Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro active against Glioblastoma

Article abstract of DOI:10.1016/j.ejmech.2016.12.036

In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US FDA and in other countries for the treatment of different diseases or are currently in phase I/II clinical trials. Herein we present the synthesis and the characterization of a novel series of pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme (GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity effect against U87 cell line with an IC₅₀value of 7.1 μM.

Full text of DOI:10.1016/j.ejmech.2016.12.036

Accepted Manuscript
Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro
active against Glioblastoma
Francesca Musumeci, Anna Lucia Fallacara, Chiara Brullo, Giancarlo Grossi, Lorenzo
Botta, Pierpaolo Calandro, Mario Chiariello, Miroslava Kissova, Emmanuele Crespan,
Giovanni Maga, Silvia Schenone
PII:
S0223-5234(16)31041-8
10.1016/j.ejmech.2016.12.036
EJMECH 9127
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 September 2016
Revised Date: 14 December 2016
Accepted Date: 17 December 2016
Please cite this article as: F. Musumeci, A.L. Fallacara, C. Brullo, G. Grossi, L. Botta, P.
Calandro, M. Chiariello, M. Kissova, E. Crespan, G. Maga, S. Schenone, Identification of new
pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro active against Glioblastoma, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.036.
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ACCEPTED MANUSCRIPT
Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro active
against Glioblastoma
Francesca Musumeci, Anna Lucia Fallacara, Chiara Brullo, Giancarlo Grossi, Lorenzo Botta,
Pierpaolo Calandro, Mario Chiariello, Miroslava Kissova, Emmanuele Crespan, Giovanni Maga and
Silvia Schenone

ACCEPTED MANUSCRIPT
Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine
kinase inhibitors in vitro active against Glioblastoma
Francesca Musumecia,1, Anna Lucia Fallacarab,1, Chiara Brulloa, Giancarlo Grossia, Lorenzo Bottac,
Pierpaolo Calandrob, Mario Chiariellod, Miroslava Kissovae, Emmanuele Crespane, Giovanni Magae
and Silvia Schenonea*
aDipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
bDipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena Via Aldo Moro 2,
53100 Siena, Italy
cDipartimento di Farmacia, Università “Federico II” di Napoli, Via D. Montesano 49, 80131 Napoli, Italy
dConsiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica and Istituto Toscano Tumori, Core
Research Laboratory, Via Fiorentina 1, 53100 Siena, Italy.
eConsiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207,
27100 Pavia, Italy
1 These authors contributed equally to the publication.
1

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Abstract
In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US
FDA and in other countries for the treatment of different diseases or are currently in phase I/II
clinical trials. Herein we present the synthesis and the characterization of a novel series of
pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme
(GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to
efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of
kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected
selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their
antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity
effect against U87 cell line with an IC50 value of 7.1 µM.
Keywords: pyrrolo-pyrimidines, Src, Glioblastoma, kinase inhibitors, small molecules
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1. Introduction
SFKs (Src family kinases) are a family of cytoplasmic tyrosine kinases (TKs) composed of eight
highly homologous members, Src, Fyn, Yes, Blk, Fgr, Hck, Lck and Lyn. They present a conserved
structure which includes a N-terminal region, two Src homology domains (SH2 and SH3), a
catalytic domain (SH1) and, finally, a short C-terminal tail [1]. These kinases are overexpressed or
hyperactivated in several pathologies, especially in cancer [2]. Among SFKs, c-Src is involved in
pathways controlling cell proliferation, migration, invasion and angiogenesis [3]. High levels of c-
Src in different tumors are generally correlated with a poor prognosis. Recently, c-Src has been
shown to be frequently hyperactivated or overexpressed also in Glioblastoma multiforme (GBM), a
brain tumor characterized by a high degree of proliferation, angiogenesis, necrosis, and
invasiveness. Src inhibition reduced GBM cell growth, viability and migration both in vitro and in
mouse models [4].
For all these reasons, c-Src is a good therapeutic target, and many small molecules Src inhibitors
have been developed for the treatment of different tumors [5]. However, because of the high
homology among the catalytic domains of some cytoplasmic TKs, Src inhibitors are active also on
other TKs, in particular on Bcr-Abl, a chimeric kinase present in chronic myeloid leukemia (CML)
[6]. In the last ten years, three dual Src/Abl inhibitors have been approved and are currently on the
market as first or second line therapy for CML. These are dasatinib, (SprycelTM, by Bristol-Myers
Squibb), bosutinib, (Bosulif®, by Pfizer), and very recently, ponatinib (Iclusig®, Ariad Pharma Ltd)
(Fig. 1) [7]. These drugs and other compounds, e.g. saracatinib (Fig. 1) are being tested in clinical
trials, alone or in combination, for the treatment of solid malignancies.
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Cl
Cl
O
HN
N
N
N
O
O
CN
H
N
N
N
S
H
N
N
N
O
OH
Cl
N
dasatinib
bosutinib
N
N
N
O
O
HN
O
O
Cl
N
N
H
N
N
N
O
N
O
N
saracatinib
ponatinib
CF3
Fig. 1. Structures of dual Src/Abl inhibitors approved or in clinical trials for solid or hematologic malignancies.
Recently, different libraries of pyrazolo[3,4-d]pyrimidines active as SFK and/or Abl inhibitors have
been synthesized [8,9]. In particular, our in house compounds 1 (Fig. 2) showed Ki values in the
nanomolar range both in enzymatic and cell assays and possessed in vivo antitumor activity on
xenograft models derived from different cell lines [10-12].
R
X
N
N
N
R2
N
1
R1
R = aliphatic or aromatic amino groups
R1 = H, CH3, Cl
R2 = H, alkyl, thioalkyl and aminoalkyl groups
X = H, F, Cl, Br
Fig. 2. In house library of pyrazolo[3,4-d]pyrimidines 1.
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On the other hand, the pyrrolo[2,3-d]pyrimidine scaffold is being extensively investigated and in
the last few years many of such compounds resulted active as kinase inhibitors. The Jak kinase
inhibitors tofacitinib 2 [13], ruxolitinib 3 [14] and oclacitinib 4 [15] (Fig. 3) have been recently
approved by the US FDA and in other countries for the treatment of rheumatoid arthritis,
myelofibrosis and canine allergic dermatitis, respectively, and other similar Jak inhibitors are in
clinical trials [16]. AEE788, 5 (Fig. 3) is a dual EGFR/VEGFR inhibitor [17,18] which has been
evaluated in phase I/II clinical trials [19] for its activity in patients with recurrent or relapsed GBM.
CCT128930, 6 [20] and AZD5363, 7 [21] (Fig. 3) inhibit Akt, a serine-threonine kinase often
deregulated in tumors, such as GBM, breast and gastric cancer. AZD5363 is currently being tested
in several clinical trials for the treatment of different solid malignancies [22]. Other recently
published pyrrolo[2,3-d]pyrimidines include leucine-rich repeat kinase-2 (LRRK2) [23], 3-
phosphoinositide dependent protein kinase-1 (PDK1) [24], Aurora kinase [25], multiple TK
receptors (VEGFR, EGFR, PDGFR) [26, 27] and Bruton's tyrosine kinase (Btk) inhibitors [28].
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HN
O S O
N
NC
N
N
N N
CN
O
N
N
N
N
H
N
N
N
N
N
H
H
2, tofacitinib
4, oclacitinib
3, ruxolitinib
OH
NH2
O
NH2
N
H
Cl
N
N
HN
Cl
N
N
N
N
N
N
N
N
H
H
N
N
N
H
6, CCT128930
5, AEE788
7, AZD5363
Fig. 3. Pyrrolo[2,3-d]pyrimidine kinase inhibitors.
Interestingly, some recently appeared derivatives are also active on Src [29-31].
On the basis of this knowledge and with the aim of investigating if the deaza-isosteres of in house
inhibitors 1 maintain the activity on Src both in enzymatic and in cell assays, we decided to
synthesize a family of pyrrolo[2,3-d]pyrimidines 8 (Fig. 4), and to test them on Src in enzymatic
assays and as antiproliferative agents on a specific cancer cell line. Some of compounds 8 are
strictly correlated with their pyrazolo[3,4-d]pyrimidine analogues 1 bearing a C2 thiomethyl group,
a C4 amino group, and a N7 2-chloro-2-phenylethyl chain, whereas other derivatives are substituted
in N7 with a benzylic chain, more easily accessible from a synthetic point of view.
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R
N
N
H3CS
N
R1
8
R = amino groups
R1 = CH2CHClC6H5,
CH2C6H4-pF
Fig. 4. General structure of pyrrolo[2,3-d]pyrimidines 8.
2. Results and Discussion
2.1. Synthesis of pyrrolo[2,3-d]pyrimidine derivatives
The synthesis of compounds 8a-c was performed starting from 6-amino-2-(methylthio)pyrimidin-
4(3H)-one 9, prepared following the procedure reported by Baker and coll. [32]. Compound 9 was
cyclized using an aqueous solution of chloroacetaldehyde in a microwave open vessel apparatus to
obtain 2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-4-ol 10 in a higher yield compared to the
literature method [33]. Then compound 10 was chlorinated in C4 to give 4-chloro-2-(methylthio)-
7H-pyrrolo[2,3-d]pyrimidine 11 following a literature method [33]. Intermediate 11 was alkylated
in N7 using 2-bromo-1-phenylethanone at room temperature in the presence of sodium hydride to
obtain compound 12. The latter was reduced to 13 with sodium borohydride in a mixture of THF
and water. Intermediate 13 was in turn treated with the Vilsmeier complex (POCl3/DMF, 1:1) in
CH2Cl2 at reflux for 8h to obtain compound 14, bearing a 2-chloro-2-phenyethyl chain in N7.
Finally, reaction of 14 with the suitable anilines in absolute ethanol at reflux for 3-5h gave the
desired compounds 8a,b in good yields, while compound 8c was obtained by treating 14 with an
excess of benzylamine in anhydrous toluene at room temperature for 24h (Scheme 1).
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O
OH
Cl
HN
H3CS
i
N
ii
N
N
N
NH2
N
H
H3CS
N
H3CS
N
H
9
10
11
Cl
Cl
N
N
iv
iii
N
N
H3CS
N
H3CS
N
13
12
OH
O
R
N
Cl
N
N
v
vi
N
8a R = NHC6H5
8b R = NHC6H4m-Cl
8c R = NHCH2C6H5
H3CS
N
H3CS
N
8a-c
14
Cl
Cl
Scheme 1. Synthetic route for the preparation of 8a-c. Reagents and conditions: (i) Chloroacetaldehyde (50% aq.
solution), AcONa, H2O, MW 100 W, 80 °C, 2 min. (ii) POCl3, TEA, 130 °C, 4h. (iii) NaH, anhyd. DMF, 0 °C, 20 min,
then 2-bromo-1-phenylethanone, from 0 °C to rt, 18h. (iv) NaBH4, THF, H2O, 0 °C, then rt, 3h. (v) POCl3/DMF,
CH2Cl2, reflux, 8h. (vi) Method A: anilines, abs. EtOH, reflux, 5h (to obtain compounds 8a,b); Method B:
benzylamine, anhyd. toluene, rt, 24h (to obtain compound 8c).
The synthesis of compounds 8d-j was performed starting from 4-chloro-2-(methylthio)-7H-
pyrrolo[2,3-d]pyrimidine 11, which was alkylated in N7 with sodium hydride and 4-fluorobenzyl
chloride in anhydrous acetonitrile at 50 °C for 2h to give 4-chloro-7-(4-fluorobenzyl)-2-
(methylthio)-7H-pyrrolo[2,3-d]pyrimidine 15. The compound was treated with the suitable amine in
DMSO at 90-130 °C to obtain the desired compounds 8d-j (Scheme 2).
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Cl
N
Cl
N
i
N
ii
N
N
H3CS
H3CS
N
H
F
11
15
8d R = nC4H9
R
N
8e R = 1-pyrrolidino
8f R = 1-piperidino
8g R = 4-morpholino
8h R = NHCH2C6H5
8i R = NHCH2CH2C6H5
8j R = NHCH2CH2C6H4p-Cl
N
N
H3CS
F
8d-j
Scheme 2. Synthetic route for the preparation of 8d-j. Reagents and conditions: (i), NaH, anhyd. CH3CN, 4-
fluorobenzyl chloride, 50 °C, 2h. (ii) amines, DMSO, 90-130 °C, 3-5h.
2.2 Molecular Modeling Studies
A molecular docking protocol has been employed in order to evaluate the ability of the new
synthesized compounds 8a-j to interact with the ATP-binding site of c-Src kinase. For this purpose,
the X-ray crystal structure of Src in a complex with a pyrazolo[3,4-d]pyrimidine derivative, 11V,
(PDB code: 4O2P) [11], previously solved and reported by us, was used for computational studies.
The 4O2P X-ray structure was shown to contain two protein molecules within the crystallographic
cell unit, referred as chain A and chain B, respectively.
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Fig 5. Left) Schematic representation of 11V ligand’s interactions within the ATP-binding site of c-
Src; Right) Binding mode of 11V in 4O2P crystal structure: panel A and panel B show chain A and
chain B, respectively. DFG-motif and Glu310 are displayed as sticks. Hydrogen bonds are reported
as yellow dashes.
Many conformational differences have been observed between the two chains especially in the αC-
helix and in the rearrangement of the activation loop (Fig. 5). The ligand 11V was found to have a
similar binding pose in both chains, but the protein binds the compound assuming two different
conformations. Indeed, in chain A (Fig. 5A) the Glu310 side chain is turned away from the ATP-
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binding site and exposed to the solvent, resulting in a rotation of the αC-helix usually observed in
the inactive conformation of the Src-family members. The chain A conformation appears very
similar to the ones of the inhibited enzyme phosphorylated on Tyr527 (PDB code: 2SRC [34]; PDB
code: 2PTK [35]). Moreover, the N-terminal amino acids of the activation loop are arranged in a
three-turns α-helix that resemble the 2SRC structure. On the contrary, in the chain B Glu310
displays its side chain projected toward the ATP-binding site forming a salt bridge with Lys295
(Fig. 5B). Another important feature is represented by the orientation of the Asp404 belonging to
the DFG motif: in chain A the side chain of Asp404 creates a steric hindrance which reduces the
size of the hydrophobic pocket I, hindering the binding of molecules with an elongated shape.
Taking into account the structural differences between the two structures and the conformational
plasticity of c-Src kinase in the presence of small molecules, both chains have been used for
molecular modeling simulations.
Docking studies have been performed by means of the docking software Glide [36]. Both chain A
and B have been prepared for the docking procedure by using the Protein Preparation Wizard
protocol [37]. The missing residues were replaced and all the loops refined according to a procedure
already reported [11].
The reliability of the docking protocol was assessed by simulating the interactions between 11V and
c-Src and then comparing the modelled complexes with the experimental structures. As a result, the
program was able to reproduce in both the chains the binding mode of 11V.
Compounds 8a-j were drawn, minimized and finally docked into the catalytic site of Src. All the
binding poses have been submitted to a MM-GBSA rescoring. After rescoring, the best docking
poses have been analysed. The best binding modes, in terms of both energies and reliability of the
poses, have been found for compounds 8a-c, whose molecular structures closely resemble the one
of the pyrazolo[3,4-d]pyrimidine 11V. In compounds 8a-c, bearing a 2-chloro-2-phenylethyl chain
in N7, the C4 substituent kept contacts with the hydrophobic region 1 (HR1) in the ATP-binding
site of Src establishing a hydrogen bond through the NH group with the Thr338 side chain.
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Moreover, a cation-π interaction occurs between Lys295 and the anilino ring maintaining the ligand
stable in its binding mode (Fig. 6A).
Fig 6. Best predicted docking poses of compounds 8b (orange, panel A), 8h (deep teal, panel B)
and 8e (pale green, panel C) within the ATP binding site of 4O2P crystal structure chain B.
Compound 8b has been scored as the best compound (Table S2) of the series thanks to the presence
of the chlorine atom in meta position on the R substituent which interacts with Asp404 and Phe405
belonging to the DFG-motif. For compounds 8a-c similar results were obtained in both the chains.
On the other hand, some differences have been observed for derivatives 8h-j between chain A and
B. In chain A, because of the conformation of the Asp404 which partially occupies the entrance of
the HR1, the size of the binding site is reduced and this feature does not allow the program to fit the
ligands into the ATP-binding site with a unique and rational binding pose. Conversely, in chain B
reasonable binding modes with high docking scores were found. In particular, after rescoring, 8h
has been predicted to have a good ∆G of binding (Table S2) forming a cation-π interaction with
Lys295 and profitable hydrophobic contacts with HRI (Fig. 6B). The presence of a substituent in
C4 position with a longer spacer seems to be unfavourable in the case of 8h, if compared with 8b,
because of steric hindrance which does not allow the formation of the hydrogen bond between the
NH group and the side chain of Thr338. The ligand pose results shifted toward the solvent with
hydrophobic interactions, with HR1 mostly stabilizing the binding mode. The negative effect of a
long C4 substituent is more evident in the case of compounds 8i and 8j which partially come out
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from the site. Derivatives 8d-g present a characteristic binding mode, probably because of the more
polar nature of their R substituents, with the N7 group projecting into the HR1 and the R substituent
being solvent exposed (Fig. 6C). Moreover, the pyrrolo-pyrimidine core was found rotated of 180
degrees with the SCH3 group projecting toward the hinge region.
As a final consideration, despite the binding poses of compounds 8a-j present common features
with those of the ligand 11V, pyrrolo-pyrimidines lack the nitrogen atom which usually interacts
with the backbone of Met341 in the pyrazolo[3,4-d]pyrimidine analogues. The absence of this
important point of contact with the hinge region makes our pyrrolo[2,3-d]pyrimidines less active
than their pyrazolo counterpart, but they open the way to the study of a new promising scaffold
which could be further optimized.
2.3. Enzymatic Assays
All synthesized compounds were initially tested in a cell free assay (see Experimental Section) to
evaluate their affinity toward isolated c-Src (Table 1).
In agreement with our computational studies, 8b resulted one of the best compounds of our small
library of pyrrolo[2,3-d]pyrimidines, showing a good inhibitory effect of the enzymatic activity
with a Ki value of 0.7 µM. As expected, 8c, which has a longer side chain as R substituent, or 8a,
which lacks the mCl substituent, did reduce the activity of the enzyme in a less efficient way than
8b. In the series 8d-j the substitution of the 2-chloro-2-phenylethyl side chain in R1 with a shorter
4-fluoro-benzyl group, which lacks the bulky Cl atom, gives the possibility to the compounds with a
longer R substituent to better accommodate into the ATP binding cleft and go deeply into the HR1.
This is confirmed by the fact that 8h also showed a good inhibitory activity against c-Src (0.5 µM),
despite the presence of a long benzyl group as R substituent. In general, for all two series, it was
observed that increasing the length of the linker at C4 position caused a high reduction of the
inhibitory power against c-Src (compare 8c with 8b and 8j with 8h). On the other hand, compound
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8d, bearing a nC4H9 substituent in C4 and a 4-fluoro-benzyl chain in N7, showed to be too small for
effectively bind the ATP cleft of Src (Ki = 3 µM).
Table 1: Structures and inhibitory activity of compounds 8a-j.
R
N
N
H3CS
N
R1
Src (Ki, µM)a
1.5±0.10
0.7±0.20
6.0±0.08
3.0±0.10
2.0±0.30
2.5±0.30
4.7±0.20
0.5±0.06
0.8±0.04
5.0±0.10
U87 (IC50, µM)
27.4±0.16
143.1±0.20
50.7±0.18
20.7±0.38
51.8±0.21
50.7±0.42
20.3±0.15
7.1±0.16
Cmpd
8a
R
R1
NHC6H5
CH2CHClC6H5
CH2CHClC6H5
CH2CHClC6H5
CH2C6H4-pF
CH2C6H4-pF
CH2C6H4-pF
CH2C6H4-pF
CH2C6H4-pF
CH2C6H4-pF
CH2C6H4-pF
NHC6H4-mCl
NHCH2C6H5
nC4H9
8b
8c
8d
8e
1-pyrrolidino
1-piperidino
4-morpholino
NHCH2C6H5
NHCH2CH2C6H5
NHCH2CH2C6H4-pCl
8f
8g
8h
8i
13.3±0.16
46.1±0.08
8j
a
Values are the mean of at least two experiments.
The compounds have been also tested on Fyn, another member of SFKs, and showed a certain
degree of activity, probably due to the similarity among the members of this family of kinases. The
most active compound is 8i which possesses a Ki value of 2 µM on this enzyme.
On the other hand, the compounds resulted inactive when tested on a small panel of kinases,
including c-Kit, Abl, AblT315I, Flt3 and EGFR, demonstrating a degree of selectivity toward c-Src.
2.4 Cytotoxicity Assays on U87 GB cell line
The compounds have been successively tested on U87 GB cell line, that has been chosen because of
Src involvement in this aggressive tumor [38].
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The most active compound was 8h, with an IC50 value of 7.1 µM after 72h, while the other
derivatives resulted less active with IC50 values higher than 10 µM (Fig. 7).
Fig. 7. Viability of U87 cell line evaluated at 72h is reported for the most active compound 8h and
for 8b and 8e as examples of less active compounds of each series.
3. Conclusions
A series of pyrrolo[2,3-d]pyrimidines has been synthesized and tested for their activity against c-
Src. Molecular docking studies together with a MM-GBSA rescoring approach revealed our
compounds possess profitable features for the binding to the catalytic site of c-Src, being able to act
as ATP-competitive inhibitors of the enzyme. Compounds 8a-j have been tested on a small panel of
kinases. As a result, our pyrrolo[2,3-d]pyrimidines demonstrated a certain selectivity for c-Src,
since they did not show any activity against other tyrosine kinases, with the only exception of 8i
which turned out moderately active against Fyn.
The compounds have been finally tested in vitro for their cytotoxicity on U87 GB cell line and 8h
demonstrated to be active with an IC50 value of 7.1 µM.
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The inadequacy of the current therapies against GBM raises the need of new drugs able to inhibit
the growth of this aggressive tumor. In this contest, our pyrrolo[2,3-d]pyrimidine derivatives stand
as a new promising scaffold with a good potency against GBM and therefore worthy of further
investigation.
4. Experimental
4.1 Chemistry. Starting materials were purchased from Aldrich-Italia (Milan, Italy). Melting points
were determined with a Büchi 530 apparatus and are uncorrected. IR spectra were measured in KBr
1
13
with a Perkin-Elmer 398 spectrophotometer. H and C NMR spectra were recorded on a Varian
Gemini 200 (200 MHz) instrument. Chemical shifts are reported as δ (ppm) relative to TMS as the
internal standard, J in Hz. 1H patterns are described using the following abbreviations: s = singlet, d
= doublet, t = triplet, q = quartet, q = quintet, s = sextet, m = multiplet, and br s = broad singlet.
TLC was carried out using Merck TLC plates silica gel 60 F254. Chromatographic purifications
were performed on columns packed with Florisil®, 100-200 mesh, or with Fluka silica gel 60 Å,
220-440 mesh, for flash technique. Analyses for C, H, N and S were within ±0.3% of the theoretical
value. Mass spectra (MS) data were obtained using an Agilent 1100 LC/MSD VL system (G1946C)
with a 0.4 mL/min flow rate using a binary solvent system of 95:5 methanol/water. UV detection
was monitored at 254 nm. MS were acquired in positive and negative modes, scanning over the
mass range 50-1500. The following ion source parameters were used: drying gas flow, 9 mL/min;
nebulizer pressure, 40 psig; drying gas temperature, 350 °C. All target compounds possessed a
purity of ≥95% as verified by elemental analyses by comparison with the theoretical values.
Microwave apparatus: CEM Discover (CEM Corporation, Matthews, NC, USA), single-mode
microwave oven, max emitted power 300 Watt, temperature control by optical fiber, magnetic
stirring of the sample, air cooling system.
4.1.1 2-(Methylthio)-7H-pyrrolo[2,3-d]pyrimidin-4-ol (10).
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Chloroacetaldehyde (50% aqueous solution, 1 mL, 7.87 mmol) was added to a suspension of 6-
amino-2-(methylthio)pyrimidin-4(3H)-one 9 (0.5 g, 3.18 mmol) and sodium acetate (0.88 g, 10.69
mmol) in water (12.5 mL) and the mixture was heated at 80 °C for 2 min in a microwave oven
(open vessel mode, 100 W). After cooling to room temperature, acetone (3 mL) was added and the
obtained solid was filtered. Grey solid (yield 71%, lit. 55%), constituted by a mixture of the C4 keto
1
1
enolic forms, as shown by IR and H NMR data [33]. Mp: 265-267 °C. H NMR (DMSO): δ 3.26
(s, 3H, SCH3), 7.61-7.65 (m, 1H, H-5), 7.75-7.90 (m, 1H, H-6), 9.59 (br s, 1H disappears with
D2O), 9.92 (br s, 1H disappears with D2O). IR (KBr) cm-1: 3330-2900 (OH), 3220 (NH), 1667
(C=O). MS: m/z [M+1]+ 182. Anal. calcd. for C7H7N3OS, C 46.39, H 3.89, N 23.19, S 17.69, found
C 46.51, H 3.96, N 23.15, S 17.68.
4.1.2 4-Chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine (11).
POCl3 (5 mL, 53.7 mmol) and triethylamine (0.2 mL, 1.43 mmol) were added to 2-(methylthio)-
7H-pyrrolo[2,3-d]pyrimidin-4-ol 10 (0.10 g, 0.55 mmol); the mixture was heated at 130 °C for 4h
and then cooled to room temperature. The excess of POCl3 was removed by distillation under
reduced pressure. Ice was then carefully added to the residue and the suspension was extracted with
diethyl ether (3 x 20 mL). The organic phase was washed with H2O (10 mL), dried on MgSO4,
filtered and concentrated under reduced pressure. The crude was purified by column
chromatography (Florisil®, 100-200 mesh), using diethyl ether as the eluent to afford the pure
product 4-chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine (77%, lit. 43% ) [33]. Mp: 207-209
°C. 1H NMR (CDCl3): δ 2.67 (s, 3H, SCH3), 6.56-6.59 (m, 1H, H-5), 7.21-7.23 (m, 1H, H-6). IR
(KBr) cm-1: 3412 (NH), 3122 (CH), 1614 (C=C). MS: m/z [M+1]+ 201. Anal. calcd. for C7H6N3ClS,
C 42.11, H 3.03, N 21.05, S 16.06, found C 42.29, H 3.38, N 21.14, S 16.36.
4.1.3 2-[4-Chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-1-phenylethanone (12).
17

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Sodium hydride (60% dispersion in mineral oil, 0.04 g, 1 mmol) was added in small portions at 0
°C to a solution of 4-chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine 11 (0.2 g, 1 mmol) in
anhydrous DMF and the mixture was stirred at room temperature for 20 min. Then 2-bromo-1-
phenylethanone (0.3 g, 1 mmol) was added dropwise at 0 °C and the reaction was stirred at room
temperature for 18h. Then the mixture was poured into water (20 mL) and extracted with
ethylacetate (3 x 10 mL); the organic phases were washed with water (2 x 10 mL), dried on MgSO4,
filtered and concentrated under reduced pressure. The crude was purified by column
chromatography (Silica gel 220-440 mesh), using CH2Cl2 as the eluent to afford the pure product as
a white solid (47%). Mp: 157-159 °C. 1H NMR (CDCl3): δ 2.65 (s, 3H, SCH3), 5.65 (s, 2H, CH2N),
6.60 (d, J = 3.6, 1H, H-5), 7.11 (d, J = 3.6, 1H, H-6), 7.52-7.56, 7.64-7.67 and 8.03-8.05 (3m, 5H
Ar). IR (KBr) cm-1: 1694 (C=O). MS: m/z [M+1]+ 319. Anal. calcd. for C15H12N3OClS, C 56.69, H
3.81, N 13.22, S 10.09, found C 56.65, H 3.83, N 12.97, S 10.07.
4.1.4 2-[4-Chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-1-phenylethanol (13).
A solution of sodium borohydride (0.26 g, 6.9 mmol) in water (1 mL) was slowly added at 0 °C to a
solution of 2-[4-chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-1-phenylethanone 12 (0.31
g, 1 mmol) in THF (6 mL), and the reaction was stirred at room temperature for 3h. Then the
mixture was poured in water (20 mL) and extracted with diethyl ether (3 x 10 mL); the organic
phases were washed with water (2 x 10 mL), dried on MgSO4, filtered and concentrated under
reduced pressure to obtain an oil which crystallized by adding a 1:1 mixture of ethylic ether and
1
petroleum ether (bp 40-60 °C) to give a white solid (69%). Mp: 157-159 °C. H NMR (CDCl3):
δ
2.65 (s, 3H, SCH3), 3.91 (br s, 1H, OH disappears with D2O), 4.32-4.64 (m, 2H, CH2N), 5.10-5.25
(m, 1H, CHOH), 6.48 (d, J = 3.6, 1H, H-5), 7.03 (d, J = 3.6, 1H, H-6), 7.23-7.47 (m, 5H Ar). IR
(KBr) cm-1: 3500-3200 (OH). MS: m/z [M+1]+ 321. Anal. calcd. for C15H14N3OClS, C 56.33, H
4.41, N 13.14, S 10.03, found C 56.08, H 4.73, N 13.04, S 9.98.
18

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4.1.5 4-Chloro-7-(2-chloro-2-phenylethyl)-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine (14).
The Vilsmeier complex, previously prepared from POCl3 (1.65 mL, 17.6 mmol) and DMF (1.28 g,
17.6 mmol) was added to a suspension of 2-[4-chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-
7-yl]-1-phenylethanol 13 (0.56 g, 1.76 mmol) in CH2Cl2 (10 mL). The mixture was refluxed for 8h.
After cooling to room temperature, the mixture was washed with H2O (2 x 20 mL), dried on
MgSO4, filtered, and concentrated under reduced pressure. The crude oil was purified by column
chromatography (Silica gel 220-440 mesh), using diethyl ether as the eluent, to afford the pure
product as a white solid (92%). Mp: 87-88 °C. 1H NMR (CDCl3): δ 2.65 (s, 3H, SCH3), 4.66-4.80
(m, 2H, CH2N), 5.29-5.34 (m, 1H, CHCl), 6.47 (d, J = 3.6, 1H, H-5), 6.99 (d, J = 3.6, 1H, H-6),
7.22-7.50 (m, 5H Ar). MS: m/z [M+1]+ 339. Anal. calcd. for C15H13N3Cl2S, C 53.26, H 3.87, N
12.42, S 9.48, found C 53.23, H 3.81, N 12.18, S 9.15.
4.1.6 General procedure for compounds 8a,b.
The appropriate aniline (2 mmol) was added to a solution of 4-chloro-7-(2-chloro-2-phenylethyl)-2-
(methylthio)-7H-pyrrolo[2,3-d]pyrimidine 14 (0.33 g, 1 mmol) in absolute ethanol (10 mL) and the
reaction mixture was stirred at reflux for 5h. After cooling, a solid precipitated. It was filtered,
washed with H2O, and recrystallized from absolute ethanol (10 mL) to give the final products as
white solids.
4.1.6.1
7-(2-Chloro-2-phenylethyl)-2-(methylthio)-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-
1
amine (8a). Yield 66%, mp: 157-159 °C. H NMR (CDCl3): δ 2.65 (s, 3H, SCH3), 4.72-5.22 (m,
2H, CH2N), 5.39-5.66 (m, 1H, CHCl), 7.00-7.76 (m, 12H, H-5 + H-6 + 10Ar), 8.40 (br s, 1H, NH
disappears with D2O). IR (KBr) cm-1: 3000-2830 (NH). 13C NMR (MeOD): δ 168.96, 150.48,
146.94, 141.15, 138.23, 129.39, 129.08, 128.86, 127.54, 124.04, 123.66, 120.80, 101.88, 100.01,
60.17, 53.07, 14.82. MS: m/z [M+1]+ 396. Anal. calcd. for C21H19N4ClS, C 63.87, H 4.85, N 14.19,
S 8.12, found C 63.81, H 4.62, N 14.38, S 7.99.
19

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4.1.6.2
N-(3-Chlorophenyl)-7-(2-chloro-2-phenylethyl)-2-(methylthio)-7H-pyrrolo[2,3-
1
d]pyrimidin-4-amine (8b). Yield 61%, mp: 147-149 °C. H NMR (CDCl3):
δ
2.66 (s, 3H, SCH3),
4.64-5.23 (m, 2H, CH2N), 5.41-5.67 (m, 1H, CHCl), 7.00-7.81 (m, 11H, H-5 + H-6 + 9Ar), 9.76 (br
13
s, 1H, NH disappears with D2O). IR (KBr) cm-1: 3000-2800 (NH). C NMR (MeOD): δ 168.96,
150.48, 146.94, 144.13, 138.23, 134.43, 130.17, 129.39, 128.86, 127.54, 124.04, 122.58, 120.89,
120.18, 101.88, 100.01, 60.17, 53.07, 14.82. MS: m/z [M+1]+ 430. Anal. calcd. for C21H18N4Cl2S, C
58.74, H 4.23, N 13.05, S 7.47, found C 58.76, H 4.37, N 13.25, S 7.11.
4.1.7
N-Benzyl-7-(2-chloro-2-phenylethyl)-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (8c).
Benzylamine (0.4 g, 4 mmol) was added to a solution of 4-chloro-7-(2-chloro-2-phenylethyl)-2-
(methylthio)-7H-pyrrolo[2,3-d]pyrimidine 14 (0.3 g, 1 mmol) in anhydrous toluene (5 mL) and the
mixture was stirred at room temperature for 24h. Then, the organic phase was washed with H2O (2
x 10 mL), dried on MgSO4, and concentrated under reduced pressure. The crude oil was purified by
column chromatography (Silica gel 220-440 mesh), using diethyl ether as the eluent. The obtained
oil crystallized as a yellow solid by adding a 1:1 mixture of diethyl ether and petroleum ether (bp
40-60 °C) (81%). Mp: 106-107 °C. 1H NMR (CDCl3): δ 2.65 (s, 3H, SCH3), 4.65-4.71 (m, 4H, CH2N
+ CH2Ar), 5.05 (br s, 1H, NH disappears with D2O), 5.33-5.35 (m, 1H, CHCl), 6.45 (d, J = 3.6, 1H,
H-5), 6.96 (d, J = 3.6, 1H, H-6), 7.18-7.50 (m, 10H Ar). IR (KBr) cm-1: 3100-2900 (NH). 13C NMR
(MeOD): δ 169.80, 153.68, 148.47, 139.85, 138.23, 129.39, 128.86, 128.43, 127.59, 127.54,
126.92, 124.04, 103.94, 100.01, 60.17, 53.07, 43.38, 14.82. MS: m/z [M+1]+ 410. Anal. calcd. for
C22H21N4ClS, C 64.61, H 5.18, N 13.70, S 7.84, found C 64.38, H 5.08, N 13.87, S 7.78.
4.1.8 4-Chloro-7-(4-fluorobenzyl)-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine (15).
Sodium hydride (60% dispersion in mineral oil, 0.1 g, 2.5 mmol) was slowly added to a solution of
4-chloro-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine 11 (0.19 g, 1 mmol) in anhydrous acetonitrile
20

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freshly distilled (5 mL) and the mixture was stirred at room temperature for 20 min. Then 4-
fluorobenzyl chloride (0.14 g, 1 mmol) solved in anhydrous acetonitrile (5 mL) was added
dropwise. The mixture was heated at 50 °C for 2h, then cooled to room temperature and filtered to
eliminate the little amount of solid obtained. The solution was then evaporated under reduced
pressure and the crude was purified by column chromatography (Florisil®, 100-200 mesh), using
diethyl ether as the eluent to afford pure 4-chloro-7-(4-fluorobenzyl)-2-(methylthio)-7H-
pyrrolo[2,3-d]pyrimidine 15 as light yellow solid (36%). Mp: 99-100 °C. 1H NMR (CDCl3): δ 2.65
(s, 3H, SCH3), 5.36 (s, 2H, CH2N), 6.53 (d, J = 3.6, 1H, H-5), 6.96-7.04 and 7.14-7.24 (2m, 5H,
4Ar + H-6). MS: m/z [M+1]+ 309. Anal. calcd. for C14H11N3ClFS, C 54.63, H 3.60, N 13.65, S
10.42, found C 54.41, H 3.69, N 13.42, S 10.15.
4.1.9 General procedure for the synthesis compounds 8d-j.
The suitable amine (5 mmol) was added to a solution of 4-chloro-7-(4-fluorobenzyl)-2-
(methylthio)-7H-pyrrolo[2,3-d]pyrimidine 15 (0.31 g, 1 mmol) in DMSO (5 mL) and the mixture
was heated at 90-130 °C (the temperature depended on the boiling point of the amine) for 3-5h,
monitoring by TLC the product formation. Then the mixture was cooled to room temperature,
poured in ice-water (50 mL), extracted with CH2Cl2 (3 x 20 mL), dried on MgSO4, filtered and
concentrated under reduced pressure. The crude was purified by column chromatography (Florisil®,
100-200 mesh), using CHCl3 as the eluent to afford the pure products.
4.1.9.1 N-Butyl-7-(4-fluorobenzyl)-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (8d).
1
Yield: 42%, (yellow oil). H NMR (CDCl3): δ 0.89 (t, J = 7.2, 3H, CH3), 1.37 (sx, J = 7.2, 2H,
CH2CH3), 1.59 (quint, J = 7.2, 2H, CH2CH2CH3), 2.52 (s, 3H, SCH3), 3.53 (q, J = 7.2, 2H,
CH2NH), 5.04 (br s, 1H, NH disappears with D2O), 5.21 (s, 2H, CH2N), 6.22 (d, J = 3.6, 1H, H-5),
6.65 (d, J = 3.6, 1H, H-6), 6.84-6.94 and 7.07-7.21 (2m, 4H Ar). IR (KBr) cm-1: 3382 (NH). 13C
NMR (MeOD): δ 170.01, 162.67, 154.48, 147.83, 132.42, 130.45, 125.95, 115.11, 103.01, 101.64,
21

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50.49, 43.71, 30.87, 20.23, 14.82, 14.02. MS: m/z [M+1]+ 345. Anal. calcd. for C18H21N4FS, C
62.76, H 6.15, N 16.27, S 9.31, found C 62.58, H 6.45, N 16.21, S 9.08.
4.1.9.2 7-(4-Fluorobenzyl)-2-(methylthio)-4-pyrrolidin-1-yl-7H-pyrrolo[2,3-d]pyrimidine (8e).
1
Yield: 52%, (yellow solid), mp 149-150 °C. H NMR (CDCl3): δ 1.86-2.00 (m, 4H, 2CH2 pyrr.),
2.51 (s, 3H, SCH3), 3.66-3.78 (m, 4H, 2CH2N pyrr.), 5.22 (s, 2H, CH2N), 6.39 (d, J = 3.4, 1H, H-5),
13
6.61 (d, J = 3.4, 1H, H-6), 6.83-6.96 and 7.07-7.21 (2m, 4H Ar). C NMR (MeOD): δ 167.66,
162.67, 155.30, 151.11, 132.42, 130.45, 123.66, 115.11, 110.00, 101.76, 65.59, 50.49, 45.92, 14.82.
MS: m/z [M+1]+ 343. Anal. calcd. for C18H19N4FS, C 63.13, H 5.59, N 16.36, S 9.36, found C
63.25, H 5.69, N 16.27, S 9.06.
4.1.9.3 7-(4-Fluorobenzyl)-2-(methylthio)-4-piperidin-1-yl-7H-pyrrolo[2,3-d]pyrimidine (8f).
1
Yield: 46%, (yellow solid), mp 73-74 °C. H NMR (CDCl3): δ 1.51-1.72 (m, 6H, 3CH2 pip.), 2.50
(s, 3H, SCH3), 3.74-3.88 (m, 4H, 2CH2N pip.), 5.23 (s, 2H, CH2N), 6.34 (d, J = 3.6, 1H, H-5), 6.65
(d, J = 3.6, 1H, H-6), 6.83-6.98 and 7.08-7.21 (2m, 4H Ar). 13C NMR (MeOD): δ 167.66, 162.67,
155.30, 151.11, 132.42, 130.45, 123.66, 115.11, 110.00, 101.76, 50.49, 48.07, 25.08, 23.42, 14.82.
MS: m/z [M+1]+ 357. Anal. calcd. for C19H21N4SF, C 64.02, H 5.94, N 15.72, S 9.00, found C
64.24, H 5.94, N 15.70, S 9.07.
4.1.9.4 7-(4-Fluorobenzyl)-2-(methylthio)-4-morpholin-4-yl-7H-pyrrolo[2,3-d]pyrimidine (8g).
1
Yield: 51%, (yellow solid), mp 115-116 °C. H NMR (CDCl3): δ 2.50 (s, 3H, SCH3), 3.75 (t, J =
5.4, 4H, 2 CH2N morph.), 3.87 (t, J = 5.4, 4H, 2 CH2O morph.), 5.24 (s, 2H, CH2N), 6.33 (d, J =
3.6, 1H, H-5), 6.69 (d, J = 3.6, 1H, H-6), 6.84-6.98 and 7.06-7.23 (2m, 4H Ar). 13C NMR (MeOD):
δ 168.25, 162.67, 153.43, 151.84, 132.42, 130.45, 123.66, 115.11, 108.99, 101.76, 50.49, 49.09,
26.01, 14.82. MS: m/z [M+1]+ 359. Anal. calcd. for C18H19N4FSO, C 60.32, H 5.34, N 15.63, S
8.95, found C 60.15, H 5.44, N 15.63, S 8.70.
4.1.9.5 N-Benzyl-7-(4-fluorobenzyl)-2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (8h).
Yield: 36%, (yellow solid), mp 95-96 °C. 1H NMR (CDCl3): δ 2.51 (s, 3H, SCH3), 4.76 (d, J = 5.8,
2H, CH2NH), 5.22 (s, 2H, CH2N), 6.17 (d, J = 3.6, 1H, H-5), 6.62 (d, J = 3.4, 1H, H-6), 6.83-6.97
22

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and 7.07-7.38 (2m, 9H Ar), 7.73 (br s, 1H, NH disappears with D2O). IR (KBr) cm-1: 3228 (NH).
13C NMR (MeOD): δ 169.91, 162.67, 153.45, 147.86, 139.85, 132.42, 130.45, 128.43, 127.59,
126.92, 125.95, 115.11, 103.57, 101.64, 50.49, 43.38, 14.82. MS: m/z [M+1]+ 379. Anal. calcd. for
C21H19N4FS, C 66.64, H 5.06, N 14.80, S 8.47, found C 66.52, H 5.18, N 14.64, S 8.17.
4.1.9.6
7-(4-Fluorobenzyl)-2-(methylthio)-N-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (8i). Yield: 37%, (brown solid), mp 61-68 °C. 1H NMR (CDCl3): δ 2.56 (s, 3H, SCH3), 2.92
(t, J = 6.8, 2H, CH2Ar), 3.81 (q, J = 6.8, 2H, CH2NH), 5.07 (br s, 1H, NH disappears with D2O),
5.21 (s, 2H, CH2N), 6.15 (d, J = 3.4, 1H, H-5), 6.64 (d, J = 3.4, 1H, H-6), 6.83-6.98 and 7.06-7.33
13
(2m, 9H Ar). IR (KBr) cm-1: 3317 (NH). C NMR (MeOD): δ 170.01, 162.67, 154.48, 147.83,
139.08, 132.42, 130.45, 129.19, 128.83, 126.13, 125.95, 115.11, 103.01, 101.64, 50.49, 43.75,
35.64, 14.82. MS: m/z [M+1]+ 393. Anal. calcd. for C22H21N4FS, C 67.32, H 5.39, N 14.27, S 8.17,
found C 67.34, H 5.60, N 14.36, S 8.00.
4.1.9.7
N-[2-(4-Chlorophenyl)ethyl]-7-(4-fluorobenzyl)-2-(methylthio)-7H-pyrrolo[2,3-
1
d]pyrimidin-4-amine (8j). Yield: 31%, (yellow solid), mp 141-143 °C. H NMR (CDCl3): δ 2.55
(s, 3H, SCH3), 2.90 (t, J = 6.8, 2H, CH2Ar), 3.83 (q, J = 6.8, 2H, CH2NH), 5.15 (br s, 1H, NH
disappears with D2O), 5.25 (s, 2H, CH2N), 6.18 (d, J = 3.4, 1H, H-5), 6.67 (d, J = 3.4, 1H, H-6),
13
6.85-7.01 and 7.10-7.37 (2m, 8H Ar). IR (KBr) cm-1: 3310 (NH). C NMR (MeOD): δ 170.01,
162.67, 154.48, 147.83, 136.26, 132.64, 132.42, 130.45, 130.05, 129.35, 125.95, 115.11, 103.01,
101.64, 50.49, 43.75, 35.64, 14.82. MS: m/z [M+1]+ 428. Anal. calcd. for C22H20N4ClFS, C 61.89,
H 4.72, N 13.12, S 7.51, found C 61.67, H 4.57, N 13.10, S 7.33.
4.2 Molecular Modeling
4.2.1 Proteins Preparation. Crystal structures of c-Src in complex with 11V (PDB code: 4O2P) has
been previously published by our group and used for the present study [11]. After removal of bound
ligand, both chain A and chain B were prepared by using the Protein Preparation Wizard [36]
workflow, from Schrödinger Suite. The ionization and tautomeric states of His, Asp, Glu, Arg and
23

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Lys were adjusted to match a pH of 7.4. Next, optimization of the hydrogen-bonding network was
obtained by reorienting hydroxyl and thiol groups, amide groups of Asn and Gln, and His ring.
Finally, the systems were refined by running a restrained minimization (OPLS3 force field) which
was stopped when RMSD of heavy atoms reached 0.30 Å, the specified limit by default.
4.2.2 Ligand Preparation. All compounds were drawn and minimized using Maestro 10.4 and
Macromodel 11.0 (Schrödinger suite) [37], respectively. Furthermore, Ligprep 3.6 [37] was used to
predict ionization and tautomeric states for the ligands using a pH of 7±0.5.
4.2.3 Docking Studies. Docking simulations were performed using the Glide program [36] within
the ATP binding sites of Src. The prepared protein systems were used to generate the receptor grids,
and no scaling was done for van der Waals radii of nonpolar receptor atoms. A grid box of default
size was centered on the X-ray ligand. No constraints were included during grid generation while
the rotation of the hydroxyl groups was allowed. After grid preparation, compounds were flexibly
docked and scored using the Glide standard-precision (SP) mode, treating the proteins as rigid.
Docking experiments were performed using 0.80 factor to scale van der Waals radii of the nonpolar
ligand atoms with partial atomic charge less than 0.15.
4.2.4 MM-GBSA. For the MM-GBSA analysis Prime 4.2 [37] program has been used. The binding
free energy (∆Gbind) for each compound was calculated using the following equation:
Eq 1.
∆Gbind = ∆GMM + ∆Gsovl + ∆GSA
where ∆GMM is the difference in the minimized energies between the Src-ligands complexes and the
sum of the energies of the apo enzyme and the free compound. ∆Gsolv is the difference in the GBSA
solvation energy of the Src-ligand complexes and the sum of the solvation energies for the unbound
enzyme and inhibitor. ∆GSA is the difference in surface area energies for the complexes and the sum
of the surface area energies for the apo-Src and unbound inhibitor.
24

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5. Biological Assays
5.1 In vitro kinase inhibition assays
Chemicals were purchased from Sigma-Aldrich, pure ATP was from Promega and P33-g-ATP was
from Hartman Analytic. Recombinant full length, HIS6-tagged Src and Fyn were purchased from
Merk Millipore; recombinant GST tagged, C-terminal fragment (696-end), EGFR was also from
Merk Millipore; recombinant GST-HIS6 C-terminal fragment (544-976) Kit was purchased from
ProQinase.
Assays conditions. Fyn and Src reactions were performed in 50 mM MOPS/NaOH, pH 7.0, 0.1 mM
EDTA, 0.0013% or 0.00087% NP40 respectivelly, 0.1 mM Na3VO4, 10% DMSO, 3 mM
MnCl2/MgCl2, 100 µM ATP/[γ-33P]ATP, 250 µM of the Src substrate peptide
KVEKIGEGTYGVVYK, and 30 ng active enzyme.
Kit and Flt3 reaction were performed in 60 mM HEPES/NaOH pH 7.5, 0.0013% NP40, 3 mM
Na3VO4, 10% DMSO, 3 mM MgCl2/MnCl2, 1.2 mM DTT, 50 mg/ml PEG6.000, 20 µM and 2 µM
ATP/[γ-33P]ATP respectively. For only Kit reaction, 100 µM HER2 substrate peptide
GGMEDIYFEFMGGKKK and 50 ng of active enzyme were used, while, for Flt3 reaction, 50 µM
peptide substrate KVEKIGEGTYGVVYK and 50 ng of enzyme were used.
EGFR reaction were performed in 20 mM HEPES/NaOH pH 7.5, MOPS/NaOH, pH 7.0, 0.1 mM
EDTA, 0.01% NP40, 5% glycerol, 0.1 mM 2-mercaptoethanol, 1 mg/ml BSA, 10 mM
MnCl2/MgCl2, 0.8M (NH4)SO4, 16 µM ATP/[γ-33P]ATP, 400 µM angiotensin 2 (substrate) and 35
ng of active enzyme.
To avoid plastic adsorption of enzymes and peptide, all reactions were performed using protein
low-binding tubes (LoBind, Eppendorf). All reactions were performed in 10 ml at 30 °C for 10 min.
Reactions were stopped by adding 5 ml of phosphoric acid 0.5%. Aliquots (10 mL) were transferred
on P30 Filtermat (PerkinElmer), washed five times with 75 mM phosphoric acid for 5 min and once
with acetone for 4 min. The filter was dried and transferred to a sealable plastic bag, and
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scintillation cocktail (4 mL) was added. Spotted reactions were read in a scintillation counter
(Trilux, PerkinElmer).
ID50 values were obtained according to Equation (2), where v is the measured reaction velocity, V is
the apparent maximal velocity in the absence of inhibitor, I is the inhibitor concentration, and ID50
is the 50% inhibitory dose.
Eq. 2
v = V/{1+(I/ID50)}
5.2 Cell viability
In vitro experiments were carried out using the Glioblastoma cell line U87 MG. Cells were
purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were
cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS, 2 mM L-
glutamine and 10000 units/mL Penicillin/Streptomycin at 37 °C in 5% CO2 atmosphere.
Briefly, 2.5 × 104 U87 MG cells were plated in 12-well plates and incubated with compounds in
DMSO at increasing concentrations (0.1, 1.0, 10 and 100 µM) for 24, 48 and 72h at 37 °C in 5%
v/v CO2. Cell number and viability were evaluated using Z2 Coulter Counter (Beckman Coulter).
IC50 (drug concentration that determined the 50% of growth inhibition) was calculated by GraphPad
Prism 6.0 software using the best fitting sigmoid curve.
Acknowledgments
Lead Discovery Siena s.r.l. is acknowledged for financial support.
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