New insight into nucleo α-amino acids – Synthesis and SAR studies on cytotoxic activity of β-pyrimidine alanines

Article abstract of DOI:10.1016/j.bioorg.2020.103864

Three series of the β-pyrimidine alanines, including willardiine - a naturally occurring amino acid, were prepared from the L-serine-derived sulfamidates. Compounds 3b, 4a and 4b demonstrated antiproliferative activity toward the studied cancer cell lines, albeit the effect of these compounds on human brain astrocytoma MOG-G-CCM cells was more significant than on human neuroblastoma SK-N-AS cells. The cytosine analog of willardiine, compound 4b, reduced viability of MOG-G-CCM cells with EC₅₀ = 36 ± 2 μM, more effectively than AMPA antagonist GYKI 52466. Willardiine showed possible capability of affecting invasiveness of glioblastoma U251 MG cells with no effect on their viability and morphology. Compound 3d, the ethyl ester of willardiine, featured activity toward binding domain hHS1S2I of the GluR2 receptor. Docking analysis revealed that the location mode of compound 3d at the S1S2 domain of hGluR2 (PDB ID: 3R7X) might differ from that of willardiine.

Full text of DOI:10.1016/j.bioorg.2020.103864

BioorganicChemistry100(2020)103864
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New insight into nucleo α-amino acids – Synthesis and SAR studies on
cytotoxic activity of β-pyrimidine alanines
Jolanta Ignatowskaa, Ewa Mironiuk-Puchalskaa, Piotr Grześkowiaka, Patrycja Wińskaa,
Monika Wielechowskaa, Maria Bretnera, Olena Karatsaib, Maria Jolanta Rędowiczb,
⁎
Mariola Koszytkowska-Stawińskaa,
a Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland
b Laboratory of Molecular Basis of Cell Motility, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three series of the β-pyrimidine alanines, including willardiine - a naturally occurring amino acid, were pre-
pared from the L-serine-derived sulfamidates. Compounds 3b, 4a and 4b demonstrated antiproliferative activity
toward the studied cancer cell lines, albeit the effect of these compounds on human brain astrocytoma MOG-G-
CCM cells was more significant than on human neuroblastoma SK-N-AS cells. The cytosine analog of willardiine,
Willardiine
Nucleo amino acids
Brain tumors
Anticancer agents
Sulfamidates
compound 4b, reduced viability of MOG-G-CCM cells with EC50 = 36
2 μM, more effectively than AMPA
antagonist GYKI 52466. Willardiine showed possible capability of affecting invasiveness of glioblastoma U251
MG cells with no effect on their viability and morphology. Compound 3d, the ethyl ester of willardiine, featured
activity toward binding domain hHS1S2I of the GluR2 receptor. Docking analysis revealed that the location
mode of compound 3d at the S1S2 domain of hGluR2 (PDB ID: 3R7X) might differ from that of willardiine.
Glutamate receptor
1. Introduction
nervous system or some peripheral tissues [13,14]. Experiments with
glutamate ionotropic receptor (iGluR) antagonists revealed their ability
Nucleo amino acids are defined as α-amino carboxylic acids with
pyrimidine or purine nucleic acid bases in the side chain, attached to
the amino acid fragment via the N1 (pyrimidine) or N9 (purine) atom
[1]. Great attention has been paid to β-pyrimidine alanines (Fig. 1A)
due to interactions of willardiine or 5-halowillardiines with glutamate
receptors [2,3]. Identification of these interactions resulted in the em-
ployment of willardiine or 5-halowillardiines in studies on activity of
the mammalian brains [4] and development of the new class of gluta-
mate receptor modulators [5]. On the other hand, the hydrogen-
bonding properties of 5-methylwillardiine (Fig. 1A) were used for ob-
taining self-replicating systems acting as catalysts in the Friedel-Crafts
reaction [6]. Ability of β-nucleo alanines to form peptide systems led to
de novo designed peptide nucleic acids [7] or β-nucleo alanine-labeled
proteins [8] capable of recognizing nucleobases in nucleic acids or
penetrating into cells [9] without cytotoxic effects. Novel conjugates
with promising properties were also obtained from β-pyrimidine ala-
nines and natural peptides, such as human insulin [10], tylosin anti-
biotics [11] or dermorphine [12].
to decrease proliferation and migration of the selected cancer cells
[13,15]. For this reason iGluR antagonists have received great attention
as potential anticancer therapeutics. On the other hand, glutamate, α-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and 5-
fluorowillardiine (Fig. 1A, R = F) acting as AMPA GluR agonists were
reported to increase cell proliferation, migration and invasion of the
selected iGluRs-expressing cancers [15,16]. However, the reported
growth-stimulating effect of 5-fluorowillardiine seemed not to be the
general feature of β-pyrimidine alanine-derived AMPA GluR agonists.
Recently we have reported that willardiine had no effect on viability of
human brain astrocytoma cells MOG-G-CCM expressing the AMPA
GluR1 subunit [17]. We also identified the willardiine derivatives 1
(Fig. 1B) to be more active against human brain astrocytoma cells
MOG-G-CCM than GYKI 52466, the AMPA GluR1 subunit antagonist
[17]. These findings have revealed that studies on potential of β-pyr-
imidine alanines in anticancer drugs research and development are
entirely justifiable. To the best of our knowledge, anticancer effect of β-
pyrimidine alanines with modified amino acid moiety has not yet been
reported.
Glutamate signaling system has recently been suggested as the
therapeutic target in the treatment of tumors derived from the central
Herein, we report the highly efficient synthesis of three series of β-
⁎ Corresponding author.
E-mail address: mkoszyt@ch.pw.edu.pl (M. Koszytkowska-Stawińska).
https://doi.org/10.1016/j.bioorg.2020.103864
Received 6 January 2020; Received in revised form 15 April 2020; Accepted 17 April 2020
Availableonline15May2020
0045-2068/©2020TheAuthors.PublishedbyElsevierInc.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
Fig. 1. (A) β-Pyrimidine alanines of medicinal interest. (B) The prepared in our laboratory β-pyrimidine alanines active against human brain astrocytoma cells MOG-
G-CCM expressing the AMPA GluR1 subunit [17]. (C) The target compounds of the current project.
pyrimidine alanines 2, 3 and 4 (Fig. 1C) and their potential cytotoxic
activity. Having in mind complementarity between the target-based
drug discovery (TDD) approach and the phenotypic drug discovery
(PDD) approach [18], we examined compounds 2–4 in terms of their in
vitro effect of on selected tumors, including tumors expressing the
AMPA GluR1 subunit, and then of their in vitro AMPA GluR2 subunit-
binding activity. The in vitro AMPA GluR2 subunit-binding assays were
correlated with the ligand-receptor binding interactions found from
docking analysis. Further, we evaluated in vitro effect of willardiine on
viability and motility of highly malignant human glioblastoma U251
MG cells in order to examine a scope of the findings on effect of will-
ardiine on the AMPARs expressing tumors.
most efficient approach to the Nα-acylated acids 2c, 3c and 4c, re-
spectively (Table 1, entries 8–10) [26]. This method was reproducible
in terms of both the yield and the specific rotation value [α] of products
(see, Experimental, Section 4.1). The utility of this method was con-
firmed by a good agreement in the specific rotation values [α] of
compound 2c and the authentic sample (Table 1, entry 8). Our two-step
approach involving preparation of the uracil derivative 3a followed by
its hydrolysis provided not yet reported optically active compound 3c
in the total yield of 51% (Table 1, entry 9). Next, the Pd-catalyzed
hydrogenolysis (Scheme 1, reaction conditions (iv)) was employed for
the preparation of ester 2d from compound 2a (Table 1, entry 11), 5-
methylwillardiine hydrochloride 2e from compound 2b or free 5-me-
thylwillardiine from compound 2c (Table 1, entry 12 or 13, respec-
tively), and willardiine from compound 3b (Table 1, entry 14). This
method was not useful for obtaining ester 3d because of the hydro-
genation of the uracil ring of substrate 3a under the reaction conditions
[27]. In contrast, the Pd-catalyzed hydrogen transfer technique
(Scheme 1, reaction conditions (v)) was sufficiently mild to obtain
compound 3d by the selective and efficient removal of the Cbz group
from the same substrate 3a (Table 1, entry 15) [28]. The optical rota-
tion values [α] determined for 5-methylwillardiine and willardiine
obtained from the hydrogenolysis featured good agreement with those
reported in the literature (Table 1, entry 13 and 14, respectively). Our
two-step approach involving preparation of the thymine derivative 2a
followed by its hydrogenolysis provided not yet reported optically ac-
tive 5-methylwillardiine ethyl ester 2d in the total yield of 82%
(Table 1, entry 11). Further, when employed uracil, the approach let us
to obtain willardiine ethyl ester 3d in the total yield of 47% and de-
termine its not yet reported optical rotation value [α] (Table 1, entry
15).
2. Results and discussion
2.1. Synthesis
The with a N-protected serine lactone under basic conditions has
been reaction of a nucleobase the most often used method for the
preparation of β-nucleo alanines [19]. However this method has got
severe disadvantages, such as (a) the use of the environmentally
harmful metal salts (NaH or K2CO3) [2,19], (b) poor reproducibility
[9,20], (c) poor mass balance and laborious workup of the reaction
mixture from the Mitsunobu reaction required for preparation of the
starting serine lactone [21] or (d) chemical sensitivity of some serine
lactones implicating their special storage and handling conditions [22].
The another literature methods suffering from not reported [23] or
unverified [24] optical purity of the final β-nucleo alanines have also
been reported.
The N1-substitution pattern of the pyrimidine ring in b-purimidine
We envisaged sulfamidates 5 as the amino acid precursors of the
target compounds (Scheme 1) since we needed a fast and convenient
method for production of the target compounds. The preliminary NaH-
mediated reaction between sulfamidate 5a and thymine gave un-
satisfactory results (Scheme 1, reaction conditions (i); Table 1, entry 1)
because of low yield and low specific rotation value [α] of the reaction
product when compared with the reference sample obtained from the
esterification of the authentic free acid prepared in accordance with the
protocol of Geotti-Bianchini [25] (for the esterificaction protocol see
Experimental, subsection 2). On the contrary, the treatment of sulfa-
midate 5a with thymine (Scheme 1, reaction conditions (ii)), after prior
silylation of thymine with N,O-bis(trimethylsilyl)acetamide, afforded
product 2a with the specific rotation value [α] being in good agreement
with that of the reference sample (Table 1, entry 2). Therefore, the N,O-
bis(trimethylsilyl)acetamide-promoted alkylation was chosen for pre-
paration of compounds 2b; 3a,b and 4a,b (Table 1, entries 3–7). Sig-
nificant differences in reactivity of sulfamidates 5 or the used nucleo-
bases were not noted in this reaction, except for the reaction between
sulfamidate 5b and N4-benzoylcytosine producing product 4b in 52%
yield (Table 1, entry 7). The 1 M aq NaOH-promoted hydrolysis of
compounds 2a, 3a and 4a (Scheme 1, reaction conditions (iii)) was the
alanines obtained from our approach was confirmed from the 1H–13
C
HMBC NMR spectrum of compounds 2a and 2c prepared in accordance
with the reaction conditions (ii) or (ii) and (iii) (Scheme 1), respec-
tively. In the 1H–13C HMBC NMR spectrum of compound 2a the fol-
lowing interactions were diagnostic for establish the N-1 substitution of
its pyrimidine ring (Fig. 2): H-3 (3.68 and 4.16 ppm)
↔ C-2′
(150.9 ppm), H-3 (3.68 and 4.16 ppm) ↔ C-6′ (141.7 ppm) and H-6′
(7.38 ppm) ↔ C-3 (47.9 ppm). For the 1H–13C HMBC NMR spectrum of
compound 2a, see Supplementary Material (Section 3.3). Further, the
1H–13C HMBC NMR spectrum of compound 2c obtained from the NaOH
promoted hydrolysis of compound 2a revealed interactions of the H-3
protons specific to the N1-substitution pattern of its pyrimidine ring
(Fig. 2): H-3 (3.62 ppm and 4.21 ppm) ↔ C-2′ (150.9 ppm) and H-3
(3.62 ppm and 4.21 ppm) ↔ C-6′ (141.9 ppm). For the 1H–13C HMBC
NMR spectrum of compound 2c, see Supplementary Material (subsec-
tion 3.8). The same interactions were detected in the 1H–13C HMBC
NMR spectrum of the authentic sample of compound 2c obtained from
the alkylation of thymine with the serine lactone [25]. For the 1H–13
C
HMBC NMR spectrum of the reference compound 2c, see
2

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
Scheme 1. Preparation of the target com-
pounds 2–4. Nucleobase = Thymine, Uracil
or N4-Benzoylcytosine. Reaction conditions.
(i) (1) Thymine, NaH, DMF, room tempera-
ture, 30 min; (2) 5a, DMF, room tempera-
ture, 1 day. (ii) (1) Thymine (or uracil or N4-
Benzoylcytosine),
N,O-bis(trimethylsilyl)
acetamide, acetonitrile, room temperature,
1 h; (2) 5, acetonitrile, room temperature,
1–3 days. (iii) 1 M aq NaOH, THF, 1,4-di-
oxane, room temperature, 1 day. (iv) H2
(baloon), Pd/C, MeOH, room temperature,
2–5 h. (v) Cyclohexene, Pd/C, EtOH, reflux,
7 h.
Supplementary Material (Section 4.6).
activity toward cancer cell lines derived from CNS (Table 3): SK-N-AS –
human neuroblastoma (not expressing the AMPA GluR1 and GluR2
subunits) and MOG-G-CCM – human brain astrocytoma (expressing the
AMPA GluR1 subunit) [36]. Willardiine was used as the negative con-
trol [17], and GYKI 52,466 (AMPA antagonist) was used as the positive
control in theses assays. Whereas willardiine showed no effect on via-
bility of human brain astrocytoma MOG-G-CCM cells at the con-
centration of 200 µM, its amino acid-protected derivative 3b decreased
The green reaction metrics were calculated for our protocols leading
to willardiine or compound 2e (Table 2). The two-step preparation of
these compounds from O-Bn-sulfamidate 5b featured higher yield and
higher Carbon Efficiency (CE) and Reaction Mass Efficiency (RME) than
the literature methods employing N-Boc-serine lactone [2,33]. The
avoidance of metal salt wastes, corrosive trifluoroacetic acid and toxic
pyridine [34] in our protocol is also worth emphasizing.
viability of these cells at the concentration of 100 µM, i.e. 5
1% of
viable cells relative to control cells was determined. The same effect
was also observed for compounds 4a and 4b i.e. when the amino acid-
protection of willardiine was accompanied by the replacement of its
2.2. In vitro assays
The synthesized compounds were evaluated for their cytotoxic
Table 1
Preparation of β-pyrimidine alanines 2–4.
Entry
Substrate(s); Reaction conditions
Product, Isolated yield
Specific rotation value [α]
1
2
Thymine, 5a; i
Thymine, 5a; ii
2a, 28%,
2a, 85%
[α] −15.9 (c 0.11, MeOH)
[α] −88.2 (c 0.11, MeOH)
[α] −85.6° (c 0.11, MeOH)a
[α] −109.1 (c 0.12, MeOH)
[α] −111.0 (c 0.11, MeOH)
[α] −64.9 (c 0.11, MeOH)
[α] −90.1 (c 0.12, MeOH)
[α] −34.3 (c 0.11, CH2Cl2)
[α] −86.7 (c 0.20, MeOH)
[α] −85.1 (c 0.20, MeOH)b
[α] −91 (c 0.2, MeOH) [25]
[α] −99.5 (c 0.21, MeOH)c
[α] −88.3 (c 0.10, MeOH)
[α] + 11.9 (c 0.10, MeOH)d
[α] −12.8 (c 1.0, 1 M HCl)
[α] −16.7 (c 1.0, 1 M HCl)[α] −15 (c 1.0, 1 N HCl) [29]
[α] –23.7 (c 0.90, 1 M HCl)
[α] −21 (c 1.0, 1 N HCl) [30]
[α] + 1.9 (c 0.11, MeOH)e
3
4
5
6
7
8
Uracil, 5a; ii
3a, 62%
4a, 61%
2b, 69%
3b, 78%
4b, 52%
2c, 83%
N4-Benzoylcytosine, 5a; ii
Thymine, 5b; ii
Uracil, 5b; ii
N4-Benzoylcytosine, 5b; ii
2a; iii
9
3a; iii
4a; iii
2a; iv
2b; iv
2c; iv
3b; iv
3c, 83%
10
11
12
13
14
4c, 53%
2d, 96%
2e, 87%
5-methylwillardiine,76%
willardiine, 98%
15
a
b
c
d
e
3a; v
3d, 75%
From the reference sample obtained from the esterification of the authentic free acid prepared in accordance with the protocol of Geotti-Bianchini [25].
For the authentic free acid prepared in accordance with the protocol of Geotti-Bianchini [25].
Racemate was reported [31].
Racemate was reported [32].
A specific rotation value [α]) was not reported [28].
3

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
Table 3
The viability of MOG-G-CCM and SK-N-AS cell lines after 48 h treatment with
the examined compounds. The MTT-based assay.
Compound
CLogPa MOG-G-CCM cell line
SK-N-AS cell line
Viability/%b,c EC50/μMd Viability/%b,c EC50/μMd
2a
93
39
100
98
100
83
5
2
5
97
84
91
90
93
100
28
92
86
4
1
5
3
6
3
2b
2c
3
3
2d
4
3
2e
3a
5
3b
2.11
1
89
6
4
3
4
165
12
3c
100
90
1
2
3d
5
4a
2.55
3.71
0
0
64
36
13
2
1
2
84
60
8
4b
2
6
13
4c
90
101
83
3
4
82
–
3
5
willardiine
−3.37
2e
GYKI 52,466 3.45
86
Fig. 2. The 1H–13C interactions observed in the 1H–13C HMBC NMR spectra of
compounds 2a and 2c obtained from our method. For the 1H–13C HMBC NMR
spectra see, Supplementary Material (subsection 3.3 and 3.8, respectively).
a
b
c
From ChemBioOffice 2010.
At 100 μM.
The percent of viable cells relative to control cells and represent the
mean
SD.
Table 2
d
e
Determined for the most active compounds.
At 200 µM.
Reaction metrics for the current method vs the reaction metrics for the litera-
ture methods operating in the synthesis of willardiine or compound 2e.
Amino acid-building block, Method
Reaction metricsa/%
growth-stimulating effect of AMPA receptor agonists (including 5-
fluorowillardiine [15]) on cancer cells. These observations along with
the ambiguity in literature regarding the effects of willardiine and its
derivatives on proliferation and migration of various cancer cells
[13–17] urged us to examine effects of willardiine on highly malignant
human glioblastoma U251 MG cells (Figs. 3–5). The Transwell™ mi-
gration assay was performed to check influence of willardiine on ability
of the cells to migrate through porous membrane. As shown in Fig. 3,
willardiine at 500 µM concentration seemed to decrease the migration
of glioblastoma U251 MG cells.
Yield
AE
CE
RME
Preparation of willardiine
sulfamidate 5b, Currentb
76
28.1
45.5
35.2
14.1
3.4
16.7
2.6
N-Boc-serine lactone, ref. [2]
N-Boc-serine lactone, ref. [33]
Preparation of 5-methylwillardiine
sulfamidate 5b, Currentd
58c
21
2.3
1.6
52
24
29.5
36.8
10.8
6.9
12.1
5.0
N-Boc-serine lactone, ref. [33]
AE = Atom Economy, CE = Carbon Efficiency, RME = Reaction Mass
Efficiency.
a
b
c
Calculated from ref. [35].
The two-step protocol with intermediate 3b is analyzed.
The highest yield among those obtained from three experiments conducted
in our laboratory (the obtained yields ranged from 23% to 58%).
d
The two-step protocol involving intermediate 2b as the precursor of 5-
methylwillardiine hydrochloride 2e is analyzed.
uracil ring with the cytosine one (1
0% (4a) or 2
0% (4b) of
viable cells relative to control cells was determined). Compounds 3b,
4a and 4b also demonstrated antiproliferative activity toward the stu-
died cell lines, albeit the effect of these compounds on MOG-G-CCM
cells was more significant than on SK-N-AS cells. The determined per-
cent of viable SK-N-AS cells relative to control cells was: 28
4 (3b),
4
1 (4a) and 6
tosine derivative 4b showed the lowest EC50 values toward both the
examined cell lines (EC50 (MOG-G-CCM cell line) = 36 2 and EC50
2 (4b). Among the active compounds, the cy-
(SK-N-AS cell line) = 60
13; for experimental GraphPad Prism plots
used for determination of the EC50 values see, Supplementary material,
subsection 1.1). Moreover, caspase-3/7 activation was observed in
MOG-G-CCM cells after 48
h treatment with compound 4b
(Supplementary material, subsection 1.2), suggesting induction of
apoptosis by this compound. The determined activities of compounds
3b, 4a and 4b, when analyzed in the light of their structure, might
indicate a relation between the activity and lipophilicity of these
compounds. The ClogP values (Table 3) supported this hypothesis, since
the most active compound 4b featured the highest ClogP value sug-
gesting its highest lipophilicity among the discussed compounds.
The observed inertness of willardiine towards human brain astro-
cytoma MOG-G-CCM cells was unforeseen in the light of reports on
Fig. 3. Effects of willardiine on migration of U251MG glioblastoma cells.
Transwell migration assay was performed for cells treated for 48 h in the pre-
sence or absence of 500 µM willardiine. Bars in upper panels, 100 µm.
4

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
Fig. 4. Viability of U251MG glioblastoma cell line after treatment with will-
ardiine (the MTS-based assay). Data are means
SD.
Fig. 6. Effects of willardiine on the level of proteins involved in cell pro-
liferation, migration and adhesion. U251MG cell lysates after 48-h culture with
500 µM willardiine were blotted with the antibodies against the respective
proteins. GAPDH was used as a protein loading control. More details in
Experimental, section 10.
glioblastoma cells with willardiine to examine the levels of the proteins
involved in cell proliferation as well as in cell migration and adhesion
(Fig. 6). Our findings revealed that willardiine even at the high con-
centration of 500 μM did not cause evident changes in the levels of the
proteins involved in regulation of cell proliferation [37], namely Erk 1/
2 and Akt and their phosphorylated (active) forms, as well as in the
levels of the proteins involved in cell migration and adhesion [38], i.e.
β-actin, vinculin, talin, and focal adhesion kinase (FAK) and its phos-
phorylated form (p-FAK). Also, there was no any substantial difference
in the level of both glutamate receptor subunits GluR1 and GluR2 in the
cells cultivated in the presence of willardiine.
Fig. 5. Morphology of U251MG glioblastoma cells after treatment with 500 µM
willardiine. (A) Images obtained with light microscope after 24-htreatment. (B)
Images obtained with fluorescent microscope after staining for actin filaments
with Alexa Fluor 488-phalloidin, and for nuclei with DAPI after 48-h treatment.
Bars, 100 µm.
2.3. Receptor binding experiments and the docking studies
However, as shown in Fig. 4 cell viability assessed by the MTS assay
was not significantly altered in the examined willardiine concentration
range (10–1000 µM) regardless the time of treatment with willardiine
(24–72 h) with respect to the control conditions. Moreover, we did not
observe evident effects of willardiine on the cell morphology and actin
cytoskeleton organization of the treated cells (Fig. 5A and B, respec-
tively). Analogous observations were made for another malignant
human glioblastoma cell line U87 MG (not shown).
The in vitro AMPA receptor-binding assays were reported as one of
the principal tools for identification of AMPA receptor modulators
being of interest as potential cognitive enhancers or agents with po-
tential for the treatment of neuropsychiatric and neurological disorders.
Since willardiine was recognized as one of the most important che-
motypes explored as potential AMPA receptor modulators [4,5,39], we
examined binding of the synthesized compounds to the ligand binding
domain hHS1S2I of GluR2 receptor (Table 4) [40]. Binding experiments
were performed with DL-α-[5-methyl-3H]-AMPA (the final concentra-
tion of 10 nM, 10.6 Ci/mmol). L-Glutamic acid, willardiine and
In order to understand the mechanisms of the observed discrepancy
in the effects of willardiine on U251MG cells, we performed western
blot analysis of cell lysates after 48
h treatment of U251MG
5

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
Table 4
(Table 5, binding mode 1), the “non-classical” location (Table 5,
binding mode 2) involved the receptor Arg96 and Thr91 in anchoring of
the compound uracil moiety, and the receptor Glu13, Tyr61, Thr174
and Tyr190 in binding of the compound amino ester group. As a result,
the binding mode 2 featured lower free-energy of binding than the
binding mode 1 (Tables 4). The “non-classical” location in the receptor
binding domain was also suggested for compound 2d (Supplementary
material, subsection 2.2). This location offered much weaker anchoring
of compound 2d than compound 3d in the receptor binding domain.
This observation was in accordance with our in vitro findings (Table 4,
entry 4 vs 9). The docking analysis reflected the low ability of com-
pound 4b to bind hGluR2 receptor observed in our in vitro assays
(Table 4). The suggested 4b-hGluR2-S1S2 interactions involved the
receptor Glu193, Pro89 and Thr143 (Table 5).
In vitro binding to hHS1S2I binding domain of GluR2 receptor and the docking-
predicted free-energies of binding in the binding domain of hGluR2 receptor
(hGluR2-S1S2/L-glutamate crystal structure, PBD entry 3R7X [42]).
Entry Compound
Bound [3H]AMPA
EC50
ΔGbind [kcal/mol]b
[%]a
[µM]
2a
2b
2c
2d
2e
3a
3b
3c
3d
98.9
60.0
110.7
34.8
4.1
2.80
2.43
3.2
0.45
−6.5
0.70
6.12
−7.8c
102.8
76.7
91.8
13.3
17.7
0.45
0.74
0.21
14.86
−6.9 (binding mode
1)d−7.2 (binding
mode 2)d
4a
97.2
21.3
75.2
5.9
12.75
4.84
4.39
3. Conclusions
4b
48.70
−2.4
4c
willardiine
0.05
3.22
1.66
−8.5 [17]
nd
We developed efficient method for the preparation β-pyrimidine
alanines, including willardiine. Our method offered: (i) stereoselective
formation of the reaction products, (ii) reproducible individual reaction
steps and (iii) high values of the Green Chemistry reaction metrics.
The obtained data suggest that willardiine does not affect viability
and morphology of human glioblastoma U251 MG cells and U87 MG
cells and does not cause significant changes in the level (and activity) of
the proteins involved in processes crucial for migration (and invasive-
ness) such as cell proliferation and adhesion. Our observation that there
is a decrease in cell migration of the examined cancer cells indicate that
this compound could affect invasiveness of the cancer cells. Thus, fur-
ther studies are needed to elucidate the observed inhibition in mi-
gratory potential of the tumor cells after the willardiine treatment.
Willardiine was shown to be a useful core for designing compounds
active in vitro against human brain astrocytoma cells MOG-G-CCM and
human neuroblastoma cells SK-N-AS. Our findings on in vitro cytotoxic
activity of compounds 3b, 4a and 4b have shed new light on β-pyr-
imidine alanines and their potential as antitumor agents. We showed
that structural modifications of the parent willardiine devoid of cyto-
toxic activity led to the antiproliferative agents towards both tumors
expressing AMPA GluR1 subunit (such as human brain astrocytoma
cells MOG-G-CCM) and tumors not expressing AMPA GluR1 and GluR2
subunits (such as human neuroblastoma cells SK-N-AS). Our study
provides a convincing example that the structure-based approach
starting from an inactive lead compound (willardiine) can be employed
to design new anti-tumor agents.
L-glutamic acid 0.9
0.11
a
Percent of the remaining DL-α-[5-methyl-3H]-AMPA bound to hHS1S2I in
the in vitro radioligand-displacement assays at the compound concentration of
100 µM.
b
From the docking experiment with the hGluR2-S1S2/L-glutamate crystal
structure (PBD entry 3r7x).
c
d
For free 5-methylwillardiine.
See, Table 5. nd = not determined.
compound 2e were employed as the binding positive controls [41].
Binding activity of compound 3d with EC50 = 14.86 µM suggested a
tolerance of the receptor for the O-ethylation of the carboxylic group
(Table 4, entry 9 vs 13). On the other hand, the Nα-acylation of both
compound 2e and willardiine in the form of compounds 2c and 3c,
respectively, led to a dramatic decrease in their binding activity
(Table 4, entry 2 vs 5 and entry 8 vs 13; respectively). Taking into ac-
count these findings, low binding affinity of the N,O-protected deriva-
tives of willardiine or compound 2e was not surprising. Interestingly,
the previously mentioned relationships between the protection level of
the amino acid moiety and the compound binding activity was not
observed in the series of the cytosine derivatives 4a-c. Among these
compounds, the Nα-Cbz-O-benzyl ester 4b showed much higher binding
affinity to the receptor than its de-benzylated counterpart 4c (Table 4,
entry 11 vs 12). Further, compound 4b was the most active among the
examined N,O-protected derivatives of both the ethyl esters and the
benzyl esters series.
Our in vitro receptor-binding assays with the synthesized com-
pounds showed that the most active cytotoxic compounds 3b, 4a and
4b were not the most active ones in the radioligand binding displace-
ment studies. These findings, when considered in the light of the rela-
tively high lipophilicity of the compounds endowed cytotoxic activity
(based on their CLogP), might suggest the following possibilities: (i)
compounds 3b, 4a and 4b did not bind to the AMPA receptor or they
bound to the receptor at other site than the APMA-binding domain, or
(ii) the observed cytotoxic activity of these compounds resulted from
their ability to penetrate the tumor cell membrane. Additional studies
are required for elucidation of this issues.
The performed competitive radioligand binding assays solely de-
termined a relative affinities of the tested compounds for binding to the
receptor at the AMPA-binding site. They did not show a character
(agonistic or antagonistic) of the detected ligand-receptor interactions.
In order to rationalize our findings on the in vitro hHS1S2I binding
assays free-energies of binding were predicted from docking of will-
ardiine, 5-methylwillardiine, and compounds 2d, 3d and 4b in the
binding domain of hGluR2 receptor [42] (Table 4, ΔGbind). Although
the relative potency of willardiine and 5-methylwillardiine to bind
AMPA receptor was established by Roberts et al. in 1995 [41], to the
best of our knowledge, a mode of the 5-methylwillardiine-AMPA re-
ceptor binding was not proposed in the literature or reported in RCSB
PDB [43]. Our docking studies suggest the lack of binding interactions
between the receptor Thr143 and Tyr190 and the compound 5-me-
thylwillardiine when compared with willardiine (Supplementary ma-
terial, subsection 2.3 vs 2.1, respectively). We believe that these find-
ings might explain a weaker binding of 5-methylwillardiine to the
receptor than binding of willardiine. Compound 3d - the wiliardiine
ethyl ester - was capable of location at the receptor binding domain in
two modes (Tables 4 and 5). While the willardiine-like location was
supported solely by the interaction between the receptor Thr143
The in vitro receptor-binding assays showed tolerance of hGluR2
receptor on structural modifications of willardiine at the amino acid
moiety. The docking of ethyl ester of willardiine 3d suggested that
structural modifications of the parent compound might imply an unu-
sual mode of the ligand location in the receptor binding domain. This
assumption might be useful for designing novel ligands of the AMPA
receptor.
6

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
Table 5
Proposed binding modes of compounds 3d and 4b at the GluR2-hS1S2 binding domain and distances of potential ligand–protein H-bonds (≤3.3 Å).a.
Compound 3d, binding mode 1
Residue
Atomic group
(C1)O11
Distance/Åb
(Thr143)O
(Thr143)O
2.2
2.1
(N6)H
Compound 3d, binding mode 2
Residue
Atomic group
(N2)H
Distance/Åb
(Glu13)Oε2
(Tyr61) OηH
(Thr174)Oγ1
2.9
2.5
1.5
2.1
2.1
2.1
1.9
2.0
(N2)H
(N2)H
(Thr174)Oγ1
(Tyr190) OηH
(Arg96)Nη2H
(Thr91)NH
H
(C1)O11
(C1)O11
(C7)O
(C7)O
(Thr91)Oγ1
(N6)H
Compound 4b
Residue
Atomic group
(N7)H
Distance/Åb
(Glu193)Oε2
(Pro89)C]O
(Thr143)NH
2.0
3.2
2.7
(C5)O
(C7.2)O
a
b
Colors: C(ligand) = green, C(protein residue) = light blue, N = dark blue, O = red, H-bond = gray.
From the docking experiment.
4. Experimental section
4.1. General
Reagents for the synthesis were purchased from commercial sources
GYKI 52,466 was obtained from Tocris. Apo-ONE(R) Homogeneous
Caspase −3/7 Assay was obtained from Promega. DL-α-[5-methyl-3H]-
AMPA (55,4 Ci/mmol) was purchased from Perkin Elmer. Other sol-
vents, reagents and chemicals were purchased from POCH and Sigma-
Aldrich.
and used as obtained. Anhydrous acetonitrile was obtained in ac-
cordance with the literature procedure. Progress of the conducted re-
actions was monitored by TLC on Merck silica gel 60 F254 plates. Spots
were visualized by exposure to UV light or KMnO4 stain. Column
chromatography was performed using silica gel (200–400 mesh,
Merck). The NMR spectra were measured on a Varian VNMRS spec-
trometer (1H NMR at 500 MHz, 13C NMR at 125 MHz). 1H and 13C
chemical shifts (δ) are reported in parts per million (ppm) relative to
the solvent signals: CDCl3, δH (residual CHCl3) 7.26 ppm, δC 77.16 ppm;
DMSO‑d6 δH (residual DMSO‑d5) 2.50 ppm, δC 39.52 ppm; D2O, δH
(residual HOD) 4.79 ppm; signals are quoted as “d” (doublet),”t” (tri-
plet), “q” (quartet), “m” (multiplet), “br s” (broad singlet), and “dd”
(doublet of doublets). Coupling constants J are reported in Hertz. The
1H–13C HMBC NMR (Heteronuclear Multiple Bond Correlation) spectra
MOG-G-CCM – human brain astrocytoma and SK-N-AS – human
neuroblastoma cell lines were purchased from ECACC General Cell
Collection. MOG-G-CCM and SK-N-AS were cultured in Ham’s F10:
DMEM (1:1), DMEM with 1% Non Essential Amino Acids (NEAA) media
(Lonza), respectively. All media were supplemented with 10% fetal
bovine serum (EuroClone), 2 mM L-glutamine and antibiotics (100 U/
ml penicillin, 100 µg/mL streptomycin). Cells were cultured in 75 cm2
cell flasks (Sarstedt), in a humidified atmosphere of CO2/air (5/95%) at
37 °C. Human glioblastoma U251MG cell line was from Cell Lines
Service, Germany. Cells were cultivated at 37 °C with 5% CO2 in
Dulbecco's Modified Eagle Medium (DMEM; Gibco, New York, USA)
supplemented with GlutaMAX-1, 10% heat-inactivated fetal bovine
serum (FBS; Gibco, New York, USA) and antibiotics, 1% penicilin/
streptomycin solution (Gibco, New York, USA).
were measured on
a
Varian VNMRS spectrometer (1H NMR at
500 MHz). TOF-HRMS (ESI) measurements were performer with a Q-
Exactive Thermo Scientific spectrometer Optical rotation was measured
on an Anton-Paar MCP 150 polarimeter; [α]D values are given in 10−1
deg cm2 g−1; concentration (c) is given in g/100 cm3. Volatiles were
distilled off under reduced pressure on a rotating evaporator.
4.2. Preparation of compound 2a from sulfamidate 5a uner the NaH
conditions
Sodium hydride (60% in mineral oil, 40 mg, 1.00 mmol) was added
to a mixture of thymine (125 mg, 0.99 mmol) and dry DMF (7 ml).
Reaction mixture was stirred at room temperature for 2 h and cooled to
−78 °C. A solution of sulfamidate 5a (310 mg, 0.94 mmol) in DMF
Dimethyl sulphoxide (DMSO), Molecular Biology grade, used as a
solvent for all stocks of the chemical agents, was obtained from Roth.
7

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
(3 ml) was added within 5 min. The reaction mixture was stirred at
−78 °C for 45 min., then overnight at room temperature and poured
into water (30 ml). 2 M HCl was added to pH 1 (universal paper in-
dicator). The mixture was extracted with AcOEt (3 × 50 ml). Extracts
were combined and washed with water (30 ml) and dried (MgSO4).
Volatiles were distilled off under reduced pressure. The solid residue
(0.5 g) was subjected to column chromatography (hexane-AcOEt, 1:1,
v/v, then AcOEt) of afford compound 2a as a white solid (0.1 g, 28%).
atmosphere. A mixture of suflamidate 5a or 5b (0.97 mmol) and dry
acetonitrile (2.5 ml) was added. Unless otherwise given, the reaction
mixture was stirred at room temperature for 1 day. Water (2 ml) and
AcOEt (10 ml) were added and the mixture was stirred at room tem-
perature for 1 h. Volatiles were distilled off under reduced pressure. The
residue was subjected to column chromatography to afford the corre-
sponding product.
3
[α] −15.9 (c 0.11 in MeOH). δH(500 MHz; DMSO‑d6): δ 1.16 (t, JHH
4.4.1. (S)-Ethyl 2-(((benzyloxy)carbonyl)amino)-3-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)propanoate 2a
4
7.0 Hz, 3H); 1.70 (d, JHH 1.5 Hz, 3H), 3.68 and 4.16 (part AB of ABX
2
3
3
3
system, JAB 13.8 Hz, JAX 9.0 Hz, JBX 5.5 Hz, 2H), 4.10 (q, JHH
Compound 2a was obtained from thymine and sulfamidate 5a
(320 mg, 0.973 mmol). Column chromatography (AcOEt) afforded
compound 2a as a white solid (310 mg, 85%). mp 160–162 °C. [α]
−88.2 (c 0.11 in MeOH). 1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 1.16
7.1 Hz, 2H); 4.38–4.43 (part X of ABX system, 1H); 5.01 and 5.04 (AB
2
4
quartet, JAB 12.5 Hz, 2H); 7.25–7.40 (m, 6H); 7.83 (d, JHH 8.5 Hz,
1H); 11.38 (br s, 1H, NH).
3
(t, JHH 6.7 Hz, 3H, CH3-Et), 1.70 (s, 3H, CH3-5′), 3.68 and 4.16 (part
4.3. Preparation of the reference compound 2a from benzyl (S)-(2-
oxooxetan-3-yl)carbamate. [25]
AB of ABX system, 2JAB 13.7, 3JAX 9.2 Hz, 3JBX 6.0 Hz, 2H, H-3), 4.10 (q,
3JHH 6.7 Hz, 2H, CH2-Et), 4.37–4.43 (X part of ABX system, 1H, H-2),
5.01 and 5.04 (AB quartet, 2JAB 12.5, 2H, CH2-Cbz), 7.26–7.40 (m, 6H,
3
Step 1. Sodium hydride (60% in mineral oil, 85 mg, 2.08 mmol) was
added to a mixture of thymine (265 mg, 2.28 mmol) and dry DMF
(19 ml). Reaction mixture was stirred at room temperature for 2 h and
cooled to −78 °C. A solution of (S)-(2-oxooxetan-3-yl)carbamate
(460 mg, 2.08 mmol) in DMF (5 ml) was added dropwise within 1 h.
The cooling bath was removed and the reaction mixture was stirred at
room temperature for 18 h. Volatiles were distilled off under reduced
pressure. The residue (1.255 g) was dissolved in water and acidified
with 2 M HCl to pH 2 and extracted with ethyl acetate (3 × 80 ml). The
extracts were combined and dried (MgSO4). Volatiles were distilled off
under reduced pressure and the residue (0.74 g) was subjected to
column chromatography (DCM:MeOH:AcOH, 97:3:0.1, v/v/v then
90:10:0.1, v/v/v) to give (S)-2-(((benzyloxy)carbonyl)amino)-3-(5-me-
thyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propanoic acid 2c as a
white solid 0.265 g (36%). [α] −85.1 (c 0.20 in MeOH). 1H NMR
(500 MHz, DMSO‑d6): δ 1.68 (s, 3H); 3.58 and 4.24 (AB part of ABX
H-Ph and H-6′), 7.84 (d, JHH 8.5 Hz, 1H, NH-2), 11.30 (br s, 1H, NH-
3′). 13C NMR (125 MHz, DMSO‑d6, 25 °C): δ 11.9 (CH3-5′), 13.9 (CH3-
Et), 47.9 (C-3), 52.2 (C-2), 61.1 (CH2-Et), 65.6 (CH2-Cbz), 108.2 (C-5′),
127.5 (C-Ph), 127.8 (C-Ph), 128.3 (C-Ph), 136.8 (C-Ph), 141.7 (C-6′),
150.9 (C-2′), 155.9 (Cbz-C]O), 164.2 (C-4′), 169.7 (C-1). Signals were
assigned from the 1H–13C HMBC NMR spectrum (ESI, subsection 3.3).
HRMS m/z [M + H]+, calcd. for C18H22N3O6 376.1509; found
376.1500. Elemental analysis: C, 57.59%; H, 5.64%; N, 11.19% calcd.
for C18H21N3O6; found C, 57.54%; H, 5.45%; N, 10.91%.
4.4.2. (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)propanoate 2b
Compound 2b was obtained from thymine and sulfamidate 5b
(400 mg, 1.02 mmol). Column chromatography (AcOEt) afforded
compound 2b as a white solid (308 mg, 69%). mp 158–161 °C. [α]
−64.9 (c 0.11 in MeOH). 1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 1.67
(d, 4JHH 1.5 Hz, 3H, 3′–CH3), 3.72 and 4.20 (AB part of ABX system 2JAB
2
3
3
system, JAB 13.6 JAX 10.1 Hz JBX 4.7 Hz, 2H), 4.30–4.35 (X part of
ABX system, 1H); 4.97 and 5.02 (AB quartet, 2JAB 10.0, 2H); 7.27–7.35
(m, 6H); 7.48 (br s, 1H); 11.25 (s, NH, 1H). 13C NMR (125 MHz,
DMSO‑d6): δ 12.0; 48.7; 52.7; 65.4; 107.8; 127.4; 127.8; 128.4; 137.0;
142.0; 150.9; 156.0; 164.3; 171.3. Step 2. Thionyl chloride (0.1 ml,
1.37 mmol) was added drop by drop to a mixture of (S)-2-(((benzyloxy)
carbonyl)amino)-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)propanoic acid 2c (85 mg, 0.24 mmol) and ethanol (8 ml) cooled in
ice-water bath (-5 ÷ 0 °C). The mixture was stirred at the cooling bath
for 30 min. The bath was removed and the mixture was left at room
temperature overnight. Volatiles were distilled off under reduced
pressure. The residue was dissolved in AcOEt (30 ml) and washed with
aqueous saturated solution of NaHCO3 (15 ml) and water (15 ml), brine
(15 ml) and dried (MgSO4). Volatiles were distilled off under reduced
pressure to give (S)-ethyl 2-(((benzyloxy)carbonyl)amino)-3-(5-methyl-
3
3
14.0, JAX 9.0 Hz, JBX 5.5 Hz, 2H), 4.47–4.52 (part X of ABX system,
2
1H), 5.02 and 5.03 (AB quartet, JAB 12.5, 2H), 5.12 and 5.15 (AB
quartet, 2JAB 12.5, 2H), 7.45–7.21 (m, 10H), 7.90 (d, 3JHH 8.6 Hz, 1H),
11.30 (br s, 1H, NH). 13C NMR (125 MHz, DMSO‑d6, 25 °C): δ 11.9,
48.0, 52.3, 65.6, 66.5, 108.2, 127.5, 127.8, 127.9, 128.1, 128.3, 128.4,
135.5, 136.7, 141.7, 150.9, 156.0, 164.2, 169.6. HRMS m/z: [M + H]+
calcd. for C23H23N3O6 438.1665; found 438.1659.
4.4.3. (S)-Ethyl
2-(((benzyloxy)carbonyl)amino)-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propanoate 3a
Compound 3a was obtained from uracil and sulfamidate 5a
(220 mg, 0.669 mmol). Column chromatography (AcOEt) afforded
compound 3a as a white solid (150 mg, 62%). mp 127–130 °C. [α]
−109.1 (c 0.12 in MeOH). 1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 1.17
3
2
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propanoate 2a as
a
white
(t, JHH 7.0 Hz, 3H), 3.70 and 4.20 (part AB of ABX system, JAB 13.5,
3 3
solid (75 mg, 83%). [α] −85.6° (c 0.11 in MeOH). 1H NMR (500 MHz,
3JAX 9.0 Hz, JBX 6.0 Hz, 2H), 4.11 (q, JHH 7.0 Hz, 2H), 4.39–4.4 (X
3
4
2
DMSO‑d6): δ 1.16 (t, JHH 7.1 Hz, 3H); 1.70 (d, JHH 1.5 Hz, 3H), 3.68
and 4.16 (part AB of ABX system, 2JAB 13.8 Hz, 3JAX 9.0 Hz, 3JBX 5.5 Hz,
part of ABX system, 1H), 5.01 and 5.04 (AB quartet, JAB 12.5, 2H),
5.52 (d, 3JHH 7.7 Hz, 1H), 7.25–7.40 (m, 5H), 7.45 (d, 3JHH 7.7 Hz, 1H),
3
3
2H), 4.10 (q, JHH 7.1 Hz, 2H); 4.38–4.43 (part X of ABX system, 1H);
7.85 (d, JHH 8.6 Hz, 1H, NH), 11.31 (s, 1H, NH). 13C NMR (125 MHz,
2
5.01 and 5.04 (AB quartet, JAB 12.5 Hz, 2H); 7.30–7.38 (m, 6H); 7.84
DMSO‑d6, 25 °C): δ 13.9, 48.1, 52.1, 61.2, 65.6, 100.8, 127.9, 127.6,
128.4, 136.8, 145.9, 150.9, 156.0, 163.6, 169.7. HRMS m/z: [M + H]+
calcd. for C17H20N3O6 362.1352; found 362.1347. Elemental analysis:
C, 56.51%; H, 5.30%; N, 11.63% calcd. for C17H19N3O6; found C,
56.50%; H, 5.25%; N, 11.66%.
(d, JHH 8.5 Hz, 1H, NH); 11.31 (br s, 1H, NH). 13C NMR (125 MHz,
4
DMSO‑d6): δ 11.9, 13.9, 47.9, 52.2, 61.10, 65.6, 108.2, 127.5, 127.8,
128.4, 136.8, 141.7, 150.9, 156.0, 164.2, 169.7.
4.4. General procedure for the preparation of compounds 2a,b, 3a,b and
4a,b from sulfamidates 5 under the BSA-conditions
4.4.4. (S)-Benzyl
2-(((benzyloxy)carbonyl)amino)-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propanoate 3b
A mixture of the nucleobase (thymine, uracil, or N4-benzoylcyto-
sine; 0.99 mmol), N,O-bis(trimethylsilyl)acetamide (BSA, 1.98 mmol
for thymine or uracil, or 1.2 mmol for N4-benzoylcytosine) and dry
acetonitrile (2 ml) was stirred at room temperature for 1 h under argon
Compound 3b was obtained from uracil and sulfamidate 5b (1.23 g,
3.14 mmol). Column chromatography (AcOEt) afforded compound 3b
as a white solid (1.03 g, 78%). mp 135–137 °C. [α] −90.1 (c 0.12 in
MeOH). 1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 3.75 and 4.24 (AB part
8

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
2
3
3
3
of ABX system JAB 13.8, JAX 9.5 Hz, JBX 5.0 Hz, 2H), 4.48–4.53 (X
7.68 (d, JHH 9.6 Hz, 1H, NH-2), 11.28 (br s, 1H, NH-3′), 13.07 (br s,
1H, OH-1). 13C NMR (125 MHz, DMSO‑d6, 25 °C): δ 11.9 (5′–CH3), 48.3
(C-3), 52.2 (C-2), 65.5 (CH2-Cbz, 108.0 (C-5′), 127.4 (C-Ph), 127.8 (C-
Ph), 128.4 (C-Ph), 136.9 (C-Ph), 141.9 (C-6′), 150.9 (C-2′), 156.0 (Cbz-
2
3
part of ABX system, 1H), 5.02 and 5.04 (AB quartet, JAB 13.0, 2H),
2
4
5.13 and 5.16 (AB quartet, JAB 12.5, 2H), 5.49 (dd, JHH 7.8, JHH
3
2.0 Hz, 1H), 7.08–7.61 (m, 10H), 7.45 (d, JHH 7.8 Hz, 1H), 7.91 (d,
3JHH 8.7 Hz, 1H), 11.31 (s, 1H, NH). 13C NMR (125 MHz, DMSO‑d6,
25 °C): δ 48.2, 52.2, 65.7, 66.6, 100.8, 127.5, 127.8, 127.9, 128.2,
128.3, 128.4, 135.5, 136.7, 145.9, 150.9, 156.0, 163.6, 169.5. HRMS
m/z: [M + H]+ calcd. for C22H22N3O6 424.1509; found 424.1503.
C]O), 164.3 (C-4′), 171.1 (C-1). Signals were assigned from the 1H–13
C
HMBC NMR spectrum (ESI, subsection 3.8). HRMS m/z: [M + H]+
calcd. for C16H18N3O6 348.1196; found 348.1190.
4.5.2. 2-(((Benzyloxy)carbonyl)amino)-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propanoic acid 3c
4.4.5. (S)-Ethyl 3-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-2-(((benzyloxy)
carbonyl)amino)propanoate 4a
Compound 3c was obtained from ester 3a (130 mg, 0.36 mmol) as a
Compound 4a was obtained from N4-benzoylcytosine and sulfami-
date 5a (220 mg, 0,669 mmol). The reaction mixture was stirred for
3 days. Column chromatography (AcOEt) afforded compound 4a as a
white solid (190 mg, 61%). mp 163–165 °C. [α] −111.0 (c 0.11 in
white solid (100 mg, 83%) after column chromatography (CH2Cl2
:
MeOH/ 7:3, v/v). [α] −99.5 (c 0.21 in MeOH) [31]. 1H NMR
(500 MHz, DMSO‑d6, 25 °C): δ 3.46 and 4.32 (AB part of ABX system
2JAB 13.4, JAX 10.2 Hz, JBX 4.0 Hz, 2H), 4.12–4.17 (X part of ABX
3
3
MeOH). 1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 1.18 (t, JHH 7.0 Hz,
3
2
3
system, 1H), 4.99 and 4.93 (AB quartet, JAB 15.0, 2H), 5.41 (d, JHH
7.8 Hz, 1H), 6.93 (d, 3JHH 7.7 Hz, 1H), 7.27–7.35 (m, 5H), 7.50 (d, 3JHH
7.7 Hz, 1H), 11.14 (br s, 1H, NH). 13C NMR (125 MHz, DMSO‑d6,
25 °C): δ 50.3, 54.0, 65.2, 100.0, 127.4, 127.7, 128.3, 137.2, 146.3,
3H), 3.84 and 4.40 (part AB of ABX system, 2JAB 13.0, 3JAX 9.5 Hz, 3JBX
5.0 Hz, 2H), 4.13 (q, 3JHH 7 Hz, 2H), 4.53–4.57 (part X of ABX system,
2
1H), 5.00 and 5.04 (AB quartet, JAB 12.0, 2H), 7.22–8.02 (m, 12H),
11.20 (br s, 1H, NH). 13C NMR (125 MHz, DMSO‑d6, 25 °C): δ 14.0,
21.1, 50.3, 51.8, 61.2, 65.7, 95.8, 127.6, 127.8, 128.3, 128.4, 128.5,
132.7, 133.2, 136.8, 150.7, 155.1, 156.0, 163.3, 167.5, 169.8, 172.1.
HRMS (ESI) m/z: [M + H]+ calcd. for C24H25N4O6 465.1774; found
465.1768. Elemental analysis: C, 60.88%; H, 5.32%; N, 11.33% calcd.
for 2C24H24N4O6·H2O; found C, 60.93%; H, 5.06%; N, 11.84%.
151.0, 155.9, 163.9, 171.1. HRMS m/z: [M
+
H]+ calcd. for
C
15H15N3O6 334.1039; found 334.1034.
4.5.3. (S)-3-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-2-(((benzyloxy)
carbonyl)amino)propanoic acid 4c
Compound 4c was obtained from ester 4a (135 mg, 0.291 mmol) as
a white solid (67 mg 53%). A small amount of benzoic acid as impurity
was detected in the 13C NMR spectrum. [α] −88.3 (c 0.10 in MeOH).
1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 3.80 and 4.44 (AB part of ABX
4.4.6. (S)-Benzyl 3-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-2-(((benzyloxy)
carbonyl)amino)propanoate 4b
Compound 4b was obtained from N4-benzoylcytosine and sulfami-
date 5b (170 mg, 0.43 mmol). The reaction mixture was stirred for
3 days. Column chromatography (AcOEt) afforded compound 4b as a
white solid (229 mg, 52%). mp 149–152 °C. [α] −34.3 (c 0.11 in
CH2Cl2). 1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 3.88 and 4.43 (AB part
2
3
3
system, JAB 15.0 JAX 10.0 Hz JBX 5.0 Hz, 2H), 4.49–4.53 (X part of
ABX system, 1H), 4.99 and 5.02 (AB quartet, 2JAB 15.0, 2H), 7.25–8.02
(m, 14H), 11.29 (br s, NH, 1H). 13C NMR (125 MHz, DMSO‑d6, 25 °C): δ
50.4, 51.7, 65.5, 95.6, 127.5, 127.7, 128.3, 128.4, 128.5, 128.6 (ben-
zoic acid), 129.2 (benzoic acid), 130.7 (benzoic acid), 132.7, 132.8
(benzoic acid), 133.2, 136.9, 150.8, 154.8, 156.0, 163.1, 167.3, 171.2.
HRMS m/z: [M + H]+ calcd. for C22H21N4O6 437.1461; found
437.1456.
2
3
3
of ABX system, JAB 13.5, JAX 9.6 Hz, JBX 5.0 Hz, 2H), 4.62–4.66 (X
2
part of ABX system, 1H), 5.02 and 5.03 (AB quartet, JAB 12.5, 2H),
2
5.14 and 5.17 (AB quartet, JAB 12.5, 2H), 7.25–8.02 (m, 19H), 11.21
(br s, 1H, NH). 13C NMR (125 MHz, DMSO‑d6, 25 °C): 50.2, 51.8, 65.7,
66.6, 95.8, 127.5, 127.8, 127.9, 128.1, 128.3, 128.4, 128.4, 128.4,
132.7, 133.1, 135.5, 136.7, 150.7, 155.1, 156.0, 163.3, 167.2, 169.6.
HRMS m/z: [M + H]+ calcd. for C29H27N4O6 527.1931; found
527.1925. Elemental analysis: C, 66.15%; H, 4.98%; N, 10.64% calcd.
for C29H26N4O6; found C, 65.94%; H, 4.89%; N, 10.58%.
4.6. General procedure for the preparation of compounds: willardiine, 5-
methylwillardiine, 2d and 2e
A mixture of compound 2a (or 2b or 2c or 2c, Pd/C (10%) and
MeOH was treated with hydrogen under the balloon pressure at room
temperature. Progress of the reaction was monitored by TLC (CHCl3/
MeOH, 7:3, v/v) (or CHCl3/MeOH, 9:1, v/v for hydrogenation of
compound 2a). A solid precipitated during the reaction. The mixture
was worked-up as given below.
4.5. General procedure for preparation of compounds 2c, 3c and 4c
A mixture of compound 2a (or 3a or 4a, 0.346 mmol), THF (2.4 ml)
and 1,4-dioxane (3.6 ml) was added dropwise to aqueous NaOH (1 M,
1.6 ml). The mixture was stirred at room temperature overnight and
water (30 ml) was added. The mixture was extracted with CH2Cl2
(15 ml). Aqueous phase was acidified with 2 M HCl to pH 1 (universal
paper indicator). The mixture was extracted with AcOEt (3 × 25 ml).
The combined organic phases were dried (MgSO4) and volatiles were
distilled off under reduced pressure to obtain compound 2c (or 4c). The
residue from the reaction of compound 3a was subjected to column
chromatography to give compound 3c.
4.6.1. 2-Amino-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propanoic
acid (willardiine)
Willardiine was obtained from hydrogenation of compound 3b
(692 mg, 1.63 mmol) in MeOH (110 ml) in the presence of Pd/C (10%,
70 mg) for 4 h. The reaction mixture was filtrated through a Celite pad.
The Celite pad was transferred to a round-bottomed flask and sonicated
with MeOH (50 ml) for 15 min. The mixture was filtered through a
Celite pad. The Celite pad was washed with MeOH (2 × 50 ml). The
filtrates were discarded and the Celite pad was then washed with water
(60 ml). Volatiles were distilled off from the filtrate to give willardiine
(321 mg, 98%, a white solid, mp 204–205 °C). [α] –23.7 (c 0.90 in 1 M
HCl). Lit. [α] = -21 (c 1.0 in 1 N HCl) [30]. Lit. [α] −24.4 (c 1.16 in
6 M HCl) [2]. 1H NMR (500 MHz, D2O, 25 °C): δ 4.26 and 4.39 (part AB
4.5.1. 2-(((Benzyloxy)carbonyl)amino)-3-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propanoic acid 2c
Compound 2c was obtained from ester 2a (130 mg, 0.346 mmol) as
a white solid (0.1 g, 83%). mp 178–180 °C. [α] −86.7 (c 0.20 in
MeOH). Second run of the reaction gave compound 2c in 80% yield, [α]
−86.3 (c 0.21 in MeOH). Lit. [α] −91 (c 0.2, MeOH) [25]. 1H NMR
2
3
3
of ABX system, JAB 15.2 Hz, JAX 6.2 Hz, JBX 4.6 Hz, 2H), 4.43–4.45
4
3
3
(500 MHz, DMSO‑d6, 25 °C): δ 1.69 (d, JHH 1.0 Hz, 3H, 5′–CH3), 3.62
(part X of ABX system, 1H), 5.78 (d, JHH 7.9 Hz, 1H); 7.56 (d, JHH
7.9 Hz, 2H). 13C NMR (125 MHz, D2O, 25 °C): δ = 47.9, 52.2, 102.3,
and 4.21 (AB part of ABX system 2JAB 13.5, 3JAX 10 Hz 3JBX 5.0 Hz, 2H,
H-3), 4.34–4.40 (X part of ABX system, 1H, H-2), 4.99 and 5.03 (AB
146.8, 152.7, 166.4, 169.0. HRMS m/z: [M
+
H]+ calcd. for
2
quartet, JAB 10.0, 2H, CH2-Cbz), 7.24–7.38 (m, 6H, H-Ph and H-6′),
C7H10N3O4 200.0671; found 200.0666.
9

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
4.6.2. Ethyl 2-amino-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)propanoate 2d
163.7, 173.2. HRMS m/z: [M + H]+ calcd. for C9H13N3O4 228.0984;
found 228.0979.
Compound 2d (was obtained from compound 2a (150 mg,
0.40 mmol) in MeOH (20 ml) in the presence of Pd/C (10%, 20 mg) for
2 h. The reaction mixture was filtrated through a Celite pad. The Celite
pad was transferred to a round-bottomed flask and sonicated with
MeOH (50 ml) for 15 min. The mixture was filtered through a Celite
pad. The Celite pad was washed with MeOH (2 × 50 ml). The filtrates
were collected and volatiles were distilled off. The oleaginous residue
was subjected to column chromatography (CHCl3-MeOH, 9:1, v/v) to
give product 2d (90 mg, 96%, a white solid, mp 140–144 °C dec.).
[α] + 11.9 (c 0.10 in MeOH) [32]. 1H NMR (500 MHz, DMSO‑d6,
25 °C): δ 1.16 (t, 3JHH 7.2 Hz, 3H), 1.73 (d, 4JHH 1.4 Hz 3H), 1.92 (br s,
2H, NH), 3.54–3.61 (AB part of ABX system, 2H), 3.81–3.90 (X part of
4.8. MTS cell viability assay
The dynamics of cell growth was analyzed by the MTS assay
(Promega, USA). Cells were seeded in 96-well culture plates at a density
of 8 × 103 cells per well in a regular DMEM medium. The cultivation
medium was changed 18 h after plating; Next, cells were washed twice
with PBS and a fresh experimental medium with or without willardiine
(10–1000 µM concentration). Incubation with willardiine lasted for 24,
48 and 72 h. Then MTS test was performed for 1 h and the absorbance
was measured using Tecan SUNRISE XFluor4 plate reader at λ 490 nm.
Cell growth was calculated as a percentage of viable cells after treat-
ment by assuming 100% viability for the absorbance recorded in the
control conditions.
3
3
ABX system, 1H), 4.05 (q, JHH 7.2 Hz, 2H), 7.46 (d, JHH 1.4 Hz, 1H),
11.23 (br s, 1H, NH). 13C NMR (125 MHz, DMSO‑d6, 25 °C): δ 11.9,
13.9, 50.8, 52.9, 60.5, 107.6, 142.4, 151.0, 164.3, 173.6. HRMS m/z:
[M + H]+ calcd. for C10H16N3O4 242.1141; found 242.1135.
4.9. Fluorescent microscopy
4.6.3. 2-Amino-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
propanoic acid (5-methylwillardiine)
Cells were seeded on the cover glasses and cultured for 48 h with
500 µM willardiine. After treatment cells were fixed in 4% paraf-
ormaldehyde for 15 min and washed twice with PBS. To visualize actin
filaments, cells were stained for 20 min with Alexa 488-conjugated
phalloidin (diluted 1:40 in PBS; Invitrogen, USA), washed three times
with PBS, and mounted by Vectashield anti-fade reagent with DAPI
(Vector Laboratories, USA). Digital images of the selected areas were
taken using Nikon eclipse Ti-U fluorescent microscope equipped with
10x objective and DS-Qi2 camera.
5-Methylwillardiine was obtained from hydrogenation of compound
2b (75 mg, 0.22 mmol) in MeOH (14 ml) in the presence of Pd/C (10%,
20 mg) for 3 h. The mixture was filtrated through a Celite pad. The
Celite pad was transferred to a round-bottomed flask and sonicated
with MeOH (50 ml) for 15 min. The mixture was filtered through a
Celite pad. The Celite pad was washed with MeOH (2 × 50 ml). The
filtrates were discarded and the Celite pad was then washed with water
(60 ml). Volatiles were distilled off from the filtrate to give product
(35 mg, 76%, a white solid). [α] −15.4 (c 1.0 in 1 M HCl); [α] −16.7
(c 1.0 in 1 M HCl). Lit. [α] = -15 (c 1 in 1 N HCl) [29]. Lit. [α] = -10 (c
1.15 in NHCl) [44]. 1H NMR (500 MHz, DMSO‑d6, 25 °C): δ 1.74 (d,
4.10. Transwell migration assay
To assess cell migration, the 6.5-mm diameter polycarbonate filters
with 8-μm pore size Transwell migration inserts (Corning, New York,
USA) were used. Cells were treated with 500 µM willardiine for 48 h.
After trypsinization, cells were washed twice with serum-free medium,
and 3 × 104 cells were seeded on the upper chamber and allowed to
migrate for 8 h through the filter to the lower chamber containing
DMEM media supplemented with 10% FBS. After that, cells were fixed
by 4% paraformaldehyde for 15 min and stained with 0.5% crystal
violet for 10 min at room temperature. Non-migrated cells were re-
moved from the upper chamber using a cotton swab. Cells were finally
detected quantitatively using the above mentioned microscope.
2
3
4JHH 1 Hz, 3H), 4.03 (AB part of ABX system, JAB 14.0, JAX 7.6 Hz,
4
1H), 4.15–4.30 (AB part and X part of ABX system, 2H), 7.52 (d, JHH
1 Hz, 1H), 8.60 (br s, NH, 2H), 11.34 (s, NH, 1H). 13C NMR (125 MHz,
DMSO‑d6, 25 °C): δ 12.1, 47.0, 50.8, 108.9, 141.3, 151.4, 164.4, 168.6.
HRMS m/z: [M
+
H]+ calcd. for C8H12N3O4 214.0828; found
214.0825.
4.6.4. 2-Amino-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
propanoic acid hydrochloride 2e
Compound 2e (137 mg, 87%, a white solid) was obtained from
hydrogenation of compound 2b (277 mg, 0.63 mmol) in MeOH (30 ml),
AcOEt (10 ml) and CH2Cl2 (10 ml) in the presence of Pd/C (10%,
40 mg) for 5 h. The mixture was filtrated through a Celite pad. The
Celite pad was transferred to a round-bottomed flask and sonicated
with MeOH (50 ml) for 15 min. The mixture was filtered through a
Celite pad. The Celite pad was washed with MeOH (2 × 50 ml). The
filtrates were discarded and the Celite pad was then washed with 2 M
HCl (40 ml). Volatiles were distilled off from the filtrate to give com-
pound 2b (137 mg, 87%, a white solid). [α] −12.8 (c 1.0 in 1 M HCl).
4.11. Immunoblotting
Western blot analyses were conducted using whole cell protein ex-
tracts. After treatment cell monolayer was washed three times in ice-
cold PBS and lysed in RIPA Buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% IGEPAL, 50 mM NaF,
2 mM Na3VO4, 1 mM PMSF, protease inhibitors and phosphatase in-
hibitors cocktailes (Roche, Germany)) at ice for 20 min. Cell extracts
were centrifugated at 12000 rpm at 4 °C for 20 min and supernatants
were used for the study. Protein concentration was determined by the
Bradford method (Bio-Rad, USA). For western blot analysis proteins
were separated on 10% SDS-polyacrylamide gel (SDS-PAGE) and
transferred onto nitrocellulose membrane (Amersham, Germany). The
membranes were blocked with 5% milk or 5% BSA in TBS with 0.2%
Triton X-100 (TBS-T) and probed with t primary and secondary anti-
bodies. Primary antibodies used in analyses wereagainst GluR1
(ab109450, Abcam, UK), GluR2 (32–0300, Invitrogen, USA), p-Erk1/2
and Erk1/2 (4094 and 4695, respectively, Cell Signaling, USA), p-Akt
and Akt (9271 and 9272, respectively, Cell Signaling, USA), p-FAK and
FAK (8556 and 3285, respectively, Cell Signaling, USA), vinculin
(V4505, Sigma-Aldrich, USA), talin (sc-7534, Santa Cruz
Biotechnology, USA), β-actin (A3854, Sigma-Aldrich, USA). Anti-mouse
and anti-rabbit secondary antibodies from Millipore (USA) were used.
4.7. Preparation of ethyl 2-amino-3-(2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)propanoic 3d
A mixture of compound 3a (120 mg, 3.3 mmol), ethanol (6 ml),
cyclohexene (2.8 ml) and Pd/C (10%, 30 mg) was refluxed under argon
atmosphere for 7 h. The mixture was cooled to room temperature and
filtered through a Celite pad. The pad was washed with ethanol (30 ml).
Volatiles were distilled off from the combined filtrates. The residue was
subjected to column chromatography (CHCl3:MeOH, 9:1, v/v) to give
product as a white solid (56 mg, 75%). [α] + 1.9 (c 0.11 in MeOH)
3
[28]. 1H NMR (500 MHz, CDCl3, 25 °C): δ 1.29 (t, JHH 7.1 Hz, 3H),
1.74 (bs, 2H, NH2), 3.74–3.84 (m, 2H), 4.05–4.09 (m, 1H), 4.16–4.26
3
3
(m, 2H), 5.67 (d, JHH 7.9 Hz, 1H), 7.28 (d, JHH 7.9 Hz). 13C NMR
(125 MHz, CDCl3, 25 °C): δ 14.3. 52.1, 53.4, 62.0, 101.8, 145.8, 151.1,
10

J. Ignatowska, et al.
BioorganicChemistry100(2020)103864
Also, detection of GAPDH with the relevant antibody (MAB374,
Millipore, USA) was used as a protein loading control. Subsequently,
the blots were washed with TBS-T, and the HRP-bound secondary an-
tibody was detected using the ECL reagent (Millipore, USA).
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Declaration of Competing Interest
None.
Acknowledgements
This work was financed by National Science Center (NCN), Poland,
Project No 2015/17/B/ST5/00547. The contribution of Piotr
Grześkowiak was financed by National Centre for Research and
Development (NCBiR), Poland, Project No POWR.03.02.00-00-1007/
16-00 (POWER 2014-2020). This work was financially supported by
Warsaw University of Technology, Warsaw, Poland. The part of biolo-
gical assays (M.J.R. and O.K.) were performed with the support from
the European Union's Horizon 2020 Research and Innovation
Programme under the Marie Skłodowska-Curie grant agreement No.
665735 granted to the Nencki Institute, Warsaw, Poland.
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