Synthesis, biological evaluation and docking studies of new pyrrolo[2,3-d] pyrimidine derivatives as Src family-selective tyrosine kinase inhibitors

Article abstract of DOI:10.3109/14756366.2012.715288

In this study, the synthesis and potential enzyme interactions of new Pyrrolo[2,3-d]pyrimidine derivatives along with their inhibitory activity against SFK enzymes such as Fyn, Lyn, Hck, and c-Src were reported. The results indicated that compounds were s

Full text of DOI:10.3109/14756366.2012.715288

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(5): 1080–1087
2013 Informa UK, Ltd.
©
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.715288
RESEARCH ARTICLE
Synthesis, biological evaluation and docking studies of new
pyrrolo[2,3-d] pyrimidine derivatives as Src family-selective
tyrosine kinase inhibitors
1
1
2
3
Sebla Dincer , Kadir Taylan Cetin , Arzu Onay-Besikci , and Süreyya Ölgen
1
2
Department of Chemistry, Faculty of Science, Ankara University, Tandoğan, Ankara, Turkey, Department
3
of Pharmacology, Faculty of Pharmacy, Ankara University, Tandoğan, Ankara, Turkey, and Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandoğan, Ankara, Turkey
Abstract
In this study, the synthesis and potential enzyme interactions of new Pyrrolo[2,3-d]pyrimidine derivatives along with
their inhibitory activity against SFK enzymes such as Fyn, Lyn, Hck, and c-Src were reported. The results indicated
that compounds were slightly active of tested SFK enzymes in comparison with PP2 for Fyn, A-419259 for Lyn
and CGP77675 for c-Src. Compound N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)-4-
(
3,4-dimethoxyphenyl)butanamide (5) was identified as a non-selective slight inhibitor against Fyn, Lyn and c-Src.
However, compounds did not show any inhibitory effects on Hck. Docking studies were performed to analyze the
binding mode of compounds against SFKs. The best interaction was obtained between compound 5 and the active
site of Fyn and c-Src enzymes in comparison with reference compounds PP2 and CGP77675, respectively.
Keywords: Pyrrolopyrimidine derivatives, inhibition of SFK, docking analysis, synthesis, selectivity
Introduction
9
–11
and melanomas . Src also plays prominent roles in
invasion, tumour progression and development of
metastasis, therefore Src is a promising target for cancer
Src family kinases (SFKs) are involved in a broad
spectrum of cellular processes, such as cell growth,
1
,2
12–15
differentiation, survival, adhesion, and migration .
Many evidences showed that abnormal SFK signalling
participates in the pathogenesis of many diseases
including cancers, osteoporosis, inflammation-mediated
bone loss, rheumatoid arthritis, Alzheimer’s and
Parkinson’s disease . e activation of SFK in human
cancers may occur through a variety of mechanisms that
include domain interaction and structural remodelling
in response to various activators or upstream kinases
theraphy . Lck is a positive activator in T-cell signalling
(TCR), and its activation is involved in autoimmune
diseases such as rheumatoid arthritis, organ transplant
rejection and asthma. TCR signals initiated by Lck
ultimately lead to gene regulation events triggering
cytokine release, proliferation and survival of antigen-
specific T cell thereby amplifying specific immune
3–6
1
6
responses . Hck plays an important role in signalling
1
7
process of inflammation . Hck is a phagocyte-specific
proto-oncogene of the Src family and is expressed as
two isoforms, p59Hck and p61Hck. It plays a critical role
in Bcr/Abl-chronic myeloid leukaemia and is able to
transform fibroblasts in vitro. However, the tumourigenic
activity of Hck and the respective oncogenic functions
7
and phosphatases . e Src family of cytoplasmic protein
tyrosine kinases includes Src, Lck, Fyn, Lyn, Hck, Fgr,
Blk, Yrk, and Yes . e prototype member of SFKs, c-Src
8
(Src) is frequently overexpressed and activated in a
variety of epithelial and non-epithelial cancers such as
colorectal, breast, ovarian, lung and pancreatic cancer
18
of Hck isoforms have not been examined . Lyn is
Address for Correspondence: Prof. Dr. Süreyya Ölgen, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University,
Tandogan, Ankara, 06100 Turkey. Tel.: +90 (312) 2033073. Fax: +90 (312) 213 1081. E-mail: olgen@ankara.edu.tr
(
Received 06 June 2012; revised 13 July 2012; accepted 14 July 2012)
1
080

Pyrrolopyrimidine derivatives as SFK inhibitors 1081
phosphorylated in a KIT-independent manner in
neoplastic mast cells (MCs) and it was shown that
inhibitors such as dasatinib and bosutinib disrupt Lyn-
driven oncogenic signalling in neoplastic MC . Among
the SFKs, excess Fyn activity in the brain is associated with
conditions such as Alzheimer’s and Parkinson’s diseases.
at nanomolar levels in osteoporosis and cancer models,
1
6,29
bothinvitroandinvivo(Figure1) .Otherpyrrolo[2,3-d]
3
0
pyrimidine compound PKI166 , inhibits c-Src with an
19
31,32
IC value of 130 nM and A-419259
inhibits c-Src, Lck
5
0
and Lyn with IC values of 9, <3 and <3 nM, respectively
5
0
(Figure 1). Compound (S)-6-(4-(2-(dimethylamino)

erefore inhibition of Fyn kinase may help counteract
ethoxy)phenyl)-5-phenyl-N-((tetrahydrofuran-2-yl)
20
16
these nervous system disorders . Elevated levels of Fyn
in fibroblast cells exhibited a cancer-like phenotype
with increased anchorage-independent growth and
prominent morphologic changes. Fyn is overexpressed in
various cancers, including glioblastoma, head, neck, and
prostate cancer. It is also involved in melanoma induces
morphogenic transformation, alteration of mitogenic
signals and stimulation of cell growth and proliferation .
Most of the kinase activity inhibitors possess at least two
aromatic rings, which interfere with ATP binding
Several small molecule inhibitors of SFKs are currently
investigated in clinical trials. ese Src inhibitors
include heterocyclic ATP analogs, pyrazolo[2,3-d]
pyrimidines, pyrrolo[2,3-d]pyrimidines, pyrido[2,3-d]
pyrimidines, quinolines, and olomucines . Here, the
information about SFK enzymes inhibitors pyrrolo and
pyrazolo[3,4-d]pyrimidine were summarized, since both
skeletons are bioisosteres. Pyrazolo[3,4-d]pyrimidine
derivatives, PP1 and PP2 belong to traditional ATP
analogues (Figure 1), which could inhibit c-Src, Lck and
Fyn enzymes of SFK, showing IC values in the range
methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
was
found as Lck inhibitor with IC of <10 nM (Figure 1).
5
0
In this work, we have prepared new Pyrrolo[2,3-d]
pyrimidine derivatives and evaluated for their inhibitory
activities and selectivities against SFKs, namely Fyn, Lyn,
Hck and c-Src. (Scheme 1). For this aim, structurally dif-
ferent type of amide derivatives are randomly designed
to find new promising candidate molecules for specific
SFKs inhibition. Subsequently, docking analyses were
performed to evaluate binding properties and establish-
ing relationships between biological activity-binding
affinities of compounds.
2
1
2
2,23
.
Materials and methods
24
Chloroacetonitrile, methylformate, 2,6-diamino-4-hy-
droxy pyrimidine, sodium methoxide, sodium acetate, 10%
Pd/C, 4-(3,4-dimethoxyphenyl)butyric acid, (4-methyl-
thio)benzoic acid, 5-methylthiophene-2-carboxylic acid,
(4-chlorophenoxy)acetic acid (from Sigma-Aldrich, St.
Louis, MO, USA); ethanol, hydrochloric acid, isopropyl
alcohol, sodium hydroxide, sulphuric acid, dimethylsulf-
oxide (from Merck, Darmstadt, Germany), Fyn, Lyn, and
Hck kinases (Millipore, MA, USA), c-Src kinase (Invitrogen,
NY, USA) and ProFluor Src-family kinase assay kit, 8 plate
(Promega, Madison, USA) were purchased. Melting points
were measured with a capillary melting point apparatus
5
0
2
2,25–28
of 4–6 nM
. Amide derivatives of PP1 and PP2,
which bearing a C3 un-substituted phenyl ring were
found potent inhibitor of Fyn with similar potency, with
IC50 values of 6.5 nM . Pyrrolopyrimidine compounds
CGP76030 and CGP77675, were reported to possess
significant inhibitory activities against c-Src, Lck and Yes
28
Figure 1. Pyrazolopyrimidine and pyrrolopyrimidine derivatives as SFK inhibitors.
©
2013 Informa UK, Ltd.

1
082 S. Dincer et al.
Scheme 1. Synthesis of pyrrolo[2,3-d]pyrimidine amide derivatives.
(
Electrothermal 9100; Essex, UK). e Nuclear Magnetic
added as dropwise over 30 min at 0°C. e reaction mix-
ture was stirred at 0°C for 3 h. Chloro(formyl) acetoni-
trile 1 was obtained as an oily compound (6.3 g), which
was used without further purification for next step
(Scheme 1). NaOAc (0.101 mol, 8.29 g) and 2,6-diamino-
4-hydroxy pyrimidine 2 (0.051 mol, 6.37 g) was mixed
and heated at 100°C. Compound 1 in water was added to
this mixture and it was refluxed for 5 h. e yellow solid
was collected and dried in vacuo oven at 50°C for 24 h.
1
Resonance ( H-NMR) spectra were recorded on Varian
Mercury 400 NMR spectrometer for 400 MHz (Varian
Inc., Palo Alto, CA, USA). e chemical shift values were
expressed in parts per million (ppm) relative to tetra-
methylsilane as an internal standard and signals were
reported as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet). Mass spectra were recorded on a Waters
ZQ Micromass LC-MS spectrometer (Waters Corporation,
Milford, MA, USA). Perkin Elmer S-55 Spectrofluorimetry
3
3
1
Compound 3 was obtained with 36%. M.p. >300°C; H-
NMR (400 MHz, DMSO-d , δ, ppm): 6.40 (s, 2-NH , 2H),
(from Perkin Elmer, Shelton, CT, USA) was used to measure
6
2
fluorescence of phosphorylation reaction for enzyme activ-
ity studies. Analytical TLC was carried out on Merck 0.2mm
precoated silica gel (60 F-254) aluminium sheets (from
Merck, Darmstadt, Germany), with visualization by irra-
diation with a UV lamp. e flash column chromatography
was accomplished on silica gel 60 (230–400 mesh, Merck,
Darmstadt, Germany). Elemental analysis was taken on a
Leco-932 CHNS-O analyzer (St. Joseph, MI, USA).
7.60 (s, 7-CH, 1H), 10.70 (s, 8 NH, 1H), 11.98 (s, 3-NH,
1H); IR (KBr, cm ): 3450, 3400, 3100 (N-H signals), 2240
(C≡ N), 1620 (C=C); ESI-MS: m/z, 176.3 (M+1).
−
1
2-Amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-
4(3H)-one (compound 4, PreQ1)
PreQo was suspended in 50 mL MeOH and 50 mL of 1 N
HCl. Ten percentage of Pd/C was added. e compound
was treated with a hydrogenation gas (3 atm) at room tem-
perature and stirred for 24 h. e precipitate was purified
by silica gel column chromatography. Compound 4 was
5
-(Cyano)-2-aminopyrrolo[2,3-d]pyrimidin-4(3H)-one
(
PreQ , compound 3)
0
1
Methyl formate was added to a stirred mixture of NaOCH3
obtained with Yield 95%. M.p. 220–225°C; H NMR (400
in anhydrous toluene at 0°C and chloroacetonitrile was
MHz; DMSO-d , δ, ppm): 4.00 (2H, s, CH -NH ), 6.46 (2H,
6
2
2
Journal of Enzyme Inhibition and Medicinal Chemistry

Pyrrolopyrimidine derivatives as SFK inhibitors 1083
s, 2-NH ), 6.78 (1H, s 7-CH), 10.58 (1H, s, 8-NH), 11.21
(C=C); LC/MS (ESI, m/z): 330.9 (M+1, 100%); HPLC-RT:
4.75 min (pure single peak); C H N O S calcd. C 54.70, H
4.59, N 21.26; found C 54.98, H 4.33, N 21.45.
2
−
1
(
1H, s, 3-NH); IR (KBr, cm ): 3440, 3340, 3225 (N-H sig-
nals), 1610 (C=C); LC/MS, (ESI, m/z): 180.5 (M+1), 163.0
-NH , %100).
1
5
15
5
2
(
3
N-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]
pyrimidin-5-yl)methyl]-5-methylthiophene-2-
carboxamide (7)
The general synthesis of 2-amino-pyrrolo[2,3-d]
pyrimidin-4(3H)-one-5-(substituted) amide derivatives
1
(
compounds 5–8)
Yield 0.118 g, 35%; m.p. 260–262°C; H NMR (400 MHz,
One millimolar appropriate acid was dissolved in DMF
and 1.2 mmol CDI was added at 0°C. e mixture was
stirred at room temperature for 1 h. One millimolar
DMSO-d , δ, ppm): 2.46 (3H, s, thiophene-CH ), 4.44
6
3
(2H, d, J = 5.2 Hz, -CH -NH-C=O), 6.15 (2H, s, 2-NH ),
2
2
6.57 (1H, d, J = 2.0 Hz, CH-7), 6.83 (1H, dd, J = 4.0, 1.2
Hz, thiophene-H4′), 7.49 (1H, d, J = 4.0 Hz, thiophene-
PreQ was added and the mixture was stirred at 50°C
1
for 12–24 h. e solvent was removed in vacuo and
the resulting residue was purified by column chro-
matography on silica gel (CH Cl /MeOH, 9.5:0.5) to
H5′), 9.19 (1H, t, J = 5.2 Hz, -CH -NH-C=O), 10.52 (1H, s,
2
1
3
8-NH), 10.91(1H, s, 3-NH); C NMR (δ, ppm): 14.8 (CH ),
3
36.2 (-C-NH-), 99.2 (C-5), 114.6 (C-7), 115.8 (C-6), 127.0
(C-4′), 128.4 (C-5′), 138.4 (C-1′), 144.9 (C-9), 152.5 (C-2),
2
2
provide compounds 5–8. Purity of compounds were
determined by HPLC and RT (Retention Time) were
given as below. Chromatographic conditions; column
−
1
152.9 (C-3′), 160.7 (C-4), 161.2 (NH-C=O); IR (KBr, cm ):
3400, 3340, 3130 (N-H signals), 1640 (C=O Amide), 1600
(C=C); LC/MS (ESI, m/z): 304.7 (M+1, 100%); HPLC-RT:
4.15 min (pure single peak); C H N O S calcd. C 55.03, H
C18 (Isterra) 5 mm × 25 cm, mobile phase 15 H O: 65
2
CH CN: 10 MeOH: 10 formic acid 0.1% in CH CN, flow
3
3
13 13
5
2
rate: 0.55 mL/min, temperature: 25°C.
4.01, N 21.39; found C 54.76, H 3.69, N 21.50.
N-((2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]
pyrimidin-5-yl)methyl)-4-(3,4-dimethoxy phenyl)
butanamide (5)
N-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]
pyrimidin-5-yl)methyl]-2-(4-chlorophenoxy)
acetamide (8)
1
1
Yield 0.086 g, 40%; m.p. 256–258°C; H NMR (400
Yield 0.065 g, 33%; m.p. 200–202°C; H NMR (400 MHz,
MHz, DMSO-d , δ, ppm): 1.77 (2H, m, J = 7.6 Hz, CH -
DMSO-d , δ, ppm): 4.36 (2H, d, J = 6.0 Hz, CH -NH-C=O),
6
2
6
2
CH -CH ), 2.09 (2H, t, J = 7.6 Hz, O=C-CH -CH ), 2.47
4.49 (2H, s, -O-CH -C=O), 6.10 (2H, s, 2-NH ), 6.53 (1H, d,
2
2
2
2
2
2
(
3
2H, t, J = 7.6 Hz, -CH -CH -Ar), 3.70 (3H, s, Ar-OCH ),
J = 2.0 Hz, CH-7), 7.00 (2H, d, J = 9.2 Hz, Ar-H3′,5′), 7.35
2
2
3
.72 (3H, s, Ar-OCH ), 4.28 (2H, d, J = 5.2 Hz, -CH -NH-
(2H, d, J = 9.6 Hz, Ar-H2′,6′), 8.88 (1H, t, J = 5.6 Hz, -CH -
3
2
2
13
C=O), 6.09 (2H, s, 2-NH ), 6.48 (1H, d, J = 2.4 Hz, CH-7),
NH-C=O), 10.52 (1H, s, 8-NH), 10.87 (1H, s, 3-NH); C
2
6
.64 (1H, dd, J = 8.0, 2.0, Ar-H2′), 6.75 (1H, d, J = 2.0 Hz,
NMR (δ, ppm): 35.3 (-C-NH-), 67.1 (CH OPh), 99.3 (C-5),
2
Ar-H6′), 6.82 (1H, d, J = 8.0 Hz, Ar-H3′), 8.19 (1H, t, J =
114.5 (C-7), 117.4 (C-6), 115.5 (C-2′), 115.9 (C-6′), 129.8
5
3
.2 Hz, -CH -NH-C=O), 10.35 (1H, s, 8-NH), 10.85 (1H, s,
2
(C-5′), 129.9 (C-3′), 135.8 (C-9), 153.0 (C-2), 153.2 (C-4′),
1
3
−1
-NH); C NMR (δ, ppm): 27.9 (-C-C-C), 34.9 (O=C-C-
157.1 (C-1′), 160.5 (C-4), 167.3 (NH-C=O); IR (KBr, cm ):
C-C), 35.7 (-C-C-C-Ar), 35.7 (-C-NH-), 56.0 (Ar-OCH ),
3420, 3310, 3150 (N-H signals), 1640 (C=O Amide), 1600
3
+
5
1
6.2 (Ar-OCH ), 99.0 (C-6′), 112.5 (C-5), 112.8 (C-3′),
(C=C); LC/MS (ESI, m/z): 348.8 (M , 100%); HPLC-RT:
3
14.4 (C-7), 116.2 (C-6), 120.7 (C-2′), 134.9 (C-1′), 147.6
4.79 min (pure single peak); C H N O Cl.H O calcd. C
1
5
14
5
3
2
(
1
C-4′), 149.3 (C-5′), 152.2 (C-9), 152.9 (C-2), 160.2 (C-4),
49.26, H 4.41, N 19.15; found C 49.13, H 4.68, N 19.27.
−
1
71.9 (NH-C=O); IR (KBr, cm ): 3450, 3375, 3320, 3125
(
(
N-H signals), 1630 (C=O Amide), 1600 (C=C); LC/MS
ESI, m/z): 386.8 (M+1, 100%); HPLC-RT: 3.54 min (pure
In vitro assay for Src tyrosine kinase activity
Kinase activity (Src-family kinases, Hck, Lyn, Fyn, and
c-Src) and the effect of the molecules were determined by
ProFluor Src-Family Kinase Assay Kit (Promega). A titra-
tion assay was performed for each kinase to determine
the amount of the enzyme that results in approximately
80% phosphorylation as suggested by the manufacturer .
e compounds were dissolved in 10% DMSO and tested
at 1, 10 and 100 µM concentrations. Briefly, the mol-
ecules were mixed with a reaction buffer that included a
specific substrate for Src-family kinases (R110), a control
substrate and the kinase. e reaction was initiated by
the addition of ATP. After incubating the 96-well reaction
plate at 22°C for 1 h, protease solution was added to each
well and the plate was incubated for 1 h. e fluorescence
of the liberated R110 was read at an excitation wave-
length of 485 nm and emission wavelength of 530 nm.
e strength of fluorescence is inversely correlated with
single peak); C H N O , calcd. C 59.21, H 6.02, N 18.17;
found C 59.57, H 6.28, N 18.00.
1
9
23
5
4
N-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]
pyrimidin-5-yl)methyl]-4-(methylthio) benzamide (6)
Yield 0.030 g, 16%; m.p. 238–240°C; H NMR (400 MHz,
34
1
DMSO-d , δ, ppm): 4.25 (3H, s, SCH ), 4.46 (2H, d, J = 5.6
6
3
Hz, -CH -NH-C=O), 6.11 (2H, s, 2-NH ), 6.55 (1H, d, J =
2
2
2
.0 Hz, CH-7), 7.28 (2H, d, J = 8.4 Hz, Ar-H3′,5′), 7.77 (2H,
d, J = 8.4 Hz, Ar-H2′,6′), 9.27 (1H, t, J = 5.2 Hz, -CH -NH-
2
1
3
C=O), 10.51 (1H, s, 8-NH), 10.88 (1H, s, 3-NH); C NMR
(
(
1
δ, ppm): 14.8 (SCH ), 36.4 (-C-NH-), 99.2 (C-5), 114.6
3
C-7), 115.8 (C-6), 125.7 (C-3′), 125.9 (C-5′), 128.1 (C-2′),
28.2 (C-6′), 131.4 (C-1′), 142.9 (C-9), 152.5 (C-2), 152.9
−1
(
3
C-4′), 160.7 (C-4), 165.5 (NH-C=O); IR (KBr, cm ): 3450,
400, 3190, 3145 (N-H signals), 1700 (C=O Amide), 1600
©
2013 Informa UK, Ltd.

1
084 S. Dincer et al.
the kinase activity. e Src kinase activity is measured
as the difference between the total activity of no vehicle
which are inversely correlated with the kinase activ-
ity. For the primary assay, compounds were tested
against Lyn, Fyn, Hck and c-Src of SFK enzymes at 0.1,
0.01 and 0.001 mM concentrations. In these concentra-
tions range, compounds inhibited the enzymes Fyn
(DMSO) and the activity of enzyme in the presence of
DMSO. Percentage inhibition value of compounds was
determined as the mean of triplicate measurements.
(
Figure 2), Lyn (Figure 3) and c-Src (Figure 4) by 18–46,
Molecular modelling
For this study, X-ray crystal structure of Fyn-saturosporine
complex (PDB entry 2DQ7), Lyn-PP2 complex (PDB entry
12–42, and 14–71%, respectively. ese results indicated
that compounds are slightly active inhibitors of the tested
2ZV9), Hck-PP1 complex (PDB entry 1QCF), and c-Src-
CGP77675 complex (PDB entry 1YOL) were downloaded
from Protein Data Bank (PDB). Solvents of molecules were
deleted and hydrogen atoms were added to obtain the
docking grid the active site was defined using Autogrid.
e grid size was set to 40×40×40 points with grid spac-
ing of 0.375 Å. e grid box was placed on the centre of
the ligand from the corresponding crystal structure
complexes. 2D structures of inhibitors were established
by using ChemBioDrawUltra 11.0 and minimized with
ChemBio3DUltra 11.0. To be exported to Autodock, the
minimized structures were converted into the mol2 file
format. e structures of proteins and ligands were com-
bined using the Autodock 4.2.1 and Vina 1.0.2 software
package (e Scripps Research Institute, La Jolla CA,
USA). e rigid root and rotatable bonds were defined
using ADT. e reliability of the docking protocol was first
tested by simulations of the binding mode of known SFK
inhibitors (PP1, PP2, CGP77675, and A419259). e results
were ranked by the scoring functions of ligand-fit, and ana-
lyzed for relatively lower binding energy, and potential in
forming hydrogen bonds with SFK kinases. e ligand was
fully optimized inside the binding site during the docking
simulations. e conformation with the lowest predicted
binding free energy of the most occurring binding modes
in SFKs active pockets were evaluated to binding proper-
ties and relationships between biological activity-binding
affinities of compounds. Representation binding modes
of compounds were shown by using PyMOL Molecular
Graphics System (DeLano Scientific LLC, 2008).
Figure 2. e inhibitory effect of the molecules at 0.1, 0.01 and
0
.001 mM concentrations on Fyn activity.
Figure 3. e inhibitory effect of the molecules at 0.1, 0.01 and
0.001 mM concentrations on Lyn activity.
Results and discussion

e starting compound 1 was synthesized with the
reaction of methyl formate and chloroacetonitrile in
anhydrous toluene (Scheme 1). To obtain compound
3
(PreQ ), 2,6-diamino-4-hydroxy pyrimidine 2 was
0
reacted with compound 1 in presence of sodium acetate.
PreQ (compound 4) was prepared by reduction of cyano
1
3
3
group of PreQ to amine using H /Pd in 1 M HCl . e
0
2
synthesis of amide derivatives of pyrrolo[2,3-d]pyrimi-
dine (compounds 5–8) were achieved by the reaction of
PreQ and corresponding acid derivatives in presence of
1
1
,1′-carbonyldiimidazole (CDI) in DMF. e structural
1
elucidations of compounds were established by H-NMR,
IR, HPLC and MS. e proton numbering for NMR inter-
pretation is indicated on the formula in Scheme 1.

e inhibitory activities of compounds were evalu-
Figure 4. e inhibitory effect of the molecules at 0.1, 0.01 and
0.001 mM concentrations on c-Src activity.
ated via measurement the strength of fluorescence,
Journal of Enzyme Inhibition and Medicinal Chemistry

Pyrrolopyrimidine derivatives as SFK inhibitors 1085
Table 1. Inhibition of SFKs (c-Src, Fyn and Lyn) by compounds 5–8 and reference compounds.
b
b
b
%
c-Src inhibition
% Fyn inhibition
% Lyn inhibition
0.01
0
.1
0.01
mM
0.001
mM
0.1
mM
0.01
mM
0.001
mM
0.1
0.001
mM
a
Comp. No
IC50 values
mM
mM
mM
5
54 ± 4
50 ± 4
50 ± 1
21 µM)
38 ± 4
35 ± 2
32 ± 1
32 ± 1
25 ± 1
17 ± 2
c
(
6
7
14 ± 4
71 ± 2
4 ± 4
25 ± 2
39 ± 2
46 ± 3
41 ± 0
30 ± 4
33 ± 5
28 ± 5
28 ± 2
42 ± 4
40 ± 1
16 ± 2
21 ± 3
12 ± 2
12 ± 3
53 ± 6
16 µM)
c
(
8
60 ± 1
50 ± 2
40 ± 6
36 ± 3
31 ± 8
18 ± 0
28 ± 1
20 ± 1
13 ± 2
PP2
A-419259
0.5 nM (Fyn)
0.3 nM (Lyn)
CGP77675 0.4 nM (c-Src)
a
Activity data of reference compounds were calculated as nM level.
Average (from three independent experiments) % inhibitory effects of compounds at 0.1, 0.01 and 0.001 mM concentrations.
b
c
IC50 values of compounds were given as µM level.
inhibition (Table 1) was obtained with compound 6 as
shown by an inhibition rate of 46 and 42% at 0.1mM con-
centration, respectively. Moreover, compound 5 showed
Fyn inhibitions rates of 35 and 32% at 0.01 and 0.001 mM
concentrations, respectively. On the other hand, com-
pound 5 possessed weaker inhibitory effect on Lyn as
shown by 25 and 17% inhibitions at 0.01 and 0.001 mM
concentrations, respectively. To evaluate the inhibitory
activities at lower concentrations, the data confirmed
that compound 5 had higher inhibition profiles against
Fyn and Lyn. is result revealed that compound 5 was
more potent than the other compounds. As seen in Figure
4
, compound 7 showed 53% c-Src inhibition 0.01 mM
concentration (IC was determined as 16 µM), whereas
5
0
compound 5 showed 50% c-Src inhibition at much lower
0
.001 mM concentration (IC was determined as 21 µM).
50
e results indicate that compound 5 is slightly inhibi-
tor of all three kinases, which suggest compound 5 has
a non-selective inhibitory properties. While sulphur
containing compounds 6 and 7 have similar inhibitory
potencies against Fyn and Lyn, compound 6 has not
any inhibition against c-Src. However, compound 7 and
8
are better selective inhibitors of c-Src. Overall, these
findings suggest that the features of substituents on fifth
position might have significant influence on the inhibi-
tory potencies and selectivity profiles of compounds. It
is noteworthy to insert new substituents on this position
to design more potent and selective pyrrolopyrimidine
compounds against SFKs.
Binding conformation and the degree of fit into the
SFKs (c-Src, Fyn, Lyn and Hck) of the compounds were
evaluated by using Autodock 4.2.1 and Vina 1.0.2. e
program successfully reproduced the X-ray coordinates
of the inhibitor binding with RMSD values ranging from
Figure 5. (A) Comparison of compound 5 and PP2 (orange) with
Fyn binding site; (B) Comparison of compound 5 and CGP77675
(
green) with c-Src binding site.
1–3 Å and exhibited a similar accuracy with the same
compounds when docked into the SFK binding sites.
Each docked compound was assigned a score according
to its binding mode onto the binding site of enzymes. e
results suggest that ligands with highest binding affinities
have a better chance of interaction with enzymes and have
SFK enzymes in comparison with PP2 (IC = 0.5 nM) for
50
Fyn, A-419259 (IC50 = 0.3 nM) for Lyn and CGP77675
IC = 0.4 nM) for c-Src. Compounds did not show any
(
5
0
inhibitory properties against Hck. e best Fyn and Lyn
©
2013 Informa UK, Ltd.

1
086 S. Dincer et al.
also a good probability of acting as inhibitors. From the
interaction mode of the compounds with the predicated
active site, it has been found that only compound 5 makes
three hydrogen bonds with Fyn. One of the hydrogen
bound was observed between the H (NH-3 of pyrolopy-
rimidine ring) and the O (OH) backbone of ASP130 with
the distance of 1.8 Å. A couple of hydrogen bonds formed
between the amino group at 2nd position of pyrrolopy-
rimidine ring and C=O of ASP130 with the distances of 2.2
Å and 3.8 Å (Figure 5A). All compounds were found to bind
into the active site of Fyn similar to PP2, which is known
as most potent inhibitor of Fyn but none of them are over-
lapped well with PP2, including compound 5 (Figure 5A).
Figure 5B depicted the interactions of compound 5 and
CGP77675 with the active site of c-Src. Compound 5 was
found to bind into the ATP binding site of c-Src similar to
the crystallized inhibitor CGP77675 but none of hydro-
gen bonds involving the pyrrolopyrimidine nucleus and
the hinge region were determined. Compounds were not
docked well onto the Lyn and Hck in comparison with
potent known inhibitors A419259 and PP1, respectively.
10. Edwards J. Src kinase inhibitors: an emerging therapeutic
treatment option for prostate cancer. Expert Opin Investig Drugs
2
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1. Rothschild SI, Gautschi O, Haura EB, Johnson FM. Src inhibitors in
lung cancer: current status and future directions. Clin Lung Cancer
2010;11:238–242.
12. Sakamoto M, Takamura M, Ino Y, Miura A, Genda T, Hirohashi S.
Involvement of c-Src in carcinoma cell motility and metastasis. Jpn
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3. Sawyer TK. Cancer metastasis therapeutic targets and drug
discovery: emerging small-molecule protein kinase inhibitors.
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5. Saad F, Lipton A. SRC kinase inhibition: targeting bone metastases
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a
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e docking studies suggested that the flexible or longer
side chains of our compounds at 5th position might be
the reason for weak binding property and activity in com-
parison with the reference compounds. As conclusion of
above results, it was impossible to identify for each inhibi-
tor a molecular portion that interacts with the same pocket
of the SFKs binding site, since inhibitors showed quite dif-
ferent binding modes.
1
9. Gleixner KV, Mayerhofer M, Cerny-Reiterer S, Hörmann G, Rix U,
Bennett KL et al. KIT-D816V-independent oncogenic signaling
in neoplastic cells in systemic mastocytosis: role of Lyn and Btk
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0. Resh MD. Fyn, a Src family tyrosine kinase. Int J Biochem Cell Biol
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target in cancer. Cancer 2010;116:1629–1637.
2
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1
Declaration of interest
22. Belsches-Jablonski AP, Demory ML, Parsons JT, Parsons SJ. e src
pathway as a therapeutic strategy. Drug Discov. Today: er. Strate.

is work was partially supported by grants from the
2
005;2:313–321.
University of Ankara Research Fund (BAP-09B3336002).
23. Noble ME, Endicott JA, Johnson LN. Protein kinase
inhibitors: insights into drug design from structure. Science
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substrate binding site inhibitors. Curr Opin Investig Drugs
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