Design, synthesis and characterization of N9/N7-substituted 6-aminopurines as VEGF-R and EGF-R inhibitors

Article abstract of DOI:10.1016/j.ejmech.2008.04.012

In this study we report on the design, synthesis and biological characterization of novel N₉ or N₇ arylethanone-substituted 6-aminopurines and 6-methoxypurines, respectively, as EGF-R and VEGF-R inhibitors. The compounds were initial

Full text of DOI:10.1016/j.ejmech.2008.04.012

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1788e1793
http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and characterization of N9/N7-substituted
6-aminopurines as VEGF-R and EGF-R inhibitors
a
a
a
b
Christian Peifer a, , Stefanie Buhler , Dominik Hauser , Katrin Kinkel , Frank Totzke ,
*
¨
Christoph Schachtele b, Stefan Laufer a
¨
a Department of Pharmacy, Eberhard-Karls-University, Auf der Morgenstelle 8, D-72076 Tu¨bingen, Germany
b ProQinase GmbH, Breisacherstraße 117, D-79106 Freiburg, Germany
Received 1 February 2008; received in revised form 8 April 2008; accepted 15 April 2008
Available online 30 April 2008
Abstract
In this study we report on the design, synthesis and biological characterization of novel N9 or N7 arylethanone-substituted 6-aminopurines and
6-methoxypurines, respectively, as EGF-R and VEGF-R inhibitors. The compounds were initially profiled in a panel of 24 cancer-relevant pro-
tein kinases. Dependent on the regio-substitution of the purine core we found inhibition activity for EGF-R and VEGF-R with IC50 values in the
mM range. The two novel N9/N7 2-(6-amino-purine)-1-(1H-indole-3-yl)ethanone derivatives were characterized in an enhanced panel of 78
kinases showing the N9 derivative to also inhibit MNK1 and IRR while the N7 isomer was found to be specific for VEGF-R2.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: VEGF-R; EGF-R; Protein kinase inhibitors; N7/N9-Substituted 6-aminopurines
1. Introduction
cancer-relevant PKs. Furthermore, two novel compounds
have been tested in a specificity profile over 78 PKs.
Small molecule inhibitors of the receptor tyrosine kinases
VEGF-R (vascular endothelial growth factor receptor) [1]
and EGF-R (epidermal growth factor receptor) [2,3] are in par-
ticular of pharmaceutical interest because these protein
kinases (PKs) are considered to be validated drug targets in
angiogenesis and cancer [4]. In recent publications we pre-
sented compounds combining the purine system from the orig-
inal cosubstrate ATP and phenyl moieties in order to explore
possible interactions with the different regions of the ATP
binding site in several disease-related protein kinases [5,6].
In this communication we report on the design, synthesis
and results of our SAR investigation directed at the N9/N7-
regiospecific substitution of the 6-aminopurine scaffold (I
and II, Scheme 1) as VEGF-R/EGF-R inhibitors and the bio-
logical characterization of the compounds in a panel of 24
2. Results and discussions
We designed the scaffolds I and II on the basis of the mod-
elled binding mode of compound 1 (belonging to scaffold I) in
the ATP pocket of VEGF-R2 (Fig. 1). Herein, the pose of 1
involves H-bond interactions of the 6-aminopurine moiety ad-
dressing the hinge-region carbonyl of Cys917. In contrast to
the situation in ATP where N1 is accepting an H-bond from
the protein, in this binding mode an H-bond is accepted by
N7 of the adenine scaffold (Fig. 1). The N9 indolylethanone
substitution is directed towards the hydrophobic pocket I
(HPI) where the indole moiety forms pep stacking interac-
tions to the Phe1045 residue of the DFG motif [7]. However,
in particular this interaction towards HPI was considered to
determine activity and selectivity of the inhibitor for VEGF-
R2/3 [8]. Furthermore, in this binding mode of 1 the hydro-
phobic region II (HRII) is not yet addressed and, therefore,
leaving chemical space for optimization. To prove the
* Corresponding author. Tel.: þ49 7071 29 75278; fax: þ49 7071 29 5037.
E-mail address: christian.peifer@uni-tuebingen.de (C. Peifer).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.012

C. Peifer et al. / European Journal of Medicinal Chemistry 44 (2009) 1788e1793
1789
O
reaction conditions. The N9/N7-regiospecific-substituted
isomers were isolated by flash chromatography and subse-
quently heated with concentrated ammonium hydroxide
solution in a Berghof B25 pressure reactor to yield the cor-
responding 6-aminopurine derivatives (I/II, Fig. 3). In light
of the aminolysis of 6-chloropurine derivatives, catalytic
amounts of Cu(I)I decreased the yields and side products
occurred [11]. The reaction of adequate N9/N7-precursors in
a methanolic solution of ammonia gave compounds 7 and
8 [12] as major products besides minor amounts of 3 and
4 [13e16].
NH2
NH2
N
N
R
N
7
N
N
N
9
N
O
N
R = indolyl, phenyl, pyridyl
R
Scheme 1. N9/N7 regioselective-substituted 6-aminopurine scaffolds I and II.
Compound
9 was obtained under basic conditions
(K2CO3) by the reaction of 6-chloropurine and 2-bromo-
1H-indol-3-ylethanone in non-distilled DMF which contains
dimethylamine. Hence, in a one-pot reaction both the alkyl-
ation of N9 according to the general scheme as well as the
nucleophilic substitution of purine-C6 by dimethylamine
occurs.
concept of the inhibitor design as a lead structure the N9/N7-
regiospecific-substituted 6-aminopurines 1, 2 and related ana-
logues were synthesized and evaluated for biological activity.
3. Chemistry
The synthesis for both I and II starts from 6-chloro-9H-
purine. Due to the tautomeric situation in the purine nucleus,
modifications of the purine scaffold in this study by base cat-
alyzed SN reaction with 2-bromo-1-arylethanone derivatives
leads mainly to N9-substituted purines with only 5e10% of
N7-substituted purines. Vice versa, major regioselective N7-
substitution was conveniently afforded by use of the auxiliary
cobaloxime [9] CH3Co(DH)2OH2 whereas N9-substitution
occurred only in 5e12% (Fig. 2) [10].
Accordingly, in this study the reaction performed well for 2-
bromo-1H-indol-3-ylethanone and 2-bromo-1-phenylethanone
to yield compounds 1e4 (Fig. 3). Unexpectedly, the cobaloxime
strategy was not successful for 2-bromo-1-pyridylethanones 5
and 6 and hence only the N9-substituted derivatives were
obtained. Due to the additional pyridine nitrogen the coba-
loximeepurine complex was probably not stable using these
4. Biological evaluation
These nine compounds have been evaluated for their
potency against 24 cancer-relevant protein kinases belonging
to different families of the kinome (Table 1) [17]. Consistent
with the modelling hypothesis,
1 inhibited VEGF-R2
(IC50 ¼ 81 mM) and slightly more potently blocked the epider-
mal growth factor receptor (EGF-R, IC50 ¼ 35 mM; a modelled
binding mode of 1 in EGF-R [18] comparable to VEGF-R2 is
available in the Supporting information). Concerning SAR, the
involvement of the amino-function of 1 in ligandeprotein
interactions to these PKs is supported by complete loss of
the biological activity of aminomethylated derivative 9 in
the panel. Inhibition in the mM range for VEGF-R2 was also
found for 2, 6 and 7 whereas EGF-R is inhibited by 2, 3, 5
and 6. Compounds 1 and 2 inhibited VEGF-R2 but not the
highly homologous VEGF-R3. In contrast, compound 7 shows
an IC50 of 25 mM for VEGF-R2 and 45 mM for VEGF-R3 with
specificity over the other kinases of this panel. In light of the
modelled binding mode of 1 (Fig. 1) this result was unex-
pected since the amino-function in 1 (H-bond donor) is
replaced in 7 by a methoxy group (H-bond acceptor). Further-
more, the corresponding N7 regioisomer 8 was not inhibiting
any of the PKs. However, the inhibition of 7 indicates an alter-
native binding mode in VEGF-R for this compound which was
not predicted by our modelling studies. Interestingly, depen-
dent on the regio-substitution of the 1H-indol-3-ylethanone
moiety, compound 1 (N9) inhibited EGF-R more potently
than VEGF-R2 whereas the converse situation is true for 2
(N7, VEGF-R2 over EGF-R).
However, in order to reveal their potential as multikinase
inhibitors [19] 1 and 2 were additionally characterized in an
enhanced specificity panel of 78 PKs at a concentration of
10 mM [20] (data available in the Supporting information).
Herein, 1 inhibited MNK1 [21] (MAP kinase signal-
integrating kinase, 73 ꢀ 3% inhibition at 10 mM) and IRR
[22] (insulin-receptor related receptor, 51 ꢀ 1% inhibition at
Fig. 1. Modelled binding mode of 1 in the crystallographically determined
ATP binding pocket of VEGF-R2 (PDB 1Y6A [7]). Compound 1 forms key
H-bonds to Cys917 (hinge-region) and pep stacking interactions to
Phe1045 in the hydrophobic pocket I; Val914 (gatekeeper).

1790
C. Peifer et al. / European Journal of Medicinal Chemistry 44 (2009) 1788e1793
Cl
N
N
90°C/ΔP/NH3
Val914
K2CO3/DMF
I
N
N
2-bromo-1-
arylethanone
O
Cl
N
N
R
R
N
N
H
Cl
N
O
90°C/ΔP/NH3
K2CO3/ACN/
N
N
CH3Co(DH)2OH2
N
2-bromo-1-
arylethanone
II
Fig. 2. Synthesis of purine derivatives with regioselective substitution of the scaffold at N9 (I) and at N7 (II) by the use of auxiliary cobaloximeeH2O complex.
10 mM) while 2 not significantly blocked by any of the 78 PKs
in this panel.
Thus, compounds 2 and 7 are interesting lead structures
for the development of potent and selective VEGF-R2
inhibitors.
(1.55 mmol) was slowly added and the progress of the reaction
was monitored by TLC (ethylacetate 9/ethanol 1). After com-
pleteness of the reaction 20 ml H2O was dropped in and the
mixture extracted by ethylacetate (3 ꢁ 30 ml), the organic
phase separated, dried over Na2SO4 and evaporated. The prod-
uct was purified by flash chromatography.
5. Experimental section
5.2. General method for N7 alkylation of
6-chloro-9H-purine
5.1. General method for N9 alkylation of
6-chloro-9H-purine
Methylaquacobaloxime (0.5 g; 1.55 mmol) was dissolved
in 10 ml absolute acetonitrile and stirred in the dark. To this
solution, 6-chloro-9H-purine (0.24 g; 1.55 mmol) was added
to form the purineecobaloxime complex which precipitates
K2CO3 (0.21 g; 1.55 mmol) was suspended in a solution of
0.24 g (1.55 mmol) 6-chloro-9H-purine in 10 ml DMF and
stirred for 30 min. Then 2-bromo-1-arylethanone reagent
Compound
scaffold
R
1
2
3
I
II
I
3-indolyl
3-indolyl
phenyl
phenyl
5
6
I
I
2-pyridinyl
3-pyridinyl
CH3
H3C
N
CH3
O
N
O
N
N
CH3
O
N
N
N
N
N
H
N
N
N
N
N
7
8
9
O
O
Fig. 3. Set of test compounds 1e9.

C. Peifer et al. / European Journal of Medicinal Chemistry 44 (2009) 1788e1793
1791
Table 1
IC50 values (mM) of test compounds in a profiling panel of 24 cancer related
PKs
(s, 2H); 7.09e7.38 (m, 4H), 7.39e7.62 (m, 1H); 7.98e8.27
(m, 3H); 8.62 (s, 1H). 13C NMR DMSO-d6; 50 MHz, d
[ppm] ¼ 49.16, 112.71, 113.80, 118.73, 121.37, 122.50,
123.51, 125.61, 134.79, 136.88, 142.38, 150.31, 152.74,
156.30, 187.50. MS: m/z ¼ 293 (M þ Hþ) HRMS
C15H12N6O: 292.10651 calc. 292.10723. Melting point:
362.3 ꢂC.
PK
1
2
3
4
5
6
7
8
9
AKT1
ARK5
e
e
e
69
82
e
e
e
35
e
95
e
e
e
81
e
e
e
e
e
e
e
85
e
e
e
e
e
e
e
e
e
82
e
e
e
e
82
30
e
e
e
e
e
e
e
95
e
e
e
e
e
e
e
e
e
75
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
89
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
93
e
e
e
e
75
58
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
25
45
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
Aurora-A
Aurora-B
B-RAF-VE
CDK2/CycA
CDK4/CycD1
COT
5.3.2. 2-(6-Amino-7H-purine-7-yl)-1-(1H-indole-3-yl)
ethanone (2)
The compound was synthesized following general proce-
dures for N7 alkylation of 6-chloro-9H-purine and SN reaction
(yield 81.7%). 1H NMR DMSO-d6; 200 MHz, d [ppm] ¼ 5.93
(s, 2H), 6.76 (s, 2H), 7.09e7.36 (m, 2H), 7.52 (s, 1H), 7.96e
8.39 (m, 3H), 8.55 (1H). 13C NMR DMSO-d6; 50 MHz, d
[ppm] ¼ 52.54, 112.48, 112.73, 113.56, 121.42, 122.52,
123.53, 125.66, 135.08, 136.86, 147.42, 151.97, 152.43,
160.09, 187.95. MS: m/z ¼ 293 (M þ Hþ). HRMS
C15H12N6O: 292.10467 calc. 292.10723. Melting point:
288.3 ꢂC.
EGF-R
EPHB4
ERBB2
FAK
IGF1-R
SRC
VEGF-R2
VEGF-R3
FLT3
INS-R
MET
PDGFR-beta
PLK1
SAK
5.3.3. 2-(6-Amino-9H-purine-9-yl)-1-phenylethanone (3)
The compound was synthesized following general proce-
dures for N9 alkylation of 6-chloro-9H-purine and SN reaction
(yield 63.6%). 1H NMR DMSO-d6; 200 MHz, d [ppm] ¼ 5.86
(s, 2H), 7.25 (s, 2H), 7.56e7.66 (m, 2H, C3), 7.70e7.78 (m,
1H), 8.07e8.0 (m, 3H), 8.12 (s, 1H). 13C NMR DMSO-d6;
50 MHz, d [ppm] ¼ 49.4, 118.70, 128.46, 129.40, 134.51,
134.58, 142.00, 150.30, 152.84, 156.31, 193.20. MS: m/z ¼
253 (Mþ). HRMS C13H11N5O: 253.09748 calc. 253.096335.
Melting point: 292.4 ꢂC (decomposition).
TIE2
CK2alpha1
‘e’ indicates no significant inhibition up to 100 mM.
as an orange solid. Acetonitril 5 ml and K2CO3 (0.21 g;
1.55 mmol) were added and the mixture was stirred at room
temperature. After 30 min the 2-bromo-1-arylethanone reagent
(1.55 mmol) was added and the progress of the reaction was
monitored by TLC (ethylacetate 9/ethanol 1). After complete-
ness of the reaction the solvent was evaporated and 20 ml of
4 M NaOH was dropped in. The mixture was extracted by di-
chloromethane, the organic phase separated, dried over
Na2SO4 and evaporated. The product was purified by flash
chromatography.
5.3.4. 2-(6-Amino-7H-purine-7-yl)-1-phenylethanone
(4) [16]
The compound was synthesized following general proce-
dures for N7 alkylation of 6-chloro-9H-purine and SN reaction
1
(yield 35%). H NMR DMSO-d6; 200 MHz, d [ppm] ¼ 6.16
(s, 2H), 6.79 (s, 2H), 7.56e7.66 (m, 2H), 7.68e7.78 (m,
1H), 8.01e8.08 (m, 2H), 8.18 (s, 1H), 8.19 (s). 13C NMR
5.3. General method for SN reaction of N9N7-alkylated
6-chloro-purines
DMSO-d6; 50 MHz,
d
[ppm] ¼ 53.21, 112.45, 128.63,
129.17, 134.38, 134.56, 147.14, 151.84, 152.48, 160.05,
193.77. MS: m/z ¼ 254 (M þ Hþ). HRMS C13H11N5O:
253.09697 calc. 253.096335. Melting point: 244.6 ꢂC.
A Berghof B25 high pressure reactor was charged with the
N9 or N7-alkylated 6-chloro-purine derivative and 20 ml of
concentrated NH3. The mixture was heated to 90 ꢂC and the
progress of the reaction was monitored by TLC (ethylacetate
9/ethanol 1). After completeness of the reaction 20 ml H2O
was added and the mixture extracted by ethylacetate
(3 ꢁ 30 ml), the organic phase separated, dried over Na2SO4
and evaporated. The product was purified by flash
chromatography.
5.3.5. 2-(6-Amino-9H-purine-9-yl)-1-(pyridine-2-yl)
ethanone (5)
The compound was synthesized following general proce-
dures for N9 alkylation of 6-chloro-9H-purine and SN reaction
(yield 88.9%). 1H NMR DMSO-d6; 200 MHz, d [ppm] ¼ 5.93
(s, 2H), 7.25 (s, 2H), 7.74e7.83 (m, 1H), 7.97e8.14 (m, 4H),
8.81e8.87 (m, 1H). 13C NMR DMSO-d6; 50 MHz, d
[ppm] ¼ 49.44, 118.73, 122.20, 129.12, 138.36, 142.05,
149.88, 150.29, 151.37, 152.84, 156.30, 194.12. MS: m/z ¼
254 (Mþ). HRMS C12H10N6O: 254.09411 calc. 254.09158.
Melting point: 268.3 ꢂC.
5.3.1. 2-(6-Amino-9H-purine-9-yl)-1-(1H-indole-3-yl)
ethanone (1)
The compound was synthesized following general proce-
dures for N9 alkylation of 6-chloro-9H-purine and SN reaction
(yield 80.0%). 1H NMR DMSO-d6; 200 MHz, d [ppm] ¼ 5.63

1792
C. Peifer et al. / European Journal of Medicinal Chemistry 44 (2009) 1788e1793
5.3.6. 2-(6-Amino-9H-purine-9-yl)-1-(pyridine-3-yl)
ethanone (6)
Theassayfor all enzymes contained 60 mM HEPESeNaOH,
pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 mM Na-orthovanadate,
1.2 mM DTT, 50 mg/ml PEG20000, 1 mM [g-33P]-ATP
(approx. 5 ꢁ 105 cpm/well), recombinant protein kinase
(50e400 ng). Depending on the kinase, the following sub-
strate proteins were used: AKT1 (GSK3/14-27), ARK5 (auto-
phosphorylation), Aurora-A, Aurora-B (Tetra(LRRWSLG)),
B-Raf-VE (MEK1 KM), CDK2/CycA (histone H1), CDK4/
CycD1 (Rb-CTF), CK2-A1 (casein), EGF-R, EPHB4,
ERBB2, FAK, IGF1-R, SRC, VEGF-R2, VEGF-R3 (poly
(Glu,Tyr) 4:1), FLT3, INS-R, MET, PDGFRbeta (poly
(Ala,Glu,Lys,Tyr) 6:2:5:1), PLK1 (Casein), SAK (autophos-
phorylation), TIE2 (poly(Glu,Tyr) 4:1), COT (autophosphory-
lation). The IC50 values were measured by testing 10
concentrations of compounds in singlicate. As a parameter
for assay quality, the Z0-factor for the low and high controls
of each assay plate (n ¼ 8) was used (see Supporting informa-
tion). The final DMSO concentration in the assay was 1%.
The reaction cocktails were incubated at 30 ꢂC for 80 min.
The reaction was stopped with 50 ml of 2% (v/v) H3PO4,
plates were aspirated and washed two times with 200 ml of
0.9% (w/v) NaCl. Incorporation of 33Pi was determined with
a microplate scintillation counter (Microbeta Trilux, Wallac).
All assays were performed with a BeckmanCoulter/Sagian ro-
botic system.
The compound was synthesized following general proce-
dures for N9 alkylation of 6-chloro-9H-purine and SN reaction
(yield 62.8%). 1H NMR DMSO-d6; 200 MHz, d [ppm] ¼ 5.92
(s, 2H), 7.25 (s, 2H), 7.8 (m, 1H), 7.9e8.1 (m, 4H); 8.8 (s,
1H). MS: m/z ¼ 254 (Mþ). HRMS C12H10N6O: 254.09232
calc. 254.09158.
5.3.7. 2-(6-Methoxy-9H-purine-9-yl)-1-phenylethanone (7)
The compound was synthesized following general proce-
dures for N9 alkylation of 6-chloro-9H-purine. The SN reaction
was performed with methanolic ammonia to yield 21% of 1 and
57.6% of 7. 1H NMR Aceton-d6; 200 MHz, d [ppm] ¼ 4.15 (s,
3H), 6.02 (s, 2H), 7.58e7.68 (m, 2H), 7.71e7.80 (m, 1H),
8.13e8.20 (m, 2H), 8.25 (s, 1H), 8.44 (s, 1H). 13C NMR Ace-
ton-d6; 50 MHz, d [ppm] ¼ 49.35, 53.36, 120.76, 127.99,
128.88, 133.98, 134.46, 143.96, 151.54, 152.84, 160.80,
191.80. MS: m/z ¼ 268 (Mþ). HRMS C14H12N4O2:
268.09713 calc. 268.0960. Melting point: 156.5 ꢂC.
5.3.8. 2-(6-Methoxy-7H-purine-7-yl)-1-phenylethanone
(8) [12]
The compound was synthesized following general proce-
dures for N7 alkylation of 6-chloro-9H-purine. The SN reaction
was performed with methanolic ammonia to yield 35% of 2 and
The specificity testing of compounds 1 and 2 was
performed by the Division of Signal Transduction Therapy,
University of Dundee, Scotland. Details of the kinase assay
panel are available in the Supporting information.
1
49.5% of 8. H NMR methanol-d4; 200 MHz, d [ppm] ¼ 3.35
(s, 2H), 4.00 (s, 3H), 7.56e7.65 (m, 2H), 7.69e7.78 (m, 1H),
8.08e8.14 (m, 2H), 8.37 (s, 1H), 8.56 (s, 1H). 13C NMR meth-
anol-d4; 50 MHz, d [ppm] ¼ 48.33, 53.32, 127.71, 128.67,
128.67, 133.92, 134.13, 147.20, 151.62, 157.51, 160.30,
192.41. MS: m/z ¼ 268 (Mþ). HRMS C14H12N4O2:
268.09647 calc. 268.0960. Melting point: 165.7 ꢂC.
5.5. Molecular modelling
All modellings were performed on a RedHat Linux system.
For visualization and building the structures SYBYL 7.2 was
used. The Connolly surface was calculated using the MOL-
CAD module in Sybyl and coloured according to the lipophi-
licity (from very hydrophobic to hydrophilic corresponds to
brown over green to blue). Compound 1 was docked into
the active site of VEGFR-2 and EGF-R using the FlexX dock-
ing program [23]. The FlexX scoring function was applied
during the placement and construction phase of the ligands
and DrugScore for the final ranking [24]. The 3D coordinates
of the VEGFR-2 catalytic core in complex with a 2-anilino-5-
aryl-oxazole inhibitor were taken from the Brookhaven Pro-
tein Databank PDB code 1Y6A [7] and of the EGF-R catalytic
domain from PDB 1XKK [18].
5.3.9. 2-[6-(Dimethylamine)-9H-purine-9-yl]-1-(1H-indole-
3-yl)ethanone (9)
The compound was obtained following general procedures
for N9 alkylation of 6-chloro-9H-purine in DMF to yield 10%
of 2-(6-Chloro-9H-purine-9-yl)-1-(1H-indole-3-yl)ethanone
and 42.3% of 3. 1H NMR DMSO-d6; 200 MHz, d [ppm] ¼ 3.37
(s, 6H), 5.68 (s, 2H), 7.14e7.28 (m, 2H), 7.49e7.56 (d, 1H),
8.05e8.12 (d, 1H), 8.13e8.18 (2H), 8.65 (s, 1H), 12.12 (s, 1H).
13C NMR DMSO-d6; 50 MHz. d [ppm] ¼ 30.87, 50.10, 112.70,
113.55, 118.89, 121.29, 122.78, 123.81, 125.29, 134.44,
136.58, 141.29, 150.60, 152.05, 154.49, 187.52. MS: m/z ¼ 321
(M þ Hþ). HRMS C17H16N6O: 320.13728 calc. 320.13853.
The HPLC retention times and purity data of all compounds
are available in the Supporting information.
5.4. Selectivity profiling of compounds by IC50 values
using 24 protein kinases
Acknowledgements
We are grateful to BERGHOF Products & Instruments
GmbH, Lab-Technik, Harretstr.1, D-72800 Eningen, Germany
for technical support by the high pressure reactor BR-25. The
specificity testing of compounds 1 and 2 over 78 PKs was
performed by the Division of Signal Transduction Therapy,
University of Dundee and is gratefully acknowledged.
A proprietary protein kinase assay (33PanQinaseÒ Activity
Assay) was used for measuring the kinase activity of the 24
protein kinases. All kinase assays were performed in 96-well
FlashPlatesTM from Perkin Elmer/NEN (Boston, MA, USA)
in a 50 ml reaction volume.

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