2-Anilino-4-(benzimidazol-2-yl)pyrimidines - A multikinase inhibitor scaffold with antiproliferative activity toward cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2012.04.007

2-Anilino-4-(benzimidazol-2-yl)-pyrimidines, synthesized by reaction of a readily available benzimidazole-substituted enaminone with suitable arylguanidines, were shown to inhibit four cancer-related protein kinases (Aurora B, PLK1, FAK, and VEGF-R2). The most potent derivative exhibited antiproliferative activity for several cancer cell lines of the NCI in vitro cell line panel in submicromolar concentrations. Both the anilinopyrimidine structure and the substitution pattern at the aniline ring appear to be important for the protein kinase inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2012.04.007

European Journal of Medicinal Chemistry 53 (2012) 254e263
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-Anilino-4-(benzimidazol-2-yl)pyrimidines e A multikinase inhibitor scaffold
with antiproliferative activity toward cancer cell lines
Renate Determann a, Jan Dreher a, Knut Baumann a, Lutz Preu a, Peter G. Jones b, Frank Totzke c,
,
Christoph Schächtele c, Michael H.G. Kubbutat c, Conrad Kunick a
*
a Technische Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany
b Institut für Anorganische und Analytische Chemie, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
c ProQinase GmbH, Breisacher Straße 117, 79106 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Anilino-4-(benzimidazol-2-yl)-pyrimidines, synthesized by reaction of
a
readily available
Received 6 February 2012
Received in revised form
5 April 2012
Accepted 6 April 2012
Available online 14 April 2012
benzimidazole-substituted enaminone with suitable arylguanidines, were shown to inhibit four cancer-
related protein kinases (Aurora B, PLK1, FAK, and VEGF-R2). The most potent derivative exhibited anti-
proliferative activity for several cancer cell lines of the NCI in vitro cell line panel in submicromolar
concentrations. Both the anilinopyrimidine structure and the substitution pattern at the aniline ring
appear to be important for the protein kinase inhibitory activity.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Anilinopyrimidine
Benzimidazol
Cancer
COMPARE
Multikinase inhibitor
1. Introduction
As far as clinical data are available, unwanted side-effects of mul-
tikinase inhibitors are in fact frequent [12,13], but in the light of the
Protein kinases are involved in a multiplicity of biochemical
mechanisms regulating division, growth, and death of cells. Aber-
rant hyperactivity of protein kinases is frequently observed in
cancer tissues. Since the launch of imatinib as a drug against chronic
myelogenous leukemia more than ten years ago, the inhibition of
tumor-related protein kinases has been established as a general
principle in cancer treatment [1e3]. At the beginning of the protein
kinase inhibitor era, a narrow selectivity profile was judged
a desirable inhibitor property, mainly because inhibition of “off-
target protein kinases” was considered as equivalent to a risk of
unwanted side-effects. This general point of view is no longer an
unchallenged paradigm. In contrast, it is now accepted that inhib-
iting more than one protein kinase can be beneficial because the
tumor is attacked concomitantly at several relevant targets. More-
over, if a single cancer-related kinase becomes drug-resistant,
a multi-targeted drug will still act on the remaining array of
target kinases [4e6]. Along these lines, the recently launched kinase
inhibitor drugs dasatinib [7], sunitinib [8], sorafenib [9], and pazo-
panib [10,11] inhibit a broad array of cancer-related protein kinases.
poor therapeutic alternatives are acceptable in individual cases.
In the course of our studies directed at identifying novel protein
kinase chemotypes [14e16], we have previously reported the
annulated 2-anilinopyrimidines of type 1 (Chart 1) [17]. 1 acts as
a dual inhibitor of the cancer-related protein kinases VEGF-R2 and
PLK1 and inhibits the in vitro growth of distinct cancer cell lines in
micromolar concentrations. Based on docking studies, it was
assumed that the anilinopyrimidine partial structure in 1 is the
molecular motif binding to the hinge region of the ATP-binding site
in protein kinases [17]. 2-Anilinopyrimidines are among the
most frequent kinase inhibitor motives in the pdb database [18] and
are frequently mentioned in patents describing protein kinase
inhibitors that are claimed to be anticancer agents. A search for
novel 4-aryl-2-anilinopyrimidines identified benzimidazol-2-yl-
substituted derivatives of the general structure 2 as hitherto
unknown. The synthesis of this new chemotype and its inhibitory
activity on cancer-related protein kinases are reported here.
2. Chemistry
The general synthetic procedure toward the desired 2-anilino-
4-(benzimidazol-2-yl)-pyrimidines 2 is outlined in Scheme 1.
* Corresponding author. Tel.: þ49 531 391 2751; fax: þ49 531 391 2799.
E-mail address: c.kunick@tu-braunschweig.de (C. Kunick).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.007

R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
255
structures of all new 2-anilino-4-(benzimidazol-2-yl)pyrimidines 2
were supported by IR, 1H NMR and 13C NMR spectra. In the 1H NMR
spectra of 2, the singlet of the methyl group appeared near 4.3 ppm.
The protons in 5- and 6-positions of the pyrimidine ring caused
a characteristic pattern of two doublets near 7.6 and 8.6 ppm,
respectively, characterized by a coupling constant of 5 Hz. A
prominent signal in the 13C spectra of all congeners 2 appeared near
159 ppm which was assigned to the tertiary carbon in position 6 of
the pyrimidine ring.
As outlined above, the anilinopyrimidine substructure was
included in the title compounds 2 as a general hinge-binding
element to the ATP-binding site in protein kinases. For compar-
ison, we also synthesized the related pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-dione 7, which, because of the lack of a hydrogen bond
acceptoredonor motif, was considered unable to bind in a fashion
similar to the anilinopyrimidines 2. As shown in Scheme 1, 7 was
Fig. 1. X-ray structure of compound 7. The molecule is planar to within an r.m.s.
deviation of 0.045 A. Color code: hydrogen (white), carbon (gray), nitrogen (blue),
oxygen (red). Visualization by UCSF Chimera [22]. For details refer to the Supporting
Information. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
ꢀ
prepared by refluxing the enaminone
4 with 6-amino-1,3-
dimethyluracil 6 in acetic acid. Because 6 is an ambident reagent
with nucleophilic properties both at the exocyclic amino group
and at the ring carbon C-5, the reaction with the bis-electrophile 4
may lead to either regioisomer 7 or 8 (Chart 2). While one-
dimensional 1H NMR and 13C NMR spectra of the reaction
product were consistent with both structures, a 1H/13C-correlated
two-dimensional NMR experiment (HMBC) favored structure 7 due
to a cross correlation found between C(4) and H(5) of the pyrido
[2,3-d]pyrimidine ring. Finally the structure was unambiguously
identified as 7 by a single crystal X-ray structure analysis (Fig. 1).
The workup of all products 2ae2j and 7 comprised crystallization
2-Acetyl-1-methyl-1H-benzimidazole (3) [19] was treated with
dimethyl formamide dimethyl acetal (DMF-DMA), following
a procedure published by the group of Farag [20], who has also
demonstrated the suitability of the resulting enaminone 4 as
a reaction partner for guanidine, thus generating 2-amino-4-
(benzimidazol-2-yl)-pyrimidines related to 2 but lacking the N-aryl
substituent [21]. In an analogous manner, 4 reacted with N-phe-
nylguanidinium nitrates 5 in the presence of sodium hydroxide in
refluxing propan-2-ol to furnish the desired compounds 2aej. The
Fig. 2. Proposed binding mode of 2j (carbon atoms and bonds depicted green) in the ATP-binding pocket of PLK1 (source of the protein structure: PDB-file 2OWB [27]). The highest
scored pose generated by the docking program FRED [46] is shown. Visualization was carried out using PyMOL [47]. OMEGA was used for conformer generation [48]. Hydrogen
bonds are indicated by yellow dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

256
R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
Fig. 3. Solvent accessible surface of the ATP-binding site of PLK1 (PDB-file 2OWB [27]) with docked 2j. Docking was performed with FRED [46]. Visualization was performed with
MOE [49].
procedures involving polar solvents such as ethanol or DMF/water
mixtures. This led to a considerable loss of material, as is reflected
by the modest to moderate yields given in the experimental part.
digit micromolar IC50 values on at least one of the tested kinases.
Aurora B, FAK, PLK1 and VEGF-R2 proved to be particularly sensitive
to the test compounds (Table 1). Since these four kinases are
involved in correlated processes of cancer development and
maintenance, their concerted inhibition could be useful in the
treatment of malignancies. One may note that 2ge2j are not
promiscuous kinase inhibitors, since some of the test kinases (e.g.
AKT1, refer to Table 1) were not inhibited by the test compounds.
The serine/threonine kinase Aurora B belongs to the group of
mitotic kinases and is involved in the attachment of chromosomes
and establishing correct bi-orientation. The enzyme acts as both
a cytokinesis kinase and a spindle checkpoint kinase, and also
3. Biological evaluation and discussion
A radiometric protein kinase assay (33PanQinaseÒ Activity
Assay) was used for measuring the kinase inhibition activity of the
novel compounds on 11 recombinant cancer-related protein
kinases (AKT1, ARK5, Aurora B, AXL, FAK, IGF1-R, MET, PLK1, PRK1,
SRC, VEGF-R2). Whereas 2ae2f and 7 failed to inhibit any of the
tested kinases in concentrations below 10 mM, 2ge2j showed single
Chart 1. Anilinopyrimidines with protein kinase inhibitory properties.

R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
257
H3C
O
N
N
N
O
N
O
N
N
O
CH3
N
CH3
N
N
N
CH3
H3C
CH3
7
8
Chart 2. 1,3-Dimethyl-7-(1-methyl-1H-benzimidazol-2-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione 7 and its hypothetical regioisomer 8.
Scheme 1. Synthesis of 2-anilino-4-(benzimidazol-2-yl)pyrimidines
2 and of 1,3-dimethyl-7-(1-methyl-1H-benzimidazol-2-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione 7.
Reagents and conditions: (i) DMF-DMA, 115e120 ꢀC, 2.5 h, 76%, (ii) NaOH, propan-2-ol, reflux, 12e30 h, 12e55% (2d: the free base 5d was used; 2j: conditions for synthesis:
150 ꢀC, 1.5 h, 34%) (iii) acetic acid, reflux, 8 h, 35%. For substituents R1 and R2 refer to Table 1.
mw,
Table 1
Protein kinase inhibition by 2aej and 7 compared to 1 and to standard agents sorafenib and sunitinib (IC50 values [m
M]).a
R1
R2
AKT1
Aurora B
FAK
0.64
PLK1
VEGF-R2
0.073
1
e
e
>100
>100
>100
41
>100
>100
75 ꢁ 21
>100
>100
>100
>100
>100
>10
3.0
5.7
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
H
H
H
H
eOH
Cl
eOH
e
H
eCH3
Cl
>100
>100
>100
>100
>100
62 ꢁ 25
11.1 ꢁ 2.9
5.5 ꢁ 0.2
7 ꢁ 2.3
6.0 ꢁ 0.2
>100
1.8
38 ꢁ 23
60 ꢁ 21
13.4 ꢁ 3.7
58
>100
25
69
24
>100
>100
>100
15 ꢁ 1
12 ꢁ 1
3.7 ꢁ 0.4
6.0 ꢁ 0.1
1.2 ꢁ 0.2
>100
38
69
Morpholino
eOC2H5
eOCH3
eOH
H
eOH
eOCH3
e
>100
27 ꢁ 10
11.4 ꢁ 3.6
5.2 ꢁ 1.1
10.4 ꢁ 2.7
3.4 ꢁ 0.8
92
>100
44 ꢁ 19
9.2 ꢁ 1.9
3.2 ꢁ 0
7.5 ꢁ 2.0
7.2 ꢁ 0.3
85
2j
7
Sorb
Sunc
e
e
>10
1.6
>10
>10
0.022
0.070
e
e
>10
1.5
a
Values with range given represent the mean of two independent determinations. Values without range indicate a single measurement.
Sor ¼ sorafenib, mean values of three independent experiments.
b
c
Sun ¼ sunitinib, single experiment.

258
R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
phosphorylates chromatin proteins [23]. Because overexpression of
Aurora B has been detected in particular cancer tissues [24], several
small molecules inhibiting Aurora B are currently being developed
as antitumor drugs [25].
inhibitors sorafenib and sunitinib. As shown in Table 2, the aver-
aged cytotoxicity values of 2j, sorafenib, and sunitinib are similar
(2.4, 2.4, and 2.0 mM, respectively). However, the interindividual
susceptibility of distinct cell lines spans 2 orders of magnitude after
treatment with 2j. In contrast, the standard compounds sorafenib
and sunitinib inhibited most of the cell lines by single digit
micromolar concentrations. A comparison of the results in the
IVCLS found with 2j and 1 shows a similarity of patterns. This
correlation is not surprising considering the structural relationship
of 1 and 2j and the fact that both 1 and 2j inhibit PLK1 and VEGF-R2
(Table 2).
Similarity of patterns in the IVCLS is quantified by the COMPARE
program [42,43]. COMPARE calculates a pairwise correlation coef-
ficient (PCC) for compounds tested in the IVCLS. A PCC ¼ 1 indicates
that two compared patterns are completely identical. It was sug-
gested that PCC values >0.6 indicate similar pharmacological
mechanisms of compared test compounds [44]. Moreover, it has
been shown that subtle structural modifications within compound
families can be clustered by PCC thresholds down to >0.35 [45].
The results listed in Table 3 show a moderate correlation
between 1 and 2j, documented by a PCC ¼ 0.493. Interestingly, the
multikinase inhibitor sunitinib displays a somewhat similar pattern
compared to 1 and 2j (PCCs ¼ 0.437 and 0.41, respectively). In
contrast, sorafenib appears to be completely unrelated not only to
test compounds 1 and 2j, but also to the second established mul-
tikinase inhibitor sunitinib.
Based on the limited data presented here, speculations
regarding structureeactivity relationships in the compound class
appear premature. However, the data collected in Table 1 show that
a small oxygen-containing substituent in position 3 or 4 of the
phenyl ring (derivatives 2fe2j) seems to be important for kinase
inhibition. Compounds lacking such a substituent (2aec) or
compounds with a larger substituent (2d, 2e) exhibit only low
activity. To rationalize these observations, we created a model of
the proteineligand interaction by docking 2j into the ATP-binding
pocket of PLK1 (pdb-file 2OWB [27]). A reasonable pose was
produced in which the anilinopyrimidine structure is placed in the
hinge region, forming a pair of hydrogen bonds to Cys133 (for
similar characteristic hinge-binding modes refer to [18]). Further-
more, hydrogen bonds are possible between the ligand’s methoxy
group and the guanidine portion of Arg136 at the entrance of the
Similar to the Aurora family members, PLK1 (polo like kinase 1)
is important for several mitotic events, e.g. centrosome maturation,
kinetochore assembly, orchestration of microtubule nucleation, and
cytokinesis. PLK1 overexpression has been determined in a broad
variety of human cancers [26]. Although PLK1 function is crucial for
normal cell division, PLK1 inhibition has been suggested as
a promising concept for cancer therapy [26,27]. The observation
that p53-deficient cancer cells are especially vulnerable to PLK1
inhibition [28] led to the concept of selectively targeting p53-
deficient tumors with PLK1 inhibitors. Clinical trials with PLK1
inhibitors as anticancer drugs are currently underway [29].
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase
regulating many physiological processes, e.g. tissue proliferation,
cell survival and cell motility. Furthermore, FAK is involved in
promotion of angiogenesis and tissue invasion [30]. Similar to PLK1,
FAK interacts closely with p53. It was recently reported that FAK
overexpression and p53 mutations are highly correlated in human
breast cancer [31]. Various other tumors also show upregulation
of FAK, often correlated with tumor malignancy [30]. Using
a knockout mouse model, it was demonstrated that endothelial FAK
is essential for tumor growth and tumor-associated angiogenesis.
Mice lacking endothelial-specific FAK expression showed reduced
tumor angiogenesis and tumor growth [32]. FAK activity also
interferes with signaling cascades downstream from VEGF-R2 (see
below). Upon activation by VEGF, Src kinase is activated, which in
turn activates FAK, thus promoting endothelial cell migration [33].
Besides its role in tumorigenesis and angiogenesis, increased FAK
expression is related to metastasis. Consequently, FAK inhibitors
have been developed as anticancer agents [34].
The vascular endothelial growth factor receptor 2 (VEGF-R2) is
a tyrosine kinase involved in tumor vascularization. To grow
beyond a certain magnitude, a solid tumor needs a supply of oxygen
and nutrients through blood capillaries. [35] For induction of
angiogenesis, VEGF and other growth factors are secreted from
hypoxic tumor tissue. Activation of VEGF receptors (VEGF-Rs) on
vascular endothelial cells leads to formation of new tubular struc-
tures, resulting in tumor vascularization. VEGF-R2 is one of the
crucial target structures of the above-mentioned multikinase
inhibitors sorafenib [9] and sunitinib [36,37] and also of other drugs
in clinical development [38e40]. Since FAK and VEGF-R2
participate in the same signaling cascade, the conjoint inhibition
of both kinases could lead to significant abrogation of tumor
vascularization.
Table 2
Antiproliferative activity (GI50 values, [mM]) of 1, 2j and the standard agents sor-
afenib and sunitinib toward selected cancer cell lines in the in vitro cell line
screening (IVCLS) of the National Cancer Institute.
Cell line
1a
2ja
Sorafenibb Sunitinibc
Taken together, the combination of Aurora B, FAK, PLK1 and
VEGF-R2 was considered as a promising target ensemble for anti-
Cell lines sensitive to 2j
SW-620 (colon cancer)
MDA-MB-435 (melanoma)
OVCAR-3 (ovarian cancer)
CAKI-1 (renal cancer)
0.44
0.62 3.2
0.19 2.0
0.93 3.2
0.86 3.2
0.47 2.5
1.3
2.0
3.2
0.63
2.0
cancer multikinase inhibitors. For
a preliminary information
0.20
0.55
0.15
0.79
regarding performance in a cellular environment, the most potent
compound 2j was screened for antiproliferative activity in the
in vitro cell line screening (IVCLS) project of the National Cancer
Institute (NCI) [41].
MCF-7 (breast cancer)
Cell lines less sensitive to 2j
EKVX (non-small cell lung cancer)
HT29 (colon cancer)
SNB-19 (CNS cancer)
UACC-257 (melanoma)
TK-10 (renal cancer)
Indeed, 2j not only inhibited the growth of several cell lines in
submicromolar concentrations, but also exhibited an explicit
selectivity pattern in the cell line panel. While the cancer cell lines
SW-620 (colon), MDA-MB-435 (melanoma), OVCAR-3 (ovarian),
CAKI-1 (renal), and MCF-7 (breast) were particularly sensitive to
the test compound, other cell lines were inhibited to a lower extent
(EKVX, HT29, SNB-19, UACC-257, TK-10). Given the manifold char-
acteristic protein kinase deregulations found in different cancer
cell lines, a selective cytotoxicity is expected for a multikinase
inhibitor. Interestingly, the degree of selectivity displayed by 2j
was more pronounced than that of the established multikinase
4.1
3.4
21
19
4.1
17
27
10
5.5
26
2.5
2.5
3.2
2.0
5.0
7.9
1.6
10
4.0
6.3
Averaged antiproliferative activity over approx. 60 cancer cell lines
MG-MIDd
1.5 2.4 2.4
2.0
a
b
c
Mean of two independent determinations.
Averaged data of three test runs.
Averaged data of four test runs.
MG-MID ¼ Meangraph midpoint, corresponds to the averaged GI50 value [
d
mM]
over all cell lines in the NCI in vitro cell line panel.

R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
259
Table 3
125-4, LiChrosphere 100 RP-18, 5
isocratic, volume of injection 10 mL; detection (DAD) at 254 and
m
m, flow rate 1.000 mL/min,
Pairwise correlation coefficients determined by a Matrix COMPARE analysis of
compounds tested in the in vitro cell line screening of the National Cancer Institute.a
280 nm; AUC % method; time of detection 15 min, net retention
time (tN), dead time (tM) related to DMSO. The indicated lmax values
refer to the UV spectra generated by the diode array detector.
Preparation of buffer A for HPLC: Triethyl amine (20 mL) and
sodium hydroxide (242 mg) were dissolved in HPLC grade water up
to a volume of 1000 mL. Subsequently, the pH of the solution was
adjusted to 2.6 by addition of concentrated sulfuric acid. Prepara-
tive HPLC: Merck LaPrep, LaPrep P110 preparative HPLC pump,
Knauer injection loop 5 mL, LaPrep P216 fraction collector, LaPrep
P311 spectrophotometer, self packing device Merck NW 25 with
a column tube 125 mm, inner diameter 25 mm, silica gel: Merck
1
2j
0.493
Sunitinib
Sorafenib
1
2j
0.437
0.410
0.068
ꢄ0.048
0.188
0.493
0.437
0.068
Sunitinib
Sorafenib
0.410
ꢄ0.048
0.188
a
GI50 values (averaged data for multiple test runs; highest tested concen-
M) were used. 58e60 cell lines were included in the calculation of
the correlation coefficient.
tration ¼ 100
m
binding pocket (Fig. 2). As shown in Fig. 3, 2j fits perfectly to the
shape of the PLK1 ATP-binding pocket. Because of the alignment of
this pose, substituents larger than methoxy could interfere with the
pillar-like structure formed by Leu59 and Arg136 at the pocket
entrance, a finding that explains the low inhibitory activity of the
derivatives 2d and 2e.
LiChrospherÒ 100 RP-18, 12
mm, flow rate 40 mL/min, detection at
254 nm. All compounds employed in biological tests were used in
ꢃ95% purity. Starting materials 3, 5d (free base) and 6 were
purchased from commercial suppliers and used without further
purification. Aryl guanidine nitrates 5aec and 5eej were prepared
according to a literature method [51].
4. Conclusion
5.1.2. 3-(N,N-Dimethylamino)-1-(1-methyl-1H-benzimidazol-2-yl)
prop-2-en-1-one (4)
2-Anilino-4-(benzimidazol-2-yl)pyrimidines 2 were discov-
ered as a group of compounds inhibiting at least four cancer-
related protein kinases, namely Aurora B, PLK1, FAK, and VEGF-
R2. For the synthesis of 2, the readily available enaminone 4
was reacted with suitable arylguanidines 5. The most potent
protein kinase inhibitor in the series (2j) inhibited several cancer
cell lines of the NCI panel in submicromolar concentrations. Both
the anilinopyrimidine structure and the substitution pattern at
the aniline ring appear to be important for the protein kinase
inhibitory activity. A molecular docking pose of 2j explained
preliminary structureeactivity trends. On the basis of these
results, a further synthesis program is warranted aiming to
increase kinase inhibition potency and to tailor ADME properties.
The simple synthetic procedure and the plausible docking mode
of 2j in PLK1 are good arguments to consider such a development
campaign.
2-Acetyl-1-methyl-1H-benzimidazole (3) (10.0 mmol, 1.74 g)
and dimethyl formamide dimethyl acetal (DMF-DMA) (34 mL)
were stirred at 115e120 ꢀC for 2.5 h. The mixture was evaporated to
dryness. The residue was crystallized from toluene/petrol ether
(1:1) to yield 76% of a yellow powder.
Mp.: 146e148 ꢀC (150 ꢀC [20]) IR (KBr): 1639 cmꢄ1 (C]O); 1H
NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 2.94 (s, 3H, CH3), 3.19 (s, 3H,
CH3), 4.11 (s, 3H, CH3), 6.25 (br s, 1H, CH), 7.26e7.30 (m, 1H, ArH),
7.33e7.37 (m, 1H, ArH), 7.60e7.62 (dd, 1H, J ¼ 8.1/1.2 Hz, ArH),
7.72e7.75 (dd, 1H, J ¼ 8.0/1.0, ArH), 7.82 (d, 1H, J ¼ 12.8 Hz, CH); 13C
NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 31.8, 37.1, 44.6 (CH3); 110.7
(2C), 120.1 (2C), 122.4, 123.7 (tert. C); 136.5, 141.1, 148.9, 179.3 (quat.
C); C13H15N3O (229.28); calcd. C 68.10, H 6.59, N 18.33; found C
67.90, H 6.71, N 18.20; HPLC purity: 99.9% at 254 nm and 99.9% at
280 nm; tN ¼ 3.57 min, tM (DMSO) ¼ 1.06 min (ACN/H2O; 20:80);
lmax: 247 nm, 305 nm and 363 nm.
5. Experimental protocols
5.1.3. General procedure A for the synthesis of N-aryl-4-(1-methyl-
1H-benzimidazol-2-yl)pyrimidin-2-amines 2ae2c and 2ee2i
A mixture of 3-(N,N-dimethylamino)-1-(1-methyl-1H-benzi-
midazol-2-yl)prop-2-en-1-one (4) (1.00 mmol, 229 mg), an
appropriately substituted N-arylguanidinium nitrate (5) (1.2
mmol), and sodium hydroxide (1.2 mmol, 48 mg) was refluxed in
propan-2-ol (5 mL) until the starting material 4 was no longer
detectable by TLC control. After cooling to room temperature the
precipitate was filtered by suction, washed with water and petrol
ether and purified by the method given for the particular title
compound.
5.1. Synthetic chemistry
5.1.1. General
Monomode microwave device: CEM Discover focused micro-
wave synthesis system with ChemDriver software. Melting points
(mp) were determined on an electric variable heater (Barnstead
Electrothermal IA 9100) and were not corrected. IR spectra were
recorded as KBr discs on a Thermo Nicolet FT-IR 200. 1H NMR
spectra and 13C NMR spectra were recorded on a Bruker Avance II-
600, solvent DMSO-d6 if not stated otherwise, internal standard
tetramethylsilane, signals in ppm (
determined on a CE Instruments FlashEA 1112 elemental analyzer
(Thermo Quest).
d
scale). Elemental analyses were
5.1.3.1. 4-(1-Methyl-1H-benzimidazol-2-yl)-N-phenylpyrimidin-2-
amine 2a. Preparation following General Procedure A (reaction
time 15.5 h) and purification by crystallization from ethanol yielded
a colorless powder (46.3%).
Mass spectra were recorded on a double-focused sector field
mass spectrometer Finnigan-MAT 95. Accurate measurements
were conducted according to the peakmatch method using per-
fluorokerosene (PFK) as an internal mass reference; (EI)-MS: ioni-
zation energy 70 eV. TLC: Polygram Sil G/UV254, MachereyeNagel,
40 mm ꢂ 80 mm, visualization by UV illumination (254 nm).
Column chromatography: silica gel 60 (Merck), column width
2.5e3 cm, column height 15e30 cm. Purity was determined by
HPLC using the following devices and settings [50]: LaChrom Elite
(Merck/Hitachi), pump L-2130, autosampler L-2200, diode array
detector L-2450, organizer box L-2000; column Merck LiChroCART
Mp.: 200e204 ꢀC; IR (KBr): 3250 cmꢄ1 and 3187 cmꢄ1 (NH); 1H
NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 4.29 (s, 3H, N-CH3), 7.02 (tt,
1H, J ¼ 7.4/1.1 Hz, ArH), 7.30e7.36 (m, 3H, ArH), 7.38e7.40 (m, 1H,
ArH), 7.65 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H), 7.71 (d, 1H, J ¼ 7.7 Hz,
ArH), 7.76e7.77 (m, 3H, ArH), 8.65 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H),
9.81 (s, 1H, NH); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 32.8
(CH3); 110.9, 111.0, 119.6, 119.7, 121.9 (2C), 122.6, 123.8, 128.4 (2C),
159.0 (tert. C); 137.1, 139.9, 141.8, 147.7, 157.4, 159.5 (quat. C);
C18H15N5 (301.35); calcd. C 71.74, H 5.02, N 23.24; found C
71.49, H 4.98, N 23.10; HPLC: 99.9% at 254 nm and 99.9% at

260
R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
280 nm; tN ¼ 3.04 min, tM (DMSO) ¼ 1.02 min (ACN/H2O; 60:40);
lmax: 279 nm.
Mp.: 164e165 ꢀC; IR (KBr): 3429 cmꢄ1
3195 cmꢄ1 (NH); 1H NMR (DMSO-d6, 600 MHz):
,
3252 cmꢄ1 and
(ppm) ¼ 1.33 (t,
d
3H, J ¼ 7.3 Hz, O-CH2-CH3), 4.00 (q, 2H, J ¼ 7.2 Hz, O-CH2-CH3), 4.25
(s, 3H, N-CH3), 6.90e6.93 (m, 2H, ArH), 7.29e7.32 (m, 1H, ArH),
7.37e7.39 (m, 1H, ArH), 7.58 (d, 1H, J ¼ 5.1 Hz, pyrimidine-H),
overlapped by 7.60 (m, 2H, ArH), 7.69 (d, 1H, J ¼ 8.2 Hz, ArH),
7.75e7.77 (dt, 1H, J ¼ 1.0/8.0 Hz, ArH), 8.59 (d, 1H, J ¼ 5.1 Hz,
pyrimidine-H), 9.59 (s, 1H, NH); 13C NMR (DMSO-d6, 150.9 MHz):
5.1.3.2. 4-(1-Methyl-1H-benzimidazol-2-yl)-N-(4-methylphenyl)pyr-
imidin-2-amine (2b). Preparation following General Procedure A
(reaction time 20 h) and purification by crystallization from ethanol
yielded a yellow powder (24.4%).
Mp.: 211e212 ꢀC; IR (KBr): 3431 cmꢄ1 and 3265 cmꢄ1 (NH);
NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 2.28 (s, 3H, CH3), 4.28 (s, 3H,
d
(ppm) ¼ 14.7, 32.8 (CH3); 63.0 (sec. C), 110.2, 111.0, 114.1 (2C),
N-CH3), 7.14e7.15 (m, 2H, ArH), 7.30e7.33 (m, 1H, ArH), 7.37e7.40
(m, 1H, ArH), 7.62 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H), overlapped by
7.62e7.63 (m, 2H, ArH), 7.69e7.71 (m, 1H, ArH), 7.77 (td, 1H, J ¼ 1.0/
8.1 Hz, ArH), 8.62 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H), 9.70 (s, 1H, NH);
119.7, 121.8 (2C), 122.6, 123.7, 159.0 (tert. C); 132.7, 137.1, 141.8,
147.8, 153.9, 157.4, 159.7 (quat. C); C20H19N5O (345.41); calcd. C
69.55, H 5.54, N 20.28; found C 68.64, H 5.48, N 20.02; MS (EI): m/z
(%) ¼ 345 [M]þꢅ (85); HRMS (EI): m/z [M]þꢅ calcd. 345.15896, found
345.15956; HPLC: 99.6% at 254 nm and 99.7% at 280 nm;
13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 20.3, 32.8 (CH3); 110.6,
111.0 (2C), 119.7 (2C), 122.6, 123.8, 128.9 (2C), 159.0 (tert. C); 130.8,
137.1, 137.3, 141.8, 147.8, 157.4, 159.5 (quat. C); C19H17N5 (315.37);
calcd. C 72.36, H 5.43, N 22.21; found C 72.19, H 5.25, N 22.09; HPLC:
99.9% at 254 nm and 99.9% at 280 nm; tN ¼ 4.69 min, tM (DMSO) ¼
1.02 min (ACN/H2O; 60:40); lmax: 240 nm, 281 nm and 300 nm.
tN ¼ 3.71 min, tM (DMSO) ¼ 1.02 min (ACN/H2O; 60:40); lmax
:
243 nm and 298 nm.
5.1.3.6. N-(4-Methoxyphenyl)-4-(1-methyl-1H-benzimidazol-2-yl)
pyrimidin-2-amine (2f). Preparation following General Procedure A
(reaction time 15 h) and purification by crystallization from ethanol
yielded a yellow powder (51.5%).
5.1.3.3. 4-(1-Methyl-1H-benzimidazol-2-yl)-N-(4-chlorophenyl)pyr-
imidin-2-amine (2c). Preparation following General Procedure A
(reaction time 15 h) and purification by crystallization from ethanol
yielded a yellow powder (31.3%).
Mp.: 185e189 ꢀC; IR (KBr): 3432 cmꢄ1
,
3253 cmꢄ1 and
(ppm) ¼ 3.75 (s,
3193 cmꢄ1 (NH); 1H NMR (DMSO-d6, 600 MHz):
d
3H, O-CH3), 4.25 (s, 3H, N-CH3), 6.92e6.95 (m, 2H, ArH), 7.29e7.32
(m, 1H, ArH), 7.37e7.39 (m, 1H, ArH), 7.58 (d, 1H, J ¼ 5.0 Hz,
pyrimidine-H), 7.61 (m, 2H, ArH), 7.70 (d, 1H, J ¼ 8.1 Hz, ArH), 7.76
(m, 1H, ArH), 8.59 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H), 9.60 (s, 1H, NH);
Mp.: 217e218 ꢀC; IR (KBr): 3260 cmꢄ1 and 3188 cmꢄ1 (NH); 1H
NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 4.29 (s, 3H, N-CH3),
7.31e7.33 (m, 1H, ArH), 7.38e7.41 (m, 1H, ArH), overlapped by 7.40
(m, 2H, ArH), 7.68 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H), 7.70e7.72 (m,
1H, ArH), 7.76e7.78 (m, 1H, ArH), 7.79e7.82 (m, 2H, ArH), 8.67 (d,
1H, J ¼ 5.0 Hz, pyrimidine-H), 9.95 (s, 1H, NH); 13C NMR (DMSO-d6,
13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 32.8, 55.1 (CH3); 110.3,
111.0, 113.6 (2C), 119.7, 121.7 (2C), 122.6, 123.8, 159.1 (tert. C); 132.9,
137.1, 141.8, 147.8, 154.6, 157.4, 159.7 (quat. C); C19H17N5O (331.38);
calcd. C 68.87, H 5.17, N 21.13; found C 68.56, H 5.21, N 21.06; HPLC:
99.5% at 254 nm and 99.6% at 280 nm; tN ¼ 5.00 min, tM (DMSO) ¼
1.02 min (ACN/H2O; 50:50); lmax: 299 nm.
150.9 MHz):
d
(ppm) ¼ 32.8 (CH3); 111.0, 111.3, 119.7 (2C), 120.9,
122.7, 123.8, 128.3 (2C), 159.0 (tert. C); 125.3, 137.1, 139.0, 141.8,
147.6, 157.4, 159.2 (quat. C); C18H14ClN5 (335.37); calcd. C 64.38, H
4.20, N 20.86; found C 64.27, H 4.17, N 20.68; HPLC: 99.8% at 254 nm
and 99.9% at 280 nm; tN ¼ 5.32 min, tM (DMSO) ¼ 1.02 min (ACN/
H2O; 60:40); lmax: 278 nm, 301 nm.
5.1.3.7. 4-{[4-(1-Methyl-1H-benzimidazol-2-yl)pyrimidin-2-yl]
amino}phenol (2g). Preparation following General Procedure A
(reaction time 19 h). The precipitate was crystallized twice from
ethanol to yield a yellow powder (55.6%).ꢄ1
5.1.3.4. 4-(1-Methyl-1H-benzimidazol-2-yl)-N-[4-(morpholin-4-yl)
phenyl]pyrimidin-2-amine (2d). A mixture of 3-(N,N-dimethyla-
mino)-1-(1-methyl-1H-benzimidazol-2-yl)prop-2-en-1-one
Mp.: 270e271 ꢀC; IR (KBr): 3426 cm (OH), 3246 cmꢄ1 and
(4)
3115 cmꢄ1 (NH); 1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 4.23 (s,
(234 mg, 1.02 mmol) and N-[4-(morpholin-4-yl)phenyl]guanidine
(5d, free base) (266 mg, 1.21 mmol) was refluxed in propan-2-ol
(5 mL) for 12 h. After cooling to room temperature the precipitate
was filtered by suction, washed with water and petrol ether and
purified by crystallization from ethanol to yield a yellow powder
(53%).
3H, N-CH3), 6.74e6.77 (m, 2H, ArH), 7.29e7.31 (m, 1H, ArH),
7.36e7.38 (m, 1H, ArH), 7.46 (m, 2H, ArH), 7.55 (d, 1H, J ¼ 5.0 Hz,
pyrimidine-H), 7.69 (d, 1H, J ¼ 8.0 Hz, ArH), 7.76 (m, 1H, ArH), 8.56
(d, 1H, J ¼ 5.0 Hz, pyrimidine-H), 9.18 (s, 1H, OH), 9.46 (s, 1H, NH);
13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 32.7 (CH3); 109.9,
111.0, 114.9 (2C), 119.7, 122.4 (2C), 122.6, 123.7, 159.0 (tert. C);
131.2, 137.1, 141.8, 147.8, 152.9, 157.3, 160.0 (quat. C); C18H15N5O
(317.35); calcd. C 68.13, H 4.76, N 22.07; found C 67.76, H 4.96, N
21.78; HPLC: 99.9% at 254 nm and 99.9% at 280 nm; tN ¼ 3.78 min,
tM (DMSO) ¼ 1.03 min (ACN/buffer A; 35:65); lmax: 241 nm and
300 nm.
Mp.: 210e212 ꢀC; IR (KBr): 3435 cmꢄ1
,
3256 cmꢄ1 and
(ppm) ¼
3186 cmꢄ1 (NH); 1H NMR (DMSO-d6, 600 MHz):
d
3.05e3.07 (m, 4H, 2 ꢂ CH2), 3.74e3.76 (m, 4H, 2 ꢂ CH2), 4.26 (s,
3H, N-CH3), 6.93e6.96 (m, 2H, ArH), 7.29e7.32 (m, 1H, ArH),
7.37e7.39 (m, 1H, ArH), 7.56 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H),
overlapped by 7.57e7.58 (m, 2H, ArH), 7.70 (d, 1H, J ¼ 8.2 Hz,
ArH), 7.75e7.77 (dt, 1H, J ¼ 1.0/8.0 Hz, ArH), 8.58 (d, 1H, J ¼ 5.0 Hz,
pyrimidine-H), 9.55 (s, 1H, NH); 13C NMR (DMSO-d6, 150.9 MHz):
5.1.3.8. 3-{[4-(1-Methyl-1H-benzimidazol-2-yl)pyrimidin-2-yl]
amino}phenol (2h). Preparation following General Procedure A
(reaction time 20 h) and purification by crystallization from ethanol
yielded a yellow powder (52.2%).
d
(ppm) ¼ 32.9 (CH3); 48.8 (2C), 66.2 (2C) (sec. C); 110.1, 111.0, 115.4
(2C), 119.7, 121.1 (2C), 122.6, 123.7, 159.0 (tert. C); 132.1, 137.1, 141.8,
146.6, 147.9, 157.4, 159.7 (quat. C); C22H22N6O (386.45); calcd. C
68.38, H 5.74, N 21.75; found C 68.31, H 5.54, N 21.57; HPLC: 99.8% at
254 nm and 99.7% at 280 nm; tN ¼ 2.98 min, tM (DMSO) ¼ 1.02 min
(ACN/buffer A; 50:50); lmax: 301 nm.
Mp.: 224e225 ꢀC; IR (KBr): 3435 cmꢄ1 (OH), 3246 cmꢄ1 and
3190 cmꢄ1 (NH); 1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 4.30 (s,
3H, N-CH3), 6.41e6.43 (m, 1H, ArH), 7.10 (t, 1H, J ¼ 7.8 Hz, ArH),
7.13e7.15 (m, 1H, ArH), 7.30e7.33 (m, 1H, ArH), 7.35 (t, 1H,
J ¼ 2.0 Hz, ArH), 7.38e7.40 (m, 1H, ArH), 7.63 (d, 1H, J ¼ 5.2 Hz,
pyrimidine-H), 7.72 (m, 1H, ArH), 7.78 (m, 1H, ArH), 8.65 (d, 1H,
J ¼ 5.2 Hz, pyrimidine-H), 9.35 (s, 1H, OH), 9.71 (s,1H, NH); 13C NMR
5.1.3.5. N-(4-Ethoxyphenyl)-4-(1-methyl-1H-benzimidazol-2-yl)pyr-
imidin-2-amine (2e). Preparation following General Procedure A
(reaction time 22.5 h). The precipitate was crystallized twice from
ethanol to yield a yellow powder (47.0%).
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 32.7 (CH3); 106.5, 109.0, 110.4,
110.8, 111.0, 119.7, 122.7, 123.8, 129.1, 159.0 (tert. C); 137.1, 141.0,

R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
261
141.8,147.8,157.4,157.5,159.4 (quat. C); C18H15N5O (317.35); calcd. C
68.13, H 4.76, N 22.07; found C 67.85, H 4.86, N 21.87; HPLC: 99.8%
at 254 nm and 99.9% at 280 nm; tN ¼ 3.86 min, tM (DMSO) ¼
1.06 min (ACN/H2O; 25:75); lmax: 278 nm and 300 nm.
(s, 3H, N-CH3), 4.43 (s, 3H, N-CH3), 7.69e7.74 (m, 2H, ArH),
7.96e7.98 (dd, 1H, J ¼ 7.5/1.2 Hz, ArH), 8.14 (d, 1H, J ¼ 7.9 Hz, ArH),
8.22 (d, 1H, J ¼ 8.1 Hz, pyridine-H), 8.80 (d, 1H, J ¼ 8.1 Hz,
pyridine-H); Because of poor solubility in common solvents,
a standard 13C spectrum could not be recorded. Therefore, a 13C-
DEPT-NMR was recorded after addition of trifluoroacetic acid,
which is reported here: 13C-DEPT-NMR (DMSO-d6, 150.9 MHz):
5.1.3.9. 2-Chloro-4-{[4-(1-methyl-1H-benzimidazol-2-yl)pyrimidin-
2-yl]amino}phenol (2i). Preparation following General Procedure A
(reaction time 30 h). Purification by a single crystallization from
ethanol and subsequent preparative HPLC (ACN/H2O, 45:55) yiel-
ded a yellow powder (12.0%).
d
(ppm) ¼ 28.5, 29.9, 34.2 (CH3); 113.6, 115.6, 120.3, 126.8, 127.2,
139.4 (tert. C) (quat C not shown); C17H15N5O2 (321.34); calcd. C
63.54, H 4.71, N 21.79; found C 63.32, H 4.84, N 21.65; HPLC: 97.8% at
254 nm and 95.9% at 280 nm; tN ¼ 2.16 min, tM (DMSO) ¼ 1.03 min
(ACN/H2O; 50:50); lmax: 353 nm.
Mp.: 239e241 ꢀC; IR (KBr): 3403 cmꢄ1 (OH), 3253 cmꢄ1 and
3185 cmꢄ1 (NH); 1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 4.27 (s,
3H, N-CH3), 6.95 (d, 1H, J ¼ 8.7 Hz, ArH), 7.30e7.32 (m, 1H, ArH),
7.37e7.39 (m, 1H, ArH), overlapped by 7.41 (d, 1H, J ¼ 2.4 Hz, ArH),
7.61 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H), 7.71 (d, 1H, J ¼ 8.2 Hz, ArH),
7.76 (dt, 1H, J ¼ 1.1/8.0 Hz, ArH), 7.81 (d, 1H, J ¼ 2.4 Hz, ArH), 8.61 (d,
1H, J ¼ 5.0 Hz, pyrimidine-H), 9.62 (s, 1H, OH), 9.82 (s, 1H, NH); 13C
5.2. Biological assays
5.2.1. In vitro kinase assays
A radiometric protein kinase assay (33PanQinaseÒ Activity
Assay) was used for measuring the activity of the protein kinases.
NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 32.7 (CH3); 110.6, 111.0,
116.3, 119.7, 120.2, 122.6, 123.8, 159.1 (tert. C); 118.9, 121.6, 132.3,
137.1, 141.8, 147.8, 148.2, 157.4, 159.6 (quat. C); C18H14ClN5O
All assays were performed with
robotic system and 96-well FlashPlatesÔ from Perkin Elmer
(Boston, MA, USA) in a 50 L reaction volume. The reaction
cocktail was pipetted in 4 steps in the following order: 20 L of
assay buffer; 5 L of ATP solution (in H2O); 5 L of test compound
(in 10% DMSO); 10 L of substrate/10 L of enzyme solution
(premixed). The assay for all enzymes contained 60 mM
HEPESeNaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 M Na-
33P]-
a Beckman Coulter/Sagian
(351.79); MS (EI): m/z (%) ¼ 351 [M]þ (100); HRMS (EI): m/z
m
ꢅ
[M ꢄ H]þ calcd. 350.08086, found 350.07982; HPLC: 99.9% at
m
ꢅ
254 nm and 99.7% at 280 nm; tN ¼ 2.94 min, tM (DMSO) ¼ 1.06 min
(ACN/buffer A; 45:55); lmax: 242 nm and 301 nm.
m
m
m
m
5.1.3.10. 2-Methoxy-5-{[4-(1-methyl-1H-benzimidazol-2-yl)pyr-
imidin-2-yl]amino}phenol (2j). In a thick-walled sealed microwave
vessel, a mixture of 3-(N,N-dimethylamino)-1-(1-methyl-1H-ben-
zimidazol-2-yl)prop-2-en-1-one (4) (1 mmol, 229 mg), N-(3-
hydroxy-4-methoxyphenyl)guanidinium nitrate (5j) (293 mg,
1.20 mmol), and sodium hydroxide (1.2 mmol, 48 mg) in propan-2-
ol (5 mL) were heated to 150 ꢀC for 1.5 h in a mono mode synthesis
microwave device (150 W, maximum pressure 250 psi). After
cooling to room temperature, the precipitate was filtered by
suction, washed with water and petroleum ether and crystallized
from ethanol. Subsequent purification by preparative HPLC (ACN/
H2O, 40:60) yielded a yellow powder (34%).
m
orthovanadate, 1.2 mM DTT, 50 mg/mL PEG20000, 1 mM [g-
ATP (approx. 5 ꢂ105 cpm per well). The following substrates
were used [substrate amount in square brackets given in ng/
50 mL]: GSK3(14-27) [2000] for AKT1; tetra(LRRWSLG) [500] for
Aurora B; Poly(Glu,Tyr)4:1 [125] for FAK and VEGF-R2; RBER-
CHKtide [2000] for PLK1.The reaction cocktails were incubated at
30 ꢀC for 80 min. The reaction was stopped by adding 50
m
L of 2%
(v/v) phosphoric acid. Plates were aspirated and washed twice
with 200
L of 0.9% (w/v) NaCl. Incorporation of 33P was deter-
m
mined with a microplate scintillation counter (Microbeta Trilux,
Wallac). For calculation of IC50 values, data were generated for
comparison without enzyme in the presence of substrate (“low
control”). Moreover, data with enzyme but without inhibitor
were generated (“high control”). The difference between high
and low control was taken as 100% enzyme activity. The residual
activities for each concentration and the compound IC50 values
were calculated using Quattro Workflow V3.0.6 (Quattro
Research GmbH, Munich, Germany; www.quattro research.com).
The fitting model for the IC50 determinations was “Sigmoidal
response (variable slope)” with parameters “top” fixed at 100%
and “bottom” at 0%. The fitting method used was a least squares
fit. In case of solubility problems, outliers in the higher concen-
tration range were removed to generate more realistic sigmoidal
curves for IC50 calculation. As parameter for assay quality, the Z0-
factor [52] was calculated for the low and high controls of each
plate. For the projects reported here, Z0-factors were >0.5, indi-
cating an excellent assay quality.
Mp.: 181e183 ꢀC; IR (KBr): 3397 cmꢄ1 (OH), 3245 cmꢄ1 (NH); 1H
NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 3.75 (s, 3H, O-CH3), 4.27 (s,
3H, N-CH3), 6.88 (d, 1H, J ¼ 8.7 Hz, ArH), 7.05e7.07 (dd, 1H, J ¼ 8.6/
2.5 Hz, ArH), 7.28 (d, 1H, J ¼ 2.4 Hz, ArH), 7.30e7.32 (m, 1H, ArH),
7.37e7.39 (m, 1H, ArH), 7.57 (d, 1H, J ¼ 5.0 Hz, pyrimidine-H), 7.71
(d, 1H, J ¼ 7.9 Hz, ArH), 7.76 (d, 1H, J ¼ 7.9 Hz, ArH), 8.59 (d, 1H,
J ¼ 5.0 Hz, pyrimidine-H), 8.96 (s,1H, OH), 9.49 (s,1H, NH); 13C NMR
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 32.7, 55.9 (CH3); 108.6, 110.2,
110.8, 111.0, 112.5, 119.7, 122.6, 123.7, 159.0 (tert. C); 133.5, 137.1,
141.8, 143.2, 146.3, 147.9, 157.4, 159.7 (quat. C); C19H17N5O (347.37);
calcd. C 65.70, H 4.93, N 20.16; found C 65.32, H 4.98, N 19.85; HPLC:
99.9% at 254 nm and 99.9% at 280 nm; tN ¼ 3.16 min, tM (DMSO) ¼
1.02 min (ACN/buffer A; 40:60); lmax: 303 nm.
5.1.4. 1,3-Dimethyl-7-(1-methyl-1H-benzimidazol-2-yl)pyrido[2,3-
d]pyrimidine-2,4-(1H,3H)-dione (7)
A mixture of 3-(N,N-dimethylamino)-1-(1-methyl-1H-benzi-
midazol-2-yl)prop-2-en-1-one (4) (229 mg, 1.00 mmol) and 6-
amino-1,3-dimethyluracil (6) (155 mg, 1.00 mmol) in acetic acid
(8 mL) was refluxed for 8 h. After cooling to room temperature,
a precipitate formed, which was filtered off by suction. The filtrate
was poured into aqueous 5% sodium acetate solution (25 mL). A
precipitate formed, which was filtered off and combined with the
precipitate from the reaction mixture. The combined solids were
crystallized from a DMF/water mixture (7:1) to yield colorless
needles (35.3%).
5.2.2. In vitro cell line screening
The in vitro cell line screening was performed at the American
National Cancer Institute (Bethesda, U.S.A.) as described before [14].
In brief, approx. 60 human tumor cell lines are incubated with test
compounds for 48 h in five concentration levels (10ꢄ8 to 10ꢄ4 M).
Cells are fixed and the protein mass is stained with sulforhodamine
B (SRB) solution. After measuring the absorbance at 515 nm the
percentage of growth is calculated using the different absorbance
measurements [time zero (Tz), control growth (C), and growth in
the presence of drug (Ti)]. Further details of the test method have
been published [41,42,53,54].
Mp.: 303e304 ꢀC; IR (KBr): 1702 cmꢄ1 and 1654 cmꢄ1 (C]O);
1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 3.40 (s, 3H, N-CH3), 3.73

262
R. Determann et al. / European Journal of Medicinal Chemistry 53 (2012) 254e263
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settings proposed by Kirchmair et al. [55] (HQS stands for maxconfs
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5.4. COMPARE analysis
Using the routine accessible on the NCI web site (http://dtp.nci.
nih.gov/compare/), the matrix COMPARE analysis was performed
using GI50 (growth inhibition 50%) data including the following
compounds (NSC designator; number of included cell lines and
number of independent experiments given in brackets): 1 (NSC
743304; 58 cell lines, two experiments); 2j (NSC 750416; 60 cell
lines; two experiments); sorafenib tosylate (NSC 747971; 60 cell
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Crystal data: Triclinic, space group P1, a ¼ 8.0722(3),
ꢀ
b ¼ 8.9740(4), c ¼ 10.0591(4) A,
a
¼ 88.817(4),
b
¼ 86.437(3),
3
ꢀ
ꢀ
g
¼ 81.945(4) , Z ¼ 2, V ¼ 720.04 A . Data were recorded at 100 K on
an Oxford Diffraction Xcalibur diffractometer using mono-
chromated Mo Ka radiation (l
¼ 0.71073 A, 2qmax 56.6ꢀ). The
ꢀ
structure was refined on F2 using the program SHELXL-97 (G.M.
Sheldrick, University of Göttingen, Germany). Methyl groups were
idealized as rigid groups allowed to rotate but not tip; other H
atoms were included with a riding model. The final wR2 was 0.078,
with a conventional R1 of 0.038 for 220 parameters and 3564
independent reflections. Complete data (excluding structure
factors) have been deposited at the Cambridge Crystallographic
Data Centre under the number CCDC 860815. These data can be
obtained free of charge from www.ccdc.cam.ac.uk/data_request/cif.
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Acknowledgments
Funding by a grant of the European Commission (Contract No
LSHB-CT-2004-503467; to F. T., C. S., M. H. G. K., and C.K.) is
gratefully acknowledged. The authors thank the National Cancer
Institute for evaluating compounds 1 and 2j in the NCI’s in vitro cell
line screening (IVCLS) project. We are grateful to Mr. A. Weinkauf
for recording the X-ray data.
Appendix A. Supplementary data
Supplementary data associated with this article (details of X-ray
structure determination) can be found, in the online version, at doi:
10.1016/j.ejmech.2012.04.007. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
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