Design, synthesis, and in vitro activity of pyrazine compounds

Article abstract of DOI:10.3390/molecules24234389

Despite the fact that there are several anticancer drugs available, cancer has evolved using different pathways inside the cell. The protein tyrosine phosphatases pathway is responsible for monitoring cell proliferation, diversity, migration, and metabolism. More specifically, the SHP2 protein, which is a member of the PTPs family, is closely related to cancer. In our efforts, with the aid of a structure-based drug design, we optimized the known inhibitor SHP099 by introducing 1-(methylsulfonyl)-4-prolylpiperazine as a linker. We designed and synthesized three pyrazine-based small molecules. We started with prolines as cyclic amines, confirming that our structures had the same interactions with those already existing in the literature, and, here, we report one new hydrogen bond. These studies concluded in the discovery of methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride as one of the final compounds which is an active and acceptable cytotoxic agent.

Full text of DOI:10.3390/molecules24234389

molecules
Article
Design, Synthesis, and In Vitro Activity of
Pyrazine Compounds
Panagiotis Parsonidis 1, Mahammad Shaik 2, Athanasia Panagiota Serafeim 1, Ioanna Vlachou 1,
Vasiliki Daikopoulou 1 and Ioannis Papasotiriou 3,
*
1
RGCC S.A. Industrial Area of Florina, GR53100 Florina, Greece;
parsonidis.panagiotis@rgcc-genlab.com (P.P.); athanasia.serafeim@rgcc-genlab.com (A.P.S.);
vlachou.ioanna@rgcc-genlab.com (I.V.); daikopoulou.vasiliki@rgcc-genlab.com (V.D.)
Medicinal Chemistry Division, Rgcc India Private Limited, Hyderabad 500090, India;
shaik.mahammad@rgcc-india.com
RGCC International GmbH, Baarerstrasse, 956301 Zug, Switzerland
Correspondence: office@rgcc-genlab.com or papasotiriou.ioannis@rgcc-international.com;
Tel.: +30-2385041950
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 12 November 2019; Accepted: 29 November 2019; Published: 1 December 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Despite the fact that there are several anticancer drugs available, cancer has evolved
using different pathways inside the cell. The protein tyrosine phosphatases pathway is responsible
for monitoring cell proliferation, diversity, migration, and metabolism. More specifically, the
SHP2 protein, which is a member of the PTPs family, is closely related to cancer. In our efforts,
with the aid of a structure-based drug design, we optimized the known inhibitor SHP099 by
introducing 1-(methylsulfonyl)-4-prolylpiperazine as a linker. We designed and synthesized
three pyrazine-based small molecules. We started with prolines as cyclic amines, confirming
that our structures had the same interactions with those already existing in the literature,
and, here, we report one new hydrogen bond. These studies concluded in the discovery of
methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride as one of the final
compounds which is an active and acceptable cytotoxic agent.
Keywords: structure-based drug design; pyrazine compounds; protein tyrosine phosphatases
1. Introduction
The definition of cancer is that it is a disease caused by an uncontrolled division of abnormal cells
in a part of the body. According to the World Health Organization [1], 9.6 million people worldwide
are estimated to have died from cancer in 2018, making cancer the second most deadly disease globally.
The most common cancers are lung (2.09 million cases), breast (2.09 million cases), and colorectal
(1.8 million cases). Thus, discovery of novel drugs for the treatment of cancer patients is vital.
The activation of the PTP’s (Protein tyrosine phosphatases) pathway in human cancers has
triggered the development of a variety of pharmacological inhibitors targeting catalytic sites of the
SHP2 cascade. The SHP2 protein belongs to the protein tyrosine phosphatases group of enzymes. It is
a non-receptor protein which is constituted from two SH2 (N-terminal Src homology 2) domains, a
PTP domain, and a C-terminal tail. The activity of SHP2 is auto-inhibited, as the N-SH2 domain is
binding with the PTP domain [2]. The upregulation expression of the PTPN11 gene that encodes SHP2
protein exists in melanoma [3,4], liver cancer [5,6], and lung cancer [7].
More specifically, the SHP2 oncogene activates the RAS-ERK signaling pathway, so it can regulate
cancer cell survival and proliferation [
8,9]. In 2016, Fortanet et al. [10] revealed a new allosteric
binding site of SHP2 with X-ray analysis. After structure-based drug design, they synthesized a small
Molecules 2019, 24, 4389; doi:10.3390/molecules24234389
www.mdpi.com/journal/molecules

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molecule, SHP099. The same molecule was used by Chen’s group [11], who reported that receptor
tyrosine kinases-driven cancer cells depend on SHP2 survival. One year later, LaRochelle et al. [12
]
followed an alternative route to find allosteric inhibitors by making use of a partially active cancer
mutant, SHP2F285S. In recent years, many different attempts have been performed for the discovery of
new SHP2 inhibitors [13]. Cyanoacrylamides [14], 6-amino-3-methylpyrimidinones [15], substituted
thiazoles [16], and fused bicyclic compounds [17] have shown interesting results in SHP2 inhibition
(Figure 1).
In order to evaluate and determine the utility of substituted pyrazine compounds, such as
SHP2 inhibitors, we built a pharmacophore model using the highly selective SHP2 allosteric
inhibitor SHP099-SHP2 complex. In the present work, we designed three small molecules using
structure-based drug design. The substituted pyrazine compounds incorporated with novel 1-(methyl
sulfonyl)-4-prolylpiperazine, as a linker to amides and sulphonamides, and the cytotoxic effect on
different human cancer cell lines were examined.
O
Cl
H2N
Cl
HN
NH2
N
N
Br
N
N
S
N
N
Compound 12
CF3
SHP099
Compound 23
NH2
NH2
S
N
N
NH
Cl
N
H2N
NH2
O
H
N
O
N
N
N
N
O
N
N
O
SHP394
SHP389
Figure 1. The Tyrosine phosphatase SHP2 allosteric inhibitors according to the literature.
2. Results
2.1. Structure Retrieval and Validation
The three-dimensional structure of SHP2 was downloaded from the Research Collaboratory
for Structural Bioinformatics (RCSB) Protein Data Bank and used for the structure-guided design of
low molecular weight organic compounds [18]. The crystal structure (PDB ID: 5EHR) was solved
through X-ray crystallography with a resolution of 1.7 Å and contained 526 amino acids which covered
87.8% of the canonical protein sequence (UniProtKB ID: Q06124) [11]. The quality of the crystal
structure was validated using the SWISS-MODEL Structure Assessment tools [19]. Ramachandran
plot analysis showed that 96.62% of the residues were within energetically favored regions, while only
0.85% (VAL505, LYS324, GLU313, GLY115) were Ramachandran outliers (Figure 2A) [20]. The outliers
were found adjacent to the gaps in the protein structure. The Qualitative Model Energy Analysis 4
(QMEAN4) global score of 5EHR indicates that the overall quality of the structure was good when
compared to experimental structures of similar size (Figure 2B) [21]. Assessment of the local quality of
the structure showed that only a few compounds were characterized by low quality (Figure 2C).

Molecules 2019, 24, 4389
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A
B
C
Figure 2.
ψ
(
A
) Ramachandran plot analysis to assess the chemical correctness of backbone
angles of SHP2 structure 5EHR. ( ) QMEAN4-based comparison plot. The structure of 5EHR is
) QMEANDisCo-based local quality plot. The threshold of 0.6 is indicated
ϕ and
B
indicated using a red star. (
C
by a black line below which low-quality residues are found.
The allosteric inhibitor SHP099 was co-crystallized with the protein structure of SHP2. The SHP099
(Chemical name: 6-(4-azanyl-4-methyl-piperidin-1-Yl)-3-(2,3-bis(chloranyl)phenyl)pyrazin-2-amine) is
a highly potent and selective allosteric inhibitor of SHP2 with an IC50 value of 0.071 µM. The inhibitor
binds to the interface of the N-terminal SH2, C-terminal SH2, and PTP domains and stabilizes an
auto-inhibited conformation of SHP2 [11]. We used the above protein structure for the structured-guided
design of novel allosteric SHP2 inhibitors.
2.2. Small Molecule Docking
An in silico study based on molecular docking was implemented to explore the interactions
between the newly synthesized small molecules and the allosteric binding site on SHP2. Docking
was performed against the 3D structure of SHP2, and the docking scores and binding poses of the
compounds were obtained. Based on a co-crystallized ligand, SHP099, several compounds were
designed in order to improve the binding affinity. To enhance the affinity between the binding
pocket and the compounds, the third ring was changed while the aminopyrazine moiety and the
dichlorophenyl moiety were kept identical to the reference compound. That design path was followed,
because the two last moieties had the strongest bonds in the binding pocket, while the third part
could be improved. Compounds 10
, 18, and 19 (Scheme 1) showed scores comparable to that of the
co-crystallized ligand, SHP099. The models of the protein–ligand complexes reveal the interactions
between compounds 10, 18, and 19 and the surrounding residues of the binding pocket (Figure 3).
All three compounds form a hydrogen bond with Glu250 of SHP20s PTP domain and a hydrogen bond
with Thr218 that lies on the loop between domains C-SH2 and PTP. Hydrophobic interactions between

Molecules 2019, 24, 4389
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the dichlorophenyl ring and residues of the PTP domain are shared with Thr218 which lies on the loop
between domains C-SH2 and PTP. Hydrophobic interactions between the dichlorophenyl ring and the
surrounding residues further aid the protein–ligand interactions.
A
B
Glu250
Glu250
Thr218
Arg111
Phe113
D
C
Glu250
Glu250
Thr218
Thr218
Figure 3.
(
A
) Docked conformation of SHP099, (
B) docked conformation of compound 10, (C) docked
conformation of compound 18, and (
D) docked conformation of compound 19. The residues in grey
form hydrogen bonds with the compound.
19
10
18
Scheme 1. Compounds after docking evaluation.
2.3. Synthetic Strategy
The synthesis of compound 10 (Scheme 2) started with the Suzuki–Miyaura coupling between
(2,3-dichlorophenyl) boronic acid ( ) and 3-bromo-6-chloropyrazin-2-amine ( ), affording intermediate
) in 74% yield, and then protection of amine on pyrazine with di-tert-butyl dicarbonate gave the
2
1
(3

Molecules 2019, 24, 4389
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di-protected intermediate (
4
) in 60% yield. Then, a SNAr reaction on this biphenyl intermediate with
l-proline methyl ester (5) provided the proline ester intermediate (6) with 47% yield. Ester hydrolysis
with base in homolytic mixture of water and THF (tetrahydrofuran) ended up with acid intermediate
(7) in 54% yield. Amide coupling with l-proline methyl ester (
5) resulted in proline amide intermediate
(8
) with 49% yield. Final deprotection using 4 M HCl in MeOH gave a 36% yield of compound
9,
which, upon reduction with LAH (lithium aluminum hydride) in THF, afforded compound 10 as free
base after acid/base extraction with very low yield (13%).
HO
OH
H2N
N
N
Cl
B
Cl
Cl
N
N
Br
Pd(PPh3)4
2M Na2CO3,
THF/H2O, reflux,N2
74%
Cl
NH2
Cl
Cl
1
2
3
O
O
Cl
N
O
O
O
O
O
O
N
N
N
Cl
O
O
NH
N
N
N
N
HCl
5
Boc2O
O
Cs2CO3, DMSO, 85 oC
Et3N, DMAP, R.T.
60%
O
Cl
Cl
Cl
4
6
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
NH
O
LiOH·H2O
HCl
5
N
OH
N
O
O
THF/H2O, R.T.
HATU, DIPEA, DMF, R.T
O
Cl
Cl
O
Cl
Cl
8
7
HCl
H2N
N
N
HCl
H2N
N
N
N
1 M LAH in THF
0 oC to R.T.
N
N
N
Methanolic HCl
R.T.
O
Cl
O
Cl
OH
Cl
O
Cl
10
9
Scheme 2. Synthesis of compound 10.
The synthesis of compounds 18 and 19 (Scheme 3) started with the sulphonamide synthesis on
tert-butyl piperazine-1-carboxylate (11) with methane sulfonyl chloride and trifluoromethane sulfonic
anhydride and ended up with intermediates 12 and 13. Deprotection with 4 M methanolic HCl
resulted in intermediates 14 and 15 with 50% and 58% yields, respectively. Amide coupling between
(6-((ditert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)proline (7) and intermediates 14
and 15 resulted in intermediates 16 and 17 in 40% and 45% yields, accordingly. Final deprotection of
amine on pyrazine gave the final compound 18 with a 40% yield and compound 19 with a 35% yield.

Molecules 2019, 24, 4389
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R
N
R
N
H
N
i. Et3N, DCM, 0 oC to R.T.
ii. DIPEA, DCM, 50 o
4 M HCl in MeOH, N2
N
N
C
N
H.HCl
O
O
O
O
14 R = SO2CH3; 50%
15 R = SO2CF3; 58%
i. 12 R = SO2CH3; 45%
ii. 13 R = SO2CF3; 38%
11
R
N
O
O
O
O
O
O
ClH.HN
O
N
O
N
N
N
N
N
N
N
N
OH
HATU, DIPEA, DMF, R.T.
O
O
N
R
Cl
Cl
Cl
Cl
16 R = SO2CH3; 40%
17 R = SO2CF3; 45%
7
ClH.H2N
N
N
ClH.H2N
N
N
N
F
4M HCl in MeOH, R.T. N2
F
O
N
N
O
O
N
F
N
O
S
N
S
Cl
O
Cl
O
Cl
Cl
18
40%
19
35%
Scheme 3. Synthesis of compounds 18 and 19.
The synthesis of compound 10 was performed by another synthetic route in order to increase
the final yield. We succeeded to synthesize two different intermediates which were tested for their
biological activity. However, we did not receive the final product (compound 10) with this synthetic
route. The synthesis of
intermediate ( ) with l-prolinol (17) and which provided the pyrazine ortho prolinol compound
a 60% yield. Compound on deprotection with 4 M methanolic in MeOH gave compound as yellow
colored hydrochloride salt with a 45% yield.
5
and
6
(Scheme 4) started with an SNAr reaction on the Boc protected biphenyl
4
5
with
5
6
OH
O
O
O
O
O
O
N
O
N
H
N
N
N
O
N
N
N
Cl
L-Prolinol (20)
OH
Cs2CO3, DMSO, 85 o
C
Cl
Cl
Cl
Cl
4
21
ClH.H2N
N
N
N
4M HCl in MeOH
R.T.
OH
Cl
Cl
22
Scheme 4. Synthesis of intermediates 21 and 22.

Molecules 2019, 24, 4389
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2.4. Biological Effect of Intermediates (21, 22) in Human Cancer Cell Lines
Cytotoxic effect of the first two intermediates on MCF7, MDA-MB-231, HCT116, and MOR was
evaluated using the MTT assay. Time- (24, 48, 72 h) and dosage- (0.001–100 M) dependent inhibition of
the viability of cancer cells was estimated. According to our results, at 48 h, MCF7 showed a statistically
significant decrease in viability at 0.1 M (30%) and at 0.01 M (42%) of intermediate 21. At 48 h, there
was a significant effect on HCT116 viability with 0.1 M (17%) and 0.01 M (16%) of intermediate 21.
µ
µ
µ
µ
µ
Intermediate 21 did not have any statistically significant inhibition in cell proliferation of MDA-MB-231
and MOR at all time points. There was no statistically significant result on MCF7 after treatment with
intermediate 22 at all time points. Human breast adenocarcinoma cell line MDA-MB-231 appeared
to have significantly reduced viability at 72 h with 1
µM (20%) and 0.1
µM (15%) of intermediate 22.
There was a significant effect on HCT116 viability at 48 h with 1
µM (23%) and at 72 h with 0.1 µM
(31%) of intermediate 22. The significant effect on MOR cell proliferation observed at 24 h with 0.1
(25%) and with 0.01 µM (23%) of intermediate 22.
µM
2.5. Biological Effect of Intermediate 9 and final 10 in Human Cancer Cell Lines
Intermediate
the effect they might have on the four human cancer cell lines. Intermediate
effect on MCF7 proliferation at 24 h with 0.1 M (17%) with CVE, at 48 h with 0.001 µM (27%) with
MTT and with 0.001 µM (9%) with CVE, and at 72 h with 0.001 M (12%), 0.01 M (11%), and 1 µM
(9%) MTT and with 0.1 M (7%) and 1 µM (10%) SRB. The two concentrations that are highlighted in
bold (i.e., 0.001 and 1 M) showed significant effects at the same time point at two of the three assays.
There was no significant effect of intermediate on MDA-MB-231 at all time points and all assays.
Human colon cancer cell line HCT116 showed significant reduced viability after the treatment with
intermediate only at the MTT assay at 24 h with 0.01 M (18%) and 0.1 M (16%). Intermediate
significantly decreased the viability of MOR at 24 h with 0.001 M (20%) and 0.1 M (13%) MTT, at 48 h
with 0.001 M (19%), 0.01 µM (16%) MTT, and 0.01 µM (13%) SRB, and at 72 h with 0.001 M (30%),
0.01 µM (31%), 0.1 µM (18%) MTT, with 0.1 µM (9%) CVE and with 0.01 µM (25%) SRB. Similarly, the
9
and final 10 compoundswere tested with MTT, SRB, and CVE assays to determine
9
(Table 1) had a significant
µ
µ
µ
µ
µ
9
9
µ
µ
9
µ
µ
µ
µ
concentrations highlighted in bold (i.e., 0.01 and 0.1 µM) showed significant effects at the same time
point for two of the three assays.
Table 1. Cytotoxicity (%) results of intermediate 9 against human cancer cell lines according to viability
assays (i.e., MTT, SRB, CVE). There were no statistically significant effects against the MDA-MB-231
cell line at any assay and at any time point.
24h
SRB
48h
72h
SRB
Intermediate 9
MCF7
MTT
-
CVE
MTT
SRB
CVE
MTT
CVE
0.001 µM
(12%),
0.01 µM
0.1 µM
(7%),
1 µM
(10%)
0.1 µM
(17%)
0.001 µM
(27%)
0.001
µM
-
-
-
(9%)
(11%), 1
(9%)
µM
0.01 µM
(18%), 0.1
(16%)
HCT116
MOR
µ
M
-
-
-
-
-
-
-
-
-
0.001 µM
(19%),
0.01 µM
(16%)
0.001 µM
(30%),
0.01 µM
(31%)
0.001 µM
(20%), 0.1
(13%)
0.01 µM
(13%)
0.01 µM
(25%)
0.1 µM
(9%)
µM
Final compound 10 (Table 2) demonstrated a significant effect on MCF7 viability at 24 h with
0.001 M (13%) and 0.1 M (20%) MTT and 1 M (10%) CVE, at 48 h with 0.01 M (6%) CVE, and at
72 h with 0.001 M (8%), 0.01 M (11%), 0.1 M (15%), and 1 M (8%) MTT. The effect on MDA-MB-231
viability was observed at 24 h with 0.01 M (16%) and 0.1 M (30%) MTT and at 72 h with 0.1 M (13%)
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ

Molecules 2019, 24, 4389
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MTT. The MTT was the only assay that gave statistically significant results on HCT116 proliferation at
24 h with 0.001 M (19%), 0.01 M (14%), 0.1 M (32%) and at 72 h with 0.01 M (25%) and 0.1
(22%). No significant effect with the CVE assay was observed on MOR viability. The MTT showed
significant effects at 24 h with 0.001 M (17%), 0.01 M (28%), and 0.1 M (27%) and at 48 h with
0.01 M (31%) and 0.1 M (23%), and at 72 h with 0.001 M (25%), 0.01 M (38%), and 0.1 M (33%).
With the SRB assay, the only significant effect was at 48 h with 1 µM (24%).
µ
µ
µ
µ
µM
µ
µ
µ
µ
µ
µ
µ
µ
Table 2. Cytotoxicity (%) results of intermediate 10 against human cancer cell lines according to viability
assays (i.e., MTT, SRB, CVE).
24 h
SRB
48 h
SRB
72 h
SRB
Intermediate 10
MCF7
MTT
CVE
MTT
CVE
MTT
CVE
0.001 µM (8%),
0.01 µM (11%),
0.1 µM (15%),
1 µM (8%)
0.001
µ
M (13%),
1 µM
(10%)
0.01 µM
(6%)
-
-
-
-
-
0.1 µM (20%)
0.01 µM (16%),
0.1 µM (30%)
MDA-MB-231
HCT116
-
-
-
-
-
-
-
-
-
-
0.1 µM (13%)
0.01 µM
-
-
-
0.001 µM (19%),
0.01 µM (14%),
0.1 µM (32%)
(25%), 0.1
(22%)
µM
-
0.001 µM
(25%),
0.01 µM
0.01
(31%),
0.1
(23%)
µ
M
0.001 µM (17%),
0.01 µM (28%),
0.1 µM (27%)
1 µM
(24%)
MOR
-
-
-
-
-
µM
(38%), 0.1
(33%)
µ
M
Determination of apoptosis by flow cytometry.
Intermediate 9 and final molecule 10 were also assessed for their biological activity on the four
human cancer cell lines with flow cytometry by Annexin V and 7-AAD double staining. Annexin
V positive cells indicated early apoptotic cells, 7-AAD positive cells indicated necrotic cells, double
positive cells represented the late apoptotic cells, and the double negative cells indicated the living
cells. The concentrations that appeared (Table 3) to have a significant effect on MCF7 cell viability at
the same time point in two of the three assays were at 48 h with 0.001
intermediate . The percentages of untreated cells were 88.48% for living cells, 0.41% for early apoptotic,
1.84% for late apoptotic, and 1.3% for dead cells. After treatment with 0.001 M of intermediate 9, the
percentages of cells were 86.53% for living cells, 0.38% for early apoptotic, 1.38% for late apoptotic, and
2.34% for dead cells. Generally, an increasing dose of intermediate led to an increasing percentage of
apoptosis and necrosis. At 72 h, the percentages of untreated cells were 78.21% for living cells, 1.62%
for early apoptotic, 6.45% for late apoptotic, and 1.52% for dead cells. After treatment with 1 M of
intermediate , the percentages of cells were 78.21% for living cells, 1.51% for early apoptotic, 5.91%
for late apoptotic, and 2.34% for dead cells.
µM and at 72 h with 1 µM of
9
µ
9
µ
9
Table 3. Apoptosis results in MCF7 after the treatment with intermediate 9.
MCF7
Untreated (48h)
0.001 µM (48 h)
Untreated (72h)
1 µM (72 h)
Living Cells
88.48%
0.41%
1.84%
1.3%
86.53%
0.38%
1.38%
2.34%
78.21%
1.62%
6.45%
1.52%
78.21%
1.51%
5.91%
2.34%
Early Apoptotic
Late Apoptotic
Dead Cells
Similarly, the concentrations that appeared (Table 4) to have a significant effect on MOR cell
viability at the same time point in two of the three assays were at 48 h with 0.01 M and at 72 h
with 0.01 M and 0.1 M of intermediate . At 48 h, the percentages of untreated cells were 43.11%
for living cells, 9.99% for early apoptotic, 33.85% for late apoptotic, and 6.05% for dead cells. After
µ
µ
µ
9

Molecules 2019, 24, 4389
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treatment with 0.01
µM of intermediate
9
, the percentages of cells were 42.53% for living cells, 7.75%
for early apoptotic, 29.62% for late apoptotic, and 13.14% for dead cells. At 72 h, the percentages of
untreated cells were 61.21% for living cells, 4.56% for early apoptotic, 20.64% for late apoptotic, and
9.23% for dead cells. After treatment with 0.01
64.78% for living cells, 4.59% for early apoptotic, 12.06% for late apoptotic, and 13.35 for dead cells.
After treatment with 0.1 M of intermediate , the percentages of cells were 61.26% for living cells,
µM of intermediate 9, the percentages of cells were
µ
9
5.63% for early apoptotic, 15.22% for late apoptotic, and 12.76% for dead cells.
Table 4. Apoptosis results in MCF7 after the treatment with intermediate 9.
MOR
Untreated
0.01 µM (48 h) Untreated
0.01 µM (72 h) Untreated
0.1 µM (72 h)
Living Cells
43.11%
9.99%
33.85%
6.05%
42.53%
7.75%
29.62%
13.14%
61.21%
4.56%
20.64%
9.23%
64.78%
4.59%
12.06%
13.35%
61.21%
4.56%
20.64%
9.23%
61.26%
5.63%
15.22%
12.76%
Early Apoptotic
Late Apoptotic
Dead Cells
On the other hand, final molecule 10 appeared (Table 5) to have significant cytotoxic activity
against MDA-MB-231 at 0.1 M with MTT assay. At 24 h, the percentages of untreated cells were
69.6% for living cells, 3.02% for early apoptotic, 14.24% for late apoptotic, and 2.39% for dead cells.
After treatment with 0.1 M of final compound 10, the percentages of cells were 56.79% for living cells,
µ
µ
3.16% for early apoptotic, 27.72% for late apoptotic, and 2.52% for dead cells.
Table 5. Apoptosis results in MDA-MB-231 after the treatment with final molecule 10.
MDA-MB-231
Living Cells
Early Apoptotic
Late Apoptotic
Dead Cells
Untreated
0.1 µM (24 h)
69.6%
3.02%
14.24%
2.39%
56.79%
3.16%
27.72%
2.52%
3. Discussion
The aim of our study was to determine the utility of substituted pyrazine compounds such as
SHP2 inhibitors. We built a pharmacophore model using the highly selective SHP2 allosteric inhibitor
SHP099–SHP2 complex. A set of three novel compounds was discovered using a structure-based drug
design. Compared with the existing inhibitors, the new compounds not only had a similar ability
to be bound in the allosteric binding site of SHP2, but also a new hydrogen bond was determined
during this binding mode. The modulation of existing inhibitors and designing new inhibitors of
SHP2 are really crucial if we consider that SHP2 is a therapeutic target due to the fact of its importance
in known oncogenic pathways and emerging role in immuno-oncology. According to the literature,
there are amino pyrazines, such as kinase inhibitors [11,16,22–25], which have different biological
data depending on their structures. Hence, in this work, we have focused on allosteric inhibition
of SHP2–PTP catalytic sites through small molecules, particularly in amino pyrazine compounds
with different novel cyclic amine rings with substitutions. Initially, we started with condensed ring
systems. We started with prolines as cyclic amines; through in silico modelling, we came to know that
the interactions are as reported in the literature with one new hydrogen bond. Also, we noticed the
space available with the ortho position of proline for functionalization. Then, we designed structures
with different substitutions at the 2 and 3 positions of the prolines followed by a chain of amides
with substitutions.
As result of our efforts, we synthesized three novel pyrazine small molecular prolinamides
and sulphonamides as allosteric inhibitors of SHP2 which were determined by structure-based drug
design. The new compounds contained the 1-(methylsulfonyl)-4-prolylpiperazine group as a linker.
The final compound (10) and some of its intermediates (compounds 9, 21, 22) were evaluated for

Molecules 2019, 24, 4389
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their cytotoxic effect on different human cancer cell lines, and they were examined by four different
biological assays. Generally, an increasing dose of the intermediates led to an increasing percentage of
apoptosis and necrosis. Concerning the final compound 10, after treatment with 0.1 µM, the percentages
of MDA-MB-231 human breast adenocarcinoma cells were 56.79% for living cells, 3.16% for early
apoptotic, 27.72% for late apoptotic, and 2.52% for dead cells. The methodology described to identify
allosteric inhibitors also avoided the drug discovery challenges related to the polarity and homology
of the orthosteric binding pocket of SHP2. Finally, the compounds 18 and 19 will be evaluated for their
cytotoxic effect in different cancer cell lines, and the compound 10 will be modified in order to achieve
a better effect, as the results we presented in this work encourage us to further modify our structures.
4. Materials and Methods
4.1. Small Molecule Docking
The molecular docking studies on SHP2 were performed using ICM version 3.8-7c (Molsoft LLC,
San Diego, CA, USA) on a 2.0 GHz Intel Xeon Gold 6138 processor. The X-ray structure of SHP2 (PDB
ID: 5EHR) included two copies of the protein, and chain A was chosen as the receptor for docking. The
protein was prepared for docking through ICM’s automated protocol. This process includes: the global
optimization of hydrogens, the optimization of the orientation of His, Pro, Asn, Gln, and Cys residues,
and the deletion of water molecules that are not tightly bound to the protein structure. The binding
pocket was defined around the co-crystallized ligand, SHP099, by creating a box around the ligand
with a margin of 3. The box defines the potential energy maps and the selected margin value was
enough, in this case, to encompass the whole binding site. The co-crystallized ligand, SHP099, was
removed before docking. The chosen docking approach involved a rigid receptor and fully flexible
ligands. A table of compounds was imported into ICM and docked against the allosteric binding
pocket of SHP2 with an effort of 2. The effort value (ranges from 1 to 10) determines the “thoroughness”
which relates to the length of the simulation’s sampling time. Conformational sampling uses the
Monte Carlo minimization procedure, and the scoring is according to the ICM scoring function [26–28].
A docking score was assigned to each docked compound and used to assess the predicted interaction.
4.2. Chemistry
4.2.1. General Information
One milligram of synthesized compound was dissolved in 1 mL of a methanol/water mixture
(90/10), filtered through a 0.45
(1260 Infinity Series HPLC, 6120 Quadrupole MSD, Agilent Technologies Inc., Richardson, TX, USA)
was carried out with the use of a reverse phase column, Zorbax RX-C8 (5 M, 250 mm 4.6 mm,
µM filter, and inserted in an amber glass vial. The LC/MS analysis
µ
×
Agilent Technologies, Santa Clara, CA, USA) maintained at 40 ◦C. Chromatograms were integrated
and analyzed using OpenLAB Chemstation (version M8301AA, Revision C.01.07 Agilent Technologies
Inc., Richardson, TX, USA). The solvent system used was: (A) aqueous solution of 0.1% ammonium
acetate and (B) solution of 0.1% ammonium acetate in methanol. The separation was carried out at a
0.4 mL/min flow rate with isocratic elution of 10% solvent A and 90% solvent B for 20 min. The injection
volume was 10 µL and ultraviolet (UV) detection was carried out at 278, 280, and 325 nm. Mass
spectrometry analysis was performed with a 6120 Series Quadrupole System (Agilent Technologies,
Santa Clara, CA, USA) equipped with an electrospray ionization source (ESI) operating in negative
mode by scan analysis with a 100–1000 m/z scan range. Proton and carbon nuclear magnetic resonance
(1H and 13C-NMR) spectra were recorded with dimethylsulfoxide-d6 (in DMSO-d6) on the following
instrument: Bruker (1H at 400 MHz and 13C-NMR at 100 MHz, Milano, Italy).

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4.2.2. Experimental Procedures and Analytical Data
All reactions were carried out under N2 atmosphere unless specified.
Methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride (10): To a clean dry
round-bottom flask, which was purged with nitrogen to remove water traces and other particles,
3-bromo-6-chloropyrazin-2-amine (1.0 eq) was added and dissolved in dry THF (10 volumes). Then,
(2,3-dichlorophenyl) boronic acid (1.3 to 1.5 eq) was added and stirred for 10 min under inert
atmosphere. The reaction mass was degassed using a nitrogen inlet and vacuum outlet for 15 min.
Tetrakis (triphenylphosphine) palladium (0) (0.1 eq) was added and stirred for 15 min. After the
addition of a solution of 2 M Na2CO3, the reaction mixture was slowly heated to reflux. The process
of the reaction was evaluated by TLC and LCMS. After the solution was stirred for 4 h, it was
cooled to RT. Ethyl acetate and water were added, and the reaction mixture was stirred for another
10 min. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate.
The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation
of the solvent, the crude product was purified by flash chromatography (0% to 50% gradient of ethyl
acetate/n-hexane) to get 6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-amine (
3
) as a yellow crystalline
solid. MS m/z 274.10/276.11 (M + H)+.
To a cooled solution (0 ◦C) of 6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-amine (
3) (1.0 eq) in DCM
(10 volumes), triethyl amine (2.5 eq) was added and stirred for 10 min. Then, Boc anhydride (1.5
eq) and DMAP (0.1 eq) were slowly added and stirred for 2 h at room temperature. The reaction
mixture was diluted with DCM and washed with water and brine solution. The organic layer was
dried over sodium sulphate, filtered, and concentrated to get crude compound as a solid. The crude
solid was purified by flash chromatography (0% to 15% ethyl acetate/n-hexane) to get di-tert-butyl-
(6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-yl)carbamate (
(ES-, -100 PEAK).
4) as a white amorphous solid. MS m/z 374.1
To a solution of di-tert-butyl-(6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-yl)carbamate (1.0 eq) in
DMSO (10 volumes) was added cesium carbonate (2.5 eq) and stirred for 10 min. Then proline
◦
methyl ester (
5
) (1.5 eq) was slowly added and stirred for another 8 h at 85 C. The reaction
mixture was diluted with ethyl acetate and washed with water and brine solution. The organic
layer was dried over sodium sulphate, filtered, and concentrated to get crude compound as a solid.
The crude solid was purified by flash chromatography (0% to 50% ethyl acetate/n-hexane) to get
(6-((di-tert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolinate (
6) as a white solid.
MS m/z 567.24 (M + H)+.
To a cold solution (0 ◦C) of (6-((di-tert-butoxycarbonyl) amino)-5-(2,3-dichlorophenyl)
pyrazin-2-yl)prolinate (
6
) (1.0 eq) in THF (10 volumes), methanol (5 volumes) and “LiOH H2O
·
(2.0 eq.) were added were stirred for 10 min. The reaction mixture was warmed to room temperature
and stirred overnight. After the completion of the reaction, the solvents were evaporated. Water was
added to the crude mass acidified with 6 N HCl until pH = 4. The aqueous layer was extracted with
ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate,
filtered, and concentrated to get crude compound as a solid. The crude mass was washed with ether
and dried to get (6-((di-tert-butoxycarbonyl) - amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)proline (
7) as
a white solid. MS m/z 551.13/553.11 (M − H)−.
To a clean dry round-bottom flask, which was purged with nitrogen to remove water traces and
other particles, a solution of (6-((di-tert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)
proline (1.0 eq) was added in DMF(10 volumes). Then L-proline methyl ester (5) (2.0 eq) in DMF
was added and stirred for 10 min. Finally, HATU (2.0eq) and DIPEA (3.0eq) were added and stirred
overnight. The reaction mixture was diluted with ethyl acetate and washed with water and brine
solution. The organic layer was dried over sodium sulphate, filtered, and concentrated to get crude
compound as a solid. The crude solid was purified by flash chromatography (0% to 5% MeOH/DCM)
to get methyl (6-((di-tert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate (8)
as a colorless liquid.

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To a cooled solution (0 ◦C) of (6-((ditert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)
prolylprolinate ( ) (1.0 eq) in MeOH, 4M HCl was added in methanol (10 volumes) and stirred for
8
10 min. The reaction mixture was warmed to room temperature and stirred overnight. The reaction
mixture was distilled with methanol and dried to get crude mass. The crude product was washed
with ether and dried to get methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate
) as a yellow solid MS m/z: 502,11 (M + H)+ 1H-NMR (400 MHz, DMSO-d6)
hydrochloride (9 δ ppm
7.68 (d, J = 7.2 Hz, 1H) 7.45 (t, J = 7.6 Hz, 1H) 7.36 (d, J = 7.2 Hz, 1H) 7.17 (s, 1H) 4.76 (d, J = 8
Hz, 1H) 4.34–4.30 (m, 2H) 3.92–3.88 (m, 2H) 3.61–3.50 (m, 4H) 2.29–2.16 (m, 2H) 2.03–1.95 (m, 4H)
1.88–1.81(m, 1H).
(1-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)pyrrolidin-2-yl)methyl)pyrrolidin-2-yl)methanol (10):
All the intermediates from
round-bottom flask, the nitrogen was purged to remove water traces and other particles and charged
with methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride ( ) dissolved
1 to 9 were prepared following the procedures above. To a clean dry
9
in dry THF. The solution was cooled to 0 ◦C, added 1M LAH in THF (4.0 eq), and stirred for 10 min.
The reaction mixture was warmed to room temperature and was stirred overnight. The reaction
was quenched with 1 N aqueous NaOH at 0 ◦C. Ethyl acetate was added to the mixture and stirred
for 15 min and then filtered on a celite pad. The filtrate was concentrated and dried to get crude
product. The crude mass was purified by flash chromatography (0 to 7% MeOH/DCM) to get
(1-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)pyrrolidin-2-yl)methyl)pyrrolidin-2-yl)methanol as
a yellow solid (18). MS m/z: 422.15 (M + H)+ 1H-NMR (400 MHz, DMSO-d6)
δ ppm 7.62 (d, J = 4.8Hz,
1H) 7.42(dd, J = 8Hz, 1H) 7.30(d, J = 6Hz, 1H) 7.19 (s, 1H) 5.69(s, 1H) 4.36(t, J = 5.2Hz, 1H) 4.08(br, 1H)
3.55(br, 2H) 3.16(br, 2H) 2.60(m, 1H) 2.40(br, 1H) 2.08–2.03(m, 1H) 1.84–1.77(m, 4H) 1.69–1.67(m, 2H)
1.67–1.51(m, 1H) 1.23(s, 1H).
(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin2yl)pyrrolidin2yl)(4-((trifluoromethyl)sulfonyl)piperazin-1-yl)
methanone hydrochloride (19): To a stirred solution of tert-butyl-piperazine-1-carboxylate (11) in DCM
(10 volumes) hunig’s base (2.5 eq) was added and stirred for 10 min. The solution was cooled
◦
to
1 hr at
−
50 C. T◦hen trifluoromethane sulphonic anhydride (1.5 eq) was slowly added and stirred for
−
50 C. The reaction mixture was diluted with DCM and washed with water and brine
solution. The organic layer was dried over sodium sulphate, filtered, and concentrated to get crude
compound as a solid. The crude solid was purified by flash chromatography to get tert-butyl
4-((trifluoromethyl)sulfonyl)piperazine-1-carboxylate (13) as an off-white solid. MS m/z: 343.30
(M + 23)+, 241.30 (M − 100)+.
To
a
cold stirred solution (at
0
◦C) of tert-butyl-4-((trifluoromethyl)sulfonyl)
piperazine-1-carboxylate (13) in MeOH, 4 M methanolic HCl (10 volumes) was slowly added
and stirred overnight at room temperature. The reaction mass was distilled with methanol
and dried to get crude mass. The reaction mixture was washed with ether and dried to get
1-((trifluoromethyl)sulfonyl)piperazine hydrochloride (15) as a light brown solid. MS m/z: 219.11
(M + H)+.
To a clean dry round-bottom flask, which was purged with nitrogen to remove water traces and
other particles, a solution of (6-((Ditert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)
proline (1.0 eq) (7) was charged in DMF (10 volumes). Then, 1-((trifluoromethyl)sulfonyl)piperazine
hydrochloride (15) (2.0 eq) in DMF was added and stirred for 10 min. Finally, HATU (2.0 eq) and DIPEA
(3.0 eq) were added and stirred overnight. The reaction mixture was diluted with ethyl acetate and
washed with water and brine solution. The organic layer was dried over sodium sulphate, filtered, and
concentrated to get crude compound as a solid. The crude solid was purified by flash chromatography
(0% to 5% MeOH/DCM) to get di-tert-butyl- (3-(2,3-dichlorophenyl)-6-(2-(4-((trifluoromethyl)sulfonyl)
piperazine-1-carbonyl)pyrrolidin-1-yl)pyrazin-2-yl)carbamate (17) as a white solid. MS m/z: 753.23
(M + H)+.

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To a cooled (at 0 ◦C) stirred solution of Di-tert-butyl (3-(2,3-dichlorophenyl)-6-(2-(4-((trifluoromethyl)
sulfonyl)piperazine-1-carbonyl)pyrrolidin-1-yl)pyrazin-2-yl)carbamate (17) in MeOH, 4 M methanolic
HCl (10 volumes) was slowly added and stirred for overnight at room temperature. The reaction mass
was distilled with methanol and dried to get crude mass. The reaction mass was washed with ether
and dried to get (1-(6-amino-5-(2,3-dichlorophenyl)pyrazine-2-yl)-pyrrolidin-2-yl)-(4-((trifluoromethyl)
sulfonyl)-piperazin-1-yl)methanone hydrochloride (19) as a yellow solid. MS m/z: 555.15 (M + H)+.
1H-NMR (400 MHz, DMSO-d6)
δ ppm 7.72 (d, J = 4Hz, 1H) 7.47 (dd, J = 7.6Hz, 1H) 7.40 (d, J = 8Hz,
1H) 7.23 (s, 1H) 4.97(d, J = 7.2Hz, 1H) 3.65 (br, 8H) 3.45 (t, J = 16Hz, 1H) 2.26 (m, 1H) 2.01(m, 3H) 1.66
(t, J = 8Hz, 1H).
(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)pyrrolidin-2-yl)(4(methylsulfonyl)piperazin-1-yl)
methanone hydrochloride (18): To a stirred solution of tert-butyl piperazine-1-carboxylate (11) in DCM (10
Volumes), triethyl amine (2.5 eq) was added and stirred for 10 min. The resulting solution was cooled
to 0 ◦C and methane sulphonyl chloride (1.5 eq) was slowly added and stirred overnight at room
temperature. The reaction mass was diluted with DCM and washed with water and brine solution.
The reaction mixture was diluted with DCM and washed with water and brine solution. The organic
layer dried over sodium sulphate, filtered, and concentrated to get crude compound as solid. Crude
solid was purified by flash chromatography (0% to 30% ethyl acetate/n-hexane) to get tert-butyl
4-(methylsulfonyl) piperazine-1-carboxylate (12) as a pale brown solid. MS m/z: 265.19 (M + H)+.
To a cold stirred solution (at 0 ◦C) of tert-butyl-4-(methylsulfonyl) piperazine-1-carboxylate (12) in
MeOH, 4 M methanolic HCl (10 volumes) was slowly added and stirred overnight at room temperature.
The reaction mass was distilled with methanol and dried to get a crude mass. The reaction mass was
washed with ether and dried to get 1-(methylsulfonyl)piperazine hydrochloride (14) as a light brown
oil. MS m/z: 165.15 (M + H)+.
To a clean dry round-bottom flask, which was purged with nitrogen to remove water traces
and other particles, (6-((Di-tert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)proline
(7
) was charged, dissolved in DMF (10Volumes) and 1-(methylsulfonyl)piperazine hydrochloride
(14) (2.0 eq) in DMF and the resulting mixture was stirred for 10 min. Finally, HATU (2.0 eq) and
DIPEA (3.0 eq) were added and stirred overnight. The reaction mass was diluted with ethyl acetate,
washed with water and brine solution. The reaction mixture was diluted with ethyl acetate and was
washed with water and brine solution. The organic layer was dried over sodium sulphate, filtered, and
concentrated to get crude compound as a solid. The crude mass was purified with flash chromatography
(0% to 5% MeOH/DCM) to get di-tert-butyl(3-(2,3-dichlorophenyl)-6-(2-(4-(methylsulfonyl)
piperazine-1-carbonyl)pyrrolidin-1-yl)pyrazin-2-yl)carbamate (16) as a white solid. MS m/z: 699.26,
701.23 (M + H)+.
To a cold stirred solution (at 0 ◦C) of di-tert-butyl (3-(2,3-dichlorophenyl)-6-(2-(4-(methylsulfonyl)
piperazine-1-carbonyl)pyrrolidin-1-yl)pyrazin-2-yl)carbamate (16) in MeOH, 4 M methanolic HCl (10
volumes) was slowly added and stirred overnight at room temperature. The reaction mass was distilled
with methanol and dried to get crude mass. The reaction mass was washed with ether and dried to
get (1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)pyrrolidin-2-yl)(4-(methylsulfonyl)piperazin-1-yl)
1
methanone hydrochloride (18) as a yellow solid. MS m/z: 499.16, 501.16 (M + H)+. H-NMR (400 MHz,
DMSO-d6) δ ppm 8.50 (br, 2H) 7.68 (d, J = 8.0Hz, 1H) 7.45–7.35 (m, 2H) 7.17 (s, 1H) 4.98 (d, J = 7.6Hz,
1H) 3.78 (br, 2H) 3.51 (br, 2H) 3.14 (br, 2H) 2.88 (s, 3H) 2.52–2.49 (m, 5H).
(3-(2,3-dichlorophenyl)-6-(2-(hydroxymethyl)pyrrolidin-1-yl)pyrazin-2-yl)carbamate (21): All the
intermediates from
flask, which was purged with nitrogen to remove water traces and other particles, Di-tert-butyl-
(6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-yl)carbamate ( ) was charged, dissolved in DMSO (10
1 to 4 were prepared as described above. To a clean dry round-bottom
4
volumes) and stirred for 10 min. Then cesium carbonate (2.5 eq) was added and stirred for 10 min.
Prolinol (1.5 eq) was slowly added and the reaction mixture was stirred for 8 h at 85 ◦C. The reaction
mixture was diluted with ethyl acetate and washed with water and brine solution. The organic
layer was dried over sodium sulphate, filtered, and concentrated to get crude compound as a solid.

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The crude solid was purified by flash chromatography (0% to 60% ethyl acetate/n-hexane) to get
Di-tert-butyl-(3-(2,3-dichlorophenyl)-6-(2-(hydroxymethyl)pyrrolidin-1-yl)pyrazin-2-yl)carbamate as
1
a colorless solid (21). H-NMR (400 MHz, DMSO-d6)
δ ppm 8.1 (s, 1H) 7.67 (d, J = 8Hz, 1H) 7.43 (t,
J = 7.6Hz, 1H) 7.29 (d, J = 8Hz, 1H) 4.84 (s, 1H) 4.1 (s, 1H) 3.58–3.54 (m, 4H) 2.03–1.95 (m, 4H) 1.28
(s, 9H).
To a cold stirred solution (at 0 ◦C) of Di-tert-butyl-(3-(2,3-dichlorophenyl)-6-(2-(hydroxymethyl)
pyrrolidin-1-yl)pyrazin-2-yl)carbamate (21) in MeOH, 4 M methanolic HCl (10 Volumes) was
slowly added and stirred overnight at room temperature. The reaction mass was distilled with
methanol and dried to get crude mass. The reaction mass was washed with ether and dried to
get (1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)pyrrolidin-2-yl)methanol hydrochloride (22) as a
1
yellow solid. H-NMR (400 MHz, DMSO-d6)
δ
ppm 7.76 (d, J = 7.6Hz, 1H) 7.49–7.43 (m, 2H) 7.39 (s,
1H) 4.12(s, 1H) 3.54–3.39 (m, 5H) 2.01–1.92 (m, 4H).
4.3. Cell Culture
Commercial cancer cell lines were purchased from ECACC (European Collection of Authenticated
Cell Cultures) (Salisbury, UK). Cells were cultured in a humidified cell incubator at 37 ◦C and 5% CO2 and
passaged when cells reached 80% confluence. The MCF7 human breast adenocarcinoma cells (luminal
type) (ECACC 86012803) were cultured in RPMI 1640 supplemented with 10% FBS, 2% l-Glutamine
and 2% NEAA. MDA-MB-231 human breast adenocarcinoma cells (triple negative) (ECACC 92020424)
were cultured in RPMI 1640 supplemented with 15% FBS and 2% l-Glutamine. HCT116 human
colorectal carcinoma cells (ECACC 91091005) were cultured in DMEM supplemented with 10% FBS
and 2% l-Glutamine. The MOR human lung adenocarcinoma cells (ECACC 84112312) were cultured
in RPMI 1640 supplemented with 10% FBS and 2% l-Glutamine. RPMI 1640 Catalog#R0883 and
l-Glutamine Catalog#G7513-100ML were obtained from Sigma–Aldrich, Darmstadt, Germany, and
FBS Catalog#FB-1001/500 was obtained from BioSera, Nuaille, France. The MEM non-essential amino
acids (NEAA) Catalog #M7145-100ML and DMEM (Dulbecco’s modified Eagle’s medium) Catalog
#D5546 were purchased from Sigma–Aldrich, Darmstadt, Germany.
4.4. Cell Viability Analysis
Viability was measured with MTT for cell-metabolism activity, with SRB and CVE for cellular
protein content. The viable cells were seeded at a density of 2
incubated at 37 ◦C and 5% CO2 for 24 h to form a cell monolayer. After 24 h of incubation, supernatant
on the monolayer was aspirated and 200 L of medium and varying concentrations of the natural
substances were added and incubated for 24, 48, and 72 h time points.
After the specific time points, 20 L of 5 mg/mL MTT Catalog #M2128 (Sigma–Aldrich, Darmstadt,
Germany) in PBS Catalog #P3813 (Sigma–Aldrich, Darmstadt, Germany) was added to each well and
incubated for 3 h at 37 ◦C and 5% CO2. Supernatants were discarded and 100
L of DMSO Catalog
×
104 (200
µL/well) in 96 well plates and
µ
µ
µ
#445103 (Carlo Erbo Reagents, Barcelona, Spain) was added and the plates were incubated for 5 min at
37 ◦C and 5% CO2 to solubilize the formazan crystals. The absorbance was measured at 560 nm, and
the reference wavelength was at 605 nm.
For the SRB assay, at the specific time points cells were fixed by adding 100
acid Catalog #91228 (Sigma–Aldrich, Darmstadt, Germany) and with incubation at 4 ◦C for 1 h. Next,
100 L of SRB solution Catalog #341738 (Sigma–Aldrich, Darmstadt, Germany) was added in the
µL trichloroacetic
µ
wells for 30 min at room temperature. After that, supernatant was discarded, cells were rinsed three
times with 1% glacial acetic acid Catalog #1.00063.1011 (Merck, Darmstadt, Germany) and were left
to air dry. Finally, the dye was solubilized with 200 µL of 10 mM Tris-base pH 10.5 Catalog #T6791
(Sigma–Aldrich, Darmstadt, Germany) and absorbance was measured at 570 nm and the reference
wavelength was at 605 nm.

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For the CVE assay, at the specific time points, supernatant was removed and cells were fixed
with 100 L 10% formalin for 20 min. Formalin was discarded and cells were left to air dry. Next,
cells were dyed with 100 L of 0.25% aqueous crystal violet solution Catalog #HT901 (Sigma–Aldrich,
Darmstadt, Germany) and left for 10 min at room temperature. Then, the supernatant was discarded,
and cells were rinsed twice with 100 L WFI (water for injection) and were left to air dry. The dye was
solubilized with 100 L of 33% glacial acetic acid Catalog #1.00063.1011 (Merck, Darmstadt, Germany).
µ
µ
µ
µ
Absorbance was measured at 570 nm and the reference wavelength was at 605 nm.
4.5. Apoptosis Assessment
Apoptosis detection was conducted with flow cytometry to determine the membrane phospholipid
phosphatidylserine exposed apoptotic cells by Annexin V-PE and 7-AAD double staining. The PE
Annexin V Apoptosis Detection Kit I Catalog #559763 (BD Biosciences, San Jose, CA, USA) was used
for carrying out the experiments by following the manufacturer’s instructions. Samples were analyzed
on FACScan Calibur (Becton Dickinson, San Jose, CA, USA) and FSC 6 Express software.
5. Conclusions
In this work we established that, with the effectiveness of structure-based drug design, we were
able to accomplish the novel 1-(methylsulfonyl)-4-prolylpiperazine group as a linker in pyrazine amine
compounds. These structures have shown a receivable anti-cancerous activity.
Author Contributions: P.P. carried out the cellular biology assays and drafted the manuscript. M.S. carried out
the synthesis of compounds and drafted the manuscript. A.P.S. performed small molecule docking and drafted
the manuscript. I.V. performed the chemical analysis and drafted the manuscript. V.D. performed the chemical
analysis and drafted the manuscript. I.P. supervised the assays and the manuscript. All authors have read and
approved the manuscript.
Funding: This research did not receive any specific grant.
Conflicts of Interest: The authors declare no potential conflicts of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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