Synthesis and in vitro EGFR (ErbB1) tyrosine kinase inhibitory activity of 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4-amines

Article abstract of DOI:10.1016/j.ejmech.2011.10.012

A series of 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4-amines have been synthesised, characterised and tested for their in vitro EGFR (ErbB1) tyrosine kinase inhibitory activity. The compounds were prepared from ethyl cyanoacetate and α-bromoacetophenones via the 2-amino-3-ethoxycarbonyl-5- aryl-pyrroles and 4-chloro-6-arylpyrrolopyrimidines. Aromatic substitution with benzylic amines was performed by conventional thermal substitution, and palladium catalysed coupling. The two methods resulted in similar yields, but the palladium coupling had the benefit of lower chemical consumption and reduced reaction times. Eight of the new compounds had IC₅₀ values in the range of 2.8-9.0 nM. Four of these have a fluorine atom positioned at sites otherwise potentially susceptible to oxidative metabolism. Structural variation of the 6-aryl group indicated that the inhibitory action was only moderately sensitive to modifications in this fragment. However, the potency depended strongly on the structure of the aromatic part of the 4-amino group, and any aromatic substitution except fluorine reduced the in vitro activity. The cellular EGFR internalization response of selected compounds was evaluated using HeLa cells. Three fluorinated derivatives had a pronounced effect in inhibiting EGFR internalization.

Full text of DOI:10.1016/j.ejmech.2011.10.012

European Journal of Medicinal Chemistry 46 (2011) 6002e6014
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Original article
Synthesis and in vitro EGFR (ErbB1) tyrosine kinase inhibitory activity
of 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4-amines
,
Svein Jacob Kaspersen a, Christopher Sørum a 1, Veronica Willassen a, Erik Fuglseth a, Eli Kjøbli b,
,
Geir Bjørkøy b, Eirik Sundby c, Bård Helge Hoff a
*
a Norwegian University of Science and Technology, Høgskoleringen 5, NO-7491 Trondheim, Norway
b Sør-Trøndelag University College, Erling Skjalgssons g 1, 7004 Trondheim, Norway
c Sør-Trøndelag University College, E. C. Dahls gate 2, 7004 Trondheim, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4-amines have been synthesised, char-
acterised and tested for their in vitro EGFR (ErbB1) tyrosine kinase inhibitory activity. The compounds
were prepared from ethyl cyanoacetate and a-bromoacetophenones via the 2-amino-3-ethoxycarbonyl-
Received 15 February 2011
Received in revised form
5 October 2011
5-aryl-pyrroles and 4-chloro-6-arylpyrrolopyrimidines. Aromatic substitution with benzylic amines was
performed by conventional thermal substitution, and palladium catalysed coupling. The two methods
resulted in similar yields, but the palladium coupling had the benefit of lower chemical consumption and
reduced reaction times. Eight of the new compounds had IC50 values in the range of 2.8e9.0 nM. Four of
these have a fluorine atom positioned at sites otherwise potentially susceptible to oxidative metabolism.
Structural variation of the 6-aryl group indicated that the inhibitory action was only moderately sensitive
to modifications in this fragment. However, the potency depended strongly on the structure of the
aromatic part of the 4-amino group, and any aromatic substitution except fluorine reduced the in vitro
activity. The cellular EGFR internalization response of selected compounds was evaluated using HeLa
cells. Three fluorinated derivatives had a pronounced effect in inhibiting EGFR internalization.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Accepted 6 October 2011
Available online 15 October 2011
Keywords:
Pyrrolopyrimidine
Tyrosine kinase
ErbB1
Pyrroles
4-Chloropyrolopyrimidines
Cancer
1. Introduction
events inside the cell that eventually leads to cell growth, prolif-
eration and differentiation. Elevated receptor tyrosine kinase
Tyrosine kinases (TK) are enzymes that bind ATP and catalyse
the transfer of -phosphate to tyrosine residues on proteins,
activity is often observed in malignant tumours [6]. The activity of
the kinases, and thereby progression of the diseases can be reduced
by administration of TK inhibitors. Several low molecular weight
inhibitors have entered the marketplace as Gefitinib, Erlotinib and
Lapatinib (Fig. 1), and still more are in clinical studies [7]. Their
mode of action is mainly by inhibition of ATP binding in the TK
domain of one or more of these growth factor receptors, resulting in
inhibition of activation and subsequent downstream signalling
events. The internalization, endocytosis and lysosomal degradation
of EGFR are all part of a process which is dependent on ligand-
induced activation of the EGF receptor [8]. Monitoring EGFR
internalization therefore acts as a potential additional test of
bioactivity of potential inhibitors.
EGFR-TK has previously been targeted by leads based on the
pyrrolopyrimidine scaffold [9]. This led to development of PKI-166
(Fig. 1), a potent inhibitor [10,11]. The activity was found to be
dependant of the stereochemistry of the 1-phenylethanamine side
chain at position 4 of the pyrrolopyrimidine. AEE-788, a structurally
more advanced analogue have also been studied [12,13]. While
Gefitinib and Erlotinib mainly act on EGFR-TK, Lapatinib [14], PKI-
g
thereby regulating their activity and function. Several tyrosine
kinases can be targets for cancer chemotherapy, the most impor-
tant being the receptor tyrosine kinases [1]. This is a large family of
receptors including among others the epidermal growth factor
receptor (EGFR) [2,3], and the vascular endothelial growth factor
receptor (VEGF) [4].
EGFR is a transmembrane glycoprotein and is composed of
a glycosylated N-terminus, an extra-cellular ligand binding region,
a hydrophobic transmembrane region and a C-terminal intracel-
lular region, which contains a tyrosine kinase domain [5]. When
epidermal growth factors (EGF) bind to EGFR the TK domain is
activated, and this triggers a chain of different signal transduction
* Corresponding author. Department of Chemistry, Norwegian University of Science
and Technology (NTNU), NO-7491, Norway. Tel.: þ4773593973; fax: þ4773594256.
E-mail address: bhoff@chem.ntnu.no (B.H. Hoff).
1
Present address: Weifa AS, Guveveien 1, 3791 Kragerø, Norway.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.012

S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
6003
H
H
O
O
N
N
O
O
N
N
N
N
N
N
N
N
N
N
O
O
HO
F
N
H
N
N
N
N
H
O
N
N
O
H3C S
O
H3C
O
Cl
F
H3C
N
H
H
H
N
H
O
Cl
Lapatinib (TykerbTM
Gefitinib (IressaTM
)
Erlotinib (TarcevaTM
)
)
PKI-166
AEE-788
Fig. 1. The structures of EGFR-TK inhibitors.
166 [15], and AEE-788 [12] also efficiently inhibit ErbB2-TK (HER-
2). AEE-788 [12] is also described as a VEGF-TK inhibitor [12].
Development of PKI-166 has been terminated, possibly due to side
effects described as transaminase elevation, diarrhoea, nausea and
vomiting [16,17]. However, there is limited knowledge on the
bioactivity of structurally related pyrrolopyrimidines and it could
be possible to modify the toxicity profile by structural alteration. As
a first step new active compounds must be identified. Moreover,
there is evidence that TK-inhibitors are efficient antiprotozoal
agents [18e20], and that kinases are involved in developing of
leprosy [21,22]. In both cases treatment can be sought by selectively
blocking kinase activity. On this background we have undertaken
the preparation of a series of N-substituted 6-aryl-7H-pyrrolo[2,3-
d]pyrimidine-4-amines, and wish to communicate their potential
as EGFR-TK (ErbB1) inhibitors.
using palladium (II) acetate and XPhos as ligand with sodium tert-
butoxide as base (Scheme 2). The catalyst was activated by heating
a solution of palladium (II) acetate and XPhos in dry tert-butanol
containing 4 mol% distilled water for 2 min at 110 ꢀC [29]. The Pd-
catalysed reactions were performed under Schlenk conditions.
Using a 1.3 fold excess of amine full conversion was obtained in less
than 5 h. The results in term of yields for the two methods are
compared in Table 1.
Although, more convenient in terms of chemical consumption
and reaction time, also the Pd-catalysed coupling gave a mediocre
51e73% yield. The major loss in yield was related to hydrolysis of
the starting materials giving 6aec (10e30% as analysed by 1H
NMR). These by-products could be removed by basic extraction
using NaOH. The small amount of water used in the activation of
the catalyst does not account for the observed level of the hydro-
lytic by-product, indicating that both reagent dryness and handling
are crucial. Similar Pd-ligand complexes have been used in
conversion of halogenated aromatics to phenols, thus the hydro-
lysis most likely takes place via the Pd-arene complex [30]. In the
thermal amination reactions not performed under Schlenk condi-
tions, the hydrolytic by-products were not detected. More efficient
catalysts for this transformation have recently been developed [31],
and work is underway in our laboratory to optimise this reaction
further. All the products analysed for EGFR-TK activity in this study
were however prepared by the thermal route to avoid potential
palladium contaminations.
2. Chemistry
The potential inhibitor compounds were synthesised as
described in Schemes 1 and 2. Ethyl cyanoacetate (1) was reacted
with HCl saturated ethanol to yield compound 2 as a crystalline
solid [23,24]. Free basing of 2 using aq. potassium carbonate at 0 ꢀC,
followed by treatment with ammonium chloride in refluxing
ethanol gave ethyl 3-amino-3-iminopropanoate hydrochloride (3)
[24]. These steps were performed on a 75 g scale.
Based on previous reports [25e27], the aminoimidate 3 was
Finally, deprotection of the methyl ether function in 23e37 was
accomplished using BBr3 in dichloromethane. This gave a variable
26e66% yield of 46e60 as their hydrobromide salts. Possibly, losses
in yield in the demethylation step were related to side reactions as
described by Paliakov et al. [32], and by differences in the solubility
of these compounds in the extractive work-up.
then reacted separately with five
a-bromoacetophenones, 4aee,
using sodium ethoxide as base to give the pyrrolidines 5aee. The
reactions could be performed at room temperature for 12 h or at
60 ꢀC for 1.5e3 h. In gram scale, 5aee were obtained in 48e64%
yield after flash chromatography or crystallisation. The reasons for
the moderate yields were not investigated, however stability issues
have been indicated for similar 2-amino-3-ethoxycarbonylpyrroles
[25].
3. EGFR-TK inhibition and internalization
Conversion of 5 to 6 was performed by a condensation where
formamide reacts with the 1,3-aminoester function in a formic
acid/DMF mixture [27,28]. The 4-hydroxypyrrolopyrimidines 6aee
precipitated from the reaction mixture upon addition of 2-propanol
resulting in 54e74% yield. The chlorination of 6 to 7 was performed
at 90 ꢀC using neat POCl3. The reaction was high yielding, but
a drawback was the large volume in the exothermic basic quench of
residual POCl3. Alternatively, 7aee could be isolated by precipita-
tion followed by careful washing.
The activity of selected compounds towards EGFR-TK was
investigated by IC50 measurements with 10 point titration using
ATP concentration equal to Km in duplicate experiments. Based on
previous reported inhibition data of a series of 4-anilino derivatives
[33], a hydrogen bond donor in the meta or para position of the 6-
Thermal nucleophilic aromatic substitution on 7a using the
amines 8e22 gave the 4-substituted pyrrolopyrimidines 23e37,
while 7bee were reacted with the amines 8 and 9 respectively to
give 38e45, Scheme 2. All the amines used were the (R)-enantio-
mers, except 12, 14, 16 and 22 which were racemates. The thermal
amination was run with a three fold excess of amine in 1-butanol.
Heating the reaction mixture at reflux for 24 h resulted in isolated
yields from 48 to 87%. Attempted use of triethylamine (three
equivalents) as a base, did not lower the amount of amine needed
to attain full conversion.
Therefore, to reduce the amount of the chiral amines, a Buch-
waldeHartwig coupling was investigated for selected substrates
Scheme 1. Synthetic route to the 4-chloropyrrolopyrimidines 7aee.

6004
S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
Table 2
EGFR-TK inhibitory activity (IC50) of compounds 46e60.
Entry
Compound
R
R1
IC50 (nM)a
Stdb
0.6
1.8
9
145
3201
1.1
3
1.7
19
2.9
13
1
2
3
4
5
6
7
8
(R)-46
(R)-47
(R)-48
(R)-49
Rac-50
(R)-51
Rac-52
(R)-53
Rac-54
55
56
57
58
(R)-59
Rac-60
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
p-F
3.7
3.8
62
450
4608
8.0
17
9.0
95
3.3
p-Br
p-CH3
p-CF3
o-F
o-CH3
m-F
m-CH3
H
9
11
12
13
14
15
16
H
H
H
p-F
o-F
m-F
26
45
52
4.0
5
8
CH2CH3
CH3
H
0.7
806
c
1008
Scheme 2. Thermal and Pd-catalysed amination using the amines 8e22. Compounds
46e60 were prepared by demethylation using BBr3.
a
The IC50 values are based on dublicate measurements (2 ꢁ 10 datapoints).
Standard deviation.
For structure see Fig. 2.
b
c
a moderate activity as compared to their non fluorinated parent
compound 55, and their chiral analogues 47, 51 and 53.
We then went on to investigate how modifications of the R2
group in fragment A (Fig. 3) affected inhibitory activity. In this
series, position 4 of the pyrrolopyrimidine was substituted by the
amines 8 (R1 ¼ H) and 9 (R1 ¼ p-F). The fluorine containing
analogues were targeted due to the high activity of derivative 47.
The R2 substituent was changed from a phenolic eOH to eOMe,
eBr, eCN, eF and eH. The compounds tested and their in vitro
activities are summarised in Table 3. Reference data for AEE-788 is
also included for comparison [12].
Methoxy compound 23 retains good potency when compared to
PKI-166 (compound 46, Table 2, entry 1). Replacement of the
phenol with unsubstituted phenyl or p-fluorophenyl also resulted
in potent compounds, and p-cyanophenyl is also well tolerated. The
p-bromophenyl derivative 40 and the fluoro-derivatives 42e44
(Entries 6e8) are less potent by comparison. Comparing the activity
of these compounds to that of AEE-788, it is clear that the R2egroup
affects the activity in a complex manner.
The cellular responses towards some of the synthesised
compounds were then evaluated using a HeLa cell line derived from
a cervical carcinoma. These cells express a high level of EGFR [34].
Ligand binding causes receptor dimerization, induced kinase
activity and a rapid endocytosis of the receptor. To test if we could
use internalization of EGFR in HeLa cells to evaluate inhibitory
activity, we first performed a time study where the internalization
was stopped at different time intervals, and the subcellular locali-
zation of the EGFR was determined by immunostaining. Rapid
internalization of the EGFR in response to ligand binding was
observed, and the presence of 500 nM PKI-166 (Compound 46)
abolished internalization for the first 10 min after stimulation (data
not shown and Fig. 4). Following 15 and 20 min after EGFR acti-
vation there was a slight increase in EGFR internalization even in
the presence of the inhibitor. We therefore evaluated selected
compounds for their ability to interfere with internalization 10 min
Fig. 2. Structures of the compounds tested for EGFR-TK activity and the numbering
system.
aryl group (position 30 and 40 in fragment A, Fig. 2) was assumed to
be important for efficient inhibitory properties. Therefore, keeping
the 40 phenolic group in fragment A constant, fragment B (Fig. 2)
was first varied to identify possible effects on the EGFR-TK inhibi-
tory properties. The results are summarised in Table 2.
PKI-166 (46), the fluorinated derivatives 47, 51 and 53, the
benzylamine derivative 55, and the 1-phenyl-1-propanamine
derivative 59, all gave activity in the low nanomolar range. Obvi-
ously, fluorine substitution is tolerated in the aromatic ring of
fragment B, and the benzylic carbon can be substituted with
a methyl or ethyl group without compromising the activity.
Introduction of more bulky groups into the para position of the
aromatic ring (fragment B) reduced the activity considerably. This
effect was most pronounced for the racemic trifluoromethyl
derivative 50. Further, the steric requirements of the inhibitors
were challenged by activity measurements of the ortho and meta
tolyl derivatives (Entries 7 and 9) and the 1-naphthyl containing
compound 60 (Entry 16). The racemate of 52 had an IC50-value of
17, indicating that the (R)-enantiomer would be highly active.
A significant drop in activity was however observed for compound
60 containing a 1-naphthyl group in fragment B. Somewhat
surprisingly, the fluorobenzylamine compounds 56e58 only had
Table 1
Comparison of yields for the thermal and the Pd-catalysed amination.
Entry Substrate Amine
R
R1/Ar R2
Pd-coupling Thermal Product
(Yield, %)
Rac-12 CH3 p-CF3 OMe 71
(R)-21 C2H5 OMe 47
Rac-22 CH3 C10H7 OMe 57
(Yield, %)
2
3
4
5
6
7
8
7a
7a
7a
7b
7b
7c
7c
75
75
59
50
48
55
59
(R)-27
(R)-36
Rac-37
(R)-38
(R)-42
(R)-39
(R)-43
H
(R)-8
(R)-9
(R)-8
(R)-9
CH3
CH3 p-F
CH3
CH3 p-F
H
H
H
F
56
49
50
73
H
F
Fig. 3. Structural variations in the R1- and R2-group.

S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
6005
Table 3
were identified as possible drug candidates having IC50 values in
the range of 2.8e9.0 nM. Four of these contain fluorine atoms at
sites potentially susceptible to oxidative metabolism.
Effect of modification in the 6-aryl group on EGFR-TK in vitro activity (IC50, nM).
Entry
Compound
R1
R2
IC50 (nM)a
Std.b
Examination of structureeactivity relationships showed that
substituents on the 6-aryl group (fragment A) could be varied
without drastic effects on the EGFR-TK inhibition. In addition to
a phenolic group, small substituents at R2 are all well tolerated.
Decreased potency was observed by introducing larger groups such
as bromine. For fragment B, the unsubstituted benzylamine, as well
as methyl and ethyl derivatives are all potent inhibitors, while
limited substitution of the aromatic moiety is tolerated. A study of
the effect of the inhibitors on the internalization process of EGFR,
confirms that several of the fluorinated derivatives have high
activity in HeLa cells. A benefit of the fluoro-containing pyrrolo-
pyrimidines in a therapeutic setting could potentially be higher
metabolic stability due to removal of sites for aromatic hydroxyl-
ation or O-glucoronidation metabolism.
1
2
3
4
5
6
7
8
(R)-23
(R)-38
(R)-39
(R)-40
(R)-41
(R)-42
(R)-43
(R)-44
(R)-45
AEE-788
H
H
H
H
OMe
H
F
Br
CN
H
4.7
3.3
6.6
63
12
33
27
29
14
2.0
0.8
2.9
2.0
17
4
7
5
12
2
0.6
H
p-F
p-F
p-F
p-F
H
F
Br
9
CN
10c
d
a
The IC50 values are based on dublicate measurements (2 ꢁ 10 datapoints).
Standard deviation.
Data is taken from Traxler et al. [12].
For structure of R2 see Fig. 3.
b
c
d
after EGFR activation. Using representative microscopy images the
cells were scored for having most of the EGFR located at the plasma
membrane or for displaying the EGFR mainly located to intracel-
lular vesicles. It was found that the presence of 500 nM of
compound 42, 43, and 47 caused a potent inhibition of EGF induced
receptor internalization (Fig. 4). At 500 nM, these compounds all
show similar levels of inhibition of receptor internalization as
compared to PKI-166 (46). Compound 38 and 39 displayed an
intermediary effect, whereas compound 41 and 55 caused a modest
effect on EGFR internalization.
6. Experimental section
6.1. General
Ethyl cyanoacetate (1), the a-bromoacetophenones 4aec, (R)-1-
phenylethanamine (8), (R)-1-(4-bromophenyl)ethanamine (10),
benzylamine (17), (R)-1-(phenyl)-1-propanamine (21), 1-(4-
trifluoromethylphenyl)ethanone, 1-(o-tolyl)ethanone and 1-(m-
tolyl)ethanone were from Fluka. BBr3, (R)-1-(p-tolyl)ethanamine
(11),
1-(1-naphthyl)-ethanamine
(22),
tert-butanol,
2-
dicyclohexylphosphino-20,40,60-triisopropylbiphenyl, palladium(II)
4. Discussion
acetate were from SigmaeAldrich. (R)-1-(4-Fluorophenyl)ethan-
The structureeactivity relationships identified in this study are
summarised in Fig. 5.
amine (9) was from Alfa Aesar. The a-bromoacetophenones 4dee
were prepared by bromination of the corresponding acetophe-
nones using molecular bromine. NMR spectra and high resolution
mass spectra where in accordance with proposed structures. The
synthesis and spectral properties of the intermediate compound 1,
2, 3, 5a, 6a, 23e24, 28, 30, and 33e35 have been described earlier
[39]. Silica-gel column chromatography was performed using
At the benzylic carbon (R-group), hydrogen, methyl and ethyl
substituents were tolerated, whereas R1-substituents larger than
fluorine in meta and para positions are not compatible with a high
activity. Substitution of hydrogen by fluorine will generally increase
the oxidative stability of the aromatic ring and may block CYP-450
mediated hydroxylations [35e37]. Larger variance in size and
electronic properties of substituents could be tolerated in the R2-
group. PKI-166 is known to be mainly metabolised by O-glucur-
onidation [17]. The results show that a hydrogen bond donating
group as R2, is not crucial for good in vitro activity. Thus, several of
the prepared compounds lacking the phenol functionallity might
have prolonged inhibitory effect in vivo.
Preliminary docking studies revealed that compounds with IC50
values in the range of 3e86 nM, have similar conformations in the
ATP binding site. Fig. 6 shows the docked structure of compound
43, superimposed with the crystal structure complex of AEE-788
[38]. The para substituent on the 6-aryl group (fragment A) is
directed towards the entrance of the binding pocket, explaining the
minor changes in IC50 values upon variation of the R2-group,
whereas there is limited space for the aromatic amines at C-4
(fragment B).
silica-gel 60A from Fluka, pore size 40e63 mm.
6.2. Molecular modelling
The docking was performed using a receptor modified from the
2J6M structure from RCSB Protein Data Bank [38], and AutoDock
Vina [40]. The co-crystallised inhibitor (AEE788) was removed from
the initial X-ray structure, water molecules were then removed and
polar hydrogens and Gastieger charges were added using AutoDock
Tools [41]. Ligands were optimised for the energy and geometry
using MM2 force fields. In the structures of the ligands, all bonds
were treated as rotatable, except for the aromatic bonds. Docking
proceeded with an exhaustiveness value of 100 and a maximum
output of 10 structures. Visualization was done using the PyMOL
software (The PyMOL Molecular Graphics System, Version 1.3,
Schrödinger, LLC).
The activity estimated from the internalization study does not
correlate directly with activity from the in vitro EGFR testing. This
could be due to differences in cellular transport processes,
permeability, binding to other proteins or metabolic stability. The
metabolic stability and the selectivity of these new compounds are
the subject of ongoing studies.
6.3. Analyses
1H and 13C NMR spectra were recorded with Bruker Avance 400
spectrometer operating at 400 MHz and 100 MHz, respectively. 19F
NMR was performed on a Bruker Avance 400 or 500 operating at
376 or 470 MHz respectively. For 1H and 13C NMR chemical shifts
are in ppm rel. to TMS, while for 19F NMR the shift values are
relative to hexafluorobenzene. Coupling constants are in hertz.
HPLC (Agilent 110-Series) with a G1379A degasser, G1311A Quat-
pump, G1313A ALS autosampler and a G1315D Agilent detector
(230 nm) was used to determine the purity of the synthesised
5. Conclusion
A series of chiral and non-chiral 4-N-substituted 6-aryl-pyrro-
lopyrimidines have been synthesised, characterised and tested for
their in vitro EGFR-TK inhibitor properties. Eight active derivatives

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S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
Fig. 4. EGF induced EGFR internalization is potently inhibited by compound 46, 42, 43 and 47. A) Subconfluent HeLa cell cultures were starved for growth factors and left untreated
or stimulated for 10 min with EGF (100 ng/mL, final concentration) in the absence or presence of the indicated compounds (500 nM, final concentration). Immunostained EGFR
(green) and DNA (blue) was imaged using a Zeiss Axiovert 200 microscope with a 63ꢁ lens and a confocal detector. The images are representative for more than 500 randomly
selected and manually inspected cells for each condition. Scale bar, 10 mm. B) A semi quantification of internalization was performed by scoring the location of the EGFR as “plasma
membrane” or “internalized” in more than 200 randomly selected cells per condition before and following 10 min of EGF stimulation in the absence or presence of the indicated
compounds (500 nM, final concentration). The study was performed in parallel as a single run, and is shown as the relative effect on internalization as determined by two
independent blinded counts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
compounds. Conditions: a Omrisphere 5 C18 (100 ꢁ 3.0 mm)
column, flow rate 1.0 mL/min, elution starting with H2O þ 1% TFA/
acetonitrile (98/2), linear gradient elution for 15 min ending at
acetonitrile/water þ 1% TFA (90/10), then 15 min isocratic elution.
The software used with the HPLC was Agilent ChemStation.
Accurate mass determination (ESI) was performed on an Agilent
G1969 TOF MS instrument equipped with a dual electrospray ion
source, or EI (70eV) using a Finnigan MAT 95 XL. FTIR spectra were
recorded on a Thermo Nicolet Avatar 330 infrared spectrophotom-
eter. All melting points are uncorrected and measured by a Büchi
melting point instrument. Some compounds were purified by
Fig. 6. Compound (R)-43 (green) docked in the EGFR-TK ATP binding domain, co-
crystallised AEE-788 (Fig. 1) is shown in red. (For interpretation of the references to
Fig. 5. Effect of structural variation on EGFR-TK activity.
colour in this figure legend, the reader is referred to the web version of this article.)

S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
6007
preparative HPLC (Agilent 110-Series) with a G1361A pump and
G1328B detector. A Microsorb100-8 C18, 250 ꢁ 21.4mm columnwas
used with a flow rate of 21 mL/min, elution starting with H2O þ 1%
TFA/acetonitrile (98/2), linear gradient elution for 25 min ending at
acetonitrile/water þ 1% TFA (90/10), then 15 min isocratic elution.
evaporated under reduced pressure. Purification was by silica-gel
column chromatography (EtOAc/n-pentane, 7/3).
6.6.2. Preparation of the 6-aryl-7H-pyrrolo[2,3-d]pyrimidin-4-ols
(6aee)
The following is representative: to a solution of anhydrous DMF
(28 mL), formic acid (11.3 mL), ethyl 2-amino-5-phenyl-1H-pyrrole-
3-carboxylate (5b) (3.50 g, 16.57 mmol) and an excess formamide
(75 mL) were added. The mixture was heated at 150 ꢀC for 20 h.
Then 2-propanol (12 mL) was added, and the mixture was cooled to
20 ꢀC. The formed precipitate was isolated by filtration, washed
with 2-propanol (10 mL) and n-hexane (2 ꢁ 15 mL), and dried
under reduced pressure to yield a solid.
6.4. In vitro EGFR (ErbB1) activity
The activity measurements of compounds towards ErbB1 was
performed by Invitrogen using their Z0-LYTEÒ assay technology
[42]. The test compounds were diluted in 1% DMSO solution, and
subjected to a duplicate 10 points titration, using an ATP concen-
tration equal to K m. The IC50 values were calculated from activity
data with
a four parameter logistic model using SigmaPlot
6.6.3. Preparation of the 6-aryl-4-chloro-7H-pyrrolo[2,3-d]
pyrimidines (7aee)
(Windows Version 11.0 from Systat Software, Inc.)
The following is representative: 6-phenyl-7H-pyrrolo[2,3-d]
pyrimidin-4-ol (6b) (1.5 g, 7.43 mmol) and neat POCl3 (13.3 mL)
were mixed and reacted at 90 ꢀC for 3 h. The solution was cooled
with an ice-salt bath, and water (60 mL) was added. NaOH (8 M,
80 mL) was used to adjust the pH to 12. The formed precipitate was
isolated by filtration, washed with water and n-pentane and dried
to give a solid.
6.5. Immunostaining and confocal microscopy analysis
HeLa cells (ATCC CCL2) were propagated as recommended by
ATCC and seeded in 8-well coverglass slides (Nunc) for confocal
imaging. Subconfluent cultures in normal growth medium were
washed in phosphate buffer saline and incubated over night in
Eagels’s minimum essential medium supplemented with 0.1% fetal
calf serum. The different compounds were added to 500 nM final
concentration and left for 30 min at 37 ꢀC before the cultures were
placed on ice for 15 min. Recombinant EGF (Sigma) was added to
the ice cold medium to 100 ng/mL final concentration and left on
ice for 30 min. The medium was then replaced with warm medium
supplemented with 0.1% fetal calf serum and incubated for 10 min
at 37 ꢀC in the cell incubator. Internalization was stopped by fixa-
tion of the cells by adding formaldehyde to 4% final concentration
for 10 min at room temperature and the cells were permeabilized
by 10 min incubation on ice in cold methanol. Unspecific binding
was blocked by 30 min incubation in phosphate buffered saline
supplemented with 3% goat serum followed by 30 min staining of
EGFR using a 1:500 dilution of a mouse anti human EGFR antibody
(sc-101, Santa Cruz Biotechnology). Unbound antibodies were
removed by extensive washing in phosphate buffered saline and
bound antibodies detected by 30 min incubation with a 1:1000
dilution of Alexa 488 conjugated goat anti mouse IgG antibody
(Invitrogen). After extensive washing, the nuclei were visualized
using the DNA dye Draq5 (Biostatus). Images were collected using
a Zeiss Axiovert 200 microscope equipped with a LSM510 META
confocal module and processed using Canvas 11. The effect of the
different compounds on EGF induced receptor internalization was
semi quantitatively determined by scoring cells for having most of
the EGFR located at the plasma membrane or for displaying the
EGFR mainly located to intracellular vesicles. The scoring was done
by two independent blinded counts.
6.6.4. Thermal aromatic amination to 23e45
The following is representative: 4-chloro-6-(4-methoxyphenyl)-
7H-pyrrolo[2,3-d]pyrimidine (7a) (275 mg, 1.06 mmol) and (R)-1-
phenylethanamine (0.44 mL, w3.5 mmol) were added to a dry
round bottle flask containing 1-butanol (3.5 mL) under argon
atmosphere. The mixture was heated at 145 ꢀC for 24 h. The
precipitate formed upon cooling to rt. was isolated by filtration,
washed with diethyl ether (25 mL) and dried resulting in a solid.
6.6.5. Palladium catalysed aromatic amination
The following is representative: catalyst pre-activation:
Pd(OAc)2 (5 mg, 0.022 mmol), XPhos (21 mg, 0.043 mmol), sono-
ficated distilled water (1 mL) and t-BuOH (2 mL) were mixed and
heated at 110 ꢀC for 2 min, resulting in a colour change from orange
to dark green indicating formation of the active catalyst.
4-Chloro-6-phenyl-7H-pyrrolo[2,3-d]pyrimidine (7b) (110 mg,
0.48 mmol), sodium tert-butoxide (167 mg, 1.75 mmol), (R)-1-
phenyletanamine (69 mg, 0.57 mmol) and t-BuOH (1 mL) were
mixed under an argon atmosphere. The active catalyst was added
and the mixture was heated to 110 ꢀC for 4 h. After cooling the
solvent was removed under reduced pressure, and the residue was
diluted with EtOAc (20 mL) and saturated with NaOH (5%, 3 ꢁ10 mL)
to remove 6b, After drying over MgSO4 and concentration under
reduced pressure 68 mg (0.22 mmol, 46%) was obtained.
6.6.6. Demethylation
The following is representative: (R)-6-(4-methoxyphenyl)-N-(1-
phenylethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (23) was dis-
solved in dry CH2Cl2 (2 mL) under argon atmosphere. BBr3 (0.17 mL,
w1.8 mmol) in dry CH2Cl2 (1.5 mL) was added drop wise over 1 h at
6.6. General procedures
6.6.1. Preparation of the pyrroles 5aee
The following is representative: ethyl 3-amino-3-iminopropanoate
hydrochloride (3) (8.3 g, 50.0 mmol) and NaOEt (5.1 g, 75.0 mmol)
were dissolved in abs. EtOH at 0 ꢀC and stirred for 20 min under argon.
The mixture was heated to 60 ꢀC, and 2-bromo-1-phenylethanone
(4b) (5.0 g, 25.0 mmol) was added portion wise over 5 min. After
1.5 h the mixture was cooled to 20 ꢀC and the solvent was evaporated
under reduced pressure. The residue was diluted with distilled water
(20 mL) and extracted with EtOAc (3 ꢁ 80 mL). The organic layer was
washed with water (3 ꢁ 20 mL) and brine (3 ꢁ 20 mL). The combined
water fractions were back extracted with EtOAc (2 ꢁ 20 mL). The
organic phases were dried over MgSO4, and the solvent was
0
ꢀC. Then the mixture was allowed to react at 20 ꢀC for 24 h. The
reaction was quenched by addition of water (10 mL), and the
mixture was extracted with EtOAc (3 ꢁ 25 mL). The combined
organic phase was washed with brine (15 mL), dried over MgSO4
and concentrated. The resulting residue was purified by precipita-
tion from acetone (0.5 mL). The solid formed was isolated by
filtration, washed with diethyl ether (10 mL) and dried.
6.7. Ethyl 2-amino-5-aryl-1H-pyrrole-3-carboxylate, 5aee
Compound 5a was prepared as described previously [43].

6008
S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
6.7.1. Ethyl 2-amino-5-phenyl-1H-pyrrole-3-carboxylate (5b) [25,44]
Compound 5b was prepared as described in Section 6.6.1. This
gave 3.7 g (16.1 mmol, 64%) of a beige solid, mp. 101e104 ꢀC, lit.
6.8.1. 6-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ol (6b) [28]
The compound was prepared as described in Section 6.6.2. This
gave 2.1 g (9.9 mmol, 65%) of a beige solid, mp > 300 ꢀC, lit. [28].
[44]. 107 ꢀC, lit. [25]. 121e122 ꢀC 1H NMR (400 MHz, DMSO-d6)
d:
>300 ꢀC 1H NMR (400 MHz, DMSO-d6)
d
: 12.33 (s, 1H, NH), 11.84 (s,
1H, OH), 7.87 (s, 1H, H-2), 7.82 (m, 2H), 7.41 (m, 2H), 7.27 (m, 1H),
6.93 (s, 1H, H-5). 13C NMR (100 MHz, DMSO-d6)
: 158.2, 149.4,
10.72 (s, 1H, NH, H-1), 7.50e7.44 (m, 2H), 7.32e25 (m, 2H),
7.11e7.05 (m, 1H), 6.46 (d, J ¼ 2.4, 1H, H-4), 5.65 (s, 2H, NH2), 4.15 (q,
d
J ¼ 6.8, 2H), 1.25 (t, J ¼ 6.8, 3H). 13C NMR (100 MHz, DMSO-d6),
d
:
143.7, 133.1, 131.4, 128.8 (2C), 127.2, 124.6 (2C), 109.1, 99.2. HRMS
(ESI): 212.0821 (calcd C12H9N3O, 212.0818, M þ Hþ). IR (neat,
cmꢂ1): 2786, 1650, 1241, 900, 749, 693, 620.
165.4, 148.6, 132.6, 129.1 (2C), 125.4, 123.6, 122.8 (2C), 104.0, 93.9,
58.6, 15.2. HRMS (ESI): 231.1124 (calcd C13H14N2O2, 231.1128,
M þ Hþ). IR (neat, cmꢂ1): 3417, 3328, 1662, 1589, 1281, 1120, 757,
691.
6.8.2. 6-(4-Fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ol (6c)
The compound was prepared as described in Section 6.6.2
starting with ethyl 2-amino-5-(4-fluorophenyl-1H-pyrrole-3-
carboxylate (5c) (2.78 g, 11.19 mmol) and formamide (54 mL,
w1.37 mol). This gave 1.90 g (8.29 mmol, 74%) of a beige solid,
6.7.2. Ethyl 2-amino-5-(4-fluorophenyl)-1H-pyrrole-3-carboxylate
(5c)
The compound was prepared as described in Section 6.6.1
starting with ethyl 3-amino-3-iminopropanoate hydrochloride
(3) (7.3 g, 43.8 mmol), NaOEt (4.5 g, 66.8 mmol) and 2-bromo-1-
(4-fluorophenyl)ethanone (4c) (5.0 g, 23.0 mmol). The crude
product was purified by silica-gel column chromatography
(EtOAc/n-pentane, 7/3) giving 2.9 g (11.7 mmol, 51%) as a beige
mp > 300 ꢀC 1H NMR (400 MHz, DMSO-d6)
d
: 12.33 (s,1H, NH, H-7),
11.84 (s, 1H, OH), 7.90e7.82 (m, 3H, H-2, C6H4F) 7.29e7.21 (m, 2H),
6.91 (s, 1H, H-5). 13C NMR (100 MHz, DMSO-d6)
: 161.7 (d,
d
J ¼ 244.5), 158.6, 149.8, 144.2, 132.6, 128.5 (d, J ¼ 3.1), 127.0 (d,
J ¼ 8.1, 2C), 116.2 (d, J ¼ 21.6, 2C), 109.5, 99.6. 19F NMR (470 MHz
solid, mp. 164e166 ꢀC 1H NMR (400 MHz, DMSO-d6)
d
: 10.72 (s,
DMSO-d6, C6F6)
d
: ꢂ116.3 (m). HRMS (ESI): 230.0725 (calcd
1H, NH, H-1), 7.52e7.46 (m, 2H), 7.15e7.08 (m, 2H), 6.42 (d,
J ¼ 2.7, 1H, H-4), 5.65 (s, 2H, NH2), 4.12 (q, J ¼ 7.2, 2H), 1.23 (t,
C12H8N3OF, 230.0724, M þ Hþ). IR (neat, cmꢂ1): 2778, 1670, 1217,
909, 839, 802, 770, 616.
J ¼ 7.2, 3H). 13C NMR (100 MHz, DMSO-d6)
d: 165.4, 160.5 (d,
J ¼ 242.7), 148.6, 129.4 (d, J ¼ 3.2), 124.7 (d, J ¼ 7.8, 2C), 122.8,
6.8.3. 6-(4-Bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ol (6d) [45]
The compound was prepared as described in Section 6.6.2 starting
with ethyl 2-amino-5-(4-bromophenyl-1H-pyrrole-3-carboxylate
(5d) (3.66 g, 11.84 mmol) and formamide (30 mL, w1.78 mol). This
gave 2.24 g (7.72 mmol, 65%) of a brown solid, mp > 300 ꢀC 1H NMR
115.8 (d, J ¼ 20.8, 2C), 103.9, 93.9, 58.5, 15.1. 19F NMR (470 MHz
DMSO-d6, C6F6)
d
: ꢂ119.3 (m). HRMS (ESI): 249.1040 (calcd
C13H13N2O2F, 249.1039, M þ Hþ). IR (neat, cmꢂ1): 3482, 3352,
1643, 1567, 1225, 1136, 1079, 834, 798, 764.
(400 MHz, DMSO-d6)
d: 11.91 (br s, 2H, OH, NH, H-7), 7.89 (s,1H, H-2),
6.7.3. Ethyl 2-amino-5-(4-bromophenyl)-1H-pyrrole-3-carboxylate
(5d) [45]
7.79 (m, 2H), 7.60 (m, 2H), 7.00 (s,1H, H-5).13C NMR (100 MHz, DMSO-
d6) d: 158.6, 150.0, 144.5, 132.3, 132.1, 131.1 (2C), 127.0 (2C), 120.5,
The compound was prepared as described in Section 6.6.1
starting with ethyl 3-amino-3-iminopropanoate hydrochloride
(3) (8.4 g, 50.6 mmol), NaOEt (5.4 g, 79.4 mmol) and 2-bromo-1-
(4-bromphenyl)ethanone (4d) (7.3 g, 26.3 mmol). The crude
product was purified by silica-gel column chromatography
(EtOAc/n-pentane, 1/1) giving 4.96 g (16.0 mmol, 61%) as a beige
109.6,100.4. HRMS (EI): 288.9846 (calcd C12H8N3OBr, 288.9845, Mþ).
IR (neat, cmꢂ1): 2779, 1657, 1242, 908, 809, 764, 616.
6.8.4. 4-(4-Hydroxy-7H-pyrrolo[2,3-d]pyrimidin-6-yl)benzonitrile
(6e) [47]
The compound was prepared as described in Section 6.6.2
starting with ethyl 2-amino-5-(4-cyanophenyl)-1H-pyrrole-3-
carboxylate (5e) (2.66 g, 10.42 mmol) and formamide (30 mL,
w1.77 mol). This gave 1.33 g (5.63 mmol, 54%) of a grey solid,
solid, mp. 256e260 ꢀC 1H NMR (400 MHz, DMSO-d6)
d: 10.78 (s,
1H, NH, H-1), 7.48e7.43 (m, 4H), 6.54 (d, J ¼ 2.6, 1H, H-4), 5.72 (s,
2H, NH2), 4.12 (q, J ¼ 7.1, 2H), 1.24 (t, J ¼ 7.1, 3H). 13C NMR
(100 MHz, DMSO-d6)
d
: 165.3, 148.9, 131.9, 131.8 (2C), 124.8 (2C),
mp > 300 ꢀC, lit. [47] >410 ꢀC 1H NMR (400 MHz, DMSO-d6)
d: 12.57
122.5, 117.6, 105.1, 94.2, 60.2, 14.5. HRMS (EI): 308.0156 (calcd
C13H13BrN2O2, 308.0155, Mþ). IR (neat, cmꢂ1): 3477, 3353, 1639,
1620, 1589, 1542, 1069, 763.
(br s, NH, H-7), 11.94 (br s, OH), 8.02 (m, 2H), 7.93 (d, J ¼ 2.7, 1H, H-
2), 7.86 (m, 2H), 7.21 (d, J ¼ 2.2,1H, H-5). 13C NMR (100 MHz, DMSO-
d6) d: 158.6, 150.5, 145.2, 136.2, 133.2 (2C), 131.6, 125.3 (2C), 119.4,
109.8, 109.4, 102.8. HRMS (EI): 236.0698 (calcd C13H8N4O,
6.7.4. Ethyl 2-amino-5-(4-cyanophenyl)-1H-pyrrole-3-carboxylate
(5e) [46]
236.0693, Mþ). IR (neat, cmꢂ1): 3112, 2862, 2231, 1652, 1078, 772.
The compound was prepared as described in Section 6.6.1
starting with ethyl 3-amino-3-iminopropanoate hydrochloride
(3) (7.7 g, 46.2 mmol), NaOEt (5.4 g, 78.85 mmol) and 2-bromo-1-
(4-cyanophenyl)ethanone (4e) (7.3 g, 32.6 mmol). The crude
product was purified by crystallisation from acetonitrile giving
4.02 g (15.8 mmol, 48%) as a yellow solid, mp. 224e226 ꢀC, lit.
6.9. 6-Aryl-4-chloro-7H-pyrrolo[2,3-d]pyrimidine, 7aee
Compound 7a was prepared as described previously [39].
[46]. 228e229 ꢀC 1H NMR (400 MHz, DMSO-d6)
d: 11.49 (s, 1H,
6.9.1. 4-Chloro-6-phenyl-7H-pyrrolo[2,3-d]pyrimidine (7b) [28]
The compound was prepared as described in Section 6.6.3
starting from 6-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ol (6b)
(1.57 g, 7.43 mmol) and neat POCl3 (13.5 mL, 54.8 mmol). This gave
1.49 g (6.49 mmol, 87%) of a yellowish solid, mp. 255e257 ꢀC 1H
NH, H-1), 7.64e7.69 (m, 4H), 6.77 (s, 1H, H-4), 5.93 (s, 2H, NH2),
4.15 (q, J ¼ 7.1, 2H), 1.25 (t, J ¼ 7.1, 3H). 13C NMR (100 MHz,
DMSO-d6) d: 165.1, 149.8, 136.9, 133.0 (2C), 122.8 (2C), 122.0, 119.9,
108.2, 106.1, 95.0, 58.7, 15.1. HRMS (EI): 255.1003 (calcd
C14H13N3O2, 255.1002, Mþ). IR (neat, cmꢂ1): 3495, 3343, 2220,
1642, 1622, 1174, 767.
NMR (400 MHz, DMSO-d6) d: 13.03 (s, 1H, NH, H-7), 8.59 (s, 1H, H-
2), 8.02 (m, 2H), 7.51 (m, 2H), 7.42 (m, 1H), 7.11 (d, J ¼ 2.0, H-5). 13C
NMR (100 MHz, DMSO-d6) d: 153.4, 150.9, 150.3, 140.7, 130.6, 129.5
6.8. 6-Aryl-7H-pyrrolo[2,3-d]pyrimidin-4-ol, 6aee
(3C),126.4 (2C), 118.3, 95.9. HRMS (ESI): 230.0488 (calcd C12H8N3Cl,
230.0480, M þ Hþ). IR (neat, cmꢂ1): 3093, 2955, 2826, 1556, 1234,
983, 866, 751, 698.
Compound 6a was prepared as described previously [39].

S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
6009
6.9.2. 4-Chloro-6-(4-fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidine (7c)
The compound was prepared as described in Section 6.6.3
starting from 6-(4-fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ol
(6c) (1.5 g, 6.54 mmol) and neat POCl3 (11.4 mL, w45.3 mmol).
This gave 1.60 g (6.46 mmol, 99%) as a beige solid, mp. 245e247 ꢀC
1H), 7.13 (m, 2H), 4.36 (q, J ¼ 6.6, 1H), 2.35 (s, 3H), 1.65 (s, 2H, NH2),
1.35 (d, J ¼ 6.6, 3H). 13C NMR (100 MHz, CDCl3)
d: 145.6, 134.4, 130.4,
126.4, 126.3, 124.1, 46.7, 24.5, 19.0.
6.10.3. 1-(m-Tolyl)ethanamine (16) [50]
1H NMR (400 MHz, DMSO-d6)
d
: 12.98 (s, 1H, NH), 8.57 (s, 1H, H-2),
The synthesis was performed as described for 12, starting with
1-(m-tolyl)ethanone (1.00 g, 7.45 mmol), Ti(O-i-Pr)4 (4.4 mL,
w14.0 mmol), ammonia in EtOH (2 M, 18 mL, w37.3 mmol) and
NaBH4 (420 mg, 11.18 mmol). This gave after silica-gel column
8.05 (m, 2H), 7.34 (m, 2H), 7.08 (s, 1H, H-5). 13C NMR (100 MHz,
DMSO-d6)
d
: 162.9 (d, J ¼ 246.9), 153.5. 150.8, 150.2, 139.8, 128.6 (d,
J ¼ 8.4, 2C), 127.3 (d, J ¼ 3.1), 118.3, 116.4 (d, J ¼ 21.8, 2C), 95.9. 19F
NMR (470 MHz DMSO-d6, C6F6)
d
: ꢂ113.5 (m). HRMS (ESI):
chromatography (diethyl ether/MeOH/NEt3, 20/5/1) 0.81
(5.99 mmol, 80%) of 1-(m-tolyl)ethanamine (16) as an yellow oil. 1H
NMR (400 MHz, CDCl3) : 7.23 (m, 1H), 7.16 (s, 1H), 7.13 (m, 1H), 7.05
g
248.0388 (calcd C12H7N3FCl, 248.0385, M þ Hþ). IR (neat, cmꢂ1):
3134, 2964, 1742, 1498, 1232, 835, 767.
d
(m, 1H), 4.08 (q, J ¼ 6.7, 1H), 2.35 (s, 3H), 1.56 (s, 2H, NH2), 1.38 (d,
6.9.3. 6-(4-Bromophenyl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (7d)
[45]
J ¼ 6.7, 3H), 13C NMR (100 MHz, CDCl3)
d: 147.7, 138.0, 128.4, 127.5,
126.4, 122.7, 51.2, 25.6, 21.3.
The compound was prepared as described in Section 6.6.3
starting from 6-(4-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-
ol (6d) (1.55 g, 5.34 mmol) and neat POCl3 (4.96 mL,
w53.2 mmol). This gave 1.46 g (4.73 mmol, 89%) of a yellow solid,
6.11. Compounds 23e45
The synthesis and spectroscopic properties of the 6-(4-
methoxyphenyl)-N-(substituted)-7H-pyrrolo[2,3-d]pyrimidin-4-
amines 23e24, 28, 30 and 33e35 have been described recently
[39].
mp > 300 ꢀC 1H NMR (400 MHz, DMSO-d6)
d
: 13.09 (s, 1H, NH, H-7),
8.59 (s, 1H, H-2), 7.97 (m, 2H), 7.69 (m, 2H), 7.15 (s, 1H, H-5). 13C
NMR (100 MHz, DMSO-d6) : 153.5, 151.1, 150.5, 139.5, 132.4 (2C),
d
129.9, 128.3 (2C), 122.8, 118.2, 96.7. HRMS (EI): 306.9515 (calcd
306.9506, Mþ). IR (neat, cmꢂ1): 3124, 2949, 1570, 1234, 869, 770.
6.11.1. (R)-N-(1-(4-Bromophenyl)ethyl)-6-(4-methoxyphenyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (25)
6.9.4. 4-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-6-yl)benzonitrile (7e)
[47]
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-methoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (190 mg, 0.73 mmol) and (R)-1-(4-bromophenyl)
ethanamine (10) (439 mg, 2.19 mmol). This gave 245 mg
The compound was prepared as described in Section 6.6.3
starting from 4-(4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-6-yl)ben-
zonitrile (6e) (510 mg, 2.16 mmol) and neat POCl3 (5.5 mL,
w59.0 mmol). This gave 420 mg (1.65 mmol, 76%) as a yellow solid,
(0.58 mmol, 79%) of a white solid, mp. 271e273 ꢀC, ½a D20
¼ ꢂ333.0 (c
ꢃ
0.24, DMSO). 1H NMR (400 MHz, DMSO-d6)
d: 11.93 (s, 1H, NH, H-7),
mp > 300 ꢀC, lit. [47]. 296e297 ꢀC 1H NMR (400 MHz, DMSO-d6)
d:
8.03 (s, 1H, H-2), 7.76 (s, 1H, NH), 7.73 (m, 2H), 7.49 (m, 2H), 7.38 (m,
13.10 (s, 1H, NH, H-7), 8.64 (s, 1H, H-2), 8.22 (m, 2H), 7.96 (m, 2H),
2H), 7.02 (m, 2H), 6.94 (d, J ¼ 1.8, 1H, H-5), 5.44 (m, 1H), 3.80 (s, 3H),
7.37 (s, 1H, H-5). 13C NMR (100 MHz, DMSO-d6)
d: 153.6, 151.7, 151.3,
1.51 (d, J ¼ 7.0, 3H). 13C NMR (100 MHz, DMSO-d6)
d: 159.1, 155,1,
138.6, 134.9, 133.4 (2C), 126.9 (2C), 119.1, 118.1, 111.5, 98.8. HRMS
(EI): 254.0357 (calcd C13H7N4Cl, 254.0354, Mþ). IR (neat, cmꢂ1):
3126, 2956, 2228, 1570, 1238, 866, 774.
151.8, 151.7, 145.6, 134.1, 131.4 (2C), 128.8 (2C), 126.4 (2C), 124.9,
119.8, 114.9 (2C), 104.3, 94.9, 55.6, 48.8, 23.2. HRMS (EI): 422.0748
(calcd C21H19Br79N4O, 422.0737, Mþ). IR (neat, cmꢂ1): 3128, 2971,
1592, 1250, 830.
6.10. 1-Arylethanamines
6.11.2. (R)-6-(4-Methoxyphenyl)-N-(1-p-tolylethyl)-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (26)
6.10.1. 1-(4-(Trifluoromethyl)phenyl)ethanamine (12) [48]
A mixture of 1-(4-(trifluoromethyl)phenyl)ethanone (400 mg,
2.13 mmol), Ti(O-i-Pr)4 (1.25 mL, w4.25 mmol) and ammonia in
EtOH (2 M, 5.30 mL, w10.6 mmol) was stirred under argon at room
temperature for 24 h NaBH4 (120 mg, 3.19 mmol) was then added,
and the resulting mixture was stirred for another 24 h. The pH of
the reaction mixture was adjusted to pH 2 using HCl (6 M), and
washed with tert-butyl methyl ether (TBME) (3 ꢁ 20 mL). Using
NaOH (pellets) the pH was adjusted to ca 10, and the mixture was
extracted with TBME (6 ꢁ 30 mL). The combined organic phase was
dried over MgSO4, and the solvent was removed under reduced
pressure to give 210 mg (1.11 mmol, 52%) of a yellow oil. 1H NMR
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (203 mg, 0.78 mmol) and 1-(p-tolyl)ethanamine
(11) (317 mg, 2.34 mmol). This gave 245 mg (0.68 mmol, 87%) of
a white solid, mp. 260e263 ꢀC, ½a D20
ꢃ
¼ ꢂ343.0 (c 0.43, DMSO). 1H
NMR (400 MHz, DMSO-d6) d: 11.90 (s, 1H, NH, H-7), 8.04 (s, 1H, H-
2), 7.72 (d, J ¼ 8.6, 2H), 7.66 (d, J ¼ 8.2, 1H, NH), 7.31 (d, J ¼ 7.8, 2H),
7.10 (d, J ¼ 7.7, 2H), 7.02 (d, J ¼ 8.6, 2H), 6.95 (s, 1H, H-5), 5.45 (m,
1H), 3.80 (s, 3H), 2.25 (s, 3H), 1.51 (d, J ¼ 6.9, 3H). 13C NMR
(100 MHz, DMSO-d6) d: 158.6, 154.8, 151.3 (2C), 142.6, 135.3, 133.5,
128.7 (2C), 126.0 (2C), 125.9 (2C), 124.5, 114.4 (2C), 103.9, 94.6, 55.2,
48.4, 22.9, 20.6. HRMS (EI): 358.1784 (calcd C22H22N4O, 358.1788,
Mþ). IR (neat, cmꢂ1): 3183, 2975, 1588, 1245, 830.
(400 MHz, CDCl3)
d
: 7.58 (m, 2H), 7.46 (m, 2H), 4.19 (q, J ¼ 6.7, 1H),
1.38 (d, J ¼ 6.7, 3H), 1.51 (s, 2H, NH2). 13C NMR (100 MHz, CDCl3)
d:
152.1 (q, J ¼ 1.1), 129.4 (q, J ¼ 31.5), 126.5 (2C), 125.8 (q, J ¼ 3.8, 2C),
124.6 (q, J ¼ 270.9), 51.4, 25.1.
6.11.3. 6-(4-Methoxyphenyl)-N-(1-(4-(trifluoromethyl)phenyl)ethyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (27)
6.10.2. 1-(o-Tolyl)ethanamine (14) [49]
The compound was synthesised as described in Section 6.6.4
starting from 4-chloro-6-(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (165 mg, 0.64 mmol), and 1-(4-trifluoromethyl)
phenyl)ethanamine (12) (157 mg, 0.83 mmol). This gave 198 mg
(0.48 mmol, 75%) of a brownish solid, mp 274e277 ꢀC 1H NMR
The synthesis was performed as described for 12, starting with
1-(o-tolyl)ethanone (1.00 g, 7.45 mmol), Ti(O-i-Pr)4 (4.4 mL,
w14.0 mmol), ammonia in EtOH (2 M, 18 mL, w37.3 mmol) and
NaBH4 (422 mg, 11.18 mmol). The crude product was purified by
silica-gel column chromatography (diethyl ether/MeOH/NEt3, 20/5/
1) yielding 0.43 g (3.18 mmol, 43%) of 1-(o-tolyl)ethanamine (14) as
(400 MHz, DMSO-d6) d: 11.95 (s, 1H, NH, H-7), 8.02 (s, 1H, H-2), 7.84
(d, J ¼ 7.7, 1H, NH), 7.73 (d, J ¼ 8.7, 2H), 7.65 (m, 4H), 7.02 (d, J ¼ 8.9,
a light yellow oil. 1H NMR (400 MHz, CDCl3)
d: 7.46 (m, 1H), 7.22 (m,
2H), 6.95 (s, 1H, H-5), 5.52 (m, 1H), 3.79 (s, 3H), 1.55 (d, J ¼ 6.9, 3H).

6010
S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
13C NMR (100 MHz, DMSO-d6)
d: 159.1, 155.0, 151.8, 151.6, 151.0,
128.5 (2C), 127.0 (2C), 126.9, 126.3 (2C), 124.0, 114.8 (2C), 104.4, 95.1,
134.1, 127.5 (q, J ¼ 32.1), 127.2 (2C), 126.4 (2C), 125.5 (q, J ¼ 3.9, 2C),
55.6, 55.3, 30.1, 11.9. HRMS (EI): 358.1792 (calcd C22H22N4O,
124.9, 124.8 (q, J ¼ 270.1), 114.8 (2C), 104.3, 94.9, 55.6, 49.1, 23.2. 19F
358.1788, Mþ). IR (neat, cmꢂ1): 3128, 2963, 1590, 1247, 697.
NMR (376 MHz, DMSO-d6, C6F6)
d
: ꢂ62.2 (s). HRMS (ESI): 413.1595
(calcd C22H19F3N4O, 413.1584, M þ Hþ). IR (neat, cmꢂ1): 3118, 1594,
6.11.8. 6-(4-Methoxyphenyl)-N-(1-(naphthalen-1-yl)ethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (37)
1323, 1251, 1115, 1066, 827.
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (159 mg, 0.61 mmol) and 1-(naphthalen-1-yl)
ethanamine (22) (314 mg, 1.83 mmol). This gave 143 mg
(0.36 mmol, 59%) as a grey solid, mp. 278e280 ꢀC 1H NMR
6.11.4. 6-(4-Methoxyphenyl)-N-(1-o-tolylethyl)-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (29)
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (102 mg, 0.39 mmol) and 1-(o-tolyl)ethanamine
(14) (214 mg, 1.58 mmol). This gave 97 mg (0.27 mmol, 69%) as
(400 MHz, DMSO-d6)
d
: 11.93 (s, 1H, NH, H-7), 8.24 (d, J ¼ 8.2, 1H),
8.04 (s, 1H, H-2), 7.94 (m, 1H), 7.87 (m, 1H), 7.81 (m, 1H), 7.71e7.64
(m, 3H), 7.59e7.45 (m, 3H), 7.01 (m, 2H), 6.97 (s, 1H, H-5), 6.27 (m,
1H), 3.79 (s, 3H), 1.67 (d, J ¼ 6.8, 3H). 13C NMR (100 MHz, DMSO-d6)
a beige solid, mp > 300 ꢀC 1H NMR (400 MHz, DMSO-d6)
d: 11.88 (s,
1H, NH, H-7), 8.03 (s, 1H, H-2), 7.72 (d, J ¼ 9.1, 2H) 7.71 (d, J ¼ 8.6,
3H, NH, Ar), 7.12 (m, 3H), 7.01 (d, J ¼ 8.3, 2H), 6.95 (s, 1H, H-5), 5.61
(m, 1H), 3.79 (s, 3H), 2.42 (s, 3H), 1.48 (d, J ¼ 6.8, 3H). 13C NMR
d: 158.7, 154.2, 151.1, 150.8, 140.7, 133.8, 133.4, 130.6, 128.6, 127.1,
126.1,125.9 (2C),125.52,125.47,124.3,123.3,122.2,114.4 (2C),103.9,
94.8, 55.2, 45.2, 21.8. HRMS (EI): 394.1789 (calcd C25H22N4O,
394.1788, Mþ). IR (neat, cmꢂ1): 3131, 1593, 1251, 828, 775.
(100 MHz, DMSO-d6) d: 159.1, 155.1, 151.8, 151.7, 144.1, 135.1, 133.9,
130.4, 126.7, 126.3 (2C), 126.4, 125.4, 124.9, 114.8 (2C), 104.3, 95.0,
55.6, 45.8, 22.1, 19.2. HRMS (ESI): 359.1850 (calcd C22H28N4O,
359.1866, M þ Hþ). IR (neat, cmꢂ1): 3102, 2956, 1591, 1249, 825,
786, 752.
6.11.9. (R)-6-Phenyl-N-(1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidin-
4-amine (38)
The compound was prepared as described in Section 6.6.4 starting
6.11.5. 6-(4-Methoxyphenyl)-N-(1-m-tolylethyl)-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (31)
with
4-chloro-6-phenyl-7H-pyrrolo-[2,3-d]-pyrimidine
(7b)
(100 mg, 0.44 mmol) and (R)-1-phenylethanamine (8) (158 mg,
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (150 mg, 0.58 mmol) and 1-(m-tolyl)ethanamine
(16) (314 mg, 2.32 mmol). This gave 141 mg (0.39 mmol, 67%) as
1.30 mmol). This gave 68 mg (0.22 mmol, 50%) as a pink solid, mp.
248e250 ꢀC, ½a 2D0
ꢃ
¼ ꢂ284.6 (c 0.40, DMSO), purity > 97% (by HPLC).
1H NMR (400 MHz, DMSO-d6)
d
: 12.02 (s, 1H, NH), 8.05 (s, 1H, H-2),
7.82e7.78 (m, 3H, NH þ Ar), 7.46e7.42 (m, 4H), 7.33e7.27 (m, 3H),
a beige solid, mp > 300 ꢀC 1H NMR (400 MHz, DMSO-d6)
d: 11.90 (s,
7.21e7.18 (m,1H), 7.10 (s,1H, H-5), 5.50(m,1H),1.53 (d, J ¼ 6.9, 3H).13C
1H, NH, H-7), 8.03 (s,1H, H-2), 7.72 (d, J ¼ 9.1, 2H), 7.68 (d, J ¼ 8.9,1H,
NH), 7.15e7.26 (m, 3H), 7.01 (m, 3H), 6.95 (s, 1H, H-5), 5.45 (m, 1H),
3.79 (s, 3H), 2.27 (s, 3H), 1.50 (d, J ¼ 6.9, 3H). 13C NMR (100 MHz,
NMR (100 MHz, DMSO-d6) d: 155.5, 152.2, 152.0, 145.9, 133.8, 132.2,
129.4 (2C), 128.6 (2C), 127.6, 126.9, 126.5 (2C), 124.9 (2C), 104.3, 96.5,
49.1, 23.3. HRMS (ESI): 315.1619 (calcd C20H18N4, 315.1604, M þ Hþ).
IR (neat, cmꢂ1): 3231, 3053, 1575, 1552, 1306, 755, 696. A 20 mg
sample was purified by preparative HPLC for the in vitro studies.
DMSO-d6) d: 159.1, 155.2, 151.8 (2C), 146.0, 137.5, 133.9, 128.5, 127.5,
127.1, 126.3 (2C),124.9, 123.6, 114.8 (2C), 104.3, 95.0, 55.6, 49.0, 23.4,
21.6. HRMS (ESI): 359.1856 (calcd C22H28N4O, 359.1866, M þ Hþ). IR
(neat, cmꢂ1): 3126, 2972, 1594, 1459, 1252, 825, 772, 696.
6.11.10. (R)-6-(4-Fluorophenyl)-N-(1-phenylethyl)-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (39)
6.11.6. N-Benzyl-6-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (32) [27,51]
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-fluorophenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7c) (148 mg, 0.60 mmol) and (R)-1-phenylethanamine
(8) (218 mg, 1.80 mmol). This gave 109 mg (0.33 mmol, 55%) as
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (125 mg, 0.48 mmol) and phenylmethanamine (17)
(155 mg, 1.45 mmol). This gave 104 mg (0.31 mmol, 64%) as a white
a white solid, mp. 278e280 ꢀC, ½a D20
ꢃ
¼ ꢂ302.4 (c 0.50, DMSO),
purity > 98% (by HPLC). 1H NMR (400 MHz, DMSO-d6)
d
: 12.02 (s,
solid, mp 280e282 ꢀC 1H NMR (400 MHz, DMSO-d6)
d: 11.94 (s, 1H,
1H, NH, H-7), 8.05 (s, 1H, H-2), 7.79 (m, 3 H, NH þ Ar), 7.40 (m, 2H),
NH, H-7), 8.10 (s, 1H, H-2), 7.94 (t, J ¼ 5.7, 1H, NH), 7.71 (d, J ¼ 8.5,
2H), 7.47e7.41 (m, 1H), 7.37e7.30 (m, 4H), 7.24e7.21 (m, 1H), 7.01
(d, J ¼ 8.5, 2H), 6.86 (d, J ¼ 2.0, 1H, H-5), 4.73 (d, J ¼ 5.7, 2H), 3.79 (s,
7.30 (m, 4H), 7.20 (m, 1H), 7.09 (s, 1H, H-5), 5.48 (m, 1H), 1.53 d,
J ¼ 7.2, 3H). 13C NMR (100 MHz, DMSO-d6)
: 161.8 (d, J ¼ 244.5, 1C),
d
155.4, 152.2, 152.0, 145.9, 132.9, 128.9 (d, J ¼ 3.2), 128.6 (2C), 126.9,
126.8 (d, J ¼ 9.2, 2C) 126.5 (2C), 116.3 (d, J ¼ 20.4, 2C), 104.3, 96.4,
3H). 13C NMR (100 MHz, DMSO-d6)
d: 159.1, 155.9, 151.8, 151.7, 140.8,
134.1, 128.6 (2C), 127.7 (2C), 127.0, 126.4 (2C), 124.9, 114.8 (2C),
104.3, 94.8, 55.6, 43.6. HRMS (EI): 330.1487 (calcd C20H18N4O,
330.1475, Mþ). IR (neat, cmꢂ1): 3421, 3117, 1601, 1350, 1272, 728.
49.1, 23.3. 19F NMR (400 MHz, DMSO-d6, C6F6)
d
: ꢂ116.2 (m). HRMS
(ESI): 333.1516 (calcd C20H17N4F, 333.1510, M þ Hþ). IR (neat,
cmꢂ1): 3223, 3053, 1570, 836, 757, 697. A 20 mg sample was puri-
fied by preparative HPLC for the in vitro studies.
6.11.7. (R)-6-(4-Methoxyphenyl)-N-(1-phenylpropyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (36)
6.11.11. (R)-6-(4-Bromophenyl)-N-(1-phenylethyl)-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (40)
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7a) (178 mg, 0.69 mmol) and (R)-1-phenylpropan-1-
amine (21) (295 mg, 2.18 mmol). This gave 188 mg (0.52 mmol,
The compound was prepared as described in Section 6.6.4
starting
with
6-(4-bromophenyl)-4-chloro-7H-pyrrolo[2,3-d]
pyrimidine (7d) (98 mg, 0.32 mmol) and (R)-1-phenylethanamine
(8) (116 mg, 0.96 mmol). This gave 64 mg (0.16 mmol, 50%) as
75%) as a white solid, mp. 265e268 ꢀC, ½a D20
¼ ꢂ297.3 (c 0.61,
ꢃ
DMSO). 1H NMR (400 MHz, DMSO-d6)
d
: 11.89 (s, 1H, NH, H-7), 8.03
a white solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ289.6 (c 0.30, DMSO), purity
(s, 1H, H-2), 7.72 (d, J ¼ 8.7, 2H), 7.65 (d, J ¼ 8.6, 1H, NH), 7.42 (d,
J ¼ 7.5, 2H), 7.30 (m, 2H), 7.19 (m, 1H), 7.02 (d, J ¼ 8.7, 2H), 6.97 (s,
1H, H-5), 5.26 (m, 1H), 3.80 (s, 3H), 1.87 (m, 2H), 0.94 (s, 3H). 13C
96% (by HPLC). 1H NMR (400 MHz, DMSO-d6)
d: 12.08 (s, 1H, NH, H-
7), 8.07 (s, 1H, H-2), 7.84 (d, J ¼ 8.2, 1H, NH), 7.72 (m, 2H), 7.64 (m,
2H), 7.43 (m, 2H), 7.30 (m, 2H), 7.19 (m, 1H), 7.14 (s, 1H, H-5), 5.51
NMR (100 MHz, DMSO-d6)
d: 159.1, 155.7, 151.8 (2C), 145.1, 133.9,
(m, 1H), 1.53 (d, J ¼ 7.0, 3H). 13C NMR (100 MHz, DMSO-d6)
d: 155.6,

S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
6011
152.5, 152.1, 145.9, 132.6 (2C), 131.3, 131.5, 128.6 (2C), 126.9, 126.9
(2C), 126.5 (2C), 120.5, 104.3, 97.3, 49.1, 23.3. HRMS (EI): 392.0629
(calcd C20H17N4Br, 392.0631, Mþ). IR (neat, cmꢂ1): 3242, 3090,
2981, 1592, 821, 693.
d6)
d
: 12.09 (s, 1H, NH, H-7), 8.07 (s, 1H, H-2), 7.84 (d, J ¼ 8.1, 1H,
NH), 7.72 (m, 2H), 7.64 (m, 2H), 7.45 (m, 2H), 7.12 (m, 3H,
NH þ Ar), 5.49 (m, 1H), 1.52 (d, J ¼ 7.0, 3H). 13C NMR (100 MHz,
DMSO-d6)
d: 161.3 (d, J ¼ 241.7), 155.5, 152.5, 152.1, 142.0 (d,
J ¼ 2.8), 132.7, 132.3 (2C), 131.5, 128.4 (d, J ¼ 8.0, 2C), 126.9 (2C),
6.11.12. (R)-4-(4e((1-Phenylethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)benzonitrile (41) [47]
120.6, 115.3 (d, J ¼ 21.1, 2C), 104.3, 97.2, 48.6, 23.3. 19F NMR
(470 MHz, DMSO-d6, C6F6)
d
: ꢂ116.7 (m). HRMS (EI): 410.0550
The compound was prepared as described in Section 6.6.4 starting
with 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-6-yl)benzonitrile (7e)
(75 mg, 0.29 mmol) and (R)-1-phenylethanamine (8) (107 mg,
0.88 mmol). This gave 54 mg (0.16 mmol, 55%) as a yellow solid,
(calcd C20H16N4Br, 410.0537, Mþ). IR (neat, cmꢂ1): 3237, 3094,
2987, 1592, 1509, 1223, 824.
6.11.16. (R)-4-(4-((1-(4-Fluorophenyl)ethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)benzonitrile (45)
mp > 300 ꢀC, lit. [47]. 333e336 ꢀC, ½a 2D0
ꢃ
¼ ꢂ316.4 (c 0.40, DMSO),
purity: 98% (by HPLC). 1H NMR (400 MHz, DMSO-d6)
d
: 12.26 (s, 1H,
The compound was prepared as described in Section 6.6.4
starting with 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-6-yl)benzo-
nitrile (7e) (64 mg, 0.25 mmol) and (R)-1-(4-fluorophenyl)ethan-
amine (9) (105 mg, 0.75 mmol). This gave 67 mg (0.19 mmol, 76%) as
NH, H-7), 8.11 (s,1H, H-2), 7.98e7.88 (m, 5H), 7.43 (m, 2H), 7.33e7.29
(m, 3H), 7.19 (m, 1H, H-5), 5.51 (m, 1H), 1.54 (d, J ¼ 7.0, 3H). 13C NMR
(100 MHz, DMSO-d6) d: 155.8, 153.2, 152.6, 145.7, 136.5, 133.4 (2C),
131.8,128.6 (2C),126.9,126.5 (2C),125.2 (2C),119.4,109.4,104.5, 99.7,
49.2, 23.2. HRMS (EI): 339.1481 (calcd C21H17N5, 339.1478, Mþ). IR
(neat, cmꢂ1): 3373, 3116, 2960, 2223,1594,1317, 702. A 20 mg sample
was purified by preparative HPLC for the in vitro studies.
a solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ249.8 (c 0.20, DMSO), purity > 97%
(by HPLC). 1H NMR (400 MHz, DMSO-d6)
d
: 12.26 (s, 1H, NH, H-7),
8.10 (s,1H, H-2), 7.99e7.89 (m, 5H), 7.50e7.42 (m, 3H), 7.31 (s,1H, H-
5), 7.16e7.13 (m, 2H), 5.50 (m, 1H), 1.53 (d, J ¼ 6.5, 3H). 13C NMR
(100 MHz, DMSO-d6)
d: 161.4 (d, J ¼ 243.2), 153.3, 152.6, 149.5, 141.9
6.11.13. (R)-N-(1-(4-Fluorophenyl)ethyl)-6-phenyl-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (42)
(d, J ¼ 2.9), 136.4, 133.4 (2C), 131.9, 128.4 (d, J ¼ 7.8, 2C), 125.3 (2C),
119.7, 115.3 (d, J ¼ 21.5, 2C), 109.4, 104.5, 99.7, 48.6, 23.6. 19F NMR
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-phenyl-7H-pyrrolo-[2,3-d]-pyrimidine
(7b) (100 mg, 0.44 mmol) and (R)-1-(4-fluorophenyl)ethanamine
(8) (182 mg,1.31 mmol). This gave 71 mg (0.21 mmol, 48%) as a solid,
(470 MHz, DMSO-d6, C6F6)
d
: ꢂ118.1 (m). HRMS (EI): 357.1383 (calcd
C21H16N5F, 357.1384, Mþ). IR (neat, cmꢂ1): 3389, 3122, 2979, 2223,
1594, 1322, 698. A 20 mg sample was purified by preparative HPLC
for the in vitro studies.
mp. 239e240 ꢀC, ½a D20
ꢃ
¼ ꢂ234.7 (c 0.25, DMSO), purity > 94% (by
HPLC). 1H NMR (400 MHz, DMSO-d6)
d
: 12.04 (s, 1H, NH, H-7), 8.06
6.11.17. (R)-4-(4-((1-Phenylethyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-
6-yl)phenol hydrobromide (46) [27,33]
(s, 1H, H-2), 7.82e7.78 (m, 3H, NH þ Ar), 7.48e7.42 (m, 4H),
7.31e7.27 (m, 1H), 7.15e7.08 (m, 3H, H-5þAr), 5.49 (m, 1H), 1.53 (d,
The compound was synthesised as described in Section 6.6.6
starting from (R)-6-(4-methoxyphenyl)-N-(1-phenylethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (23) (61 mg, 0.18 mmol) and BBr3
(0.17 mL, 1.8 mmol). This gave 40 mg, (0.10 mmol, 54%) of a white
J ¼ 6.8, 3H). 13C NMR (100 MHz, DMSO-d6)
: 161.3 (d, J ¼ 243.5),
d
155.4,152.2,152.0,142.0 (d, J ¼ 2.9),133.9,132.3,129.4 (2C),128.3 (d,
J ¼ 8.1, 2C), 127.7, 124.9 (2C), 115.3 (d, J ¼ 21.1, 2C), 104.2, 96.5, 48.5,
23.3. 19F NMR (470 MHz, DMSO-d6, C6F6): ꢂ116.2 (m). HRMS (ESI):
333.1518 (calcd C20H17N4F, 333.1510, M þ Hþ). IR (neat, cmꢂ1): 3110,
2964, 1591, 1508, 1225, 832, 743, 688. A 20 mg sample was purified
by preparative HPLC for the in vitro studies.
solid, mp > 300 ꢀC, purity > 99% (by HPLC), ½a 2D0
¼ ꢂ207.9 (c 0.11,
ꢃ
DMSO). 1H NMR (400 MHz, DMSO-d6)
d: 12.98 (s, 1H, NH, H-7), 9.83
(br s, 1H, OH), 9.55 (br s, 1H, NH), 8.30 (s, 1H, H-2), 7.66 (m, 2H),
7.50e7.48 (m, 2H), 7.39 (m, 2H), 7.32e7.29 (m, 1H), 7.24 (s, 1H, H-5),
6.89 (m, 2H), 5.37 (m, 1H), 1.66 (d, J ¼ 6.5, 3H). 13C NMR (100 MHz,
6.11.14. (R)-6-(4-Fluorophenyl)-N-(1-(4-fluorophenyl)ethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (43)
DMSO-d6) d: 158.0, 148.8 (2C), 142.3, 141.9, 137.6, 128.6 (2C), 127.5,
126.7 (2C), 126.1 (2C), 121.2, 116.0 (2C), 102.9, 96.6, 50.9, 22.3. HRMS
(ESI): 331.1555 (calcd C20H18N4O 331.1553, M þ Hþ). IR (neat,
cmꢂ1): 3135, 1647, 1495, 758, 702.
The compound was prepared as described in Section 6.6.4
starting with 4-chloro-6-(4-fluorophenyl)-7H-pyrrolo-[2,3-d]-
pyrimidine (7c) (68 mg, 0.27 mmol) and (R)-1-(4-fluorophenyl)
ethanamine (9) (115 mg, 0.83 mmol). This gave 57 mg (0.16 mmol,
6.11.18. (R)-4-(4-((1-(4-Fluorophenyl)ethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)phenol hydrobromide (47) [27]
59%) as a white solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ243.0 (c 0.4, DMSO),
purity > 96% (by HPLC).1H NMR (400 MHz, DMSO-d6)
d
: 12.04 (s,1H,
The compound was synthesised as described in Section 6.6.6
starting from (R)-N-(1-(4-fluorophenyl)ethyl)-6-(4-methoxyphenyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (24) (149 mg, 0.41 mmol) and
BBr3 (0.40 mL, 4.2 mmol). This gave 75 mg (0.17 mmol, 43%) of a white
NH, H-7), 8.06 (s,1H, H-2), 7.81e7.78 (m, 3H, NH þ Ar), 7.47e7.44 (m,
2H), 7.32e7.27 (m, 2H), 7.15e7.10 (m, 2H), 7.03 (s, 1H, H-5), 5.49 (m,
1H), 1.53 (d, J ¼ 7.2, 3H). 13C NMR (100 MHz, DMSO-d6)
d: 161.7 (d,
J ¼ 244.5), 161.2 (d, J ¼ 241.7), 155.4, 152.2, 152.0, 142.1 (d, J ¼ 3.2),
132.9, 128.9 (d, J ¼ 3.5), 128.3 (d, J ¼ 7.8, 2C), 126.9 (d, J ¼ 8.5, 2C),
116.3 (d, J ¼ 22.2, 2C),115.2 (d, J ¼ 20.8, 2C),104.3, 96.4, 48.5, 23.3.19F
solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ142.2 (c 0.12, DMSO), purity > 99% (by
HPLC).1H NMR (400 MHz, DMSO-d6)
d
: 12.97 (s,1H, NH, H-7), 9.82 (br s,
1H, OH), 9.51 (br s,1H, NH), 8.31 (s,1H, H-2), 7.65 (m, 2H), 7.56e7.52 (m,
2H), 7.25e7.20 (m, 3H, H-5, Ar), 6.89 (m, 2H), 5.39 (m, 1H), 1.65 (d,
NMR (470 MHz, DMSO-d6, C6F6)
d
: ꢂ116.1 (m), ꢂ118.2 (m). HRMS
(ESI): 351.1422 (calcd C20H16N4F2, 351.1416, M þ Hþ). IR (neat, cmꢂ1):
J ¼ 6.7, 3H). 13C NMR (100 MHz, DMSO-d6)
: 161.5 (d, J ¼ 243.4), 158.0,
d
3433, 3110, 2972, 1583, 1495, 1221, 832, 765.
148.6 (2C), 142.5, 138.2, 137.5, 128.2 (d, J ¼ 7.0, 2C), 126.7 (2C), 121.3,
116.0 (2C), 115.4 (d, J ¼ 22.1, 2C), 102.9, 96.6, 50.2, 22.4. 19F NMR
6.11.15. (R)-6-(4-Bromophenyl)-N-(1-(4-fluorophenyl)ethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (44)
(376 MHz, DMSO-d6, C6F6)
d: ꢂ114.6 (m). HRMS (ESI): 349.1465 (calcd
C20H17FN4O, 349.1459, M þ Hþ). IR (neat, cmꢂ1): 3122, 1647, 1223, 835,
The compound was prepared as described in Section 6.6.4
starting with 6-(4-bromophenyl)-4-chloro-7H-pyrrolo-[2,3-d]-
pyrimidine (7d) (88 mg, 0.29 mmol) and (R)-1-(4-fluorphenyl)
ethanamine (9) (119 mg, 0.86 mmol). This gave 72 mg
748.
6.11.19. (R)-4-(4-((1-(4-Bromophenyl)ethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)phenol hydrobromide (48)
The compound was synthesised as described in Section 6.6.6
starting from (R)-N-(1-(4-bromophenyl)ethyl)-6-(4-methoxyphenyl)-
(0.18 mmol, 62%) as a white solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ266.4 (c
0.20, DMSO), purity > 95% (by HPLC) 1H NMR (400 MHz, DMSO-

6012
S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
7H-pyrrolo[2,3-d]pyrimidin-4-amine (25) (144 mg, 0.34 mmol) and
6.11.23. -(4-((1-(o-Tolyl)ethyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-
6-yl)phenol hydrobromide (52)
BBr3 (322 mL, 3.40 mmol). This gave 80 mg (0.16 mmol, 48%) of a white
solid, mp. 267e270 ꢀC, ½a D20
ꢃ
¼ ꢂ216.2 (c 0.44, DMSO), purity > 97% (by
The compound was synthesised as described in Section 6.6.6
HPLC).1H NMR (400 MHz, DMSO-d6)
d
: 12.94 (s,1H, NH, H-7), 9.82 (br s,
starting from (29) (97 mg, 0.27 mmol) and BBr3 (270
2.70 mmol). This gave 41 mg, (0.10 mmol, 36%) of a white solid,
mp > 300 ꢀC 1H NMR (400 MHz, DMSO-d6)
: 12.51 (s, 1H, NH, H-7),
mL,
1H, OH), 9.47 (br s,1H, NH), 8.29 (s,1H, H-2), 7.65 (m, 2H), 7.58 (m, 2H),
7.44 (m, 2H), 7.16 (s,1H, H-5), 6.88 (m, 2H), 5.37 (m,1H),1.63 (d, J ¼ 6.6,
d
3H). 13C NMR (100 MHz, DMSO-d6)
d: 158.4, 149.6, 148.2, 142.5, 142.1,
9.85 (s, 1H, OH), 8.79 (br s, 1H, NH), 8.20 (s, 1H, H-2), 7.61 (d, J ¼ 8.6,
2H), 7.46 (m, 1H), 7.32 (s, 1H, H-5), 7.19e7.18 (m, 4H), 7.07 (m, 1H),
6.86 (d, J ¼ 8.7, 2H), 5.57 (m,1H), 2.41 (s, 3H),1.56 (d, J ¼ 6.6, 3H). 13C
137.9, 131.9 (2C), 128.9 (2C), 127.2 (2C), 121.7, 121.0, 116.4 (2C), 103.4,
96.8, 50.8, 22.7. HRMS (EI): 408.0582 (calcd C20H17Br79N4O, 408.0580,
Mþ). IR (neat, cmꢂ1): 3135, 1645, 1613, 1493, 761.
NMR (100 MHz, DMSO-d6) d: 158.5, 151.7 (2C), 149.7, 142.1, 137.2,
136.1, 131.3, 127.9, 127.3 (2C), 127.0, 125.7, 122.8, 116.7 (2C), 104.2,
96.6, 47.9, 22.0, 19.7. HRMS (EI): 344.1632 (calcd C21H20N4O,
344.1632, Mþ). IR (neat, cmꢂ1): 3417, 3132, 1647, 1597, 1224, 758. A
20 mg sample was purified by preparative HPLC for the in vitro
studies.
6.11.20. (R)-4-(4-((1-(p-Tolyl)ethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)phenol hydrobromide (49)
The compound was synthesised as described in Section 6.6.6
starting from (R)-6-(4-methoxyphenyl)-N-(1-(p-tolyl)ethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (26) (87 mg, 0.24 mmol) and BBr3
(231
m
L, 2.44 mmol). This gave 47 mg, (0.11 mmol, 46%) of a white
6.11.24. (R)-4-(4-((1-(3-Fluorophenyl)ethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)phenol hydrobromide (53)
The compound was synthesised as described in Section 6.6.6
starting from (R)-N-(1-(3-fluorophenyl)ethyl)-6-(4-methoxyphenyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (30) (176 mg, 0.49 mmol) and
solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ247.0 (c 0.20, DMSO), purity > 96%
(by HPLC). 1H NMR (400 MHz, DMSO-d6)
d
: 12.77 (s, 1H, NH, H-
7), 9.59 (s, 1H, OH), 8.97 (s, 1H, NH), 8.23 (s, 1H, H-2), 7.58 (d,
J ¼ 8.4, 2H), 7.29 (m, 3H, H-5þAr), 7.10 (d, J ¼ 7.8, 2H), 6.87e6.81
(m, 2H), 5.44 (m, 1H), 2.25 (s, 3H), 1.51 (d, J ¼ 6.8, 3H). 13C NMR
BBr3 (470 mL, 4.9 mmol). This gave 123 mg, (0.29 mmol, 58%) of an off-
(100 MHz, DMSO-d6)
d
: 158.4, 155.2, 150.8 (2C), 142.4, 135.9,
white solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ195.2 (c 0.13, DMSO), purity > 99%
133.4, 128.5 (2C), 126.3 (2C), 126.1 (2C), 124.3, 114.3 (2C), 103.8,
94.6, 53.2, 22.8, 20.5 HRMS (EI): 344.1634 (calcd C21H20N4O,
344.1632, Mþ). IR (neat, cmꢂ1): 3183, 1614, 1278, 833, 763. A
20 mg sample was purified by preparative HPLC for the in vitro
studies.
(by HPLC). 1H NMR (400 MHz, DMSO-d6),
d: 12.95 (s,1H, NH, H-7), 9.84
(br s, 1H, OH), 9.52 (br, 1H, NH), 8.29 (s, 1H, H-2), 7.66 (d, J ¼ 8.7, 2H),
7.46e7.40 (m, 1H), 7.38e7.33 (m, 2H), 7.23 (s, 1H, H-5), 7.15e7.11 (m,
1H), 6.89 (d, J ¼ 8.7, 2H), 5.45 (m, 1H), 1.65 (d, J ¼ 6.8, 3H). 13C NMR
(100 MHz, DMSO-d6)
d
: 162.2 (d, J ¼ 244.5), 158.0, 148.3 (2C), 145.3,
142.8,137.4,130.6 (d, J ¼ 8.0),126.7 (2C),122.2,121.3,115.9 (2C),114.3 (d,
6.11.21. 4-(4-((1-(4-(Trifluoromethyl)phenyl)ethyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-6-yl)phenol hydrobromide (50)
The compound was synthesised as described in Section 6.6.6
starting with 6-(4-methoxyphenyl)-N-(1-(4-(trifluoromethyl)
phenyl)ethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (27) (176 mg,
J ¼ 21.1), 113.2 (d, J ¼ 22.1), 103.0, 96.5, 50.4, 22.3. 19F NMR (376 MHz,
DMSO-d6, C6F6)
d:
ꢂ112.2 (m). HRMS (ESI): 349.1466 (calcd
C20H17FN4O, 349.1459, M þ Hþ). IR (neat, cmꢂ1): 3133,1648,1589, 758,
699.
0.43 mmol) and BBr3 (410
(0.21 mmol, 48%) as a beige solid, mp. 256 ꢀC, purity > 96% (by
HPLC). 1H NMR (400 MHz, DMSO-d6)
: 13.01 (s, 1H, NH, H-7),
m
L, 4.30 mmol). This gave 99 mg,
6.11.25. 4-(4-((1-(m-Tolyl)ethyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-
6-yl)phenol hydrobromide (54)
The compound was synthesised as described in Section 6.6.6
starting from 6-(4-methoxyphenyl)-N-(1-m-tolylethyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine (31) (121 mg, 0.34 mmol) and BBr3
d
9.84 (br s, 1H, OH), 9.63 (br s, 1H, NH), 8.31 (s, 1H, H-2), 7.98 (d,
J ¼ 8.2, 2H), 7.65 (m, 4H), 7.25 (s, 1H, H-5), 6.89 (d, J ¼ 6.9, 2H),
5.47 (m, 1H), 1.67 (d, J ¼ 7.5, 3H). 13C NMR (100 MHz, DMSO-d6)
(320
solid, mp. 248e249 ꢀC, purity > 95% (by HPLC). 1H NMR (400 MHz,
DMSO-d6) : 12.59 (s, 1H, NH, H-7), 9.78 (br s, 1H, OH), 8.91 (br s,
mL, 3.39 mmol). This gave 96 mg (0.23 mmol, 66%) as a beige
d: 158.6, 154.1, 150.7, 147.8, 142.7, 138.1, 129.9 (2C), 127.2 (q,
J ¼ 3.2, 2C), 127.1 (q, J ¼ 32.0), 126.9 (2C), 124.6 (q, J ¼ 270.6),
d
121.4, 116.3 (2C), 103.2, 96.6, 48.9, 22.6. 19F NMR (376 MHz,
NH), 8.21 (s, 1H, H-2), 7.64 (d, J ¼ 8.6, 2H), 7.27 (s, 1H), 7.24 (m, 2H),
DMSO-d6, C6F6)
d
:
ꢂ61.9 (s) HRMS (ESI): 399.1410 (calcd
7.10 (s, 1H, H-5), 7.07 (m, 1H), 6.86 (d, J ¼ 8.9, 2H), 5.36 (m, 1H), 2.29
C21H17N4OF3, 399.1427, M þ Hþ). IR (neat, cmꢂ1): 3150, 1609,
1277, 761, 620. A 20 mg sample was purified by preparative HPLC
for the in vitro studies.
(s, 3H), 1.58 (d, J ¼ 6.9, 3H). 13C NMR (100 MHz, DMSO-d6)
d: 158.6,
155.4, 149.2 (2C), 143.6, 137.9, 136.7, 128.8, 128.2, 127.1, 126.9 (2C),
123.5, 122.2, 116.3 (2C), 103.7, 96.0, 50.4, 23.0, 21.2. HRMS (ESI):
345.1712 (calcd C21H20N4O, 345.1710, M þ Hþ). IR (neat, cmꢂ1):
3110, 1599, 1497, 1274, 832, 761, 700. A 20 mg sample was purified
by preparative HPLC for the in vitro studies.
6.11.22. (R)-4-(4-((1-(2-Fluorophenyl)ethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)phenol hydrobromide (51)
The compound was synthesised as described in Section 6.6.6
starting from (R)-N-(1-(2-fluorophenyl)ethyl)-6-(4-methoxyphenyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (28) (218 mg, 0.60 mmol) and
6.11.26. 4-(4-(Benzylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenol
hydrobromide (55) [27,52]
BBr3 (580
m
L, 6.0 mmol). This gave 101 mg, (0.24 mmol, 39%) of
The compound was synthesised as described in Section 6.6.6
starting from N-benzyl-6-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (32) (79 mg, 0.24 mmol) and BBr3 (220 mL,
a colourless solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ177.6ꢀ (c 0.13, DMSO),
purity > 99% (by HPLC). 1H NMR (400 MHz, DMSO-d6)
d: 12.91 (br s,
1H, NH, H-7), 9.82 (br s, 1H, OH), 9.36 (br s, 1H, NH), 8.30 (s, 1H, H-2),
7.66e7.64 (m, 2H), 7.50e7.46 (m, 1H), 7.41e7.35 (m, 1H), 7.27e7.21
(m, 3H, H-5þAr), 6.87 (m, 2H), 5.58 (m, 1H), 1.65 (d, J ¼ 6.7, 3H). 13C
2.28 mmol). This gave 46 mg (0.12 mmol, 48%) of an off-white solid,
mp > 300 ꢀC, purity: 99% (by HPLC). 1H NMR (400 MHz, DMSO-d6)
d: 13.06 (s,1H, NH, H-7), 9.65 (s,1H, OH), 9.24 (s,1H, NH), 8.13 (s,1H,
NMR (100 MHz, DMSO-d6)
d
: 160.8 (d, J ¼ 244.5), 158.0, 149.2, 147.5,
H-2), 7.60 (d, J ¼ 8.2, 2H), 7.38e7.31 (m, 4H), 7.26e7.23 (m, 1H),
142.7, 137.6, 129.7 (d, J ¼ 8.0), 128.8 (d, J ¼ 13.1), 126.8 (3C), 124.7 (d,
6.84e6.82 (m, 3H, H-5þAr), 4.74 (d, J ¼ 5.3, 2H). 13C NMR (100 MHz,
J ¼ 2.3), 121.2, 116.0 (2C), 115.7 (d, J ¼ 21.1), 103.0, 96.5, 45.8, 20.8. 19F
DMSO-d6) d: 157.6 (2C), 151.1, 147.3, 140.1, 135.3, 128.8 (2C), 127.7
NMR (376 MHz, DMSO-d6, C6F6)
d
: ꢂ116.5 (m). HRMS (ESI): 349.1465
(2C), 127.2, 126.6 (2C), 123.0, 116.2 (2C), 104.1, 64.6, 43.9. HRMS (EI):
316.1320 (calcd C19H16N4O, 316.1319, Mþ). IR (neat, cmꢂ1): 3421,
3117, 1601, 1350, 1272, 728.
(calcd C20H17FN4O, 349.1459, M þ Hþ). IR (neat, cmꢂ1): 3133, 1648,
1589, 758, 699.

S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
6013
6.11.27. 4-(4-((4-Fluorobenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-6-
yl)phenol hydrobromide (56)
116.3 (2C), 104.5, 96.1, 55.7, 30.0, 11.6. HRMS (EI): 344.1630 (calcd
21H20N4O, 344.1632, Mþ). IR (neat, cmꢂ1): 3417, 3110, 2981, 1586,
C
The compound was synthesised as described in Section 6.6.6
starting from N-(4-fluorobenzyl)-6-(4-methoxyphenyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine (33) (168 mg, 0.48 mmol) and BBr3
(0.47 mL, 4.9 mmol). This gave 114 mg (0.27 mmol, 57%) of an off-
white solid, mp 274e276 ꢀC, purity: 98% (by HPLC). 1H NMR
1234.
6.11.31. 4-(4-((1-(Naphthalen-1-yl)ethyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)phenol hydrobromide (60)
The compound was synthesised as described in Section 6.6.6
starting from 6-(4-methoxyphenyl)-N-(1-(naphthalen-1-yl)ethyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (37) (113 mg, 0.29 mmol) and
BBr3 (3 mL, 3 mmol). This gave 65 mg, (0.16 mmol, 54%) as a grey
solid, mp > 300 ꢀC, purity: 97% (by HPLC). 1H NMR (400 MHz,
(400 MHz, DMSO-d6) d: 13.01 (s, 1H, NH, H-7), 9.81 (br, 1H, OH),
9.76 (br, 1H, NH), 8.35 (s, 1H, H-2), 7.65 (d, J ¼ 8.7, 2H), 7.51e7.48 (m,
2H), 7.27e7.21 (m, 2H), 7.14 (d, J ¼ 2.0, 1H, H-5), 6.88 (d, J ¼ 8.7, 2H),
4.77 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
d
: 161.7 (d, J ¼ 243.4),
158.0, 149.4, 147.5, 142.1, 137.6, 132.7, 129.7 (d, J ¼ 8.0, 2C), 126.8
DMSO-d6) d: 12.98 (s,1H, NH, H-7), 9.82 (br s,1H, OH), 9.55 (br s,1H,
(2C), 121.2, 116.0 (2C), 115.4 (d, J ¼ 21.1, 2C), 102.8, 96.5, 44.2. 19F
NH), 8.30 (s, 1H), 8.14 (s, 1H), 8.00 (m, 1H), 7.91 (m, 1H), 7.64e7.57
(m, 5H), 7.54e7.50 (m,1H), 7.26 (s,1H, H-5), 6.89e6.87 (m, 2H), 6.09
NMR (376 MHz, DMSO-d6, C6F6)
d
: ꢂ114.3 (m). HRMS (ESI):
335.1303 (calcd C19H15FN4O, 335.1303, M þ Hþ). IR (neat, cmꢂ1):
(m, 1H), 1.78 (d, J ¼ 6.5, 3H). 13C NMR (100 MHz, DMSO-d6)
d: 156.9,
3150, 1652, 1221, 758.
154.5, 151,2, 151.1, 141.0, 134.0, 133.4, 130.6, 128.6, 127.0, 126.1, 126.0
(2C),125.5 (2C), 123.3, 122.9, 122.2, 115.7 (2C), 103.9, 94.0, 52.4, 21.8.
HRMS (EI): 380.1634 (calcd C24H20N4O, 380.1632, Mþ). IR (neat,
cmꢂ1): 3126, 1646, 1613, 1494, 1176, 761. A 20 mg sample was
purified by preparative HPLC for the in vitro studies.
6.11.28. 4-(4-((3-Fluorobenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-6-
yl)phenol hydrobromide (57)
The compound was synthesised as described in Section 6.6.6
starting from N-(3-fluorobenzyl)-6-(4-methoxyphenyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine (34) (139 mg, 0.40 mmol) and BBr3
(0.39 mL, 4.00 mmol). This gave 44 mg (0.11 mmol, 26%) of an off-
white solid, mp. 271e273 ꢀC, purity > 99% (by HPLC). 1H NMR
Acknowledgements
The group is thankful to the Anders Jahres foundation for
financial support. Roger Aarvik is thanked for technical support.
Susana Villa Gonzalez is acknowledged for HRMS experiments.
(400 MHz, DMSO-d6) d: 12.73 (br, 1H, NH, H-7), 9.78 (s, 1H, OH),
9.31 (br s, 1H, NH), 8.29 (s, 1H, H-2), 7.64 (d, J ¼ 8.7, 2H), 7.46e7.40
(m, 1H), 7.27e7.25 (m, 2H), 7.17e7.12 (m, 1H), 7.04 (s, 1H, H-5), 6.87
(d, J ¼ 8.7, 2H), 4.82 (d, J ¼ 4.9, 2H) 13C NMR (100 MHz, DMSO-d6)
d:
References
161.1 (d, J ¼ 243.4), 158.0, 149.6, 147.4, 142.2, 139.6, 137.7, 130.7 (d,
J ¼ 8.0), 126.8 (2C), 123.5, 121.2, 116.0 (2C), 114.5 (d, J ¼ 21.1), 114.4
(d, J ¼ 21.1), 103.0, 96.4, 44.3. 19F NMR (376 MHz, DMSO-d6, C6F6)
[1] D. Mukherji, J. Spicer, Second-generation epidermal growth factor tyrosine
kinase inhibitors in non-small cell lung cancer, Expert Opin. Invest. Drugs 18
(2009) 293e301.
[2] P.S. Sharma, R. Sharma, T. Tyagi, Receptor tyrosine kinase inhibitors as potent
weapons in war against cancers, Curr. Pharm. Des. 15 (2009) 758e776.
[3] R. Bose, X. Zhang, The ErbB kinase domain: structural perspectives into kinase
activation and inhibition, Exp. Cell Res. 315 (2009) 649e658.
[4] V.L. Keedy, A.B. Sandler, Inhibition of angiogenesis in the treatment of non-
small cell lung cancer, Cancer Sci. 98 (2007) 1825e1830.
[5] M.W. Pedersen, H.S. Poulsen, Epidermal growth factor receptor in cancer
therapy, Sci. Med. (Narberth, PA, U. S.) 8 (2002) 206e217.
[6] B.R. Voldborg, L. Damstrup, M. Spang-Thomsen, H.S. Poulsen, Epidermal
growth factor receptor (EGFR) and EGFR mutations, function and possible role
in clinical trials, Ann. Oncol. 8 (1997) 1197e1206.
[7] P. Traxler, Tyrosine kinases as targets in cancer therapy - successes and fail-
ures, Expert Opin. Ther. Targets 7 (2003) 215e234.
[8] I.H. Madshus, E. Stang, Internalization and intracellular sorting of the EGF
receptor: a model for understanding the mechanisms of receptor trafficking,
J. Cell Sci. 122 (2009) 3433e3439.
[9] P. Traxler, G. Bold, W.K.-D. Brill, J. Frei, Pyrrolopyrimidines and Processes for
the Preparation Thereof WO 9702266, 1996.
d: ꢂ112.5 (m). HRMS (ESI): 335.1305 (calcd C19H16FN4O, 335.1303,
M þ Hþ). IR (neat, cmꢂ1): 3133, 1609, 1496, 757.
6.11.29. 4-(4-((2-Fluorobenzyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-6-
yl)phenol hydrobromide (58)
The compound was synthesised as described in Section 6.6.6
starting from N-(2-fluorobenzyl)-6-(4-methoxyphenyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine (35) (157 mg, 0.45 mmol) and BBr3
(0.44 mL, 4.6 mmol). This gave 83 mg (0.20 mmol, 44%) of a col-
ourless solid, mp. 284e285 ꢀC, purity: 98% (by HPLC). 1H NMR
(400 MHz, DMSO-d6) d: 12.94 (br, 1H, NH, H-7), 9.82 (br s, 1H, OH),
9.60 (br s, 1H, NH), 8.35 (s, 1H, H-2), 7.64 (d, J ¼ 8.7, 2H), 7.48e7.45
(m, 1H), 7.44e7.40 (m, 1H), 7.31e7.29 (m, 1H), 7.27e7.21 (m, 1H),
7.11 (s, 1H, H-5), 6.88 (d, J ¼ 8.7, 2H), 4.86 (d, J ¼ 3.5, 2H). 13C NMR
[10] G. Caravatti, New 7H-Pyrrolo[2,3-d]Pyrimidines Inhibiting ErbB and VEGF
Receptor Tyrosine Kinases (2004) Abstracts, 36th Central Regional Meeting of
the American Chemical Society, Indianapolis, IN, United States.
(100 MHz, DMSO-d6)
d
: 160.4 (d, J ¼ 245.5), 158.0, 149.8, 147.4,
142.4, 137.6, 130.0 (d, J ¼ 7.0), 129.6, 126.8 (2C), 124.7, 123.5 (d,
J ¼ 13.1), 121.2, 116.0 (2C), 115.5 (d, J ¼ 21.1), 103.0, 96.3, 39.5. 19F
[11] M. Croyle, N. Akeno, J.A. Knauf, D. Fabbro, X. Chen, J.E. Baumgartner, H.A. Lane,
J.A. Fagin, RET/PTC-induced cell growth is mediated in part by epidermal
growth factor receptor (EGFR) activation: evidence for molecular and Func-
tional Interactions between RET and EGFR, Cancer Res. 68 (2008) 4183e4191.
[12] P. Traxler, P.R. Allegrini, R. Brandt, J. Brueggen, R. Cozens, D. Fabbro, K. Grosios,
H.A. Lane, P. McSheehy, J. Mestan, T. Meyer, C. Tang, M. Wartmann, J. Wood,
G. Caravatti, AEE788: a dual family epidermal growth factor Receptor/ErbB2
and vascular endothelial growth factor receptor tyrosine kinase inhibitor with
antitumor and antiangiogenic activity, Cancer Res. 64 (2004) 4931e4941.
[13] Y.W. Park, M.N. Younes, S.A. Jasser, O.G. Yigitbasi, G. Zhou, C.D. Bucana,
B.N. Bekele, J.N. Myers, AEE788, a dual tyrosine kinase receptor inhibitor,
induces endothelial cell apoptosis in human cutaneous squamous cell carci-
noma xenografts in nude mice, Clin. Cancer Res. 11 (2005) 1963e1973.
[14] T. Schneider-Merck, M. Trepel, Lapatinib, recent results, Cancer Res. 184
(2010) 45e59.
NMR (376 MHz, DMSO-d6, C6F6)
d: ꢂ117.1 (s). HRMS (ESI): 335.1306
(calcd C19H16FN4O, 335.1303, M þ Hþ). IR (neat, cmꢂ1): 3128, 1645,
1492, 758.
6.11.30. (R)-4-(4-((1-Phenylpropyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-6-yl)phenol hydrobromide (59)
The compound was synthesised as described in Section 6.6.6
starting from (R)-6-(4-methoxyphenyl)-N-(1-phenylpropyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine (36) (98 mg, 0.27 mmol) and BBr3
(0.26 mL, 2.77 mmol). This gave 46 mg, (0.11 mmol, 40%) of a col-
ourless solid, mp > 300 ꢀC, ½a D20
ꢃ
¼ ꢂ197.3 (c 0.23, DMSO), purity:
96% (by HPLC), 1H NMR (400 MHz, DMSO-d6)
d
: 12.56 (s, 1H, NH, H-
[15] P. Traxler, G. Bold, E. Buchdunger, G. Caravatti, P. Furet, P. Manley, T. O’Reilly,
J. Wood, J. Zimmermann, Tyrosine kinase inhibitors: from rational design to
clinical trials, Med. Res. Rev. 21 (2001) 499e512.
[16] R. Hoekstra, H. Dumez, A.L.M. Eskens Ferry, G.A. van der, S.T. Planting Andre,
H.G. de, K.C. Sizer, C. Ravera, S. Vaidyanathan, C. Bucana, I.J. Fidler, A.T. van
7), 9.87 (br s, 1H, OH), 8.90 (br s, 1H, NH) 8.19 (s, 1H, H-2), 7.64e7.62
(m, 2H), 7.50e7.48 (m, 2H), 7.37e7.33 (m, 2H), 7.27e7.23 (m, 1H),
7.15 (s, 1H, H-5), 6.88e6.85 (m, 2H), 5.24 (m, 1H), 1.88 (m, 2H), 0.94
Oosterom, J. Verweij,
epidermal growth factor receptor tyrosine kinase inhibitor, in patients with
advanced solid malignancies, Clin. Cancer Res. 11 (2005) 6908e6915.
. Phase I and pharmacologic study of PKI166, an
(t, J ¼ 7.3, 3H). 13C NMR (100 MHz, DMSO-d6)
d: 158.1, 155.3, 151.4
(2C), 144.3, 133.2, 128.5 (2C), 127.6, 127.0 (2C), 126.4 (2C), 124.7,

6014
S.J. Kaspersen et al. / European Journal of Medicinal Chemistry 46 (2011) 6002e6014
[17] T. Takada, H.M. Weiss, O. Kretz, G. Gross, Y. Sugiyama, Hepatic transportofPKI166,
an epidermal growth factor receptor kinase inhibitor of the pyrrolo-pyrimidine
class, anditsmainmetabolite, ACU154, DrugMetab. Dispos32(2004)1272e1278.
[18] K. Mensa-Wilmot, Tyrosine Kinase Inhibitors as Anti-kinetolastid and Anti-
Apicomplexan Protozoal Agents WO 2008066755, 2007.
[35] B.K. Park, N.R. Kitteringham, P.M. O’Neill, Metabolism of fluorine-containing
drugs, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 443e470.
[36] H. Böhm, D. Banner, S. Bendels, M. Kansy, B. Kuhn, K. Muller, U. Obst-
Sander, M. Stahl, Fluorine in medicinal chemistry, ChemBioChem 5 (2004)
637e643.
[19] M. Parsons, J.A. Ledbetter, G.L. Schieven, Inhibitors of Tyrosine Phosphoryla-
tion in Protozoa as Drugs EP 507256, 1992.
[20] F.J. Gamo, L.M. Sanz, J. Vidal, C. de Cozar, E. Alvarez, J.L. Lavandera,
D.E. Vanderwall, D.V.S. Green, V. Kumar, S. Hasan, J.R. Brown, C.E. Peishoff,
L.R. Cardon, J.F. Garcia-Bustos, Thousands of chemical starting points for
antimalarial lead identification, Nature 465 (2010) 305e310.
[21] N. Tapinos, M. Ohnishi, A. Rambukkana, ErbB2 receptor tyrosine kinase
signaling mediates early demyelination induced by leprosy bacilli, Nat. Med.
12 (2006) 961e966.
[22] J.M. Franklin Robin, C. Zhao, Tyrosine kinases: maiming myelin in leprosy, Nat.
Med. 12 (2006) 889e890.
[37] Y.J. Wu, C.D. Davis, S. Dworetzky, W.C. Fitzpatrick, D. Harden, H. He, R.J. Knox,
A.E. Newton, T. Philip, C. Polson, D.V. Sivarao, L.Q. Sun, S. Tertyshnikova,
D. Weaver, S. Yeola, M. Zoeckler, M.W. Sinz, Fluorine substitution can block
CYP3A4 metabolism-dependent inhibition: identification of (S)-N-[1-(4-
Fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluorophenyl)acrylamide as an
orally bioavailable KCNQ2 opener devoid of CYP3A4 metabolism-dependent
inhibition, J. Med. Chem. 46 (2003) 3778e3781.
[38] C.H. Yun, T.J. Boggon, Y. Li, M.S. Woo, H. Greulich, M. Meyerson, M.J. Eck,
Structures of lung cancer-derived EGFR mutants and inhibitor complexes:
mechanism of activation and insights into differential inhibitor sensitivity,
Cancer Cell 11 (2007) 217e227.
[23] D.J. Collins,
A
synthesis, and certain properties, of ethyl 2-amino-5-
[39] C. Soerum, N. Simic, E. Sundby, B.H. Hoff, 1H, 13C and 19F NMR data of N-
substituted 6-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amines in
DMSO-d6, Magn. Reson. Chem. 48 (2010) 244e248.
nitronicotinate, J. Chem. Soc. (1963) 1337e1339.
[24] T. Kobayashi, T. Inoue, Z. Kita, H. Yoshiya, S. Nishino, K. Oizumi, T. Kimura,
Novel 2-amino-1,4-dihydropyridine calcium antagonists. I. Synthesis and
antihypertensive effects of 2-amino-1,4-dihydropyridine derivatives having
nitroxyalkoxycarbonyl groups at 3- and/or 5-position, Chem. Pharm. Bull. 43
(1995) 788e796.
[40] A.J. Trott, O. Olson, AutoDock Vina: improving the speed and accuracy of
docking with
a new scoring function, efficient optimization and multi-
threading, J. Comput. Chem. 31 (2010) 455e461.
[41] M.F. Sanner, Python: a programming language for software integration and
development, J. Mol. Graphics Modell. 17 (1999) 57e61.
[42] B.A. Pollok, B.D. Hamman, S.M. Rodems, L.R. Makings, Optical Probes and
Assays WO 2000066766, 2000.
[43] C. Soerum, N. Simic, E. Sundby, B.H. Hoff, Magn. Reson. Chem. 48 (2010)
244e248.
[44] M.T. Cocco, C. Congiu, A. Maccioni, A. Plumitallo, M.L. Schivo, G. Palmieri,
Synthesis and biological activity of some pyrrole derivatives. I, Farmaco, Ed.
Sci. 43 (1988) 103e112.
[45] R.M. Grotzfeld, H.K. Patel, S.A. Mehta, Z.V. Milanov, A.G. Lai, D.J. Lockhart,
Pyrrolopyrimidine Derivatives and Analogs and Their Use in the Treatment
and Prevention of Diseases US 2005153989, 2005.
[25] E. Toja, A. DePaoli, G. Tuan, J. Kettenring, Synthesis of 2-amino-3-(ethox-
ycarbonyl)pyrroles, Synthesis (1987) 272e274.
[26] V. Pittala, G. Romeo, L. Salerno, M.A. Siracusa, M. Modica, L. Materia,
I. Mereghetti, A. Cagnotto, T. Mennini, G. Marucci, P. Angeli, F. Russo, 3-
Arylpiperazinylethyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione deriva-
tives as novel, high-affinity and selective alpha 1-adrenoceptor ligands, Bio-
org. Med. Chem. Lett. 16 (2006) 150e153.
[27] X. Cai, C. Qian, S. Gould, Fused Bicyclic Pyrimidines as PTK Inhibitors Con-
taining a Zinc Binding Moiety WO 2008033745, 2007.
[28] P. Traxler, P. Furet, W.K.D. Brill, Pyrrolopyrimidinderivate mit anti-
proliferativer wirkung EP 682027, 1995.
[29] B.P. Fors, P. Krattiger, E. Strieter, S.L. Buchwald, Water-mediated catalyst
preactivation: an efficient protocol for CeN Cross-coupling reactions, Org.
Lett. 10 (2008) 3505e3508.
[30] K.W. Anderson, T. Ikawa, R.E. Tundel, S.L. Buchwald, The selective reaction of
aryl halides with KOH: synthesis of phenols, aromatic ethers, and benzofu-
rans, J. Am. Chem. Soc. 128 (2006) 10694e10695.
[46] G. Bold, H.G. Capraro, G. Caravatti, P. Traxler, Preparation of 7H-pyrrolo[2,3-d]
pyrimidines as Protein Tyrosine Kinase Inhibitors US 2004248911, 2004.
[47] G. Bold, P.W. Manley, A. Martin-Kohler, U. Sequin, Oxazolo-and Fuoropyr-
imidines Asn Their Use in Medicaments Against Tumors WO 2002092603,
2002.
[48] B. Miriyala, S. Bhattacharyya, J.S. Williamson, Chemoselective reductive
alkylation of ammonia with carbonyl compounds: synthesis of primary and
symmetrical secondary amines, Tetrahedron 60 (2004) 1463e1471.
[49] G. Jones, Synthesis of some dimethyl- and ethylisoquinolines, J. Chem. Soc.
(1960) 1918e1923.
[50] G. Hou, F. Gosselin, W. Li, J.C. McWilliams, Y. Sun, M. Weisel, P.D. O’Shea, C.
y. Chen, I.W. Davies, X. Zhang, Enantioselective hydrogenation of NeH imines,
J. Am. Chem. Soc. 131 (2009) 9882e9883.
[51] X. Cai, C. Qian, S. Gould, H. Zhai, Multi-functional Small Molecules as Anti-
proliferative Agents WO 2008033747, 2007.
[31] D.S. Surry, S.L. Buchwald, Dialkylbiaryl phosphines in Pd-catalyzed amination:
a user’s guide, Chem. Sci. 2 (2011) 27e50.
[32] E. Paliakov, L. Strekowski, Boron tribromide mediated debenzylation of ben-
zylamino and benzyloxy groups, Tetrahedron Lett. 45 (2004) 4093e4095.
[33] G. Caravatti, J. Bruggen, E. Buchdunger, R. Cozens, P. Furet, N. Lydon,
T. O’Reilly, P. Traxler, Pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimi-
dine derivatives as selective inhibitors of the EGF receptor tyrosine kinase,
ACS Symposium Series 796 (2001) 231e244.
[34] R.A. Wolfe, R. Wu, G.H. Sato, Epidermal growth factor-induced down-regu-
lation of receptor does not occur in HeLa cells grown in defined medium, Proc.
Natl. Acad. Sci. U. S. A. 77 (1980) 2735e2739.
[52] D.J. Resta, K.C. Sizer, P. Traxler, Use of Organic Compounds US 20030139430,
2003.