Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4- amines as highly potent EGFR-TK inhibitors with Src-family activity

Article abstract of DOI:10.1016/j.ejps.2014.04.011

The epidermal growth factor receptor is an important target in molecular cancer therapy. Herein, the enzymatic inhibition potential of a series of chiral and non chiral pyrrolopyrimidine based derivatives have been investigated and optimised. Overall, seven new compounds were identified having enzymatic IC ₅₀ values comparable to or better than the commercial drug Erlotinib. High activity was also confirmed towards the epidermal growth factor receptor L858R and L861Q mutants. Based on calculated druglike properties, eight compounds were further evaluated towards a panel of 52 other kinases revealing interesting Src-family kinase and colony stimulating factor 1 receptor kinase inhibitory activity. Cell proliferation studies with the cell lines A431, C-33A, AU-565, K-562 and genetically engineered Ba/F3-EGFR^L858R cells also showed several molecules to be more active than Erlotinib, and thus confirming these pyrrolopyrimidines as attractive drug candidates or lead structures.

Full text of DOI:10.1016/j.ejps.2014.04.011

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European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx
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Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
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Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]
pyrimidine-4-amines as highly potent EGFR-TK inhibitors with
Src-family activity
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Svein Jacob Kaspersen a, Jin Han a,b, Kristin G. Nørsett c, Line Rydså a,1, Eli Kjøbli d, Steffen Bugge a,
8 Q1
Geir Bjørkøy c,d, Eirik Sundby b, Bård Helge Hoff a,
⇑
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a Norwegian University of Science and Technology, Department of Chemistry, Høgskoleringen 5, NO-7491 Trondheim, Norway
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b Sør-Trøndelag University College, E.C. Dahls gt. 2, NO-7004 Trondheim, Norway
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c Norwegian University of Science and Technology, Department of Cancer Research and Molecular Medicine, Prinsesse Kristinas gt. 1, NO-7006 Trondheim, Norway
d Sør-Trøndelag University College, Erling Skjalgssons gt. 1, NO-7004 Trondheim, Norway
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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The epidermal growth factor receptor is an important target in molecular cancer therapy. Herein, the
enzymatic inhibition potential of a series of chiral and non chiral pyrrolopyrimidine based derivatives
have been investigated and optimised. Overall, seven new compounds were identified having enzymatic
IC50 values comparable to or better than the commercial drug Erlotinib. High activity was also confirmed
towards the epidermal growth factor receptor L858R and L861Q mutants. Based on calculated druglike
properties, eight compounds were further evaluated towards a panel of 52 other kinases revealing inter-
esting Src-family kinase and colony stimulating factor 1 receptor kinase inhibitory activity. Cell prolifer-
ation studies with the cell lines A431, C-33A, AU-565, K-562 and genetically engineered Ba/F3-EGFRL858R
cells also showed several molecules to be more active than Erlotinib, and thus confirming these pyrrol-
opyrimidines as attractive drug candidates or lead structures.
Received 24 February 2014
Received in revised form 11 April 2014
Accepted 16 April 2014
Available online xxxx
23 Q2
Keywords:
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Pyrrolopyrimidine
Tyrosine kinase
EGFR-TK
Src-kinase
Cancer
Ó 2014 Published by Elsevier B.V.
CSF1R kinase
Erlotinib
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Druglike properties
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Chemical compounds studied in this article:
Benzylamine (PubChem CID: 7504)
1-Phenylethanamin (PubChem CID: 7408)
PKI-166 (PubChem CID: 6918403)
2-Amino-2-phenylethan-1-ol (PubChem
CID: 92466)
2-Methoxyphenylboronic acid (PubChem
CID: 2733958)
4-(Hydroxymethyl)phenylboronic acid
(PubChem CID: 2734706)
PKI-166 (PubChem CID: 6918403)
Erlotinib (PubChem CID: 176870)
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1. Introduction
⇑
Corresponding author. Tel.: +47 73593973; fax: +47 73594256.
E-mail addresses: Svein.jacob.kaspersen@chem.ntnu.no (S.J. Kaspersen), Jin.
han@chem.ntnu.no (J. Han), kristin.norsett@ntnu.no (K.G. Nørsett), line.rydsa@sin
tef.no (L. Rydså), eli.kjobli@hist.no (E. Kjøbli), steffen.bugge@chem.ntnu.no
(S. Bugge), geir.bjorkoy@hist.no (G. Bjørkøy), E.Sundby@hist.no (E. Sundby), bard.
helge.hoff@chem.ntnu.no (B.H. Hoff).
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The epidermal growth factor receptor (EGFR/HER1) and other
human epidermal growth factor receptors (HER’s) are often ampli-
fied, overexpressed or mutated in solid tumours (Hynes and
MacDonald, 2009; Soonthornthum et al., 2011; Sun et al., 2012;
Yarden and Pines, 2012). Auto phosphorylation of tyrosine residues
on these HER’s provide docking sites for mediators of downstream
Q3 66
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Present address: SINTEF Energy Research, Postbox 4761 Sluppen, NO-7465
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Trondheim, Norway.
http://dx.doi.org/10.1016/j.ejps.2014.04.011
0928-0987/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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signalling eventually leading to cellular proliferation, migration,
differentiation and survival. EGFR is mainly localised at the plasma
membrane, but is also present in cellular organelles, the nucleus
and mitochondria (Irwin et al., 2011).
Previously known pyrrolopyrimidine based EGFR-TK inhibi-
tors include PKI-166 (Traxler, 2003) and AEE-788 (Traxler
et al., 2004; Park et al., 2005). Our laboratory has recently devel-
oped highly active thienopyrimidine based EGFR-TK inhibitors
(Structure I, Fig. 1) (Bugge et al., 2014). However, based on our
earlier investigation on pyrrolopyrimidines (Kaspersen et al.,
2011), we envisioned that even more potent inhibitors with bet-
ter druglike properties could be obtained by applying design
principals revealed in the thienopyrimidine study (Bugge et al.,
2014).
Establishment of a new synthetic route, allowed for a more
elaborate investigation of substituent effects in the 6-aryl group
of the 7H-pyrrolo[2,3-d]pyrimidine scaffold. Thus, by a SAR study
and a step-wise combination of favourable substitution patterns,
several highly potent pyrrolopyrimidine based EGFR-TK inhibitors
were identified. These derivatives also possess interesting inhibi-
tory effects on Src-family of kinases, HER2, HER4 and colony stim-
ulating factor 1 receptor kinase (CSF1R). Moreover, the new
compounds also possess promising activity as compared to Erloti-
nib in genetically engineered Ba/F3-EGFRL858R cells and in human
cancer cell lines.
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The HER members can be inhibited with small molecules or
monoclonal antibodies (Nedergaard et al., 2012). Due to the heter-
ogeneity of various tumours, the usefulness of these strategies
depends on the oncogenic profile of the cancer. As single agents
epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhib-
itors as Erlotinib and Gefitinib have been found most effective in
non-small cell lung cancer leading to increased progression free
survival in specific patient groups (Koehler and Schuler, 2013;
Sgambato et al., 2012). Erlotinib and Gefitinib, which both have
activity towards wild-type EGFR and the EGFRL858R mutant, are
now approved for 1st line therapy in the clinic (Sgambato et al.,
2012). Interestingly, it has been shown that Erlotinib bind both
to the active and inactive form of EGFR (Park et al., 2012). When
targeting HER receptor kinases in a therapeutic setting, a high
overall HER inhibitory activity is likely to improve efficiency, as
there is a considerable activation by heterodimerisation and cross-
talk between the HER members (Tebbutt et al., 2013). Although
both HER3 and HER4 might be of importance, the HER2 receptor
has been most intensively studied. Inhibitors like Lapatinib have
been found efficient in HER2 positive breast cancer.
While these reversible ATP competitive inhibitors have some
adverse effects (Sugiura et al., 2013; Yoshida et al., 2013), the
major drawback is that resistance is often emerging, either by a
second mutation (EGFRT790M) or by up-regulating of alternative
signalling pathways (Chong and Jaenne, 2013). The EGFRT790M
mutant can be targeted using irreversible inhibitors. Although
these agents hold much promise (Carmi et al., 2012), they have a
challenging toxicity profile not seen for the reversible inhibitors
(Barf and Kaptein, 2012). Thus, the clinical benefit therefore still
seems somewhat limited (Sequist et al., 2010; Landi and
Cappuzzo, 2013).
If however, the cancer therapy is based on hitting two or more
targets, there is a lower risk of resistance development. The estab-
lished reversible type inhibitors are therefore investigated in com-
bination with other kinase inhibitors, chemical agents, monoclonal
antibodies and radiotherapy (Tebbutt et al., 2013; Wu et al., 2013;
Lainey et al., 2013). Alternatively, inhibitors can be developed to
possess activity towards more than one kinase. Among others,
the Src-family of kinases has been identified as viable targets (Lu
et al., 2012; Kim et al., 2009). Src’s are known to interact both with
EGFR and HER2, but also other kinases and signal transduction sys-
tems and mitochondrial functions (Irwin et al., 2011; Kim et al.,
2009; Hebert-Chatelain, 2013). Examples of Src inhibitors include
Dasatinib, Bosutinib and Saracatinib, Fig. 1.
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2. Material and methods
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2.1. Chemicals
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Compounds (R)-1-phenylethanamine ((R)-2), (R)-1-(2-chloro-
phenyl)ethan-1-amine ((R)-4), (R)-1-(2-methoxyphenyl)ethan-1-
amine ((R)-6), (R)-2-(1-aminoethyl)phenol ((R)-7), 4-nitro-1-phen-
ylethan-1-amine ((R)-13), benzylamine (17), (R)-1-(phenyl)-1-
propanamine ((R)-21), (R)-2-methoxy-1-phenylethan-1-amine
((R)-25), (S)-2-amino-2-phenylethan-1-ol ((S)-24), 1-4-(tert-
butyl)phenyl)ethanone, sodium tert-butoxide, 2,2-dimethyl-1-
phenylpropan-1-one, sodium borohydride, sodium cyanoborohy-
dride, iodomethane (2 M in tert-butyl methyl ether), palladium(II)
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acetate,
2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl
(XPhos), chloro(2-dicyclohexylphosphino-20,40,60-triisopropyl-1,10-
biphenyl)[2-(20-amino-1,10-biphenyl)]palladium(II) (XPhos Pd
G2), boron tribromide were from Sigma–Aldrich. (4-Formyl-2-
methoxyphenyl)boronic acid was from Combi-Blocks, while the
other arylboronic acids were from Sigma–Aldrich. (R)-2-Methyl-
1-phenylpropan-1-amine ((R)-22) was from ABCR, (S)-2-amino-2-
(4-fluorophenyl)acetic acid was from Fluorochem, and compound
50 was from Nanjing Pharmatechs. Compound 1a–1d were pre-
pared as described previously (Kaspersen et al., 2011). Silica-gel
column chromatography was performed using silica gel 60 Å from
Fluka, pore size 40–63
lm. Celite 545 from Fluka was also used.
O
N
N
O
O
N
N
O
O
N
OH
S
O
O
N
N
N
N
Cl
O
N
N
R
H
H
H
Erlotinib
Gefitinib
I
F
N
N
O
N
O
N
N
N
N
N
N
O
Cl
O
NC
O
O
S
O
N
O
O
O
N
N
H
H
H
H
Lapatinib
O
F
Cl
Cl
O
Saracatinib
Bosutinib
Cl
Fig. 1. Examples of reversible EGFR-TK, HER2 and dual ABL/Src kinase inhibitors.
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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2.2. Analyses
at 100 nM was used to set starting point of the IC50 titration curve,
in which three levels were used 100, 1000 or 10,000 nM. The IC50
values reported are based on the average of at least 2 titration
curves (minimum 20 data points), and were calculated from activ-
ity data with a four parameter logistic model using SigmaPlot
(Windows Version 12.0 from Systat Software, Inc.). Unless stated
otherwise the ATP concentration used was equal to Km. The
average standard deviation for single point measurements were
<4%. The inhibitory potency towards EGFR mutants was
determined in the same way.
1H and 13C NMR spectra were recorded with Bruker Avance 400
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spectrometer operating at 400 MHz and 100 MHz, respectively. 19
F
NMR was performed on
a Bruker Avance 500 operating at
564 MHz. For 1H and 13C NMR chemical shifts are in ppm rel. to
TMS or DMSO-d6, 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 Quatpump,
G1313A ALS autosampler and
a G1315D Agilent detector
(230 nm) was used to determine the purity of the synthesised
compounds. All compounds evaluated for EGFR inhibitory potency
had a purity of P95%. Conditions: Poroshell C18 (100 ꢀ 4.6 mm)
column, flow rate 0.8 ml/min, elution starting with water/acetono-
nitrile (90/10), 5 min isocratic elution, then linear gradient elution
for 35 min ending at acetononitrile/water (100/0). 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
(70 eV) using a Finnigan MAT 95 XL. Accurate mass determination
in positive and negative mode was performed on a ‘‘Synapt G2-S’’
Q-TOF instrument from Waters. Samples were ionized by
atmospheric-pressure chemical ionization (ACPI) and analysed
using an atmospheric solids analysis probe (ASAP). No chromatog-
raphy separation was used before the mass analysis. FTIR spectra
were recorded on a Thermo Nicolet Avatar 330 infrared spectro-
photometer. All melting points are uncorrected and measured by
a Stuart automatic melting point SMP40 apparatus.
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2.4.2. Ba/F3 cell reporter cell analysis
Transfected Ba/F3 cells containing expression vectors for the
L858R and T790M EGFR mutants were a kind gift from Dr. Nikolas
von Bubnoff at the Technical University of Munich, Munich,
Germany (Kancha et al., 2009). The cells were cultured in RPMI
1640 (Gibco, Invitrogen) supplemented with 10% FCS (Gibco, Invit-
rogen), 1% L-glutamine (Gibco, Invitrogen) and 0.1% Gentamycin
(Sanofi Aventis). Erlotinib was purchased from LC Laboratories
(Woburn, MA). All inhibitors were reconstituted in DMSO, and
appropriate stock solutions were prepared using cell culture med-
ium. The final percentage concentrations of DMSO were <0.2%. Pro-
liferation analysis: Ba/F3 cells (1 ꢀ 104 per well) were plated into
96-well plates. Inhibitors were added in different concentrations
as indicated. Cell growth was measured at 48 h using TACSÒ XTT
Cell Proliferation Assay (Trevigen) according to the manufacturer’s
instructions.
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2.4.3. Human cancer cell lines A-431 and K-562
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2.3. General synthetic procedures
Cell proliferation assays were performed by ReactionBiology
Corp. A-431 human epidermal carcinoma and K-562 chronic mye-
logenous leukaemia cell lines were obtained from American Type
Culture Collection (Manassas, VA). Staurosporine was obtained
from Sigma–Aldrich (Saint Louis, MI). Cell Titer-GloÒ Luminescent
cell viability assay reagent was obtained from Promega (Madison,
WI). A-431 and K-562 cells were grown in Dulbecco’s Modified
Eagle Medium (DMEM). Both cell culture mediums were supple-
mented with 10% heat-inactivated fetal bovine serum (FBS),
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2.3.1. Thermal amination of 4-chloro-pyrrolo[2,3-d]pyrimidines
Compound 1a–d or 54 (1 mmol) was mixed with the selected
amine, 2–25, (3 mmol) and n-butanol (5 ml) and agitated at
145 °C for 14–24 h. Then the mixture was cooled to 22 °C, diluted
with water (15 ml) and ethyl acetate (40 ml). After phase separa-
tion, the water phase was back extracted with more ethyl acetate
(2 ꢀ 10 ml). The combined organic phases were washed with brine
(10 ml), dried over anhydrous Na2SO4, filtered and concentrated in
vacuo, before the crude mixture was purified as specified for each
individual compound.
100 lg/ml penicillin, and 100 lg/ml of Streptomycin. Cultures
were maintained at 37 °C in a humidified atmosphere of 5% CO2
and 95% air. Compound (R)-26l, (R)-26n, 41n, (S)-48b, (S)-48e,
(S)-48l, (S)-48n, (S)-60n, Erlotinib and Staurosporine (positive con-
trol) were all dissolved in DMSO in 10 mM stock. Culture medium
(10 ll) was added to each well of 384 well cell culture plates. The
compounds were diluted in a source plate in DMSO at 3-fold serial
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2.3.2. Suzuki coupling on 6-iodopyrrolo[2,3-d]pyrimidines
The following is representative: Compound (R)-56 (300 mg,
0.825 mmol) was mixed with (2-methoxyphenyl)boronic acid
(150 mg, 0.989 mmol), fine powdered K2CO3 (398 mg, 2.88 mmol),
XPhos (20 mg, 0.040 mmol), 2nd generation XPhos precatalyst
(34 mg, 0.04 mmol), 1,4-dioxane (2 ml) and water (2 ml). The reac-
tion was then stirred at 100 °C for 1.5–18 h under nitrogen atmo-
sphere. The solvent was removed and the product was diluted
with water (20 ml) and extracted with ethyl acetate (30 ml). The
water phase was extracted with more ethyl acetate (3 ꢀ 30 ml).
The combined organic phases were washed with brine (20 ml),
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The crude mixture was purified as specified for each individual
compound. The synthesis and spectroscopic data of all new
compounds are given in the Supporting information.
dilutions starting at 10 mM, total 10 doses. The compounds
(0.25
ll) were delivered from source plate to each well of the cell
culture plates by Echo 550. Then, 15
l
l of culture medium contain-
ing 5000 cells of A-431 or K-562 were added to the wells of the cell
culture plates. The cells were incubated with the compounds at
37 °C, 5% CO2 for 72 h. 25 ll of Cell Titer Glo reagent (25 ll) was
added to each well according to the instruction of the kit. The con-
tents were mixed on an orbital shaker for 2 min and incubated at
room temperature for 10 min to stabilize luminescent signal.
Luminescence was recorded by Envision 2104 Multilabel Reader
(PerkinElmer, Santa Clara, CA). The maximum luminescence for
each cell line in the absence of test compound, but in the presence
of 0.4% DMSO, was similarly recorded after incubation for 72 h. The
number of viable cells in the culture was determined based on
quantitation of the ATP present in each culture well. The percent-
age growth after 72 h (%-growth) was calculated as follows:
100% ꢀ (luminescence t = 72 h/luminescence untreated, t = 72 h).
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2.4. Biochemical profiling of kinase inhibitors
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2.4.1. In vitro EGFR (ErbB1) and EGFR mutant inhibitory potency
The compounds were supplied in a 10 mM DMSO solution, and
enzymatic EGFR (ErbB1) inhibition potency was determined by
Invitrogen (LifeTechnology) using their Z’-LYTEÒ assay technology
(Pollok et al., 2000). All compounds were first tested for their
inhibitory activity at 100 nM in duplicates. The potency observed
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2.4.4. Human cancer cell line testing AU-565 and C-33A
Cell proliferation assays were performed by Netherlands Trans-
lational Research Center B.V (NTRC). All cell lines were licensed
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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from the American Type Culture Collection (ATCC) Manassas,
Virginia (US). Master and working cell banks (MCB and WCB) were
prepared by subculturing in ATCC-recommended media and freez-
ing according to ATCC recommended protocols (www.atcc.org).
Cell line stocks for the assays were prepared from the WCB. The
MCB, WCBs and assay stocks were prepared within respectively
3, 6 and 9 passages of the ATCC vial. Proliferation analysis: Com-
pounds were weighed on a calibrated balance and dissolved in
100% DMSO to a concentration of 10 mM. The samples were stored
at room temperature. The compound stock was diluted in 3.16-fold
steps in 100% DMSO to obtain a 10-point dilution series. This was
further diluted 31.6 times in 20 mM sterile Hepes buffer pH 7.4. A
volume of 5 ml was transferred to the cells to generate a test con-
centration range from 3.16 ꢀ 10ꢁ5 M to 3.16 ꢀ 10ꢁ9 M in duplicate.
The final DMSO concentration during incubation was 0.4% in all
wells. An assay stock was thawed and diluted in its ATCC recom-
mended medium and dispensed in a 384-well plate, depending
on the cell line used, at a concentration of 400–1600 cells per well
(ChemBioDraw Ultra 13.0, CambridgeSoft, PerkinElmer) and were
prepared using LigPrep2.2 (LigPrep, v2.2; Schrödinger, LLC). For
the computational investigation of the receptor-inhibitor struc-
tures, the energy minimized structures of 2J6M and ligands were
subsequently docked using induced-fit docking (IFD) of Schröding-
er (Sherman et al., 2006a,b; Fit Docking Protocol, 2013). The result-
ing docked poses were analyzed using Glide pose viewer tool. All
the graphical pictures were made using Maestro.
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2.5.2. Calculated druglike properties
The relative druglike properties of compounds were calculated
from IC50 values (pIC50). Note that the obtained values are assay
dependant and therefore less valid when comparing studies using
other assay formats. Equations used for LE, BEI, and SEI calculations
can be found in Meanwell (2011), while LELP = logP/LE was from
Keserue and Makara (2009). The number of heavy atoms used in
calculation of LE includes all non-hydrogen atoms, and for BEI cal-
culation the molecular weight used is in kDa. The values for polar
surface area used in calculation of SEI were estimated as described
by Ertl et al. (2000). (Online resource: http://www.daylight.com/
meetings/emug00/Ertl/tpsa.html). The calculated logP (clogP)
was determined with OSIRIS Property Explorer: http://www.
organic-chemistry.org/prog/peo/.
in 45 ll medium. For each used cell line the optimal cell density
was used. The margins of the plate were filled with phosphate-buf-
fered saline. Plated cells were incubated in a humidified atmo-
sphere of 5% CO2 at 37 °C. After 24 h, 5
was added and plates were further incubated for another 72 h.
After 72 h, 25 l of ATPlite 1Step™ (PerkinElmer) solution was
ll of compound dilution
l
added to each well, and subsequently shaken for 2 min. After
10 min of incubation in the dark, the luminescence was recorded
on an Envision multimode reader (PerkinElmer). The maximum
luminescence for each cell line in the absence of test compound,
but in the presence of 0.4% DMSO, was similarly recorded after
incubation for 72 h (luminescence untreated, t = 72 h). The percent-
age growth after 72 h (%-growth) was calculated as follows:
100% ꢀ (luminescence t = 72 h/luminescence untreated, t = 72 h).
IC50 values were calculated from % growth and compound concen-
tration with a four parameter logistic model using SigmaPlot (Win-
dows Version 12.0 from Systat Software, Inc.).
371
372
373
374
2.5.3. Gini calculations
Quantification of compounds kinase selectivity by the use of
Gini plots and Gini coefficients followed the procedure previously
reported (Graczyk, 2007).
375
376
3. Result and discussion
3.1. Design of the inhibitors
377
378
379
380
381
382
383
384
385
386
387
388
389
390
Pyrrolopyrimidines have previously been investigated as EGFR-
TK inhibitors by us and others (Traxler et al., 2004; Kaspersen et al.,
2011; Caravatti, 2004; Grotzfeld et al., 2005). However, in the case
of 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4-amines,
detailed knowledge of the effect of substitution in the 6-aryl and
the 4-amino group is limited. Moreover, our previous study on thi-
enopyrimidines as EGFR-TK inhibitors (Bugge et al., 2014) showed
both that the activity could be increased by proper substitution of
these groups, but also that the pyrrolopyrimidines had an intrinsic
higher potency than the corresponding thienopyrimidines. Thus, it
was apparent that the full potential of the pyrrolopyrimidine scaf-
fold was not yet realised.
Controls: t = 0 h signal: On a parallel plate, 45
pensed and incubated in a humidified atmosphere of 5% CO2 at
37 °C. After 24 h 5 l DMSO-containing Hepes buffer and 25 ll
ll cells were dis-
l
ATPlite 1Step™ solution were mixed, and luminescence measured
after 10 min incubation (=luminescence, t = 0 h). Positive control:
The IC50 of the reference compound doxorubicin was measured
on a separate plate. The IC50 was trended. If the IC50 is out of spec-
ification (0.32–3.16 times deviating from historic average) the
assay is invalidated. Cell growth control: The cellular doubling
times of all cell lines were calculated from the t = 0 h and t = 72 h
growth signals of the untreated cells. If the doubling time is out
of specification (0.5–2.0 times deviating from historic average)
the assay is invalidated.
As our aim was to use structure–activity relationships to iden-
tify highly potent inhibitors, we investigated effects of variations
333
2.5. Molecular modelling and calculations
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
2.5.1. Molecular modelling
H
N
R
R
4
3
The X-ray crystal structures of the protein 2J6M (Wild-type
EGFR) and protein 2IJU (EGFRT790M) were prepared using the pro-
tein preparation wizard, which is part of the Maestro software
package (Maestro, v8.5; Schrödinger, LLC, New York, NY, USA).
Bond orders and formal charges were added for het-groups, and
hydrogens were added to all atoms in the system. Water molecules
beyond 5 Å from het-groups were removed. To alleviate steric
clashes that may exist in the original PDB structures, an all-atom
constrained minimization was carried out with the Impact
Refinement module (Impref) (Impact, v5.0; Schrödinger, LLC) using
the OPLS-2005 force field. The minimization was terminated when
the energy converged or the RMSD reached a maximum cutoff
of 0.30 Å. The resulting protein structures were used in the
following docking study. Ligands were drawn using ChemBioDraw
N
N
R
N
H
R
1
Fragment B
R
2
Fragment A
Fig. 2. The study of pyrrolopyrimidine based EGFR-TK inhibitors was performed by
structural variations in the 4-amino-(Fragment A) and the 6-aryl (Fragment B)
groups.
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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5
391
392
393
394
395
396
397
398
399
400
401
402
403
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
of the pyrrolopyrimidine in both the 4-benzylamine part (Frag-
ment A) and the 6-aryl part (Fragment B), Fig. 2.
crystallisation from acetonitrile compound 53 could be isolated in
74% yield and 99% purity.
In Fragment A, the R1-substituent was modified in terms of bulk
size, and also by introducing hydrogen bond accepting and donat-
ing groups. In the aromatic part of Fragment A, our previous study
indicated size limitations in this part of the binding pocket
(Kaspersen et al., 2011). Herein, this is explored in more detail. In
addition fluorinated derivatives were included due to their general
positive effect on metabolic stability (Böhm et al., 2004; Wu et al.,
2003). The investigation of structural variations in Fragment B on
EGFR-TK potency was inspired by our recent discoveries of active
thienopyrimidine based inhibitors (structure I, Fig. 1) (Bugge
et al., 2014).
Amination of 53 was attempted using (R)-1-phenylethanamine
(2), but gave
a mixture of the protected and unprotected
analogues. Instead, the pyrrolopyrimidine 54 obtained by depro-
tection, could be transformed to give the precursors 56–59 in
56–86% non-optimised yield. Suzuki coupling at C-6 using XPhos
and 2nd generation XPhos precatalyst (Kinzel et al., 2010) was then
performed using selected arylboronic acids giving most of the
targeted products. However, applying ortho- and meta-
hydroxyphenylboronic acids, the coupling only reached 50% con-
version in 40 h. Instead, these derivatives were made by deprotec-
tion of the methoxy analogues (R)-26e and (R)-26g. The para-
hydroxymethyl derivatives (R)-26n, 41n, (S)-48n and (S)-60n were
obtained by sodium borohydride reduction of the corresponding
aldehyde derivatives (R)-26m, 41m, (S)-48m and (S)-60m.
404
3.2. Synthesis
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
To enable efficient synthesis of a compound library, two differ-
ent, but complementary routes were used. Compounds with struc-
tural variation in the 4-position (Fragment A) were derived by a
method starting from 1-aryl-2-bromoethanones giving in three
steps the 6-aryl-4-chloropyrrolopyrimidines 1a–d (Kaspersen
et al., 2011, 2012). Amination chemistry in n-butanol gave the
4,6-disubstituted pyrrolopyrimidines 26–49, with varying R, R1
and R2 substituents as shown in Scheme 1. Derivatives 26–47 were
in most cases prepared as their pure (R)-enantiomer, except 29a,
33a, 39a, 40a, 47b which were racemates, (S)-26a included as a
model to test for effect of stereochemistry, and (S)-48b–d and
(S)-49b in which the stereochemical nomenclature changes as a
result of the higher priority of the CH2AO group as compared to
the aromatic ring.
Whereas the initial strategy might be highly useful in a produc-
tion setting, it was not suited for generating a larger library of
derivatives with variations in the 6-aryl group (Fragment B). There-
fore, an alternative route was established, using Suzuki coupling to
incorporate the targeted structural variations, see Scheme 2.
First, the commercially available compound 50 was converted to
52 by standard chlorination and protection. The following iodin-
ation has previously been performed by others (Engelhardt et al.,
2010), but preparative HPLC was needed to purify the iodo
compound 53. The initial 100–500 mg scale reaction resulted in
sub-optimal conversion and formation of an unidentified
by-product. However, the chemistry improved on scaling to gram
scale. Finally the reaction was performed in a 10 gram scale. By
448
3.3. In vitro enzymatic inhibition study
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
3.3.1. Structural variation in the benzylamine part (Fragment A)
The effect of structural variations in Fragment A on EGFR-TK
potency was initially studied with para-methoxyphenyl as the C-
6 substituent and 21 structural variations in Fragment A. The
EGFR-TK enzymatic potency was determined by single point mea-
surements in duplicates at 100 nM test concentration. This was fol-
lowed by IC50 10-point titration measurements also in duplicate.
Erlotinib in the same assay had an EGFR-TK inhibitory potency of
0.4 nM. Fig. 3 highlights the most important findings with respect
to the tolerated aromatic substitution pattern in Fragment A. High
EGFR-TK potency was achieved for compounds based on (R)-1-
phenylethanamine, (R)-26a, benzylamine, 41a, and (R)-l-phenyl-
propylamine, (R)-45a. In case of compound 26a the (R)-enantiomer
was approximately 16 times more active than (S)-26a. All
compounds containing fluoro substituents maintained a high
potency. The ortho and meta methyl derivatives (rac)-29a and
(rac)-33a also showed promising activity. Due to the low IC50
values seen for the ortho fluorinated analogue (R)-27a and the
ortho-methyl substituted (rac)-29a, we also prepared and tested
the ortho-chloro, (R)-28a, -hydroxyl, (R)-31a, and the -methoxy,
(R)-30a, analogues. Whereas the former two had potency of 8.1
and 6.6 nM respectively, the methoxy derivative was considerable
less active (IC50 = 105 nM).
H
N
N
N
NH2
R1
H
R
N
N
N
H
R
+
N
R1
n-BuOH
R2
Δ
Cl
R = OMe (1a), H (1b), F (1c), Br (1d)
2-25
26-49
R2
H
H
R = OMe (a)
N
N
N
N
N
N
R
1 = CH3
R
1 = H
R2
H (41a) H (45a)
R
1 = CH2CH3
R
N
H
N
R2
=
=
R2
=
R1
H (26a)
p-F (34a)
H
OH
R =
o-F (27a) p-Me (35a) o-F (42a)
o-Cl (28a) p-OMe(36a) m-F (43a)
o-Me (29a) p-NO2 (37a) p-F (44a)
Ph
Ph
R1
=
o-OMe (30a)
p-Br (38a)
Me (26b)
H (41b)
Et (45b)
i-Pr (46b)
t-Bu (47b)
CH2OH (48b)
CH2OMe (49b)
F (48c)
Br (48d)
o-OH (31a) p-CF3 (39a)
m-F (32a) p-t-Bu (40a)
m-Me (33a)
Scheme 1. Synthetic route for preparation of the pyrrolopyrimidine derivatives 26–49.
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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H
N
H
N
SO2Ph
SO2Ph
I
N
N
N
N
N
N
POCl3
1. LDA
2. I2
PhSO2Cl
Δ
N
N
N
N
NaO-t-Bu
-78 o
C
OH
Cl
Cl
Cl
50
51
52
53
H
N
N
NH2
R1
I
N
H
H
N
N
R1
N
N
R2
ArB(OH)2
NaOH
MeOH
2, 17, 24, 55
I
XPhos, XPhos 2nd
gen. precat.
n-BuOH
Δ
Cl
R2
56-59
2 = H (2, 56)
R2 = F (17, 57)
2 = H (24, 58)
54
R
R1 = CH3
R1 = H
R
R1 = CH2OH
R
1 = CH2OH R2 = F (55, 59)
Comp.
R1
R
R2
R3
R4
R3
R4
H
26e
26f
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
OMe
OH
H
H
a)
a)
N
N
N
H
R
26g
26h
26j
H
H
OMe
N
H
H
H
OH
H
CH2OH
H
H
R1
26k
26l
H
CH2OH
CH2OH
CHO
CH2OH
CHO
CH2OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
26m
26n
41m
41n
48e
48l
OMe
OMe
OMe
OMe
OMe
H
b)
b)
R2
H
CH2OH
CH2OH CH2OH
CH2OH CHO
48m
48n
48o
60m
60n
OMe
OMe
OMe
OMe
OMe
b)
b)
CH2OH CH2OH
CH2OH
F
CH2OH CHO
CH2OH CH2OH
F
Scheme 2. Pyrrolopyrimidines synthesized by Suzuki coupling on 56–59 giving (R)-26e–h, (R)-26j–n, 41m–n, (S)-48e, (S)-48l–n and (S)-60m–n. (a) BBr3 and (b) NaBH4 in
MeOH.
(R)-26a
IC50: 4.7 nM
(S)-26a
IC50: 77 nM
(R)-27a
IC50: 1.8 nM
(R)-28a
IC50: 8.1 nM
(rac)-29a
IC50: 7.5 nM
H
N
N
N
H
N
H
N
H
N
H
N
H
OMe
N
F
Cl
CH3
(R)-30a
IC50: 105 nM
(R)-31a
IC50: 6.6 nM
(R)-32a
IC50: 4.9 nM
(rac)-33a
IC50: 14 nM
(R)-34a
IC50: 2.8 nM
(R)-35a
IC50: 14 nM
41a
IC50: 3.7 nM
N
H
N
H
N
H
N
H
N
H
N
H
N
H
O
OH
F
CH3
F
CH3
42a
IC50: 5.8 nM
43a
IC50: 7.1 nM
44a
IC50: 5.0 nM
(R)-45a
IC50: 1.8 nM
N
H
N
H
N
H
N
H
F
F
F
Fig. 3. EGFR-TK enzymatic IC50 values (nM) for selected substitution patterns. See Supporting information for single point data and standard deviations.
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

PHASCI 2998
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7
H
N
N
N
OMe
N
H
=
R2
CH3
H
F
26a
(R)-
4a
(R)-3
Me
35a
36a
37a
38a
(R)-
(R)-
(R)-
(R)-
OMe
NO2
Br
CF3
t-Bu
R2
39a
40a
(rac)-
(rac)-
Fig. 4. Effect of para substitution on EGFR-TK IC50 values (nM). IC50 values are presented in log scale. See Supporting information for single point data and standard deviations.
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
Five more para substituted derivatives (Br, NO2, OMe, CF3, t-Bu)
were assayed giving IC50 values ranging from 40 to >10,000 nM, see
Fig. 4. Inspection of these data suggests that the activity is reduced
with increase in bulk size. However, as the trifluoromethyl substi-
tuted compound was more active than the para-methoxy analogue,
the electronic content of the aromatic ring might also be of
importance.
Based on the excellent IC50 value of the ethyl derivative (R)-45a,
and previous discoveries on thienopyrimidines (Bugge et al., 2014)
and furopyrimidines (Peng et al., 2013), it was also decided to
investigate the effect of the alkylamine side chain on EGFR-TK
activity. In this study the 6-position of the pyrrolopyrimidine
was substituted with a phenyl ring. As seen from Table 1, the
activity was not dramatically affected by the steric bulk of the
R1-substituent.
Fig. 5. Binding of compound (S)-48b to the EGFR-TK ATP binding site based on the
Moreover, insertion of the CH2OH group in compound (S)-48b
crystal structure 2J6M (Yun et al., 2007). The compound is indicated to engage in
improved the IC50 value down to 0.7 nM. The effect seen was sim-
hydrogen bonding via the hydroxymethyl group to Asp855 in the DFG motif, by N-1
to back-bond NH of Met793 and by the pyrrolo nitrogen to the carbonyl oxygen of
Met793.
ilar to that observed in thienopyrimidine analogues (Bugge et al.,
2014), and modelling using an induced fit procedure (Sherman
et al., 2006a,b), explains this by hydrogen bonding to the DFG motif
as seen in Fig. 5.
As the presence of the hydroxymethyl group also increases the
weight and the hydrophilicity of the compounds, the relative drug-
like properties of the candidate inhibitors were evaluated using
metrics as ligand efficiency (LE), binding efficiency (BEI), surface
efficiency index (SEI), and ligand-efficiency-dependent lipophilic-
ity (LELP) (Keserue and Makara, 2009; Hopkins et al., 2004;
499
500
501
502
503
504
505
506
507
508
509
510
511
512
Abad-Zapatero and Metz, 2005; Abad-Zapatero, 2007; Leeson and
Springthorpe, 2007). The LE values of the various compounds are
shown in Fig. 6, while BEI, SEI and LELP are given in Fig. 7. LE
and BEI are measures of binding efficiency per heavy atom and
molecular weight, respectively, and the higher the better. In terms
of LE and BEI the hydroxymethyl compound (S)-48b, the methyl
analogue (R)-26b and the benzylamine based 41b were concluded
most promising.
Too high polarity might lead to poor membrane permeability.
SEI describes how dependent activity is of polar interactions.
Erlotinib and (S)-48b were found to have comparable SEI values,
while the others are less polar. On the other hand, drug candidates
being too lipophilic will have poor solubility and higher risk of
failure due to toxic effects (Meanwell, 2011). LELP is used to assess
Table 1
EGFR-TK inhibitory potency given as % inhibition at 100 nM and IC50 values (nM)
upon variation of the R1 substituent. Unless otherwise noted IC50 values are based on
duplicate measurements.
Entry
Compound
R1
EGFR inhibition (%)
IC50 (nM)
1
2
3
4
5
6
7
8
(R)-26b
41b
CH3
H
CH2CH3
CH(CH3)2
C(CH3)3
CH2OH
95
97
91
84
88
98
100
100
3.2 0.6a,b
3.8 1.1
5.4 1.5
(R)-45b
(R)-46b
(rac)-47b
(S)-48b
(S)-49b
Erlotinib
15
10
1
3
0.7 0.3b
1.8 0.1
0.4 0.1d
CH2OMe
c
a
b
c
Previously known EGFR-TK inhibitor, Kaspersen et al. (2011).
Four IC50 titrations (40 data points).
For structure see Fig. 1.
Fig. 6. Ligand efficiency (LE) for Erlotinib and pyrrolopyrimidines having variable
R1-groups.
d
Six IC50 titrations (60 data points).
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
Whereas, the small fluorine group has only minor impact on
potency, the larger and more lipophilic bromine substituent
reduced the activity more profoundly. The EGFR-TK inhibitory
activity was less affected by fluorine substitution in para position
of Fragment A as indicated by data for compound (R)-34a and
(R)-34i.
Further it was attempted to improve antagonist activity by
merging potency inducing structural groups into one molecule.
Substructure selection in Fragment A was based on a compromise
between potency and calculated druglike properties. Therefore,
scaffolds with the more lipophilic (R)-1-phenylethanamine group,
(R)-26, and the more polar hydroxymethyl group, (S)-48, were
focused. Additionally, selected fluorinated derivatives, and two
benzylamine based compounds were included due to their highly
active EGFR-TK inhibitory properties. The compounds identified
alongside their precursor molecules are shown in Fig. 9.
Ten additional inhibitors with IC50 values below 1 nM were
identified. The most active compounds possess the 4-hydroxy-
methyl-2-methoxyphenyl group at C-6 (Fragment B). The effect
of substituents on potency was not additive indicating that
enthalpy/entropy compensating effects come into play. Generally,
somewhat higher IC50 values (lower activity) were noted for the
benzylamine fragment 41 as compared to (R)-26 and (S)-48. The
docked structure of (S)-48n is shown in Fig. 10, and the main inter-
actions between the ligand and the ATP binding site of EGFR-TK is
highlighted in Fig. 11. Compound (S)-48n is indicated to bind
similar to that of (S)-48b (Fig. 5), but an additional hydrogen bond
from the hydroxymethyl group to Leu718 was seen explaining the
increased potency. Additionally, both the secondary amine func-
tion and N-3 of the pyrimidine unit were indicated to bind to water
molecules.
The druglike properties of compounds having IC50 values below
0.7 nM were calculated as described in Section 3.3.1. Due to the
potential toxicity of the aldehydes (R)-26m, 41m, (S)-48m, and
(S)-60m, these were not included. The LE values shown in Fig. 12
revealed that all compounds except (S)-60n have a more favour-
able profile than Erlotinib.
The calculated BEI, SEI and LELP values are shown in Fig. 13. BEI
values follows the trends of LE, were only (S)-60n has lower bind-
ing efficiency than Erlotinib.
Erlotinib has similar surface efficiency index as (R)-26l and
(S)-48b, while the other pyrrolopyrimidines have lower values,
which indicates that the compounds might be too polar. On the
other hand these properties allow all compounds to have ligand-
efficiency-dependent lipophilicity (LELP) within the target area,
while Erlotinib is indicated to be too lipophilic.
Fig. 7. Binding efficiency (BEI), surface efficiency index (SEI), and ligand-efficiency-
dependent lipophilicity (LELP) for derivatives having variable R1-group.
513
514
515
516
lipohilicity. Values below 10 are preferable. In terms of LELP,
(S)-48b was analysed to have a slightly better profile than
(R)-26b, 41b and Erlotinib. As a result of these analysis further
development was based on the scaffolds (R)-26, 41 and (S)-48.
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
3.3.2. Fine tuning of activity towards EGFR-TK
The fine tuning of potency was started by studying variations in
the 6-aryl group (Fragment B), with (R)-1-phenylethanamine as
the 4-amino group. The aromatic substitution pattern in the 6-aryl
group was largely based on our recent finding in the corresponding
thienopyrimidines (Bugge et al., 2014), in which high EGFR-TK
potency was observed when having ether and hydroxyl substitu-
ents attached to the aromatic ring. The compounds analysed for
EGFR-TK potency, and the previously made reference compounds
(R)-26a and (R)-34a (Section 2.3.1), (R)-26c-d and (R)-34i
(Kaspersen et al., 2011) and (R)-26i (PKI-166) (Kaspersen et al.,
2011; Caravatti et al., 2001; Hoekstra et al., 2005) are shown in
Fig. 8.
The inhibitory potency of the compounds increased consider-
ably by having a hydroxymethyl group in para or meta position,
and compounds (R)-26k and (R)-26l both had IC50 values of
0.3 nM. Improvements in IC50 values were also seen for the ortho
derivatives (R)-26e, (R)-26j and the meta hydroxyl compound
(R)-26h. The ortho hydroxyl derivative (R)-26f was considerable
less active. The root cause of the drop in activity is somewhat
unclear, but might be due to internal hydrogen bonding with the
pyrrolo nitrogen likely to affect conformation of the 6-aryl group.
Data for the previously described compounds (R)-26c-d visual-
ise important structure–activity information for the 6-aryl group.
O
HO
HO
H
N
N
N
26e
26f
26j
IC50: 0.8 nM
H
N
R
IC50: 1.1 nM
IC50: 198 nM
N
H
N
N
F
OH
CH3
N
H
OH
O
26c
IC50: 6.6 nM
CH3
26g
IC50: 1.9 nM
26h
IC50: 0.8 nM
26k
IC50: 0.3 nM
Br
F
R = OMe, (R)-34a, IC50: 2.8 nM
R = OH, (R)-34i, IC50: 3.4 nM
26d
IC50: 63 nM
(R)-26
O
OH
OH
26a
26i
26l
IC50: 0.3 nM
IC50: 4.8 nM
IC50: 2.5 nM
Fig. 8. Effect of structural variations in Fragment B on EGFR-TK potency. The data, standard deviations and single point measurement are shown in Supporting information.
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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9
H
N
N
O
O
O
N
OH
OH
O
n
m
l
e
b
N
N
N
N
H
(S)-48
OH
48n
48m
48l
48e
48b
IC50: 0.2 nM
IC50: 0.3 nM
IC50: 0.4 nM
IC50: 0.5 nM
IC50: 0.7 nM
CH3
H
26n
IC50: 0.2 nM
26m
IC50: 0.5 nM
26l
IC50: 0.3 nM
26e
IC50: 1.1 nM
26b
IC50: 3.2 nM
(R)-26
H
41
41n
IC50: 0.5 nM
41m
IC50: 2.1 nM
41b
IC50: 3.8 nM
OH
H
(S)-60
60n
IC50: 0.3 nM
60m
IC50: 0.6 nM
F
Fig. 9. Combination of fragments to increase EGFR-TK potency. Compounds (R)-26b, 41b and (S)-48b are included as reference. The IC50 data, standard deviations and single
point data for these and some additional compounds are shown in Supporting information.
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
analogues (Bugge et al., 2014), fluorine substitution was generally
well tolerated in the aromatic part. This indicates a slightly differ-
ent binding mode.
Substitution of the 6-aryl group (Fragment B) increased EGFR-
TK activity especially when having a hydroxymethyl group in the
para or meta position. Modelling indicate this to be due to a hydro-
gen bonding interaction with Leu718 (Figs. 10 and 11). For aro-
matic methoxy substituents the activity increased in the order
para < meta < ortho. In contrast, the ortho hydroxy analogue had a
low potency, whereas the meta- and para hydroxy substituted
derivatives were highly active. Possibly, this is due to conforma-
tional effects caused by intramolecular hydrogen bonding with
the pyrrole-NH. The large lipophilic bromine substituent was not
tolerated in para position as also noted for bulky lipophilic groups
Fig. 10. Binding of compound (S)-48n to the EGFR-TK ATP binding site based on the
crystal structure 2J6M (Yun et al., 2007). The compound is indicated to engage in
hydrogen bonding via the hydroxymethyl group to Asp855 in the DFG motif, by N-1
in the thienopyrimidine series of compounds (Bugge et al., 2014).
to back-bond NH of Met793 and by the para-hydroxymethyl group to Leu718.
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
3.3.4. Activity towards EGFR mutants
The most potent compounds identified in enzymatic assays
towards wild-type EGFR-TK were also evaluated towards the
EGFRL858R, EGFRL861Q and EGFRT790M mutants. The key results are
compiled in Table 2.
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
3.3.3. Structure–activity relationships
A summary of the structure–activity relationship seen base on
this and our previous study (Kaspersen et al., 2011) is shown in
Fig. 14.
All the compounds assayed were found very potent towards
both the EGFRL858R and EGFRL861Q mutants. On average, as
compared to the wild-type EGFR-TK assay, somewhat higher IC50
values were seen towards EGFRL858R, while mostly similar IC50 val-
ues were noted in the case of EGFRL861Q. As expected for reversible
inhibitors, testing towards the EGFRT790M mutant only revealed
By modifying Fragment A the activity was increased by having
hydroxymethyl or methoxymethyl as R1, which is explained by
hydrogen interactions with Thr854 and Asp855 in the DFG motif
(Figs. 10 and 11). The insertion of bulky groups as R1 does not affect
the activity to a large extent. However, when bulky substituents
were placed in para position of the aromatic ring (R2), a dramatic
drop in potency was seen. This is explained by size limitations in
this part of the binding pocket. In the aromatic part of Fragment
A substitution seems to be better tolerated in ortho than in meta
position, although there is limited number of data for meta
substituted derivatives. As opposed to the thienopyrimidine based
activity in the
lM range (Kitagawa et al., 2013). Compounds (R)-
26l and 41 showed lower inhibitory potency towards EGFRT790M
as compared to the other drug candidates. Although a more elabo-
rate study is needed to understand the SAR relationship, the lim-
ited data suggests that the activity is increased by having a
hydroxymethyl group at the chiral centre and at the para position
of the 6-aryl ring. An ortho-methoxy group on the 6-aryl ring is also
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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S.J. Kaspersen et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx
Fig. 11. Interaction map highlighting amino acids within 5 Å distance from the docked ligand. The colours indicate residue type: green – lipophilic residues; red – acidic
residues; blue – polar residues; purple – basic residues. The protein ‘‘pocket’’ is displayed with a line around the ligand, coloured with the colour of the nearest protein
residue. Ligand atoms that are exposed to solvent are marked with grey spheres. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
Fig. 13. Binding efficiency (BEI), surface efficiency index (SEI), and ligand-
efficiency-dependent lipophilicity (LELP) for derivatives (R)-26l, (R)-26n, 41n,
(S)-48b, (S)-48l, (S)-48n and (S)-60n compared to Erlotinib.
Fig. 12. Ligand efficiency (LE) for derivatives (R)-26l, (R)-26n, (S)-41n, (S)-48b,
(S)-48e, (S)-48l, (S)-48n and (S)-60n compared to Erlotinib.
643
644
645
646
647
648
649
650
651
652
653
654
terms of off-target toxic effects, but could also represent an oppor-
tunity to inhibit several misregulated kinase signalling pathways.
635
636
637
638
639
favourable. As in the wild-type EGFR-TK assay, compounds lacking
Thus, the same eight selected pyrrolopyrimidines were tested
one or more of these groups display lower activity towards
towards an extended panel of 53 kinases, and compared with the
EGFRT790M. Docking of (R)-26l, 41n and the more potent (S)-48n
corresponding profile of Erlotinib. As this represent a huge amount
in EGFRT790M confirmed this as similar binding mode and interac-
of data it is convenient to quantify the selectivity by the use of Gini
tions were seen as in wild-type EGFR-TK.
plots and Gini coefficients (Graczyk, 2007). Overall the compounds
displayed a selectivity profile similar to Erlotinib. The Gini plot,
where the cumulative fraction of kinases is plotted towards the
640
641
642
cumulative fraction of total inhibition, for Erlotinib, the most selec-
tive compound 41n and the least selective compound (S)-48l, is
shown in Fig. 15.
3.3.5. Kinase profiling
As the ATP binding site of kinases is somewhat conserved, other
kinases might be inhibited as well. This could be problematic in
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

PHASCI 2998
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Fragment B
11
R3: OMe, CH2OH > H >> OH
R4: CH2OH > OH > OMe > H
H
N
R3
R4
N
R: CH2OH > H, OH, OMe >
F > CN > Br
R
N
Fragment A
N
R1
R1: CH2OH > CH2OMe > H, Me, Et > i-Pr, t-Bu
H
R5
R6
R5: H, F > Me, Cl, OH >> OMe
R6: H, F > Me
Stereochemistry
essential
R2
R2: H, F > CH3 > Br, NO2 > CF3, OMe > t-Bu
Fig. 15. Gini plots based on % inhibition of 53 kinases at 500 nM for Erlotinib, 41n
and (S)-48l. The cumulative fraction of kinases is plotted towards the cumulative
fraction of total inhibition. Gini coefficients: Erlotinib: 0.628; 41n: 0.665; (S)-48l:
0.577. A profile represented by the straight line would be non-selective.
Fig. 14. Structure–activity relationships identified in this and previous study
(Kaspersen et al., 2011). Colour code: green: induce potency; black: minor effects;
red: reduce potency. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
The Ba/F3-EGFR cells (Kancha et al., 2009) display growth and
survival dependent on EGFR signalling. Cell proliferation study
with Ba/F3-EGFRL858R cells using the XTT assay proved to be a
highly sensitive test system for evaluating the EGFR-TK inhibitors.
Of the eight compounds assayed, seven were highly active with
IC50 values in the range of 80–172 nM, while the benzylamine
based compound 41n, was less potent than the other derivatives.
Compounds (R)-26n, (S)-48n and (S)-60n sharing the same aryl
moiety, and the para hydroxymethyl derivative (R)-26l were found
more active than Erlotinib. None of the compounds inhibited pro-
liferation of the Ba/F3-EGFRT790M cell line indicating that the cyto-
toxicity is mainly due to EGFRL858R inhibition. The A-431 cells,
which overexpress EGFR (Ullrich et al., 1984; Bravo et al., 1985),
were also sensitive towards the compounds tested. Interestingly,
the structure of the R1 group at the stereocentre was found to be
of higher importance than was the case in the Ba/F3-EGFRL858R sys-
tem. Higher activity was noted when having the hydroxymethyl
substituent at the stereocenter than was the case for the methyl
or unsubstituted derivatives. A-431 cells also contain HER2
(Meira et al., 2011; Moasser et al., 2001) and are known to be sen-
sitive to Src inhibitors (Donnini et al., 2007). However, the kinase
selectivity data does not support inhibition of these kinases to be
the cause of the observed difference in cell proliferation seen
among the compounds. Testing towards the AU-565 cell line,
which possesses low level of EGFR, but overexpress HER2, HER3,
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
However, closer inspection of the data showed that Erlotinib
and the pyrrolopyrimidines had some major differences, see
Table 3. Erlotinib was found to be a better inhibitor of the kinase
insert domain receptor (KDR) and the RET proto-oncogene receptor
tyrosine kinase (RET) than the pyrrolopyrimidines. On the other
hand, the pyrrolopyrimidines had higher degree of inhibition
towards colony stimulating factor 1 receptor kinase (CSF1R), and
the Src-family of kinase including especially feline Gardner-Ras-
heed sarcoma viral oncogene homolog (FGR), v-src avian sarcoma
(Src) and Yamaguchi sarcoma viral oncogene homolog (YES1)
kinases. Both CSF1R (Krol et al., 2013; Xu et al., 2013) and Src-fam-
ily of kinase (Kim et al., 2009) are potential targets in cancer ther-
apy. The HER2 and HER4 inhibition potency were found rather
similar, though in cases of compounds (R)-26n and (S)-48n higher
HER preference was seen.
670
3.4. Cellular assays
671
672
673
674
675
676
Eight of the new pyrrolopyrimidine based EGFR-TK inhibitors
were evaluated in two genetically modified model cells Ba/F3-
EGFRL858R and Ba/F3-EGFRT790M, and four human cancer cell lines
including A-431 (epidermoid carcinoma), AU-565 (breast adeno-
carcinoma), K-562 (leukaemia) and C-33A (cervix carcinoma).
The results are shown in Table 4.
Table 2
IC50 values (nM) of pyrrolopyrimidines towards EGFRL858R and EGFRL861Q (nM) and activity at 50
Supporting information).
l
M towards EGFRT790M (inhibition data for other derivatives are shown in the
Compounds
R1
R
R2
R3
EGFRL858R (IC50, nM)a
EGFRL861Q (IC50, nM)a
EGFRT790M (% inhibition)b
(R)-26l
(R)-26n
41n
(S)-48b
(S)-48e
(S)-48l
(S)-48n
(S)-60n
Erlotinib
CH3
CH3
H
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
H
H
H
H
H
H
H
H
F
H
0.8 0.1
0.4 0.0
0.8 0.0
1.3 0.1
0.1 0.0
0.4 0.0
0.4 0.0
0.6 0.0
0.6 0.1
0.3 0.1
0.2 0.0
0.4 0.1
1.0 0.2
0.2 0.0
0.2 0.0
0.3 0.0
0.3 0.0
0.5 0.0
44
95
66
91
94
84
97
97
95
OMe
OMe
H
OMe
H
H
CH2OH
CH2OH
CH2OH
OMe
OMe
CH2OH
c
a
b
c
IC50 value and standard deviation based on duplicate measurement.
Percent inhibition at 50
For structure see Fig. 1.
lM concentration, average of duplicate measurements.
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

PHASCI 2998
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Table 3
Activity (% inhibition) of eight pyrrolopyrimidines and Erlotinib towards selected kinases at 500 nM test concentration.
Kinase
Erlotinib
(R)-26l
(R)-26n
41n
(S)-48b
(S)-48e
(S)-48l
(S)-48n
(S)-60n
ABL1
75
38
52
78
42
16
ꢁ13
18
70
49
68
68
64
42
29
73
92
53
25
82
40
34
37
nda
33
65
67
9
75
87
76
70
92
46
29
30
31
1
78
81
7
75
73
77
92
58
49
86
25
31
25
22
13
68
75
17
66
67
65
88
53
51
73
33
27
29
28
13
47
60
14
59
65
50
79
53
79
74
36
27
30
15
17
52
64
15
59
68
84
90
48
71
84
50
44
46
38
36
74
70
26
74
81
68
82
64
87
89
58
42
38
34
37
73
77
19
66
72
73
75
59
76
79
55
46
43
35
36
66
69
32
63
68
CSF1R (FMS)
ERBB2 (HER2)
ERBB4 (HER4)
FGR
FRK (PTK5)
FYN
HCK
KDR (VEGFR2)
LCK
LYN A
LYN B
RET
SRC
61
69
YES1
HER2/HER4 average
Src average
65
38
39
56
73
57
54
54
52
47
66
49
60
64
76
62
68
58
a
Not determined.
Table 4
Summary of cell proliferation data (IC50) of pyrrolopyrimidines towards Ba/F3-EGFRL858R, A-431, AU-565, C-33A and K-562. Curve data are shown in Supporting information.
Ba/F3-EGFRL858R
nM (n = 3)
A-431
M (n = 3)
AU-565
M (n = 2)
C-33A
M (n = 2)
K-562
M (n = 3)
l
l
l
l
80
9
1.2 0.5
0.8 0.4
0.8 0.1
0.3 0.1
>31
0.8 0.1
0.8 0.2
1.1 0.3
0.7 0.0
>100
99 27
>200
2.9 0.5
>31
9.5 0.5
>100
127 29
2.5 0.4
15
17
2
1
134 22
0.4 0.1
1.2 0.1
0.8 0.0
172 46
70 22
83 13
0.5 0.1
0.5 0.3
0.8 0.1
2.8 0.1
1.9 0.2
3.4 0.2
0.5 0.0
0.4 0.0
0.8 0.1
18
16
29
2
2
1
Erlotinib
142 65
0.4 0.1
3.3 0.6
0.9 0.0
55
9
702
703
704
705
706
707
708
709
710
711
HER4 (Bacus et al., 1992; Goestring et al., 2012; Sebban et al.,
2013), revealed that six of the compounds had comparable or bet-
ter activity than Erlotinib. Best activity was again observed for
compounds based on 2-amino-2-phenylethan-1-ol as C-4 substitu-
ent (Fragment A). The most potent compound identified was
(S)-48e having an ortho-methoxy group at the 6-aryl ring. Com-
pound (R)-26l and benzyl derivative 41n had low inhibitory effect.
The C-33A cell line, which also possesses low level of EGFR, but
contains HER2 (Meira et al., 2011), was found to be highly sensitive
towards all the pyrrolopyrimidines. Seven of the compounds tested
Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011

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13
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
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719
had comparable or better activity that Erlotinib. K-562 is a leukae-
mia Bcr-Abl positive cell line, in which cell proliferation also has
been correlated with Src inhibition (Wilson et al., 2002). K-562
was considerable less sensitive towards all the compounds than
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720
4. Conclusion
721
722
723
724
725
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727
728
729
730
731
732
733
734
735
736
737
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Acknowledgements
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Susana Villa Gonzalez is acknowledged for the HRMS experi-
ments. Roger Aarvik, Christopher Sørum and Veronica Willassen
are also acknowledged for their contribution. Prof. Dr. Justus Duy-
ster and Dr. Nikolas von Bubnoff, Technical University of Munich,
Munich, Germany kindly provided EGFR Ba/F3 cells. Anders Jahres
foundation and Technology Transfer Office (TTO-NTNU Trond-
heim) are thanked for financial support.
746
Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejps.2014.04.011.
749
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Please cite this article in press as: Kaspersen, S.J., et al. Identification of new 4-N-substituted 6-aryl-7H-pyrrolo[2,3-d] pyrimidine-4-amines as highly potent
EGFR-TK inhibitors with Src-family activity. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.011