New effective inhibitors of the Abelson kinase

Article abstract of DOI:10.1016/j.bmc.2010.07.021

The effects of substituents on the aryl ring were studied by the preparation and testing of several PD173955 analogs. Inserting a single carbon atom into the C-N bond in the aniline subunit (PDC) reduced the kinase inhibition by a factor of 200. Despite its decreased affinity for Abl compared with PD173955, PDC exhibits a Ki very similar to that reported for Imatinib. Increased water solubility is also gained by replacing the thiomethyl group with an amino or glycyl moiety. For both PD173955 and PDC, the analogs with amino groups in place of the methylthio group are 10 times more inhibitory than the parent molecules. Two molecules were identified with Kis about three orders of magnitude lower than reported for Imatinib.

Full text of DOI:10.1016/j.bmc.2010.07.021

Bioorganic & Medicinal Chemistry 18 (2010) 6316–6321
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New effective inhibitors of the Abelson kinase
a
b
b
b,c
George A. Kraus a, , Vinayak Gupta , Marjan Mokhtarian , Samir Mehanovic , Marit Nilsen-Hamilton
*
a Department of Chemistry, Iowa State University, Ames, IA 50011, United States
b Molecular Express Inc., Ames, IA 50014, United States
c Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50010, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 March 2010
Revised 6 July 2010
Accepted 9 July 2010
Available online 14 July 2010
The effects of substituents on the aryl ring were studied by the preparation and testing of several
PD173955 analogs. Inserting a single carbon atom into the C–N bond in the aniline subunit (PDC) reduced
the kinase inhibition by a factor of 200. Despite its decreased affinity for Abl compared with PD173955,
PDC exhibits a Ki very similar to that reported for Imatinib. Increased water solubility is also gained by
replacing the thiomethyl group with an amino or glycyl moiety. For both PD173955 and PDC, the analogs
with amino groups in place of the methylthio group are 10 times more inhibitory than the parent mol-
ecules. Two molecules were identified with Kis about three orders of magnitude lower than reported for
Imatinib.
Keywords:
Enzyme inhibitor
Abl kinase
Ó 2010 Elsevier Ltd. All rights reserved.
Imatinib
1. Introduction
kinase, which results in toxicity for proliferating normal cells9
and its low solubility in water. In its favor, its affinity is much low-
Because tyrosine protein kinases regulate cell proliferation,
drugs that control these kinases provide important treatments
for proliferative diseases such as cancer and atherosclerosis.1
Chronic myelogenous leukemia (CML) is a hematopoietic stem cell
disease that accounts for 15% of all adult leukemias and is charac-
terized by the clonal expansion of cells carrying the Philadelphia
(Ph) chromosome.2 The Ph chromosome, resulting from the trans-
location of genes from chromosomes 9 and 22 encodes the chime-
ric protein Bcr-Abl.2,3 It is now well recognized that the Bcr-Abl
protein is both the initial cause and major driver of CML. The rear-
ranged Bcr-Abl gene encodes a constitutively active Abl kinase that
confers the property of excessive and uncontrolled proliferation to
the myelogenous cells in which it is expressed.4 Thus, this enzyme
is an attractive target for therapeutic intervention.5
er for most kinases other than those of the Src family of which Abl
is a member, and PD173955 inhibits both the active and inactive
forms of Abl. By contrast, Imatinib only inhibits the active form
of the enzyme. In addition, the Ki for inhibition of Abl by
PD173955 is very low, making it a more potent inhibitor of Abl
and a more effective inhibitor of cancer cell proliferation than
N
H
H
N
N
N
N
N
N
O
Imatinib, a phenylaminopyrimidine-derived inhibitor of the
Abelson tyrosine kinase (c-Abl) shown in Figure 1, has been a very
successful treatment for CML and other cancers. However, resis-
tance has developed in many cancer patients.6 Thus, new drugs
that inhibit Imatinib-resistant kinases are needed.
Imatinib
Figure 1.
Substituted pyrido[2,3-d]pyrimidines are potent inhibitors that
vary in their specificities for different tyrosine kinases.7 PD173955
(1), shown in Figure 2, inhibits the Abelson kinase (Abl) and the Src
and Yes tyrosine kinases that are also often up-regulated in can-
cer.8 PD173955 inhibits the proliferation of many cancer cell types,
but two limitations prevent it from being applied in a clinical set-
ting. These are its ability to inhibit kinases other than the Abl
X
Cl
N
N
R
Y
N
H
N
N
O
Cl
S
Me
N
H
N
N
O
Me
Me
PD173955 (1)
2
* Corresponding author. Tel.: +1 515 294 6342; fax: +1 515 294 0105.
E-mail address: gakraus@iastate.edu (G.A. Kraus).
Figure 2.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.021

G. A. Kraus et al. / Bioorg. Med. Chem. 18 (2010) 6316–6321
6317
Imatinib.10,12,15 Thus we speculated that, with further modifica-
tion, PD173955 may be developed as an effective inhibitor of c-
Abl to treat patients with CML and other cancers, including those
that have developed Imatinib-resistance.
various substituted phenyl acetic acid esters was successfully
performed following the method developed by Blass et al. using
KF/Al2O3 and dimethyl acetamide at room temperature to give
functionalized pyrido[2,3-d]pyrimidinones 5a–e in moderate to
excellent yields (Scheme 1).14 Oxidation of 5a–e with 2 equiv of
m-chloroperbenzoic acid in dichloromethane gave corresponding
sulfones 6a–e in excellent yields.
Initially, the conversion of sulfone 6 into analogs of PD173955
using various anilines in boiling aprotic solvents failed. Interest-
ingly, the reaction of sulfone 6 with 3-aminobenzylamine, a more
reactive nucleophile, in boiling DMF generated an adduct in good
yield (Scheme 2).
This adduct (which we term PDC because of the extra carbon
atom between the phenyl group and the secondary amino group)
was more soluble than PD173955 in aqueous solutions, when
R = –NH2 or –NHGly. Our interest in understanding the effect of
changes in the PD structure on its ability to inhibit Abl kinase led
us to synthesize various PD and PDC analogs 7a–7e, 8a–8d, 9a–
9b and 1. The reason to develop the analogs 8c and 9b was to cir-
cumvent the problem of low chemical reactivity of aromatic amine
moiety of analogs 7a–7e. The synthetic route towards the synthe-
sis of 8c and 9b consist of standard two step sequence where 7a
and 9a were reacted with bromoacetyl bromide under basic condi-
tions followed by the treatment with ammonia to get 8c and 9b in
good yields (Scheme 3).
Clarkson and Duyster showed that 1 (Fig. 2) and related com-
pounds inhibit Bcr-Abl kinase activity with greater potency than
Imatinib. Moreover, many of these compounds also inhibit kinase
domain mutants of Bcr-Abl that are resistant to Imatinib.11 With
the emerging need for kinase inhibitors that will kill cells that
express Imatinib-resistant Bcr-Abl kinase domain mutants,
PD173955 is a prime candidate for further development.
To investigate the structural elements of PD173955 that make it
a good inhibitor of the Abl kinase, we planned to synthesize ana-
logs of 1. The bicyclic ring and the halogen containing ring subunit
of PD173955 interact with several amino acid residues in the crys-
tal structure of the kinase complex.12 As discussed below, many
analogs of 2 were also synthesized. These were expected to also
be good inhibitors of the Abl kinase because the crystal structure
of Abl with PD173955 shows the methylthiophenyl ring extending
into the solution space and not interacting extensively with the
protein.12
2. Chemistry
Our synthetic route to analogs of PD173955 began from the
known aldehyde 3, readily available from the commercially avail-
able ethyl 4-chloro-2-methylthio pyrimidine-5-carboxylate by
amination, reduction and manganese dioxide oxidation.13 The
reaction of aldehyde 3 with substituted phenylacetic acid ester 4
was hampered by problems related to solubility and low yields.
3. Results and discussion
The results of inhibition of Abl kinase (Ki) are shown below in Fig-
ures 3–5. The number in parentheses after the Ki is the number of
independent experiments performed to obtain the Ki. The asterisks
After several experiments, condensation of aldehyde
3
with
X
X
X
CHO
N
N
N
m-CPBA,
CH2Cl2, RT
KF/Al2O3,
COOEt
Y
Me
O
+
Y
S
N
N
O
S
N
NH
Me
DMA, RT
S
N
N
Me
O
O
Y
Me
Me
Me
4
6
3
5
6a: X = Cl, Y = Cl; Yield = 87%
5a: X = Cl, Y = Cl; Yield = 62%
4a: X = Cl, Y = Cl
4b: X = Cl, Y = H
4c: X = H , Y = H
4d: X = F , Y = F
4e: X = Br, Y = H
6b: X = Cl, Y = H;
6c: X = H , Y = H;
6d: X = F , Y = F;
6e: X = Br, Y = H;
= 85%
= 65%
= 84%
= 82%
5b: X = Cl, Y = H;
5c: X = H , Y = H;
5d: X = F , Y = F;
5e: X = Br, Y = H;
= 73%
= 89%
= 83%
= 92%
Scheme 1.
X
R
X
n
NH2
N
N
R
Y
n N
H
N
N
Me
O
n = 1; DMF, reflux
n = 0; diglyme, reflux
Me
O
Y
S
N
N
Me
O
O
7a
: n = 1; R = -NH2; Yield = 61%
6
7b
7c
7d
7e
8a
1
: n = 1; R = -NH2
: n = 1; R = -NH2
: n = 1; R = -NH2
: n = 1; R = -NH2
: n = 1; R = -SMe
: n = 0; R = -SMe
: n = 0; R = -NH2
= 77%
= 53%
= 67%
= 48%
= 64%
= 57%
= 55%
9a
Scheme 2.

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G. A. Kraus et al. / Bioorg. Med. Chem. 18 (2010) 6316–6321
O
Cl
Cl
Br
1.
Br
N
K2CO3, CH3CN, RT
N
H
N
H2N
Cl
Cl
2. NH3, MeOH
-30 oC - RT
n N
H
N
N
Me
O
H2N
n N
H
N
N
Me
O
O
7a n = 1
9a n = 0
8c n = 1; Yield = 74%
9b n = 0; = 68%
Scheme 3.
7a: X = Cl, Y = Cl
7b: X = Cl, Y = H
7c: X = H, Y = H
7d: X = F, Y = F
7e: X = Br, Y = H
Ki = 8 x 10-9 (16)***
6 x 10-8 (6)***
2 x 10-6 (5)**
4 x 10-8 (3)**
4 x 10-8 (1)*
X
N
H2N
Y
N
N
N
Me
O
H
Figure 3.
Cl
8a: R = -SMe
8b: R = -NH(hexyl)
8c: R = -NHCOCH2NH2
8d: R = -Biotinyl
Ki = 7 x 10-8 (3)***
9 x 10-7 (3)*
N
2 x 10-8 (3)***
2 x 10-8 (1)***
R
Cl
N
N
N
O
H
Me
Figure 4.
Cl
1
: R = -SMe
Ki = 2 x 10-10 (6)***
2 x 10-11 (6)***
9a : R = -NH2
9b : R = -NHCOCH2NH2
N
2 x 10-11 (4)***
Cl
R
N
N
N
Me
O
H
Figure 5.
represent the p values determined from the Pearson product moment
correlation analysis (***, p <0.0005, **, p <0.005, *, p <0.03).
action of PD173955 with the Abl kinase identified the chloro sub-
stituents of the aryl ring as being embedded in the enzyme’s ATP-
binding pocket and held with multiple van der Waals interactions,
which contribute to the affinity of the molecule for the Abl kinase.12
The chlorine atoms limit the rotation of the aryl ring to a rotational
angle that is the same as for the central phenyl group of Imatinib.
These phenyl groups of Imatinib and PD173955 sit in the same po-
sition, similarly rotated, in the protein. The removal of one or two
chlorine atoms from 7a or their replacement with fluoro constitu-
ents would allow rotational flexibility of the compound when not
bound to the enzyme and might increase the Ki compared with
7a by increasing koff in the equilibrium between the Abl and PD
compound and the Abl–PD complex due to increased entropy of
the unbound 7a.
The much higher Ki of 7c compared with 7b, 7d, or 7e suggests
that electronic effects also play an important role in the interaction
of PDC with the enzyme. It is likely that the relevant interacting ami-
no residue might be lys271 that lies close enough to the inhibitor in
the enzyme pocket to interact with PD173955 (and presumably also
PDC) through van der Waals forces as described for the crystal struc-
ture, but which might also be close enough to interact electrostati-
cally with the aryl ring constituentsthrough its epsilon amino group.
Although most van der Waals interactions reported for
PD173955 with Abl are located around the bicyclic constituent
The 2,6-dichlorophenyl substituent 7a clearly exhibited the
strongest inhibition. Analog 8a was prepared by the reaction of
m-methylthioaniline with the corresponding sulfone (Scheme 2).
The analogs 8b–d were synthesized by modifying 7a (Scheme 3).
PD173955 was ultimately synthesized using boiling diglyme in
sealed tube, conditions much harsher than those for the synthesis
of the PDC and its analogs (Scheme 2). The activity of 1 and adducts
9a and 9b was tested using the standard protocol and the results
are shown below.
Compounds 7a–e were synthesized to probe the steric and elec-
tronic influences on the aryl ring. Molecular models clearly show
that the 2,6-dichlorophenyl group in 1 is perpendicular to the pyr-
ido[2,3-d]pyrimidine subunit as a result of nonbonded interactions
of the chlorine atoms.12 The effectiveness with which c-Abl was
inhibited by the synthesized analogs was determined from the Ki
for each compound, which increases with decreasing inhibitory
effectiveness. As shown above, compared with the 2-chlorophenyl
substituent, the monochlorophenyl and phenyl substituents have
increasingly higher Ki values. The 2,6-difluorophenyl substituent
(7d), which is sterically the same size as the phenyl group, has
about the same inhibitory efficiency as PDC with the 2-chloro
(7b) and 2-bromo (7e) substituents. Structural analysis of the inter-

G. A. Kraus et al. / Bioorg. Med. Chem. 18 (2010) 6316–6321
6319
and the dichlorophenyl group, 2 of the 10 interactions are with
amino acids that contact the methylthiophenyl segment of
PD173955. Alteration of the molecular structure in this region by
insertion of an additional carbon between the secondary amino
group and the aryl groups results in a increase in Ki of about
200-fold, which is consistent with the loss of 2 of the 10 van der
Waals interactions that hold the inhibitor in place in the protein’s
ATP-binding pocket. The 10-fold decrease in the Ki with the
replacement of the methylthio group by an amino group on either
PD or PDC probably reflects the gain in van der Waals or an electro-
static interaction with the enzyme by way of the amino group.
129.7, 128.3, 109.4, 28.7, 14.8; LRMS: 352.0, 200.0, 154.1; HRMS
calcd M+ for C15H11Cl2N3OS: 352.0073; found: 352.0074.
5.1.2.2. 6-(2-Chlorophenyl)-8-methyl-2-(methylthio)pyrido[2,3-
d]pyrimidin-7(8H)-one (5b). Yield 73%; 1H NMR (400 MHz,
CDCl3): d 8.63 (s, 1H), 7.64 (s, 1H), 7.42–7.49 (m, 1H), 7.27–7.37
(m, 3H), 3.80 (s, 3H), 2.65 (s, 3H); 13C NMR (100 MHz, CDCl3): d
173.5, 161.8, 156.5, 154.4, 135.0, 134.8, 133.8, 131.7, 131.5,
130.0, 129.9, 126.9, 109.5, 28.6, 14.7; LRMS: 318.0; HRMS calcd
M+ for C15H12ClN3OS: 317.0390; found: 317.0396.
5.1.2.3. 8-Methyl-2-(methylthio)-6-phenylpyrido[2,3-d]pyrimi-
din-7(8H)-one (5c). Yield 89%; 1H NMR (400 MHz, CDCl3): d 8.61
(s, 1H), 7.67 (s, 1H), 7.61–7.66 (m, 2H), 7.34–7.45 (m, 3H), 3.79
(s, 3H), 2.63 (s, 3H); 13C NMR (100 MHz, CDCl3): d 172.8, 162.4,
156.3, 153.9, 135.6, 132.8, 132.6, 128.9, 128.7, 128.4, 109.9, 28.5,
14.6; LRMS: 284.1, 176.1, 158.1; HRMS calcd M+ for C15H13N3OS:
283.0779; found: 283.0778.
4. Conclusion
In summary, the effects of substituents on the aryl ring were
studied by the preparation and testing of several PD173955 ana-
logs. The results are consistent with the observation from the crys-
tal structure that electronic and van der Waals forces are likely to be
involved in the interaction of inhibitor and enzyme. They also show
that inserting a single carbon atom into the C–N bond in the aniline
subunit (PDC) reduced the kinase inhibition by a factor of 200, con-
sistent with the loss of 20% of the van der Waals interactions be-
tween inhibitor and protein. Despite its decreased affinity for Abl
compared with PD, PDC (8a) exhibits a Ki very similar to that re-
5.1.2.4. 6-(2-Bromophenyl)-8-methyl-2-(methylthio)pyrido[2,3-d]-
pyrimidin-7(8H)-one (5d). Yield 83%; 1H NMR (400 MHz, CDCl3): d
8.63 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.29–7.40
(m, 2H), 7.21–7.28 (m, 1H), 3.80 (s, 3H), 2.64 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 173.5, 161.8, 156.6, 154.5, 136.9, 135.0,
133.3, 133.1, 131.6, 130.2, 127.6, 123.9, 109.5, 28.7, 14.8; LRMS:
364.0, 362.0; HRMS calcd M+ for C15H12BrN3OS: 360.9884; found:
360.9876.
ported for Imatinib [4 ꢀ 10ꢁ8 15
]
and amino-PDC (7a) is significantly
more water soluble compared with PD173955. Furthermore,
replacement of the thiomethyl group on PD173955 with either an
amino or a glycyl group resulted in a decrease in the Ki of 10-fold,
which is 1000-fold lower than the Ki reported for Imatinib.
5.1.2.5. 6-(2,6-Difluorophenyl)-8-methyl-2-(methylthio)pyrido-
[2,3-d]pyrimidin-7(8H)-one (5e). Yield 92%; 1H NMR (400 MHz,
CDCl3): d 8.64 (s, 1H), 7.72 (s, 1H), 7.30–7.40 (m, 1H), 6.93–7.04
(m, 2H), 3.81 (s, 3H), 2.64 (s, 3H); 13C NMR (100 MHz, CDCl3): d
173.9, 162.0 (d, J = 28.0 Hz), 161.2, 159.5 (d, J = 28.0 Hz), 156.7,
154.5, 136.9, 130.6 (t, J = 40.0 Hz), 122.4, 111.7 (d, J = 100.0 Hz),
109.3, 28.7, 14.7; LRMS: 320.1; HRMS calcd M+ for C15H11F2N3OS:
319.0591; found: 319.0591.
5. Experimental
5.1. Chemistry
5.1.1. Materials and instrumentation
THF was distilled from sodium benzophenone ketyl. Methylene
chloride, DMA and DMF were purchased from Aldrich. All experi-
ments were performed under an argon atmosphere. Organic ex-
tracts were dried over anhydrous MgSO4. NMR experiments were
performed with either a Varian 300 MHz or a Bruker 400 MHz
instrument. HRMS were recorded on a Kratos model MS-50 spec-
trometer and LRMS were performed with a Finnegan 4023 mass
spectrometer. Standard grade silica gel (60 Å) was used for flash
column chromatography.
5.1.3. Representative procedure for the preparation of 6a–e
To a solution of 0.500 g (1.42 mmol) of 5a in 40 mL of chloro-
form, was added 0.800 g (3.12 mmol) of 75% m-chloroperoxyben-
zoic acid. The solution was stirred at rt for 6 h. After the
completion of reaction, 2 mL of DMSO was added to the reaction
mixture to neutralize unreacted m-chloroperoxybenzoic acid and
it was further stirred for 15 min after which the reaction mixture
was diluted with methylene chloride and washed with saturated
NaHCO3, water and brine. The organic phase was dried over anhy-
drous MgSO4 and concentrated. The crude was used for the next
step without further purification.
5.1.2. Representative procedure for the preparation of 5a–e
To a stirred solution of 0.400 g (2.19 mmol) of 4-amino-2-
(methylthio) pyrimidine-5-carbaldehyde 3 and 0.638 g (2.74 mmol)
of ethyl 2-(2,6-dichlorophenyl)acetate 4a in 10.0 mL of dry DMA,
2.536 g of KF/Al2O3 (40 wt %, KF, Sigma–Aldrich catalog #316385)
was added in batches of 0.5 g every 30 min. The reaction mixture
was then stirred at rt for 24 h under argon after which it was filtered
through Celite, the residual solid was washed with methylene chlo-
ride and the combined filtrates were concentrated. Due to the poor
solubility of dichloropyridopyrimidinones derivative 5a in most sol-
vents, it was purified by recrystallization from DMF to obtain 0.480 g
(62%). Other pyridopyrimidinones were purified by column chroma-
tography over silica gel using 1:1 hexanes/EtOAc to give pure
products.
5.1.3.1. 6-(2,6-Dichlorophenyl)-8-methyl-2-(methylsulfonyl)pyr-
ido[2,3-d]pyrimidin-7(8H)-one (6a). Yield 87%; 1H NMR (300 MHz,
CDCl3): d 9.03 (s, 1H), 7.78 (s, 1H), 7.42–7.48 (m, 2H), 7.31–7.38 (m,
1H), 3.91 (s, 3H), 3.44 (s, 3H); 13C NMR (100 MHz, CDCl3): d 165.0,
160.5, 157.5, 155.5, 135.2, 135.0, 134.7, 132.9, 131.0, 128.4, 115.0,
39.5, 29.5; LRMS: 352.0, 200.0, 154.1; HRMS calcd M+ for
C15H11Cl2N3O3S: 383.9971; found: 383.9970.
5.1.3.2. 6-(2-Chlorophenyl)-8-methyl-2-(methylsulfonyl)pyrido
[2,3-d]pyrimidin-7(8H)-one (6b). Yield 85%; 1H NMR (400 MHz,
CDCl3): d 9.00 (s, 1H), 7.82 (s, 1H), 7.47–7.53 (m, 1H), 7.33–7.43
(m, 3H), 3.89 (s, 3H), 3.43 (s, 3H); 13C NMR (100 MHz, CDCl3): d
164.6, 161.2, 157.3, 155.2, 136.6, 133.8, 133.7, 133.5, 131.3,
130.7, 130.1, 127.1, 115.1, 39.4, 29.4; LRMS: 452.3, 411.1, 391.3,
350.0, 331.1, 302.1, 268.1, 249.1, 215.1; HRMS calcd M+ for
5.1.2.1. 6-(2,6-Dichlorophenyl)-8-methyl-2-(methylthio)pyrido
[2,3-d]pyrimidin-7(8H)-one (5a). Yield 62%; 1H NMR (400 MHz,
CDCl3): d 8.66 (s, 1H), 7.60 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.29
(d, J = 8.0 Hz, 1H), 3.83 (s, 3H), 2.67 (s, 3H); 13C NMR (100 MHz,
CDCl3): d 173.9, 161.1, 156.7, 154.6, 136.1, 135.6, 133.9, 130.4,
C15H12ClN3O3S: 349.0310; found: 349.0299.

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G. A. Kraus et al. / Bioorg. Med. Chem. 18 (2010) 6316–6321
5.1.3.3. 8-Methyl-2-(methylsulfonyl)-6-phenylpyrido[2,3-d]pyr-
imidin-7(8H)-one (6c). Yield 65%; 1H NMR (400 MHz, CDCl3): d
8.99 (s, 1H), 7.85 (s, 1H), 7.64–7.71 (m, 2H), 7.42–7.49 (m, 3H),
3.88 (s, 3H), 3.41 (s, 3H); 13C NMR (100 MHz, CDCl3): d 164.2,
162.0, 157.0, 154.7, 137.7, 134.7, 131.2, 129.8, 129.1, 128.7,
115.7, 39.5, 29.4; LRMS: 318.0; HRMS calcd M+ for C15H13N3O3S:
317.0390; found: 317.0396.
3H), 7.13 (t, J = 7.6 Hz, 1H), 6.77–6.59 (m, 3H), 4.62 (d, J = 5.2 Hz,
2H), 3.71 (s, 3H); 13C NMR (100 MHz, CDCl3): d 163.1, 161.3,
158.6, 155.6, 147.0, 139.9, 136.5, 133.7, 129.8, 129.6, 128.9,
128.4, 128.0, 120.4, 118.6, 118.0, 114.3, 46.0, 28.3; LRMS: 358.2,
357.2, 356.2, 252.1, 121.1, 106.1, 77.0; HRMS calcd M+ for
C21H19N5O: 357.1590; found: 357.1600.
5.1.4.4. 2-(3-Aminobenzylamino)-6-(2-bromophenyl)-8-methyl-
pyrido[2,3-d]pyrimidin-7(8H)-one (7e). Yield 48%; 1H NMR
(400 MHz, CDCl3): d 8.18 (br s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.46
(br s, 1H), 7.37–7.32 (m, 2H), 7.26–7.20 (m, 2H), 7.13 (t,
J = 7.6 Hz, 1H), 6.78–6.60 (m, 3H), 4.62 (d, J = 5.2 Hz, 2H), 3.71 (s,
3H); 13C NMR (100 MHz, CDCl3): d 162.5, 161.6, 161.3, 158.8,
156.1, 147.1, 139.9, 137.6, 135.8, 133.1, 131.9, 129.9, 129.7,
127.5, 124.3, 120.4, 118.7, 118.1, 114.4, 46.1, 28.4; LRMS: 435,
358, 356, 252, 237, 179, 106, 77; HRMS calcd M+ for C21H18BrN5O:
435.06947; found: 435.07033.
5.1.3.4. 6-(2-Bromophenyl)-8-methyl-2-(methylsulfonyl)pyrido
[2,3-d]pyrimidin-7(8H)-one (6d). Yield 84%; 1H NMR (400 MHz,
CDCl3): d 9.00 (s, 1H), 7.79 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.42 (t,
J = 8 Hz, 1H), 7.29–7.36 (m, 2H), 3.90 (s, 3H), 3.43 (s, 1H); 13C
NMR (100 MHz, CDCl3): d 164.7, 161.2, 157.3, 155.3, 138.3, 135.8,
133.7, 133.3, 131.2, 130.9, 127.8, 123.4, 115.2, 39.5, 29.5; LRMS:
457.0, 396.0, 348.0, 254.1, 215.1; HRMS calcd M+ for
C15H12BrN3O3S: 392.9783; found: 392.9792.
5.1.3.5.6-(2,6-Difluorophenyl)-8-methyl-2-(methylsulfonyl)pyr-
ido[2,3-d]pyrimidin-7(8H)-one (6e). Yield 82%; 1H NMR (400
MHz, CDCl3): d 9.02 (s, 1H), 7.90 (s, 1H), 7.37–7.47 (m, 1H), 7.03
(t, J = 8.0 Hz, 2H), 3.89 (s, 3H), 3.42 (s, 3H); 13C NMR (100 MHz,
5.1.4.5. 2-(3-Aminobenzylamino)-6-(2,6-difluorophenyl)-8-met
hylpyrido[2,3-d]pyrimidin-7(8H)-one (7d). Yield 67%; 1H NMR
(400 MHz, CDCl3): d 8.37 (br s, 1H), 7.59 (s, IH), 7.32 (dt,
J = 7.4 Hz, J = 2.0 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.97 (t, J = 8.0 Hz,
2H), 6.76 (d, J = 7.2 Hz, 1H), 6.69 (s, 1H), 6.61 (dd, J = 8.0 Hz,
J = 2.0 Hz, 1H), 4.64 (d, J = 5.2 Hz, 2H), 3.71 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 162.3, 162.0, 161.7, 161.2, 159.8, 159.0,
156.3, 147.0, 139.8, 137.6, 129.9, 120.5, 119.9, 118.7, 118.0,
114.4, 111.7, 111.5, 105.6, 46.1, 28.5; LRMS: 394.1, 393.1, 392.1,
288.1, 269.1, 121.1, 106.1, 77.0, 69.0; HRMS calcd M+ for
CDCl3):
d 164.9, 161.9 (d, J = 28.0 Hz), 160.6, 159.4 (d, J =
28.0 Hz), 157.5, 155.4, 135.7, 131.4 (t, J = 40.0 Hz), 127.8, 114.9,
111.9 (d, J = 100.0 Hz), 39.5, 29.5; LRMS: 413.1, 352.0, 333.1,
304.1, 251.1, 209.2, 121.0; HRMS calcd M+ for C15H11F2N3O3S:
351.0489; found: 351.0491.
5.1.4. Representative procedure for the preparation of 7a–e and
8a
C21H17F2N5O: 393.1401; found: 393.1404.
A stirred mixture of 0.135 g (0.352 mmol) of sulfone 6a and
0.086 g (0.704 mmol) of 3-aminobenzylamine in DMF (5 mL) was
refluxed overnight. The resultant reaction mixture was cooled to
rt and diluted with water. This aqueous solution was extracted
with ethyl acetate (3 ꢀ 15 mL). The ethyl acetate layer was sub-
jected to water wash (2 ꢀ 10 mL) and brine wash followed by dry-
ing over anhydrous MgSO4 and concentrated in vacuo to give the
crude product which was chromatographed over silica gel using
2% MeOH in CH2Cl2 to give 0.091 g (61%) of pure 7a.
5.1.4.6. 6-(2,6-Dichlorophenyl)-8-methyl-2-(3-(methylthio)ben-
zylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (8a). Yield 64%; 1H
NMR (400 MHz, CDCl3): d 8.19 (br s, 1H), 7.42–7.38 (m, 3H),
7.27–7.22 (m, 3H), 7.18–7.16 (m, 2H), 6.84 (br s, 1H), 4.68 (d,
J = 4.4 Hz, 2H), 3.71 (s, 3H), 2.46 (s, 3H); 13C NMR (100 MHz,
CDCl3): d 161.6, 158.9, 156.3, 139.4, 139.3, 136.7, 136.6, 136.0,
134.6, 129.9, 129.4, 128.2, 126.0, 125.7, 124.6, 45.9, 28.4, 16.0;
LRMS: 457, 422, 295, 188; HRMS calcd M+ for C22H18Cl2N4OS:
456.05783; found: 456.05881.
5.1.4.1. 2-(3-Aminobenzylamino)-6-(2,6-dichlorophenyl)-8-met
5.1.5. Representative procedure for the preparation of 1 and 9a
To a solution of sulfone 6a (0.187 g, 0.5 mmol) in freshly dis-
tilled diglyme (5 ml), 1,3-phenylenediamine (0.120 g, 1.1 mmol)
was added and resulting reaction mixture was refluxed for 12 h.
After the completion of reaction, most of the solvent was evapo-
rated in vacuo and residue was purified by preparative TLC to get
amine 9a (0.110 g, 55%).
hylpyrido[2,3-d]pyrimidin-7(8H)-one (7a). Yield 61%; 1H-NMR
(400 MHz, CDCl3):
d 8.27 (br s, 1H), 7.44 (s, IH), 7.40 (d,
J = 8.0 Hz, 2H), 7.25 (t, J = 8.0 Hz, 1H), 7.14 (t, J = 8.0 Hz, 1H),
6.61–6.78 (m, 3H), 4.64 (d, J = 5.2 Hz, 2H), 3.72 (s, 3H); 13C NMR
(100 MHz, CDCl3):
d 161.67, 161.19, 158.67, 156.22, 147.00,
139.75, 136.71, 135.94, 134.55, 129.87, 129.79, 128.12, 120.27,
119.82, 118.58, 118.09, 114.42, 114.32, 113.83, 46.05, 28.33;
LRMS: 427.1, 425.1, 392.1, 390.1, 287.1, 285.1, 106.1; HRMS calcd
M+ for C21H17Cl2N5O: 452.0810; found: 425.0813.
5.1.5.1. 6-(2,6-Dichlorophenyl)-8-methyl-2-(3-(methylthio)phe-
nylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (1). Yield 57%; 1H
NMR (400 MHz, CDCl3): d 8.60 (s, 1H), 7.75 (br s, 1H), 7.54–7.52
(m, 2H), 7.42–7.38 (m, 3H), 7.33–7.25 (m, 2H), 7.01 (d, J = 8.0 Hz,
1H), 3.82 (s, 3H), 2.53 (s, 3H); LRMS: 442, 407, 362, 313, 269,
203, 196, 180; HRMS calcd M+ for C21H16Cl2N4OS: 442.04219;
found: 442.04315.
5.1.4.2. 2-(3-Aminobenzylamino)-6-(2-chlorophenyl)-8-methyl-
pyrido[2,3-d]pyrimidin-7(8H)-one (7b). Yield 77%; 1H NMR (400
MHz, CDCl3): d 8.32 (br s, 1H), 7.52 (s, IH), 7.47 (t, J = 4.0 Hz, 1H),
7.30–7.36 (m, 3H), 7.14 (t, J = 8.0 Hz, 1H), 6.77 (d, J = 7.6 Hz, 1H),
6.70 (s, 1H), 6.62 (d, J = 7.6 Hz, 1H), 4.64 (d, J = 5.2 Hz, 2H), 3.71
(s, 3H); 13C NMR (100 MHz, CDCl3): d 162.5, 161.6, 158.8, 156.1,
147.0, 140.0, 135.8, 135.5, 134.0, 131.9, 129.9, 129.6, 129.5,
126.8, 118.0, 114.4, 114.2, 46.0, 28.4; LRMS: 391.1, 356.2, 251.1,
106.1, 69.0; HRMS calcd M+ for C21H18ClN5O: 391.1200; found:
391.1204.
5.1.5.2. 2-(3-Aminophenylamino)-6-(2,6-dichlorophenyl)-8-met
hylpyrido[2,3-d]pyrimidin-7(8H)-one (9a). Yield 55%; 1H NMR
(400 MHz, CDCl3): d 8.58 (s, 1H), 7.53 (s, 1H), 7.46–7.40 (m, 3H),
7.28–7.25 (m, 1H), 7.23–7.11 (m, 2H), 7.07–6.98 (br s, 1H), 6.48
(d, J = 8.0 Hz, 1H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3): d
161.6, 159.3, 159.2, 158.6, 156.1, 147.4, 139.8, 136.5, 136.5,
136.4, 136.4, 135.9, 134.4, 130.1, 128.2, 126.3, 110.7, 110.3,
106.8, 106.5, 28.8; LRMS: 412, 377, 376, 284, 242; HRMS calcd
M+ for C20H15Cl2N5O: 411.06536; found: 411.06606.
5.1.4.3. 2-(3-Aminobenzylamino)-8-methyl-6-phenylpyrido [2,3-d]-
pyrimidin-7(8H)-one (7c). Yield 53%; 1H NMR (400 MHz, CDCl3): d
8.20 (br s, 1H), 7.64 (d, J = 7.6 Hz, 2H), 7.55 (s, 1H), 7.43–7.32 (m,

G. A. Kraus et al. / Bioorg. Med. Chem. 18 (2010) 6316–6321
6321
5.2. Pharmocology
Acknowledgment
5.2.1. Kinase assays and analysis
We thank Howard A. Levine for early guidance in data analysis.
This work was funded by NIH grant R43CA-110222, STTR to Molec-
ular Express Inc., and a subcontract to Iowa State University.
The recombinant v-Abl tyrosine kinase was from EMD Calbio-
chem, Cat#102555 (San Diego, CA), its abltide substrate was from
Upstate Biochemicals (Lake Placid, NY), the adenosine 5’-triphos-
phate (ATP) disodium salt was from Sigma, and the 32P-ATP was
from MP Biomedicals (Solon, OH). The tyrosine kinase activity
was determined from the rate of incorporation of 32P into abltide
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.021.
from
and various concentrations of PD173955, PDC or their analogs in
50 mM Tris–HCl, 0.8 mM MOPS, 10 mM MgCl2, 120 M EDTA,
0.015% BRIJ35, 1 mM DTT, 50 M Abltide, 10% DMSO, (pH 7.5).
All reaction conditions were tested in triplicate. The reactions were
started by the addition of
32P-ATP (1 Ci/10ꢁ6 mmol) to a final
concentration of 100 M. They were incubated for 30 min at
c
32P-ATP. The reaction mixtures, containing 0.4 pg/ml v-Abl
References and notes
l
l
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c
l
l
30 °C and stopped by spotting on P30 filtermat glass fiber filters
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