 1.7 Hz, 1H), 8.94
(d, J ) 12.6 Hz, 1H), 11.40 (s, 1H), 12.52 (d, J ) 12.6 Hz, 1H).
Anal. (C22H22ClN3O2 ·0.16DMF·0.25H2O) C, H, N.
6-Methoxy-4-[(4-piperidin-1-ylmethyl-phenylamino)-meth-
ylene]-4H-isoquinoline-1,3-dione (19d). By the procedure de-
scribed above for 5a, 117 mg (0.50 mmol) of (4E)-6-methoxy-4-
methoxymethylene-4H-isoquinoline-1,3-dione (17d) and 4d (95 mg,
0.50 mmol) in N,N-dimethylformamide (1 mL) were reacted to give
152 mg (78%) of 19d as a yellow solid: mp 272-5 °C (dec). MS
(ESI) m/z 392.4 (M + H). 1H NMR (400 MHz, DMSO-d6) δ ppm:
1.34-1.43 (m, 2H), 1.44-1.63 (m, 4H), 2.32 (m, 4H), 3.42 (s,
2H), 3.92 (s, 3H), 6.87 (dd, J ) 8.7, 2.1 Hz, 1H), 7.33 (d, J ) 8.3
Hz, 2H), 7.50 (d, J ) 8.3 Hz, 2H), 7.54 (d, J ) 2.1 Hz, 1H), 7.95
(d, J ) 8.7 Hz, 1H), 8.85 (d, J ) 12.6 Hz, 1H), 11.13 (s, 1H),
12.49 (d, J ) 12.6 Hz, 1H). Anal. (C23H25N3O3) C, H, N.
(4Z)-6-Nitro-4-{[(4-piperidin-1-ylmethyl)phenyl]amino}meth-
yleneisoquinoline-1,3(2H,4H)-dione (19e). By the procedure
described above for 5a, 115 mg (0.46 mmol) of (4E)-4-(meth-
oxy)methylene-6-nitroisoquinoline-1,3(2H,4H)-dione (17e) was
reacted with 93 mg (0.49 mmol) of 4d to give 137 mg (73%) of
19e as a brown solid: mp 225-235 °C (dec). MS (ES-) m/z 405.2
(M - H)-1. 1H NMR (400 MHz, DMSO-d6) δ ppm: 1.39 (m, 2H),
1.50 (m, 4H), 2.35 (m, 4H), 3.46 (s, 2H), 7.36 (d, J ) 8.3 Hz, 2H),
(4Z)-6-Piperidin-1-yl-4-({[4-(piperidin-1-ylmethyl)phenyl]-
amino}methylene)isoquinoline-1,3(2H,4H)-dione (21a). A mix-
ture of 300 mg (0.68 mmol) of (4Z)-6-bromo-4-({[4-(piperidin-1-
ylmethyl)phenyl]amino}methylene)isoquinoline-1,3(2H,4H)-di-
one (19b), tris(dibenzyldeneaacetone)-dipalladium(0) (118.30 mg,
0.129 mmol), 1,3-bis(2,6-di-isopropylphenyl)imidazolium chloride
(Ipr·HCl) 78.04 (0.184 mmol), potassium t-butoxide, (194.41 mg,
1.36 mmol), and piperidine (200 mg, 2.04 mmol) was placed in a
three-neck flask. Under N2, N,N-dimethylformamide (8 mL) was
added, and the mixture was then stirred in a preheated oil bath 100
°C for 45 min. After cooling, the mixture was treated with CH2Cl2
and filtered through celite. After evaporating all the solvents, the
residue was dissolved in methylene chloride, washed three times
with sodium bicarconate solution, dried over magnissium sulfate,
and evaporated. The yellow oily residue was purified by preparative
thin layer chromatography (5:95 ) methanol:methylene chloride),
to give 21a as a yellow solid, 80 mg (25%): mp 211-212 °C. MS
(ESI) m/z 445.3 (M + 1). HRMS (ESI) m/e calcd for C27H32N4O2
445.25981, found 445.25909 (M + H)+1 1H NMR (400 MHz,
.

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3521
DMSO-d6) δ ppm: 1.38 (m, 2H), 1.48 (m, 4H), 1.61 (m, 6H) 2.32
(m, 4H), 3.42 (m, 6H), 6.87 (dd, J ) 9.2, 1.6 Hz, H), 7.26 (d, J )
1.6 Hz, 1H), 7.32 (d, J ) 8.4 Hz, 2H), 7.46 (d, J ) 8.4 Hz, 2H),
7.82 (d, J ) 9.2 Hz, 1H), 8.78 (d, J ) 12.4 Hz, 1H), 10.89 (s, 1H),
12.48 (d, J ) 12.4 Hz, 1H). Anal. (C27H32N4O2 ·0.4H2O) C, H. N:
calcd, 12.14; found, 11.23.
7.8 Hz, 2H), 8.10 (d, J ) 8.3 Hz, 1H), 8.30 (s, 1H), 8.98 (d, J )
12.7 Hz, 1H), 11.27 (s, 1H), 12.54 (d, J ) 12.7 Hz, 1H).
(4Z)-6-(2-Furyl)-4-({[4-(4-methylpiperazin-1-yl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (22b). By the procedure
described aboved for 22a, 18b (40 mg, 0.1 mmol) and 2-furanbo-
ronic acid (11.2 mg, 0.1 mmol) were reacted to give 13 mg (30%)
of 22b: LC/MS1 Rt 1.874 min, m/z 429.1 (M + 1); LC/MS2 Rt
1.89 min, m/z 429 (M + 1); HRMS 429.1925 [M + H]+ obs,
429.1921 [M + H]+ calcd. 1H NMR (300 MHz, DMSO-d6) δ ppm:
2.88 (s, 3H), 2.97-3.19 (m, 4H), 3.52-3.84 (m, 4H), 7.08 (d, J )
8.6 Hz, 2H), 7.34 (dd, J ) 7.8, 7.1 Hz, 1H), 7.47 (d, J ) 7.1 Hz,
1H), 7.54 (d, J ) 8.6 Hz, 2H), 7.78 (d, J ) 7.80 Hz, 1H), 7.86 (d,
J ) 8.2 Hz, 1H), 8.09 (d, J ) 8.2 Hz, 1H), 8.31 (s, 1H), 8.98 (d,
J ) 12.7 Hz, 1H), 11.27 (s, 1H), 12.54 (d, J ) 12.7 Hz, 1H).
(4Z)-6-(3-Furyl)-4-({[4-(4-methylpiperazin-1-yl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (22c). A mixture con-
taining 0.15 g (0.34 mmol) of 18b, 0.076 g (0.64 mmol)of
3-furanboronic acid, 0.02 g (0.017 mmol) of tris(dibenzyldeneaac-
etone)-dipalladium(0), 0.02 g (0.064 mmol) of 2-(di-t-butyl-
phosphino)-biphenyl, and 0.072 g (0.64 mmol) of sodium carbonate
in 2 mL of DMF and 0.4 mL of water was heated at 100 °C for
1.5 h. It was filtered through a pad of celite and dried up. The
residue was purified by column chromatography over silica gel
using 5% MeOH/CHCl3 as eluent to yield 0.096 mg (66.2%) of
22c as a yellow solid: mp 190-191 °C.; MS (ESI) m/z 429.2 (M
(4Z)-6-Piperidin-1-yl-4-({[4-(methylpiperazin-1-yl)phenyl]-
amino}methylene)isoquinoline-1,3(2H,4H)-dione (20a). By the
procedure described for the preparation of 21a, 18b (300 mg, 0.68
mmol) and piperidine (200 mg, 2.04 mmol) were reacted to give
70 mg (23%) of yellow solid: mp 223-224 °C. MS (ESI) m/z
445.56 (M + 1). HRMS (ESI) m/e calcd for C26H31N5O2 446.25506,
1
found 446.25472 (M + H)+1. H NMR (400 MHz, DMSO-d6) δ
ppm: 1.61 (m, 4H), 1.99 (s, 3H), 2.22 (m, 2H), 2.44-2.47 (m, 4H),
3.12-3.15 (m, 4H), 3.42 (m, 4H), 6.85 (dd, J ) 8.8, 2.0 Hz, 1H)
7.00 (d, J ) 9.2 Hz, 2H) 7.23 (d, J ) 2.0 Hz, 1H), 7.39 (d, J ) 9.2
Hz, 2H), 7.81 (d, J ) 8.8 Hz, 1H), 8.72 (d, J ) 12.8 Hz, 1H),
10.81 (s, 1H), 12.51 (d,
J ) 12.8 Hz, 1H). Anal.
(C26H31N5O2 ·0.5H2O) C, H. N: calcd, 15.35; found, 13.56.
(4Z)-6-Morpholin-4-yl-4-({[4-(piperidin-1-ylmethyl)phenyl]-
amino}methylene)isoquinoline-1,3(2H,4H)-dione (21b). By the
procedure described above for 21a, 19b (300 mg, 0.68 mmol) and
morpholine (178 mg, 2.04 mmol) were reacted to give 95 mg (31%
yield) of 21b as a yellow solid: mp 216-217 °C. MS (ESI) m/z
446.55 (M + 1). HRMS (ESI) m/e calcd for C26H30N4O3 447.23907,
found 447.23969 (M+H)+1
.
1H NMR (400 MHz, DMSO-d6) δ
+ H)+1 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.23 (s, 3H),
.
ppm: 1.38 (m, 2H), 1.48 (m, 4H), 2.32 (m, 4H), 3.38-3.4(m, 4H),
3.42 (s, 2H), 3.72-3.85 (m, 4H), 6.91 (dd, J ) 9.2, 1.8 Hz, 1H),
7.31 (d, J ) 1.8 Hz, 1H), 7.33 (d, J ) 8.4 Hz, 2H), 7.46 (d, J )
8.4 Hz, 2H), 7.86 (d, J ) 9.2 Hz, 1H) 8.80 (d, J ) 12.4 Hz, 1H)
10.96 (s, 1H) 12.48 (d, J ) 12.4 Hz, 1H). Anal. (C26H30N4O3 ·H2O)
C, H: calcd, 12.06; found, 11.44.
2.45-2.49 (m, 4H), 2.96-3.23 (m, 4H), 7.02 (d, J ) 9.1 Hz, 2H),
7.25 (d, J ) 1.01 Hz, 1H), 7.46 (d, J ) 9.1 Hz, 2H), 7.51 (dd, J )
8.3, 1.3 Hz, 1H), 7.82 (t, J ) 1.64 Hz, 1H), 8.01 (d, J ) 8.3 Hz,
1H), 8.19 (s, 1H), 8.43 (s, 1H), 8.91 (d, J ) 12.6 Hz, 1H), 11.21
(s, 1H), 12.55 (d, J ) 12.6 Hz, 1H). Anal. (C25H24N4O3 ·0.2H2O)
C, H, N.
(4Z)-6-[(4-Methyl-piperizin-1-yl)-4-({[4-(piperidin-1-ylmethyl)-
phenyl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (21c).
By the procedure described above for 21a, 19b (300 mg, 0.68
mmol) and 4-methyl-piperazine (204.41 mg, 2.04 mmol) were
reacted to give 90 mg (29% yield) of 21c as a yellow solid: mp
192-193 °C. MS (ESI) m/z 459.59 (M + 1). 1H NMR (400 MHz,
DMSO-d6) δ ppm: 1.39 (m, 2H), 1.48 (m, 4H), 2.24 (s, 3H), 2.32
(m, 4H), 2.47 (m, 4H), 3.41 (m, 6H), 6.91 (dd, J ) 9.2, 2.0 Hz,
1H), 7.29 (d, J ) 2.0 Hz, 1H), 7.33 (d, J ) 8.4 Hz, 2H), 7.47 (d,
J ) 8.4 Hz, 2H), 7.84 (d, J ) 9.2 Hz, 1H), 8.80 (d, J ) 12.4 Hz,
1H), 10.93 (s, 1H), 12.49 (d, J ) 12.4 Hz, 1H). Anal.
(C27H33N5O2 ·0.4H2O) C, H, N.
(4Z)-6-Anilino-4-({[4-(piperidin-1-ylmethyl)phenyl]amino}-
methylene)isoquinoline-1,3(2H,4H)-dione (21d). By the procedure
described above for 21a, 19b (300 mg, 0.68 mmol) and aniline
(125 mg, 1.36 mmol) were reacted to give 120 mg (39% yield) of
21d as a yellow solid: mp 234-235 °C. MS (ESI) m/z 453.2 (M +
1). 1H NMR (400 MHz, DMSO-d6) δ ppm: 1.39 (m, 2H),
1.46-1.51 (m, 4H), 2.31 (m, 4H), 3.40 (s, 2H), 6.89-7.01 (m,
2H), 7.24 (dd, J ) 8.4, 1.6 Hz, 2H), 7.33 (d, J ) 8.8 Hz, 2H), 7.37
(m, 2H), 7.4 (d, J ) 8.4 Hz, 2H), 7.47 (d, J ) 1.6 Hz, 1H), 7.87
(d, J ) 8.8 Hz, 1H), 8.55 (d, J ) 12.8 Hz, 1H), 8.66 (m, 1H),
10.98 (s, 1H), 12.29 (d, J ) 12.8 Hz, 1H). Anal. (C28H28N4O2) C,
H, N.
(4Z)-4-({[4-(4-Methylpiperazin-1-yl)phenyl]amino}methylene)-
6-thien-3-ylisoquinoline-1,3(2H,4H)-dione (22d). By the proce-
dure described above for 22c, 18b (1.0 g, 2.26 mmol) and
3-thiopheneboronic acid (0.43 g, 3.4 mmol) were reacted to yield
0.38 g (38%) of 22d as a yellow solid: mp 224-226 °C. MS (ESI)
1
m/z 445.2 (M + H)+1. H NMR (400 MHz, DMSO-d6) δ ppm:
2.23 (s, 3H), 2.41-2.48 (m, 4H), 3.02-3.20 (m, 4H), 7.01 (d, J )
9.06 Hz, 2H), 7.46 (d, J ) 8.81 Hz, 2H), 7.61 (dd, J ) 8.31, 1.51
Hz, 1H), 7.71 (dd, J ) 5.04, 2.9 Hz, 1H), 7.84 (dd, J ) 5.0, 1.3
Hz, 1H), 8.04 (d, J ) 8.31 Hz, 1H), 8.19 (dd, J ) 2.9, 1.3 Hz,
1H), 8.31 (s, 1H), 8.95 (d, J ) 12.8 Hz, 1H), 11.22 (s, 1H), 12.56
(d, J ) 12.8 Hz, 1H). Anal. (C25H24N4O2S·0.9H2O) C, H, N.
4-[(4Z)-4-({[4-(4-Methylpiperazin-1-yl)phenyl]amino}meth-
ylene)-1,3-dioxo-1,2,3,4-tetra-hydroisoquinolin-6-yl]benzalde-
hyde (22e). By the procedure described above for 22c, 18b (0.5 g,
1.13 mmol) and 4-formylphenylboronic acid (0.25 g, 1.7 mmol)
were reacted to give 0.054 g (10.2%) of 22e as a yellow solid: mp
152-153 °C, MS (ESI) m/z 467.2 (M + H)+1. 1H NMR (400 MHz,
DMSO-d6) δ ppm: 2.23 (s, 3H), 2.47 (s, 4H), 2.97-3.20 (m, 4H),
7.01 (d, J ) 8.8 Hz, 2H), 7.32-7.52 (m, 3H), 7.60 (dd, J ) 8.3,
1.5 Hz, 1H), 7.98-8.19 (m, 4H), 8.39 (s, 1H), 8.99 (d, J ) 12.8
Hz, 1H), 10.10 (s, 1H), 11.31 (s, 1H), 12.59 (d, J ) 12.8 Hz, 1H).
Anal. (C28H26N4O3 ·0.67H2O) C, H, N.
4Z-4-({[4-(4-Methylpiperazin-1-yl)phenyl]amino}methylene)-
6-(2-phenylvinyl)isoquinoline-1,3(2H,4H)-dione (22f). To a mix-
ture of cesium carbonate (39 mg, 0.12 mmol), tetrabutylammonium
bromide (32.2 mg, 0.1 mmol), tri-o-tolylphosphine (30.4 mg, 0.1
mmol) and palladium acetate (9 mg, 0.04 mmol) in N,N-dimeth-
ylformamide (1.2 mL) were added to 18b (47 mg, 0.1 mmol) and
styrene (15.6 mg, 0.15 mmol). The reaction mixture was subjected
to microwave heating at 200 °C for 120 s. The reaction mixture
was then diluted to 2 mL with N,N-dimethylformamide and purified
by C18 reverse phase HPLC. The pure fractions were combined
and concentrated to yield 6 mg (13%) of 22f as a solid. LC/MS1
Rt 2.134 min, m/z 465 (M + 1); LC/MS2 Rt 2.84 min, m/z 465
(M+1); HRMS 465.2288 [M + H]+ obs 465.2285 [M + H]+ calcd.
1H NMR (300 MHz, DMSO-d6) δ ppm: 2.89 (s, 3H), 2.97-3.19
(m, 4H), 3.53-3.90 (m, 4H), 7.11 (d, J ) 8.4 Hz, 2H), 7.33-7.46
(m, 4H), 7.53-7.59 (m, 4H), 7.66 (d, J ) 8.4 Hz, 2H), 8.01 (d, J
(4Z)-4-({[4-(4-Methylpiperazin-1-yl)phenyl]amino}methy-
lene)-6-phenylisoquinoline-1,3(2H,4H)-dione (22a). To a suspen-
sion of 18b (40 mg, 0.1 mmol) in N,N-dimethylformamide (1 mL)
was added phenyl boronic acid (12.2 mg, 0.1 mmol), followed by
60 µL of 2 M aqueous cesium carbonate and tetrakis(triphenylphos-
phine)palladium(0) (6 mg, 0.005 mmol). The reaction mixture was
subjected to microwave heating at 150 °C for 100 s. The reaction
mixture was then diluted to 2 mL with N,N-dimethylformamide
and purified by C18 reverse phase HPLC. The pure fractions were
combined and concentrated to yield 43 mg (98%) of 22a as a solid:
LC/MS1 Rt 1.918 min, m/z 439.1 (M + 1); LC/MS2 Rt 1.99 min,
1
HRMS 439.2132 [M + H]+ obs, 439.2129 [M + H]+ calcd. H
NMR (300 MHz, DMSO-d6) δ ppm: 2.87 (s, 3H), 2.97-3.19 (m,
4H), 3.52-3.84 (m, 4H), 7.10 (d, J ) 8.5 Hz, 2H), 7.29-7.37 (m,
1H), 7.45-7.56 (m, 4 H), 7.78 (d, J ) 8.3 Hz, 1H), 7.87 (d, J )

3522 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
) 8.2 Hz, 1H), 8.25 (s, 1H), 8.90 (d, J ) 12.7 Hz, 1H), 11.27 (s,
1H), 12.54 (d, J ) 12.7 Hz, 1H).
(4Z)-4-({[4-Piperidin-1-ylmethyl]phenyl}amino)methylene)-6-
pyridin-3-ylisoquinoline-1,3(2H,4H)-dione (23d). By the proce-
dure described above for 23a, 19b (300 mg, 0.68 mmol) and
3-pyridylboronic acid (166.21 mg, 1.36 mmol) were reacted to give
100 mg (34%) of 23d as a yellow solid: mp 247-248 °C. MS
(ESI) m/z 438.53 (M + 1). HRMS (ESI) m/e calcd for C27H26N4O2
(4Z)-4-({[4-(4-Methylpiperazin-1-yl)phenyl]amino}methylene)-
6-(phenylethynyl)-isoquinoline-1,3(2H,4H)-dione (22g). A mix-
ture of 18b (0.10 g, 0.23 mmol), phenylacetylene (0.03 mL, 0.27
mmol), dichlorobis(triphenylphosphine)-palladium(II) (0.04 g, 0.046
mmol), CuI (0.0043 g, 0.023 mmol), and 0.12 mL (1.14 mmol) of
triethylamine in 2 mL of N,N-dimethylformamide was added to a
10 mL round-bottom flask, sealed with a rubber septum, deaired,
and backfilled with nitrogen gas three times and wrapped around
with aluminum foil. The reaction mixture was stirred vigorously
at 70 °C in an oil bath under nitrogen. Mass spectroscopy suggested
the completion of reaction after 45 min. The reaction mixture was
filtered through celite and subsequently evaporated under high-
pressure vacuum to brown solid. The solid was dissolved in warm
chloroform and ran through a pad of florisil, which is rinsed with
200 mL of warm chloroform. The collected organic portion was
evaporated under vacuum and purified by HPLC to give 0.057 g
(54.3%) of 22g as a brown solid: mp 169-170 °C. MS (ESI) m/z
439.21286, found 439.21212 (M + H)+1 1H NMR (400 MHz,
.
DMSO-d6) δ ppm: 1.38 (m, 2H), 1.42-1.49 (m, 4H), 2.32 (m, 4H),
3.42 (s, 2H), 7.34 (d, J ) 8.1 Hz, 2H), 7.54 (m, 3H), 7.61 (d, J )
8.1 Hz, 2H), 8.13 (d, J ) 8.4 Hz, 1H), 8.27 (dd, J ) 1.6, 8.1 1H),
8.41 (m, 1H) 8.66 (d, J ) 1.6 Hz 1H), 9.05 (d, J ) 12.6 Hz, 1H),
9.11 (d, J ) 1.6 Hz, 1H), 11.37 (s, 1H), 12.53 (d, J ) 12.6 Hz,
1H). Anal. (C27H26N4O2 ·1.2H2O) C, H, N.
(4Z)-6-(4-Fluorophenyl)-4-({[4-(piperidin-1-ylmethyl)phenyl]-
amino}methylene)isoquinoline-1,3(2H,4H)-dione (23e). By the
procedure described above for 22c except replacing (di-t-butyl-
phosphino)-biphenyl with tri-t-butylphosphine, and replacing so-
dium carbonate with cesium carbonate. 19b (500 mg, 1.13 mmol)
and 4-flurophenyl boronic acid (395.27 mg, 2.83 mmol) were
reacted to give 100 mg (20%) of 23e as a yellow solid: mp 195-196
463.1 (M + H)+1 1H NMR (400 MHz, DMSO-d6) δ ppm:
.
1
°C. MS (ESI) m/z 456.3 (M + 1)+. H NMR (400 MHz, DMSO-
2.83-3.06 (m, 5H), 3.18 (s, 2H), 3.87 (m, 2H), 7.08 (d, J ) 8.8
Hz, 2H), 7.38 (d, J ) 8.3 Hz, 1H), 7.45-7.51 (m, 3H), 7.57 (d, J
) 8.8 Hz, 1H), 7.63 (dd, J ) 6.42, 2.90 Hz, 3H), 8.04 (d, J ) 8.31
Hz, 1H), 8.40 (s, 1H), 8.95 (d, J ) 12.8 Hz, 1H), 11.38 (s, 1H),
12.59 (d, J ) 12.8 Hz, 1H). Anal. (C29H26N4O2 ·2.6H2O·1.1TFA)
C, N. H: calcd, 5.13; found 4.48.
d6) δ ppm: 1.39 (m, 2H), 1.43-1.62 (m, 4H), 2.32 (m, 4H), 3.42
(s, 2H), 7.15-7.42 (m, 4H), 7.54 (d, J ) 8.4 Hz, 3H), 7.91-7.95
(m, 2H), 8.09 (d, J ) 8.4 Hz, 1H), 8.31 (s, 1H), 9.03 (d, J ) 12.4
Hz, 1H), 11.32 (s, 1H), 12.52 (d, J ) 12.4 Hz, 1H). Anal.
(C28H26FN3O2 ·0.25H2O) C, H, N.
(4Z)-6-(4-Chlorophenyl)-4-({[4-(piperidin-1-ylmethyl)phenyl]-
amino}methylene)isoquinoline-1,3(2H,4H)-dione (23f). By the
procedure described above for 23a, using the procedure described
for the preparation of 23a, 19b (300 mg, 0.68 mmol), and
4-chlorophenyl boronic acid (214.0 mg, 1.36 mmol) were reacted
to give 250 mg (79%) of 23f as a yellow solid: mp 204-205 °C.
(4Z)-6-Phenyl-4-({[4-piperidin-1-ylmethyl]phenyl}amino)-
methylene)}isoquinoline-1,3(2H,4H)-dione (23a). A mixture of
300 mg (0.68 mmol) of 19b, tetrakis(triphenylphosphine)pal-
ladium(0) (118.0 mg, 0.102 mmol), saturated aqueous sodium
carbonate (2 mL), and phenyl boronic acid (123.42 mg, 1.02 mmol)
was placed in a three-neck flask. Under N2, N,N-dimethylformamide
(8 mL) was added and the mixture was then placed in a preheated
oil bath at 120 °C for 45 min. After cooling, the mixture was treated
with CH2Cl2 and filtered through celite. After evaporating all the
solvents, the residue was dissloved in methylene chloride, washed
three times with soduim bicarconate solution, dried over magnesium
sulfate, and evaporated. The yellow oily residue was purified by
preparative thin layer chromatography (5:95 ) methanol:methylene
chloride), to give 90 mg (30% yield) of 23a as a yellow solid: mp
214-215 °C. MS (ESI) m/z 437.54 (M + 1). HRMS (ESI) m/e
1
MS (ESI) m/z 464.90 (M + 1)+. H NMR (400 MHz, DMSO-d6)
δ ppm: 1.39 (m, 2H), 1.44-1.60 (m, 4H), 2.32 (m, 4H), 3.43 (s,
2H), 7.34 (d, J ) 8.3 Hz, 2H), 7.53 (d, J ) 8.3 Hz, 2H), 7.57-7.71
(m, 3H), 7.91 (d, J ) 8.8 Hz, 2H), 8.10 (d, J ) 8.3 Hz, 1H), 8.33
(s, 1H), 9.03 (d, J ) 12.8 Hz, 1H), 11.34 (s, 1H), 12.52 (d, J )
12.8 Hz, 1H). Anal. (C28H26ClN3O2 ·0.8H2O) C, H, N.
(4Z)-6-(3-Hydroxyphenyl)-4-({[4-piperidin-1-ylmethyl]phenyl}-
amino)methylene)}isoquinoline-1,3(2H,4H)-dione (23g). By the
procedure described above for 23e, 19b (300 mg, 0.68 mmol) and
3-(4,4,5,5-teramethyl-[1,3,2]dioxaboralan-2-yl)-phenol (300 mg,
1.36 mmol) were reacted to give 120 mg (39%) of 23g as a yellow
calcd for C28H27N3O2 438.21761, found 438.21743 (M + H)+1
.
1H NMR (400 MHz, DMSO-d6) δ ppm: 1.39 (m, 2H), 1.49 (m,
4H), 2.32 (m, 4H), 3.42 (s, 2H), 7.34 (d, J ) 8.1 Hz, 2H), 7.45 (d,
J ) 7.3 Hz, 1H), 7.48-7.63 (m, 5H), 7.87 (d, J ) 8.1 Hz, 2H),
8.10 (d, J ) 7.3 Hz, 1H), 8.33 (s, 1H), 9.04 (d, J ) 12.6 Hz, 1H),
11.33 (s, 1H), 12.53 (d, J ) 12.6 Hz, 1H). Anal. (C28H27-
N3O2 ·0.8H2O) C, N. H: calcd, 6.38; found 5.91.
(4Z)-6-(3-Furyl)-4-({[4-piperidin-1-ylmethyl]phenyl}amino)-
methylene)}isoquinoline-1,3(2H,4H)-dione (23b). By the proce-
dure described above for 23a, 19b (0.16 g, 0.365 mmol) and
3-furanboronic acid (87 mg, 0.73 mmol) were reacted to give 50
mg (32%) of 23b as a yellow solid: mp 204-205 °C. MS (ESI)
m/z 427.50 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ ppm:
1.24-1.40 (m, 2H), 1.44-1.74 (m, 4H), 2.34 (m, 4H), 3.44 (s,
2H), 7.26 (s, 1H), 7.36 (d, J ) 8.4 Hz, 2H), 7.45-7.63 (m, 3H),
7.71-7.91 (m, 1H), 8.02 (d, J ) 8.4 Hz, 1H), 8.22 (m, 1H), 8.44
(m, 1H), 8.97 (d, J ) 12.6 Hz, 1H), 11.28 (s, 1H), 12.51 (d, J )
12.6 Hz, 1H). Anal. (C26H25N3O3 ·0.6H2O) C, H, N.
1
solid: mp 235-236 °C. MS (ESI) m/z 453.54 (M + 1). H NMR
(400 MHz, DMSO-d6) δ ppm: 1.39 (m, 2H), 1.47-1.49 (m, 4H),
2.33 (m, 4H) 3.43 (s, 2H), 6.85 (d, J ) 7.6 Hz, 1H), 7.2 (m, 1H),
7.26 (d, J ) 7.6 Hz, 2H), 7.3-7.35 (m, 3H), 7.47 (d, J ) 8.1 Hz,
1H), 7.54 (d, J ) 8.3 Hz, 2H), 8.08 (m, 1H), 8.27 (s, 1H), 9.03 (d,
J ) 12.6 Hz, 1H), 9.59 (s, 1H), 11.31 (s, 1H), 12.53 (d, J ) 12.6
Hz, 1H). Anal. (C28H27N3O3 ·0.5H2O) C, H, N.
(4Z)-6-(4-Hydroxyphenyl)-4-({[4-piperidin-1-ylmethyl]phenyl}-
amino)methylene)}isoquinoline-1,3(2H,4H)-dione (23h). By the
procedure described above for 23e, 19b (300 mg, 0.68 mmol) and
4-(4,4,5,5-teramethyl-[1,3,2]dioxaboralan-2-yl)-phenol (300 mg,
1.36 mmol) were reacted to give 120 mg (39%) of 23h as a yellow
1
solid: mp 244-245 °C. MS (ESI) m/z 453.54 (M + 1). H NMR
(400 MHz, DMSO-d6) δ ppm: 1.39 (m, 2H), 1.43-1.64 (m, 4H),
2.32 (m, 4H), 3.42 (s, 2H), 6.90 (d, J ) 8.4 Hz, 2H), 7.33 (d, J )
8.4 Hz, 2H), 7.47-7.54 (m, 3H), 7.73 (d, J ) 8.4 Hz, 2H), 8.04
(d, J ) 8.1 Hz, 1H), 8.23 (s, 1H), 8.61 (s, 1H), 9.00 (d, J ) 12.6
Hz, 1H), 9.70 (s, 1H), 11.26 (s, 1H), 12.52 (d, J ) 12.6 Hz, 1H).
Anal. (C28H27N3O3 ·0.1H2O·0.49TFA) C, H, N.
(4Z)-4-({[4-(Piperidin-1-ylmethyl)phenyl]amino}methylene)-
6-thien-3-ylisoquinoline-1,3(2H,4H)-dione (23c). By the procedure
describedabovefor23a,19b(500mg,1.14mmol)and3-thiophenebo-
ronic acid (300 mg, 2.28 mmol) were reacted to give 150 mg (30%)
of 23c as a yellow solid: mp 166-167 °C. MS (ESI) m/z 444.1 (M
(4Z)-6-(3-Methoxyphenyl)-4-({[4-(piperidin--ylmethyl)phenyl]-
amino}methylene)-isoquinoline-1,3(2H,4H)-dione (23i). By the
procedure described above for 22c, 19b (0.5 g, 1.136 mmol) and
3-methoxyphenylboronic acid (0.26 g, 1.7 mmol) were reacted to
give 0.096 g (18.1%) of 23i as a yellow solid: mp 169-170 °C,
MS (ESI) m/z 468.3 (M + H)+1. 1H NMR (400 MHz, DMSO-d6)
δ ppm: 1.39 (d, J ) 4.28 Hz, 2H), 1.43-1.57 (m, 4H), 2.32 (s,
4H), 3.42 (s, 2H), 3.86 (s, 3H), 6.89-7.12 (m, 1H), 7.33 (d, J )
8.3 Hz, 2H), 7.36-7.48 (m, 3H), 7.49-7.62 (m, 3H), 8.09 (d, J )
1
+ H)+1. H NMR (400 MHz, DMSO-d6) δ ppm: 1.24-1.40 (m,
2H), 1.44-1.74 (m, 4H), 2.34 (m, 4H), 3.44 (s, 2H), 7.36 (d, J )
8.4 Hz, 2H), 7.45-7.63 (m, 3H), 7.71-7.91 (m, 2H), 8.02 (d, J )
8.3 Hz, 1H), 8.22 (s, 1H), 8.44 (s, 1H), 8.97 (d, J ) 12.6 Hz, 1H),
11.28 (s, 1H), 12.51 (d,
(C26H25N3O2S·0.33H2O) C, H, N.
J ) 12.6 Hz, 1H). Anal.

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3523
8.1 Hz, 1H), 8.31 (s, 1H), 9.03 (d, J ) 12.6 Hz, 1H), 11.32 (s,
1H), 12.53 (d, J ) 12.6 Hz, 1H). Anal. (C29H29N3O3 ·0.2H2O) C,
H, N.
acetate and hexane to give 0.208 g (79%) of 2-methyl-5-(4-nitro-
phenyl)-2,5-diaza-bicyclo[2.2.1]heptane (26) as yellow crystals: mp
100-101 °C.
(4Z)-6-(4-Methoxyphenyl)-4-({[4-(piperidin-1-ylmethyl)phenyl]-
amino}methylene)-isoquinoline-1,3(2H,4H)-dione (23j). By the
procedure described above for 22c, 19b (0.5 g, 1.136 mmol) and
4-methoxyphenylboronic acid (0.26 g, 1.7 mmol) were reacted to
give 0.12 g (22.6%) of 23j as a yellow solid: mp 168-169 °C, MS
A mixture of 26 (0.19 g, 0.815 mmol) and a catalytic amount of
Pd/C in ethanol (0.2 mL) was hydrogenated at 1 atm at room
temperature overnight. It was filtered through Celite, and the organic
solution was evaporated and then treated with 10 mL of methanol.
It was then treated with hydrochloric acid in methanol, followed
by ethyl ether, and filtered to give 0.107 g (65%) of 4-(5-methyl-
2,5-diaza-bicyclo[2.2.1]hept-2-yl)-phenylamine (27) as a bluish
1
(ESI) m/z 468.2 (M + H)+1. H NMR (400 MHz, DMSO-d6) δ
ppm: 1.39 (d, J ) 4.28 Hz, 2H), 1.43-1.57 (m, 4H), 2.32 (s, 4H),
3.37-3.49 (m, 2H), 3.86 (s, 3H), 6.89-7.12 (m, 2H), 7.33 (d, J )
8.4 Hz, 2H), 7.36-7.48 (m, 2H), 7.49-7.62 (m, 3H), 8.09 (d, J )
8.31 Hz, 1H), 8.31 (s, 1H), 9.03 (d, J ) 12.6 Hz, 1H), 11.32 (s,
1H), 12.53 (d, J ) 12.6 Hz, 1H). Anal. (C29H29N3O3 ·1.2H2O) C,
N. H: calcd, 6.47; found 6.06.
solid: MS (ESI) m/z 204.2 (M - H)+1
.
A solution of 27 (0.1 g, 0.49 mmol), 17f (0.2 g, 0.6 mmol), and
N,N-dimethylformamide (0.2 mL) was heated at 90 °C overnight.
After the solvent was removed, the residue was treated with ethyl
ether and filtered to give crude product as a light-brown solid. It
was purified by HPLC to yield 97 mg (39%) of 34 as a bright-
(4Z)-6-(4-Phenoxyphenyl)-4-({[4-piperidin-1-ylmethyl]phenyl}-
amino)methylene)}isoquinoline-1,3(2H,4H)-dione (23k). By the
procedure described above for 23a, 19b (300 mg, 0.68 mmol) and
4-phenoxyphenyl boronic acid (291.1 mg, 1.36 mmol) were reacted
to give 70 mg (19%) of 23k as a yellow solid: mp 132-133 °C;
MS (ESI) m/z 529.64 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 1.39 (m, 2H), 1.47-1.49 (m 4H), 2.33 (m, 4H), 3.43 (s, 2H),
7.08-7.21 (m, 5H), 7.32 (d, J ) 8.4 Hz, 2H), 7.44 (d, J ) 8.3,
1H), 7.52-7.56 (m, 3H), 7.90 (d, J ) 8.1 Hz, 2H) 8.09 (d, J ) 8.3
Hz, 1H), 8.31 (m, 1H), 9.03 (d, J ) 12.6 Hz, 1H), 11.34 (s, 1H),
12.53 (d, J ) 12.6 Hz, 1H). Anal. (C34H31N3O3 ·1.3H2O) C, H, N.
(4Z)-1, 3-Dioxo-4-({[4-(piperidin-1-ylmethyl)phenyl]amino}-
methylene)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (23l).
A mixture of 1.00 g (2.27 mmol) of 19b, 239 mg (2.05 mmol) of
Zn(CN)2, and 394 mg (0.341 mmol) of tetrakis(triphenylphosphine)-
palladium(0) in 17 mL of DMF under N2 was heated at 100 °C in
the dark for 1.75 h.The reaction was then poured into 40 mL of
ice-water and the product was collected, washed with H2O and
Et2O, and dried. The crude product was boiled with 10% MeOH
in CHCl3 and filtered. The filtrate was washed with 2 M NH4OH
and brine, dried, and evaporated. The residue was washed with
boiling CH3CN and the insoluble material was dried to yield 200
mg (23%) of 23l as yellow-orange crystals: mp 254-256 °C (dec).
HRMS (ESI) m/e calcd for C23H22N4O2 387.18156, found 387.18121
1
orange solid: mp 210-211 °C. MS (ESI) m/z 501. H NMR (400
MHz, DMSO-d6) δ ppm: 2.16 (d, J ) 11.8 Hz, 1H), 2.39 (d, J )
11.8 Hz, 1H), 2.88 (s, 3H), 3.11 (d, J ) 10.3 Hz, 1H), 3.31 (d, J
) 8.8 Hz, 1H), 3.58 (d, J ) 8.9 Hz, 1H), 3.68 (d, J ) 10.3 Hz,
1H), 4.37 (s, 1H), 4.69 (s, 1H), 6.57 (d, J ) 8.5 Hz, 2H), 7.51 (d,
J ) 8.5 Hz, 2H), 7.57 (dd, J ) 8.3, 2.2 Hz, 1H), 7.72 (d, J ) 8.3
Hz, 1H), 8.54 (s, 1H), 8.86 (d, J ) 11.9 Hz, 1H), 11.31 (s, 1H),
12.63 (d, J ) 12.6 Hz, 1H). Anal. calcd for C22H21IN4O2 ·
1.4C2HF3O2.
(4Z)-4-({[4-(3,5-Dimethylpiperazin-1-yl)phenyl]amino}methy-
lene)-6-iodoisoquinoline-1,3(2H,4H)-dione (35). By the procedure
described above for 34, 17f 0.15 g (0.46 mmol) and 30 (0.44 g,
2.13 mmol) were reacted to give 0.15 g (63.5%) of 35 as a light-
brown solid: mp 222-223 °C. MS (ESI) m/z 503.1 (M + H)+1
.
1H NMR (400 MHz, DMSO-d6) δ ppm: 1.03 (d, J ) 6.3 Hz, 6H),
2.12 (m, 2H), 2.72-3.03 (m, 3H), 3.54 (m, 2H), 6.98 (d, J ) 8.8
Hz, 2H), 7.45 (d, J ) 8.8 Hz, 2H), 7.56 (dd, J ) 8.4, 1.13 Hz,
1H), 7.72 (d, J ) 8.4 Hz, 1H), 8.55 (s, 1H), 8.86 (d, J ) 12.6 Hz,
1H), 11.31 (s, 1H), 12.61 (d, J ) 12.6 Hz, 1H). Anal. (C22H23IN4O2)
C, H, N.
(4Z)-6-Iodo-4-[({4-[(3R,5S)-3,4,5-trimethylpiperazin-1-
yl]phenyl}amino)methylene]-isoquinoline-1,3(2H,4H)-dione (36).
By the procedure described above for 34, 17f (0.10 g, 0.30 mmol)
and 31 (0.073 g, 0.33 mmol) were reacted. It was purified by column
chromatography over silica gel using 5% MeOH/CHCl3 as eluent
to give 0.089 g (56.7%) of 36 as a light-brown solid: mp 224-225
°C. MS (ESI) m/z 517.1 (M + H)+1. 1H NMR (400 MHz, DMSO-
d6) δ ppm: 1.08 (d, J ) 6.0 Hz, 6H), 2.19 (s, 3H), 2.21-2.31 (m,
2H), 2.39 (m, 2H), 3.57 (m, 2H), 6.99 (d, J ) 9.1 Hz, 2H), 7.46
(d, J ) 9.1 Hz, 2H), 7.56 (dd, J ) 8.3, 1.5 Hz, 1H), 7.72 (d, J )
8.3 Hz, 1H), 8.55 (d, J ) 1.5 Hz, 1H), 8.86 (d, J ) 12.6 Hz, 1H),
11.31 (s, 1H), 12.60 (d, J ) 12.6 Hz, 1H). Anal. (C23H25-
IN4O2 ·0.6H2O) C, H, N.
1
(M + H)+1. H NMR (DMSO-d6) δ ppm: 1.39 (m, 2H), 1.49 (m,
4H), 2.33 (s, 4H), 3.43 (s, 2H), 7.35 (d, J ) 6 Hz, 2H), 7.60 (m,
3H), 8.13 (d, J ) 6 Hz, 1H), 8.76 (s, 1H), 9.01 (d, J ) 9 Hz, 1H),
11.58 (s, 1H), 12.46 (d, J ) 9 Hz, 1H). Anal. (C23H22-
N4O2 ·0.3CHCl3) H, C: calcd, 66.27; found, 65.63; N: calcd, 13.27;
found, 13.87.
6-Iodo-4-{[4-(5-methyl-2,5-diaza-bicyclo[2.2.1]hept-2-yl)-phe-
nylamino]-methylene}-4H-isoquinoline-1,3-dione (34). A solution
of N-Boc-2,5-diaza-bicyclo[2.2.1]heptane (1 g, 5.04 mmol) and
p-nitrobenzene (0.8 mL, 7.56 mmol) in 10 mL of acetonitrile was
heated at 100 °C overnight. After the solvent was removed, the
residue was dissolved in ethyl acetate and washed with saturated
sodium bicarbonate solution. The organic layer was washed with
brine, dried over magnesium sulfate, filtered, and evaporated to
dryness. The oil was washed with hexane and recrystallized from
ethyl acetate in hexane to give 0.83 g (52%) of 2-Boc-5-(4-nitro-
phenyl)-2,5-diaza-bicyclo[2.2.1]heptane (24) as a yellow solid: mp
193-194 °C.
A mixture of 24 (0.5 g, 1.566 mmol) in 20 mL of methanol and
1 mL of trifluoroacetic acid was stirred overnight. After the solvents
were removed, the residue was treated with sodium bicarbonate
solution and extracted with chloroform. The organic layer was dried
over magnesium sulfate, filtered, and dried up to give a yellow
solid, which upon recrystallization gave 0.251 (73%) of 5-(4-nitro-
phenyl)-2,5-diaza-bicyclo[2.2.1]heptane (25) as a yellow solid: mp
140-141 °C.
(4Z)-4-({[4-(3,4-Dimethylpiperazin-1-yl)phenyl]amino}meth-
ylene)-6-iodoisoquinoline-1,3(2H,4H)-dione (37). By the proce-
dure described above for 34, 17f (0.15 g, 0.46 mmol) and 32 (0.11
g, 0.50 mmol) were reacted to give 0.052 g (22.6%) of 37 as a
light-brown solid: mp 157-158 °C. MS (ESI) m/z 503.1 (M +
1
H)+1. H NMR (400 MHz, DMSO-d6) δ ppm: 1.34 (d, J ) 6.3
Hz, 3H), 2.68-2.83 (m, 2H), 2.89 (s, 3H), 3.57 (s, 2H), 3.78-4.02
(m, 2H), 7.08 (t, J ) 8.7 Hz, 2H), 7.54 (d, J ) 8.7 Hz, 2H), 7.58
(d, J ) 8.3 Hz, 1H), 7.73 (d, J ) 8.3 Hz, 1H), 8.56 (s, 1H), 8.87
(d, J ) 12.6 Hz, 1H), 11.34 (s, 1H), 12.59 (d, J ) 12.6 Hz, 1H).
Anal. (C22H23IN4O2 ·1.4TFA) C, H, N.
(4Z)-6-Iodo-4-[({4-[(2S,5R)-2,4,5-trimethylpiperazin-1-
yl]phenyl}amino)methylene]-isoquinoline-1,3(2H,4H)-dione (38).
By the procedure described above for 34, 17f (0.10 g, 0.30 mmol)
and 33 (0.073 g, 0.33 mmol) were reacted. After column chroma-
tography over silica gel using 5% MeOH/CHCl3 as eluent, 0.078 g
(49.7%) of 38 as a light-brown solid was obtained: mp 199-200
°C. MS (ESI) m/z 517.1 (M + H)+1. 1H NMR (400 MHz, DMSO-
d6) δ ppm: -0.07 to 0.09 (m, 3H), 0.90 (d, J ) 4.78 Hz, 3H),
1.05-1.46 (m, 5H), 2.59-3.07 (m, 4H), 7.18 (d, J ) 8.3 Hz, 2H),
7.60 (d, J ) 8.31 Hz, 3H), 7.73 (d, J ) 8.3 Hz, 1H), 8.58 (s, 1H),
A mixture of 25 (0.247 g, 1.127 mmol), paraformaldehyde (37%
in water, 0.25 mL, 9 mmol), and formic acid (0.3 mL, 7.7 mmol)
was heated at 80 °C for 3 h. It was basified with 5N NaOH solution
to pH 10 and extracted with chloroform. The organic layer was
washed with brine, dried over magnesium sulfate, filtered, and dried
up to give a yellow solid, which upon recrystallization from ethyl

3524 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Tsou et al.
8.89 (d, J ) 12.6 Hz, 1H), 11.38 (s, 1H), 12.54 (d, J ) 12.6 Hz,
1H). Anal. (C23H25BrN4O2 ·0.6H2O) C, H, N.
(4Z)-6-(3-Furyl)-4-({[2-(4-methylpiperazin-1-yl)pyrimidin-5-
yl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (45e). By the
procedure described for 45b, 44e (73.5 mg, 0.15 mmol) and 3-furan
boronic acid (33 mg, 0.3 mmol) were reacted to give 36 mg (55%)
of 45e as a yellow solid: MS (ESI) m/z 431 (M + H)+1. 1H NMR
(400 MHz, DMSO-d6) δ ppm: 2.22 (s, 3H), 2.29-2.43 (m, 4H),
3.61-3.94 (m, 4H), 7.23 (d, J ) 1.0 Hz, 1H), 7.52 (dd, J ) 8.3,
1.3 Hz, 1H), 7.82 (t, J ) 1.8 Hz, 1H), 8.00 (d, J ) 8.3 Hz, 1H),
8.14 (s, 1H), 8.41 (s, 1H), 8.68 (s, 2H), 8.78 (d, J ) 12.6 Hz, 1H),
11.24 (s, 1H), 12.09 (d, J ) 12.6 Hz, 1H). Anal. (C23H22-
N6O3 ·H2O·0.3TFA) C, H, N.
(4Z)-6-(3-Furyl)-4-({[5-(4-methylpiperazin-1-yl)pyrazin-2-
yl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (45f). By the
procedure described for 45b, 44f (500 mg, 0.41 mmol) and 3-furan
boronic acid (114 mg,, 1.02 mmol) were reacted to give 40 mg
(23%) of 45f as a yellow solid: mp 226 -227 °C. MS (ESI) m/z
431.1 (M + 1)+. 1H NMR (400 MHz, CDCl3) δ ppm: 2.37 (s,
3H), 2.50-2.64 (m, 4H), 3.35-3.74 (m, 4H), 7.41 (dd, J ) 8.0,
1.6 Hz, 1H), 7.55 (m, 1H), 7.77 (d, J ) 1.6 Hz, 1H), 7.90 (s, 1H),
7.93 (s, 1H), 7.99 (d, J ) 8.0 Hz, 1H), 8.23 (s, 1H), 8.41 (s, 1H),
9.08 (d, J ) 12.4 Hz, 1H), 11.24 (s, 1H) 12.45 (d, J ) 12.4 Hz,
1H). Anal. (C23H22N6O3 ·1.0DMF·0.6TFA) C, H, N.
(4Z)-6-Bromo-4-({[5-(4-methylpiperazin-1-yl)pyridin-2-
yl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (44a). Using
the procedure described for the preparation of 34, 1.2 g (75%) of
44a was obtained as a orange solid from 1.0 g (3.54 mmol) 17b
and 670 mg (3.54 mmol) of 41a: mp 191-192 °C. MS (ESI) m/z
444.0 (M + 1)+. 1H NMR (400 MHz, CDCl3) δ ppm: 2.38 (s, 3H)
2.49-2.71 (m, 4H) 3.13-3.38 (m, 4H) 6.90 (d, J ) 8.81 Hz, 1H)
7.29 (d, J ) 3.02 Hz, 1H) 7.38 (dd, J ) 8.56, 1.76 Hz, 1H) 7.91
(d, J ) 1.51 Hz, 1H) 8.03-8.14 (m, 2H) 8.53 (s, 1H) 9.15 (d, J )
12.09 Hz, 1H) 12.34 (d,
(C20H20BrN5O2 ·0.7H2O) C, H, N.
J ) 12.09 Hz, 1H). Anal.
(4Z)-6-(3-Furyl)-4-({[6-(4-methylpiperazin-1-yl)pyridin-3-
yl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (45b). A mix-
ture of 300 mg (0.68 mmol) of (4Z)-6-bromo-4-({[6-(4-methylpip-
erazin-1-yl)pyridin-3-yl]amino}methylene)isoquinoline-1,3(2H,4H)-
dione (44b, prepared using a similar procedure described for 18b),
Pd2(dba)3 (125 mg, 0.136 mmol), tri-tert-butylphosphine (0.13 mL,
0.64 mmol), cesium carbonate (663 mg, 1.36 mmol), and 3-furan
boronic acid (189.72 mg, 1.7 mmol) was placed in a three-neck
flask under N2, N,N-dimethylformamide (8 ML) was added, and
the mixture was then stirred in a preheated oil bath 130 °C for 30
min. After cooling, the mixture was treated with CH2Cl2 and filtered
through celite. After evaporating all the solvents, the residue was
dissolved in methylene chloride, washed three times with brine,
dried over sodium sulfate, and evaporated. The orange oily residue
was purified by silica gel chromatography (10:90 ) methanol:
methylene chloride) to give 130 mg (45%) of 45b as a yellow solid:
Biological Methods
Enzyme Assay. CDK4/cyclin D1, CDK6/cyclin D1, and
CDK2/cyclin E were expressed in insect cells (Sf9) infected
with recombinant baculovirus and partially purified using
ammonium sulfate fractionation. CDK1/cyclin B1 was pur-
chased from New England BioLabs (Beverly, MA). Test
compounds were diluted in 20% DMSO/20 mM HEPES, pH
7.5, and serial dilutions were prepared (5 concentrations;
0.005-50 µM). High-binding ELISA microtiter plates (Costar)
were coated with the kinase substrate, glutathione-S-transferase
(GST) fusion of C-terminal fragment of the retinoblastoma
susceptibility gene product (Rb). Nonspecific binding sites were
blocked with Superblock in Tris-buffered saline (TBS; Pierce).
Kinase reactions contained the test inhibitor, 200 µM ATP, 0.5
mg/mL bovine serum albumin (BSA; Sigma), and 0.1 µL of
enzyme. Reaction volumes were adjusted to 30 µL with kinase
assay buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 5%
glycerol, 10 mM 2-mercaptoethanol), and plates were incubated
at 30 °C for 1 h. Reactions were terminated by aspiration, and
nonspecific sites were blocked with blocking buffer (TBS
containing 0.1% Tween-20 and 5% nonfat dry milk). Phospho-
rylation of the substrate was detected using phospho-Rb specific
antibodies (Ser 795) (Cell Signaling Technologies) and antirabbit
IgG/horseradish peroxidase conjugates (Amersham Life Science)
using TMB (3,3′,5,5′-tetramethylbenzidine) as substrate. Colo-
rimetric reactions were stopped with 2 N sulfuric acid, and the
absorbance was measured at 450 nm. IC50 values were
determined from inhibition plots.
Cell Culture. MCF-7 cells were cultured in RPMI medium
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine
serum (Invitrogen) and 50µg/mL gentamicin (Invitrogen) at 37
°C in a humidified incubator under 5-7% CO2.
Cell Proliferation Assay. Cells were plated in 96-well plates
and cell growth was determined using sulforhodamine B (SRB), a
protein binding dye. Briefly, cells were fixed with TCA and rinsed
extensively in water. Cells were stained with 0.4% sulforhodamine
B (Sigma-Aldrich, St Louis, MO) and washed in 1% acetic acid.
Protein-associated dye was solubilized in 10 mM Tris, and the
absorbance was measured in a Victor fluorescence reader (Wallac/
Perkin-Elmer Life Sciences, Boston, MA).
1
mp 200-201 °C. MS (ESI) m/z 430.2 (M + 1)+. H NMR (400
MHz, DMSO-d6) δ ppm: 2.26 (s, 3H), 2.4-2.43 (m, 4H),
3.49-3.52 (m, 4H), 6.96 (d, J ) 9.2 Hz, 1H), 7.25 (m, 1H), 7.52
(dd, J ) 8.4, 1.4 Hz, 1H), 7.82 (m, 1H), 7.90 (dd, J ) 9.2, 2.0 Hz,
1H), 8.01 (d, J ) 8.4 Hz, 1H), 8.17 (m, 1H), 8.37 (d, J ) 2.0 Hz,
1H), 8.43 (s, 1H), 8.86 (d, J ) 12.8 Hz, 1H), 11.24 (s, 1H), 12.39
(d, J ) 12.8 Hz, 1H). Anal. (C24H23N5O3 ·0.9H2O) C, H, N.
(4Z)-6-(3-Furyl)-4-({[5-(4-methylpiperazin-1-yl)pyridin-2-
yl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (45a). By the
procedure described for 45b, 44a (2.0g, 4.52 mmol) and 3-furan
boronic acid (1.3 g, 11.3 mmol) were reacted to give 1.2g (63%)
of 45a as a yellow solid: mp 262-263 °C. MS (ESI) m/z 430.1 (M
+ 1)+. 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.23 (s, 3H),
2.45-2.49 (m, 4H), 3.08-3.25 (m, 4H), 7.21 (d, J ) 2.4 Hz, 1H),
7.53 (d, J ) 2.4 Hz, 1H), 7.55 (m, 2H), 7.83 (m, 1H), 8.03 (m,
1H), 8.11 (m, 2H), 8.44 (s, 1H), 9.13 (d, J ) 12.8 Hz, 1H), 11.30
(s, 1H), 12.45 (d, J ) 12.8 Hz, 1H). Anal. (C24H23N5O3 ·1.13H2O)
C, H. N: calcd, 15.58; found, 15.15.
(4Z)-6-(3-Furyl)-4-({[6-(4-methylpiperazin-1-yl)pyridazin-3-
yl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (45c). By the
procedure described for 45b, 44c (400 mg, 0.9 mmol) and 3-furan
boronic acid (252 mg, 2.25 mmol) were reacted to give 90 mg
(23%) of 45c as a yellow solid: mp 275-276 °C. MS (ESI) m/z
431.1 (M + 1)+. 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.23 (s,
3H), 2.37-2.47 (m, 4H), 3.45-3.66 (m, 4H), 7.22 (m, 1H), 7.51
(d, J ) 10.0 Hz, 1H), 7.58 (dd, J ) 8.0, 1.2 Hz, 1H), 7.83 (m,
1H), 7.91 (d, J ) 10.0 Hz, 1H), 8.03 (d, J ) 8.0 Hz, 1H), 8.17 (m,
1H), 8.45 (s, 1H), 9.13 (d, J ) 12.0 Hz, 1H), 11.38 (s, 1H), 12.51
(d, J ) 12.0 Hz, 1H). Anal. (C23H22N6O3 ·1.0H2O·0.1TFA) C, H,
N.
(4Z)-4-({[3-Fluoro-4-(4-methylpiperazin-1-yl)phenyl]amino}-
methylene)-6-(3- furyl)isoquinoline-1,3(2H,4H)-dione (45d). By
the procedure described for 45b, 44d (0.5 g, 1.09 mmol) and 3-furan
boronic acid (0.2 g, 1.78 mmol) were reacted to give 0.32 g (65.8%)
of 45d as a light-brown solid: mp 203-204 °C. MS (ESI) m/z 447.2
(M + H)+1. 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.23 (s, 3H),
2.48 (s, 4H), 2.90-3.09 (m, 4H), 7.07 (t, J ) 9.19 Hz, 1H),
7.21-7.37 (m, 2H), 7.54 (dd, J ) 8.3, 1.3 Hz, 1H), 7.65 (m, 1H),
7.83 (m, 1H), 8.01 (d, J ) 8.3 Hz, 1H), 8.20 (s, 1H), 8.43 (s, 1H),
8.86 (d, J ) 12.6 Hz, 1H), 11.28 (s, 1H), 12.47 (d, J ) 12.6 Hz,
1H). Anal. (C25H23FN4O3 ·1.0H2O) C, H, N.
Flow Cytometry. Cells growing in 6-well tissue culture plates
were were pulse-labeled with 10 µM bromo-deoxyuridine for
30 min. Cells were collected by trypsinization, and fixed in 80%

4-(Phenylaminomethylene)isoquinoline-1,3(2H,4H)-diones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3525
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methanol at -20 °C. After acid denaturation and permeabili-
zation, the cells were stained with antibromodeoxyuridine-FITC
conjugates (BD Biosciences, Mountain View, CA), counter-
stained with propidium iodide, and analyzed by flow cytometry
(FACSort, BD Biosciences). Data were analyzed using Lysis
II or CELLQuest software (BD Biosciences).
Preparation of Cell Extracts and Protein Immu-
noblotting. Cell lysates were prepared in lysis buffer (25 mM
Tris-Cl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40,
0.5% sodium deoxycholate) supplemented with 0.2 mM PMSF, 1
mM DTT, 0.2 mM sodium fluoride, and 1 mM sodium vanadate
(all chemical reagents were from Sigma-Aldrich, St Louis, MO).
Protein concentrations were determined by the BioRad protein
assay (BioRad, Hercules, CA). Proteins (10-20 µg) were separated
by electrophoresis on 8 or 10% polyacrylamide-SDS gels (SDS-
PAGE) and transferred to nitrocellulose. Blots were blocked in
phosphate buffered saline (PBS) or tris-buffered saline (TBS)
containing 5% skim milk (or 5% BSA) and 0.1% Tween-20 and
incubated with antibodies in the same buffer. Blots were developed
using enhanced chemiluminescence (ECL, Amersham/GE Health-
care, Piscataway, NJ). Antibodies used were: cyclin D1 and
phospho-Rb (Ser 795) (Cell Signaling Technologies, Beverly, MA),
cyclin E and cyclin B1 (Santa Cruz Biotechnologies), Rb (Santa
Cruz Biotechnologies, Santa Cruz, CA or EMD Biosciences,
Darmstadt, Germany), CDK4 (Upstate, Lake Placid, NY), CDK2,
CDK1, and p27 (Santa Cruz Biotechnologies), actin (Chemicon,
Temecula, CA), and rabbit and mouse IgG-horseradish peroxidase
conjugates (Amersham).
Acknowledgment. The authors wish to thank Drs. Tarek
Mansour, Philip Frost and John Ellingboe for their support and
encouragement. The authors are grateful to Dr. Ping-Zhong
Huang for scaling up key intermediates. We also would like to
thank members of the Wyeth Chemical Technologies group for
analytical and spectral determinations.
(21) GLIDE; Schrodinger Software: Portland, OR; www.schrodinger.com.
(22) (a) McInnes, C.; Wang, S.; Anderson, S.; O’Boyle, J.; Jackson, W.;
Kontopidis, G.; Meades, C.; Mezna, M.; Thomas, M.; Wood, G.; Lane,
D. P.; Fischer, P. M. Structural Determinants of CDK4 Inhibition and
Design of Selective ATP Competitive Inhibitors. Chem. Biol. 2004, 11,
525–534. (b) Honma, T.; Hayashi, K.; Aoyama, T.; Hashimoto, N.;
Machida, T.; Fukasawa, K.; Iwama, T.; Ikeura, C.; Iduta, M.; Suzuki-
Takahashi, I.; Iwasawa, Y.; Hayama, T.; Nishimura, S.; Morishima,
H. Structure-Based Generation of a New Class of Potent cdk4
Inhibitors: New de Novo Design Strategy and Library Design. J. Med.
Chem. 2001, 44, 4615–4627. (c) Aubry, C.; Wilson, A. J.; Jenkins,
P. R.; Mahale, S.; Chaudhuri, B.; Marechal, J.; Sutcliffe, M. J. Design,
synthesis and biological activity of new CDK4-specific inhibitors based
on fascaplysin. Org. Biomol. Chem. 2006, 4, 787–801.
Supporting Information Available: Elementary analysis data
for compounds 5a-5d, 18a, 18b, 18e-18i, 19b-19i, 20a,
21a-21d, 22c-22e, 22g, 23a-23l, 34-38, 44a, and 45a-45f. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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