A fragment-based approach for the development of g-quadruplex ligands: Role of the amidoxime moiety

Article abstract of DOI:10.3390/molecules23081874

G-quadruplex (G4) nucleic acid structures have been reported to be involved in several human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however, G4 targeting compounds still need implementation in terms of drug-lik

Full text of DOI:10.3390/molecules23081874

molecules
Article
A Fragment-Based Approach for the Development of
G-Quadruplex Ligands: Role of the
Amidoxime Moiety
1
2
1
2
1
ID
Martina Tassinari , Alberto Lena , Elena Butovskaya , Valentina Pirota , Matteo Nadai
,
2
2, ID
1, ID
Mauro Freccero , Filippo Doria * and Sara N. Richter *
1
Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy;
martina.tassinari@unipd.it (M.T.); elena.butovskaya@gmail.com (E.B.); matteo.nadai@unipd.it (M.N.)
Department of Chemistry, University of Pavia, Viale Taramelli 10, 27100 Pavia, Italy;
alberto.lena01@ateneopv.it (A.L.); valentina.pirota01@universitadipavia.it (V.P.);
mauro.freccero@unipv.it (M.F.)
2
*
Correspondence: filippo.doria@unipv.it (F.D.); sara.richter@unipd.it (S.N.R.); Tel.: +39-049-8272446 (S.N.R.)
Academic Editor: Ramon Eritja
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 2 July 2018; Accepted: 24 July 2018; Published: 27 July 2018
Abstract: G-quadruplex (G4) nucleic acid structures have been reported to be involved in several
human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however,
G4 targeting compounds still need implementation in terms of drug-like properties and selectivity in
order to reach the clinical use. So far, G4 ligands have been mainly identified through high-throughput
screening methods or design of molecules with pre-set features. Here, we describe the development
of new heterocyclic ligands through a fragment-based drug discovery (FBDD) approach. The ligands
were designed against the major G4 present in the long terminal repeat (LTR) promoter region of the
human immunodeficiency virus-1 (HIV-1), the stabilization of which has been shown to suppress viral
gene expression and replication. Our method is based on the generation of molecular fragment small
libraries, screened against the target to further elaborate them into lead compounds. We screened
150 small molecules, composed by structurally and chemically different fragments, selected from
commercially available and in-house compounds; synthetic elaboration yielded several G4 ligands
and two final G4 binders, both embedding an amidoxime moiety; one of these two compounds
showed preferential binding for the HIV-1 LTR G4. This work presents the discovery of a novel
potential pharmacophore and highlights the possibility to apply a fragment-based approach to
develop G4 ligands with unexpected chemical features.
Keywords: G-quadruplex; amidoximes/oxadiazole/pyridine; fragments; FBA; FRET-melting
1
. Introduction
G-quadruplexes (G4s) are nucleic acids secondary structures that may form in single-stranded
guanine (G)-rich sequences under physiological conditions [1–3]. Four Gs bind via Hoogsteen-type
hydrogen bonds base-pairing to yield G-quartets, which in turn stack on top of each other to form
the G4 (Figure 1). G4s are highly polymorphic, both in terms of strand stoichiometry (forming both
+
inter- and intramolecular structures) and strand orientation/topology. The presence of K cations
specifically supports G4 formation and stability [
found in telomeres, G-rich micro- and mini-satellites, up-stream to oncogene promoters and within
the ribosomal DNA (rDNA) [ 12]. Human G4 DNA motifs are over-expressed in recombinogenic
regions [13 15], which are associated with genomic damage in cancer cells. Additionally, these regions
show mutational patterns that preserve the potential to form G4 DNA structures [11]. The identification
4–6]. In the human genome G4 DNA motifs have been
7–
–
Molecules 2018, 23, 1874; doi:10.3390/molecules23081874
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1874
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of G4 binding proteins [16–18] and G4 visualization in cells with antibody-based technology [19,20]
have also provided convincing evidence of the existence of cellular G4s in vivo, prevalently in
tumours compared to normal tissues [21]. More recently, research on G4s has also focused on
prokaryotes [22] and viruses. Indeed, G4s have been found to control key viral steps [23–26] and
treatment with G4 ligands has shown to impair viral replication [27]. Due to the possibility to
target pathogenic pathways by stabilizing G4 structures, several G4 binders have been developed.
In particular, ligands targeting tumour mechanisms at the telomere and oncogene promoter level have
been reported [24]. These share an aromatic core and protonable side chains. Some of these compounds
showed interesting antitumor properties; nevertheless, only quarfloxin proceeded into Phase II clinical
trials. Unfortunately, its limited bioavailability prevented further progress [24]. The success of tumour
targeting through a G4-binding mechanism heavily relies on the sustainable identification of new and
selective chemical entities.
Figure 1. The G-quadruplex structure. (
G-tetrad, composed of four guanines linked together through Hoogsteen H-bonds; M represents the
a
) Chemical structure and (
b
) schematic illustration of a
+
monovalent cation coordinated at the centre of the tetrad. (c) Example of intramolecular G4 structure.
Fragment-based drug discovery (FBDD) is a validated drug design strategy and a successful
alternative to traditional high-throughput screening methods [28]. Fragments are defined by “the rule
of three” (Ro3) [29] as organic molecules with molecular weight (MW) in the range of 100–300 Da [30],
each with a limited number (<3) of hydrogen bond donors and acceptors and rotatable bonds.
Following detection, fragment hits can be expanded or combined to generate larger molecules with
high affinity, selectivity and more drug-like properties [31]. As the concept of fragment-based lead
generation has become established, it has also emerged that integrated approaches with fragment-based
hits, such as high-throughput screening (HTS) can be very fruitful [32]. Using this approach, the drug
vemurafenib was the first clinical candidate born out of a fragment-screening program approved by
the US Food and Drug Administration in 2011 for the treatment of metastatic melanoma [33]. Several
other fragment-derived molecules are now at the clinical stage [30].
A fragment-based approach has recently been applied to target G4 in the c-MYC promoter and
inhibition of c-MYC expression has been obtained, even though at fragment concentrations in the µM
range [34]. Selectivity towards RNA vs. DNA G4s has also been achieved by fragment expansion by
click chemistry [35].
Here the FBDD approach was used to develop new G4 ligands targeting the major DNA G4
structure present in the Human immunodeficiency virus type-1 (HIV-1) LTR (Long Terminal Repeat).
We have previously demonstrated that three mutually exclusive and functionally significant G4s can
fold within the HIV-1 LTR, that is, LTR-II, LTR-III and LTR-IV, with LTR-III being the predominant
structure [26]. LTR G4s act as negative regulators of viral transcription and their stabilization by
G4 ligands leads to a remarkable antiviral effect, thus validating the role of G4s as anti-HIV-1
target [25
,36]. In this context, the final goal of the present work was to identify selective and
efficient ligands for LTR-III, characterized by a new pharmacophore unit with favourable drug-like

Molecules 2018, 23, 1874
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properties. We preliminarily generated small libraries of molecular fragments, screening them against
the target. The resulting hits were synthetically further elaborated into lead compounds, progressively
implementing the structural complexity of the aryl- and heteroaryl-scaffold, keeping unmodified the
structural moiety identified at the preliminary pre-screening level.
2
. Results and Discussion
2
.1. Initial Screening of Low-Molecular Weight Fragment Molecules
We initially screened around 150 structurally and chemically different fragment molecules,
selected from an in-house library of commercially available or in-lab synthesized compounds.
Each member of this starting fragment family obeyed the principal criterion of fragment libraries, that
is, Ro3. All the fragments were
≥
95% pure and showed >1 mM aqueous solubility. Their interaction
with two G4 forming sequences was investigated: in particular, with the HIV-1 LTR-III sequence [26],
which modulates HIV-1 promoter activity and with the human telomeric (hTel) [37], which is likely
the most common and frequent G4 structure within human genome. Additionally, the two model G4s,
folding into different G4 conformations, allow to detect conformation-specific hits. A double stranded
(ds) oligonucleotide was also used as a non-G4 forming sequence control.
Stabilization of the fragment library on these G4 structures was assessed by the HTS
Fluorescence Resonance Energy Transfer (FRET) melting assay, using a 4000-fold excess of fragment vs.
oligonucleotide. Similar compound/oligonucleotide ratio has been previously reported for different
library investigation [38]. This preliminary evaluation allowed us to focus on a selected family of
mono and bi functionalized single aromatic and hetero-aromatic rings (Figure 2a). Of the fifteen tested
◦
fragments belonging to this class, five displayed G4 stabilization in the range of 1.0–6.0 C (Table 1,
compounds
5
,
11
,
12
,
14 and 15), without perturbation of the ds sequence except for 14, the most potent
◦
one, which induced a moderate (2.5 C) ds stabilization.
Table 1. Stabilization of LTR-III, hTel G4s and dsDNA (0.25 µM) in the presence of 4000-fold excess of
fragments (1 mM) vs. oligonucleotide, measured by FRET melting assay.
Tm 1 ± s.d. 2 ( C)
◦
∆
Fragment
Structural Complexity
LTR-III
hTel
dsDNA
1
2
3
4
5
6
7
8
9
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
1.5 ± 0.1
1.0 ± 0.1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
a
10
11
12
13
14
15
1.5 ± 0.1
1.5 ± 0.1
<1
1.5 ± 0.1
1.5 ± 0.1
<1
6.0 ± 1.5
4.5 ± 2.2
2.5 ± 0.5
1.5 ± 0.1
3.0 ± 0.5
<1
16
17
18
19
20
21
22
23
24
25
26
27
3.6 ± 0.6
1.5 ± 0.1
<1
2.5 ± 0.1
3.0 ± 0.5
<1
<1
<1
<1
n.d.
<1
n.d.
<1
<1
3
3
3
n.d.
n.d.
5.5 ± 0.5
5.5 ± 0.5
3
3
3
n.d.
n.d.
b
5.5 ± 0.5
4.1 ± 1.3
7.1 ± 0.1
4.5 ± 0.5
6.5 ± 0.5
13.0 ± 0.6
4.0 ± 0.5
2.7 ± 1.1
6.5 ± 0.8
2.5 ± 0.1
5.0 ± 0.5
14.0 ±0.7
1.1 ± 0.2
<1
<1
<1

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Table 1. Cont.
Tm 1 ± s.d. 2 ( C)
◦
∆
Fragment
Structural Complexity
LTR-III
hTel
dsDNA
28
29
30
31
32
6.0 ± 0.5
3.0 ± 0.6
10.8 ± 1.0
4.5 ± 0.5
5.4 ± 0.9
14.7 ± 1.1
<1
1.1 ± 0.2
<1
n.d.
<1
b
3
3
3
n.d.
n.d.
12.8 ± 2.0
11.8 ± 1.9
3
3
3
3
3
4
5
6
>22.1
>22.1
>22.1
>22.1
>24.1
>24.1
>24.1
>24.1
>28.0
>28.0
c
4.1 ± 0.2
14.1 ± 1.0
1
2
Variation in the oligonucleotide T
Not determinable.
m
in the presence of the selected compound.
Standard deviation.
3
Figure 2. Fragment-based approach starting from (a) mono-aryl fragments, evolving trough (b) low
level of complexity bi- and tri-aryl derivatives and (c) final tetra-heteroaryl hits.
Target stabilization by fragments 14 and 15 is likely related to the presence of a protonated side
chain under physiological conditions, which confers an additional electrostatic interaction with the

Molecules 2018, 23, 1874
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DNA. The other three active fragments
5
,
11 and 12, are more interesting from a chemical point
of view due to the presence of a particular functional group, the amidoxime, which is sometimes
considered a bioisostere for carboxylic group. Fragments 11 and 12 display an aromatic ring and one
5
,
or two amidoximes group at 1,3 positions, directly linked to a pyrido or benzo central core (Scheme 1a).
This preliminarily SAR analysis indicated that, besides the presence of a water-soluble moiety,
at least one unit of amidoxime improved ligand efficiency and potency, acting as a pharmacophore
for LTR-III.
Amidoxime has been reported to play this role on different targets. In fact, several molecules
containing the amidoxime group are biologically active: for example, they display hypotensive
activities [39] and antibacterial and antitrypanocidic properties [40]. They were also found to generate
NO in vivo, which had neuromodulatory and neurotransmitory effects. In addition, amidoxime and
its derivatives are considered prodrugs of amidines in drug design [41]. Bearing both a hydroxyimino
and an amino group at the same carbon atom, they are structurally close to amidines, amides and
hydroxamic acids, chemical groups that are often used as mimetics of guanidine with consequent
related pharmacological effects.
2
.2. Design, Synthesis and Analysis of the Polyheteroaryl Oxadiazole/Pyridine-Ligands
The most promising and versatile three hits (i.e., fragments 11 and 12, Figure 2a) selected from
the initial FRET screening were used as central scaffolds for the subsequent synthetic implementation
Figure 2b). In particular, starting from the mono-aromatic amidoxime core, we focused on the
5,
(
chain directionality of the polyaryl-structure, tuning the number of aromatic rings, adding different
heteroaryl units and amino/cationic side chains. Moreover, to confirm the key role of the amidoxime,
this moiety was replaced with a cyano-group in each structure. From a synthetic point of view, this was
a straightforward task, as the cyano moiety is the precursor of the amidoxime.
Starting from the aromatic amidoxime central core (i.e.,
3 or 44, Scheme 1), we introduced different
3
-substituted-1,2,4-oxadiazoles. The 3-methyl-1,2,4-oxadiazoles 16 and 18 (Scheme 1) were synthesized
by an efficient cyclization protocol in the presence of acetic anhydride [42]. The formal arylation at the
C3 of the 1,2,4-oxadiazole moiety was achieved in a single-step condensation protocol and subsequent
cyclodehydration, using the amidoxime 3 or 44 and the carboxylic acid derivatives 13, 38–43 (Scheme 1)
as reactants. The condensation was optimised in the presence of 1,1’-carbonyldiimidazole as a coupling
reagent to give the O-acylbenzamidoxime intermediates, which was detected by HPLC. The following
one-pot cyclodehydration of the latter was achieved without the isolation of the intermediate, yielding
the cyano-derivatives 20
synthesised according to a standard protocol in the presence of hydroxylamine.
The newly synthesized fragments 16 32, which ranged in molecular weights from 186 to
82 Da, were analysed by FRET melting assay. All the cyano and hydroxycarboxamidine-substituted
, 22, 24–26 in good yields. Finally, terminal amidoximes 27–30 and 32 were
–
3
new fragments showed stabilization of the G4 structures without significantly affecting the ds
oligonucleotide (Table 1). The addition of a single 1,2,4-oxadiazole to a pyridine core, 16, increased
◦
the stabilization to 3.6 C. The binding properties of these implemented fragments are directly related
to the number of aromatic/hetero-aromatic units in the structures (Table 1). The best three hits
◦
2
7
,
30 and 32 were able to stabilize the G4s by 10.8–14.7 C. These molecules were based on the
6-(1,2,4-oxadiazol-3-yl)pyridine-2-amidoxime template (see Scheme 1 for numbering), embedding an
additional substituted-pyridine at C8. Fragments 30 and 32 were also analysed at 100-fold excess, in the
presence of LTR-III and hTel G4s: in these conditions, 30 increased the melting temperature by 3.5 and
◦ ◦
.0 C, respectively and 32 by 5.3 and 3.5 C, respectively (Table 2). In addition, these two compounds
3
did not show any stabilization on the ds sequence (Table 2). These data suggest a dose-dependent
effect on G4 stabilization; however, no selectivity was observed between the two tested G4s. Finally,
the ortho H substituted fragments 19, 21 and 31 showed intense interference with the fluorescence
signal in FRET analysis, therefore, Tm could not be calculated.

Molecules 2018, 23, 1874
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Scheme 1. Synthetic protocol developed for the preparation of fragment compounds 18–36.
0
0
General condensation protocol: (a) 1,1 -carbonyldiimidazole (CDI), dry DMF, 16 h, r.t.; (b) 1,1 -
◦
◦
carbonyldiimidazole (CDI), dry DMF, 6 h, 150 C. (c) NaN , DMF, 16 h, 100 C; (d) Pd(PPh ) Cl , CuI,
Phenylacetylene, THF/Et N, 50 C, 16 h, under argon atmosphere; (e) NH OH/Na CO , H O, 1 h, r.t.;
◦
f) (i) Acroyl chloride, THF, 3 h, r.t., (ii) K CO , Dioxane, 6 h, 100 C; (g) Ac O, CHCl , Et N, 16 h, r.t.;
2 3 2 3 3
3
3 2
2
◦
3
2
2
3
2
(
(
h) LiOH, THF/H O, 4 h, r.t.
2
Table 2. Stabilization of LTR-III, hTel G4s and dsDNA (0.25
µM) in the presence of 100-fold excess of
fragments (25 µM) vs. oligonucleotide, measured by FRET melting assay.
Tm 1 ± s.d. 2 ( C)
◦
∆
Fragment
Structural Complexity
LTR-III
hTel
dsDNA
(
(
(
(
(
(
16)
23)
24)
29)
30)
32)
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
2.9 ± 0.3
1.1 ± 0.1
b
<1
<1
3.5 ± 0.5
5.3 ± 0.5
3.0 ± 0.5
3.5 ± 0.4
(
(
(
(
33)
34)
35)
36)
15.5 ± 0.5
14.3 ± 0.3
9.0 ± 0.2
14.1 ± 0.2
15.3 ± 0.3
14.0 ± 1.0
8.9 ± 0.1
14.6 ± 0.3
2
<1
<1
<1
<1
c
1
Variation in the oligonucleotide Tm in the presence of the selected compound. Standard deviation.
2
.3. Design, Synthesis and Analysis of the Final Lead Compounds
The third step (Figure 2c) of our implementation design aimed at the introduction of an additional
heteroaromatic triazole moiety at C13 (see Schemes 1 and 2 for numbering) bearing a cationic side
chain, to improve both the solubility and electrostatic binding to the target. The five-step synthetic

Molecules 2018, 23, 1874
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protocol shown in Scheme 2 was optimized to prepare the final ligands 35
,
36 with excellent yields
(
overall yield >80%). Starting from the bromo-derivatives
performed with ethynyltrimethylsilane. The subsequent deprotection of the trimethylsilyl (TMS)
group upon basic methanolysis yielded the terminal alkynes and in high yields (90%) and
mild conditions. The copper(I)-catalysed alkyne-azide cycloaddition (CuAAC) reaction between
-azido-N,N-dimethylpropan-1-amine and the terminal alkynes or was carried out in aqueous
4 and 45, a Sonogashira cross-coupling was
6
7
3
6
7
tert-butanol to dissolve all the reactants and implement reaction yields affording 48 and 49, which were
used for the next hydrolytic step without purification. Both the resulting carboxylic acid derivatives
50 and 51 were reacted with the amidoximes 3 and 44 according to the previously described protocol.
The two cyano derivatives 33 and 34 were converted into the final ligands 35 and 36 with two
equivalents of hydroxylamine in aqueous ethanol solution (Scheme 2 and Figure 2c). In order to
highlight the role of amidoxime, a key binding moiety, we decided to compare 35 and 36 to their cyano
analogues 33 and 34 (Figure 2c), which maintain the identical scaffold.
Scheme 2. Synthetic protocol developed for the preparation of the final compounds 37
–
40
.
◦
(
a) Pd(PPh ) Cl , CuI, PPh , TMS-CCH, dry toluene/iPr NH, 80 C, 1 h; (b) K CO , MeOH, 2 h,
3
2
2
3
2
2
3
r.t.; (c) CuSO 5H O, NaAscorbate, H O/tBuOH, 24 h, r.t.; (d) K CO , H O/THF, 24 h, reflux.;
4
2
2
2
3
2
0
◦
0
(
6
e) (i) 1,1 -carbonyldiimidazole (CDI), dry DMF, 16 h, r.t.; (ii) 1,1 -carbonyldiimidazole (CDI), dry DMF,
h, 150 C; (f) NH OH/Na CO , H O, 1 h, r.t.
2
2
3
2
The four new compounds 33
–
36 (MW 400–433 Da) showed improved stabilization properties:
◦
∆
T
m
at 100-fold excess of compound was 8.9–15.5 C (Table 2) on G4s and, at 4000-fold excess, was
◦
higher than 20 C, the maximum level measurable in this condition (Table 1). However, fragments
◦
3
3
and 34 greatly stabilized also dsDNA (
∆
T
m
> 28.0 C at 4000-fold excess of compound, Table 1).
For this reason, only fragments 35 and 36 were further investigated. In particular, the ability of
and 36 to stabilize LTR-III and hTel G4s was confirmed by circular dichroism (CD). At 10-fold
excess of compound vs. oligonucleotide, the highest ratio achievable in CD conditions, 35 and 36
3
5
◦
◦
0.7 C (Figure 3d), respectively, also inducing a
stabilized LTR-III of 1.7
±
0.5 C (Figure 3c) and 5.1
±
slight conformational change. The same compounds induced lower stabilization on hTel G4, that is,
Tm = 0.2 ± 0.1 C (Figure 3g) and 0.5 ± 0.2 C (Figure 3h), respectively.
◦
◦
∆

Molecules 2018, 23, 1874
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+
Figure 3. CD thermal unfolding analysis. CD of LTR-III G4 (1.5
) and in the presence of 10-fold excess of 35 ) and 36 ) (15
00 mM in the absence ( ) and in the presence of 10-fold excess of 35
variation is shown as a function of the wavelength; arrows indicate the spectral change from low to
µ
M) at K 100 mM in the absence
M). CD of hTel G4 (1.5
M) at K+
) and 36 ) (15 M). CD spectra
(
a
(c
(d
µ
µ
1
e
(
g
(h
µ
high temperatures. The molar ellipticity at the peak wavelength (265 nm for LTR-III G4 (b) and 290 nm
for hTel G4 (f)) is shown as a function of the temperature.
The Taq polymerase stop assay was next performed to check if these two hits were able to inhibit
polymerase progression at the G4 site. Extended LTR-III and hTel G4 forming sequences were used,
0
containing additional flanking bases at the 3 -end: a primer annealing sequence and a 5-T linker region
to separate the annealing sequence from the first G of the G4 tract. In the absence of compounds and
+
0
in the presence of 100 mM K , both G4 forming sequences stopped the polymerase at the most 3 -end
+
G-rich region, that is, the first G-rich region encountered by the enzyme, indicating that K stimulates
G4 folding (Figure 4a, lanes 7 and 16). Upon addition of increasing amounts (50–200 nM) of 35 and 36
,
the intensity of the stop bands highly increased in all templates (Figure 4a, lanes 8–13 and 17–22) along

Molecules 2018, 23, 1874
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with considerable reduction of the full-length amplicons, thus corroborating the effective stabilization
of the G4s by both compounds at nM concentrations. In contrast, all tested fragments had no effect on
a DNA template unable to fold into G4 (Figure 4a, lanes 3–4), indicating that the observed polymerase
inhibition was G4-dependent. In accordance to the FRET melting data, 36 displayed a higher G4
stabilizing effect on both templates with respect to 35. In addition, 36 was mildly selective towards
LTR-III vs. hTel (see stop bands quantification in Figure 4b). A naphthalene diimide (NDI) ligand [43
]
was used as internal control, as it is a well-characterized G4 ligand that displays no selectivity between
LTR-III and hTel G4s (Figure 4a, lanes 14 and 23).
Figure 4. Image of a typical Taq polymerase stop assay. (
by Taq polymerase in the absence (lanes 6 and 15) and presence of 100 mM K , alone (lanes 7 and 16)
a
) LTR-III and hTel templates were amplified
+
or with increasing amounts (50, 100 and 200 nM) of 35 (lanes 8–10 and 17–19) or 36 (lanes 11–13 and
2
0–22). NDI (N) 200 nM (lanes 14 and 23) was used as control for a non-selective G4-ligand. A template
(
non-G4 cnt) made of a scrambled sequence unable to fold into G4 was also used as internal control in
+
the absence (lane 1) and presence of 100 mM K , alone (lane 2) or with 200 nM of 35 (lane 3), 36 (lane 4),
N (lane 5). Lane P: unreacted labelled primer. Lane M: ladder of markers obtained by the Maxam and
Gilbert sequencing carried out on the amplified strand complementary to the template strand. Vertical
bars indicate G4-specific Taq polymerase stop sites. (b) Quantification of lanes 6–23 shown in panel
a. Quantification of stop bands corresponding to G4 and of the full-length amplification product (FL)
is shown.

Molecules 2018, 23, 1874
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To validate the preferential binding of 36 to LTR-III with respect to hTel, we performed FRET
0
0
competition assay. 5 -FAM- and 3 -TAMRA-labeled LTR-III was mixed with increasing concentrations
of the unlabelled competitor, LTR-III or hTel (Figure 5) and a constant amount of 36. Unlabelled LTR-III
◦
◦
and hTel G4 sequences displayed T
similar to allow a meaningful comparison. No significant variation of T
as the unlabelled competitor, indicating that the latter did not compete with the labelled LTR-III for
the binding to 36. On the contrary, a dose dependent decrease of T was detected in the presence of
m
of 68.6
±
0.5 C and 68.7
±
0.1 C respectively, sufficiently
m
was observed with hTel
m
LTR-III unlabelled competitor, suggesting that 36 bound both labelled and unlabelled oligonucleotides.
These data demonstrate that, in the presence of both G4s, 36 preferentially binds to LTR-III over hTel.
0
0
Figure 5. Competition analysis by FRET assay. 5 -FAM- and 3 -TAMRA-labeled LTR-III G4 (0.25 µM)
was mixed with increasing concentrations (0–32-fold excess) of unlabelled competitor LTR-III or hTel
and a constant amount (25 µM) of 36.
3
. Conclusions
A FBDD approach was successfully applied to develop compounds against a target G4.
An unanticipated moiety, the amidoxime, was found to consistently enhance compound activity.
Our data indicate that FBDD may be a valuable approach to generate new pharmacophores that
specifically recognize G4 nucleic acid structures. Considering that one of the main obstacles in
the development of G4 ligands is their size and consequent poor pharmacokinetics properties,
the presented approach, which adds up fragments that singly bind the target, indicates a possible way
to develop compounds with smaller size and more drug-like properties.
4
. Materials and Methods
4
.1. General Information
All chemicals and solvents were purchased from Sigma Aldrich (Milan, Italy) and used without
further purification. TLC analysis was carried out on silica gel (Merck 60F 254, Milan, Italy) with
visualization at 254 and 366 nm. Flash chromatography was performed with silica gel 60 (40–63 m,
µ
Merck, Milan, Italy). All anhydrous reactions were carried out under positive pressure of nitrogen or
1
argon. Elemental analysis was provided by a Carlo Erba CHN analyser (Milan, Italy). All H-NMR
and 13C-NMR spectra were recorded on a Bruker Advance 300 MHz spectrometer (Billerica, MA,
USA) using deuterated solvents and TMS as internal standard. The spectra are reported in ppm
1
13
and referenced to deuterated DMSO (2.49 ppm for H, 39.5 ppm for C) or deuterated chloroform
(
7.26 ppm for H, 77 ppm for 13C). The following abbreviations are used: singlet (s), doublet (d), triplet
1

Molecules 2018, 23, 1874
11 of 18
(t) and multiplet (m). Naked and modified oligonucleotide sequences were purchased from Sigma
Aldrich (Milan, Italy). The NMR Figures were provided in Supplementary Materials.
4
.2. Synthetic Methods
4
.2.1. Synthesis of the Fragment Family Shown in Figure 2a
Compounds
Aldrich, while the fragments
procedures [42,44–46].
1
,
2
,
4
,
6
,
7
,
3
8
,
,
9
10 and 13 were purchased from commercial sources Merck-Sigma
11 12 14 and 15 have been synthesized according to the published
,
,
,
0
(
Z)-6-Ethynyl-N -hydroxypicolinimidamide, fragment
5
: An aqueous solution (45 mL) of hydroxylamine
−
3
−3
hydrochloride (0.11 g, 1.58
×
10 mol) and Na CO (0.167 g, 1.58
×
10 mol), was added dropwise
2
3
in 30 min at r.t., to a solution of 6-ethynyl-2-pyridinecarbonitrile (0.166 g, 1.29 × 10− mol) in ethanol
3
(
95 mL). The resulting mixture was stirred at room temperature for 2 h. Then the organic solvent was
removed under vacuum in order to induce the precipitation of the product. The light brown solid was
1
filtered and dried (0.149 g, yield 71%). H-NMR (300 MHz, DMSO-d ):
2
δ
= 10.04 (s, 1H), 7.85–7.83 (m,
= 150.5, 148.9, 140.3,
37.3, 119.5, 82.8, 80.6; elemental analysis calcd (%) for C H N O: C, 59.62; H, 4.38; N, 26.07; found C,
6
13
H), 7.57–7.59 (m, 1H), 5.79 (s, 2H), 4.4 (s, 1H). C-NMR (75 MHz, DMSO-d6): δ
1
5
8
7
3
9.67; H, 4.37; N, 26.01.
4
.2.2. Synthesis of the Fragment Family Shown in Figure 2b
0
of (Z)-6-cyano-N -
6
-(5-Methyl-1,2,4-oxadiazol-3-yl)picolinonitrile,
fragment 16
mol) was solved in 15 mL of chloroform and treated
10 mol) and 0.3 mL of Et N. The resulting mixture was stirred at
:
0.3
g
−
10
3
hydroxypicolinimidamide (1.85
with 0.21 mL of Ac O (2.22
×
−
3
×
2
3
room temperature overnight. The solvent was removed under pressure and the (Z)-6-cyano-2-N-
acetoxy-amidoxime-pyridine obtained was directly treat with 5 mL of glacial acetic acid refluxing
the solution for 4 h. The neutralization of the reaction mixture with a saturated solution of sodium
bicarbonate induce the precipitation of the product. The white solid was filtered and dried under
1
◦
vacuum (0.225 g, yield 65%). H-NMR (300 MHz, CDCl , 25 C, TMS): δ = 8.35 (d, 3J(H, H) = 7.8 Hz,
3
13
1
H), 8.05 (t, 3J(H, H) = 7.8 Hz, 1H), 7.86 (d, 3J(H, H) = 7.8 Hz, 1H), 2.74 (s, 3H). C-NMR (75 MHz,
◦
CDCl , 25 C, TMS):
δ
= 179.8, 168.7, 149.7, 140.1, 136.3, 131.7, 127.8, 118.1, 14.2; elemental analysis
3
calcd (%) for C H N O: C, 58.06; H, 3.25; N, 30.09; found C, 58.04; H, 3.27; N, 30.10.
9
6
4
3
6
-(5-Vinyl-1,2,4-oxadiazol-3-yl)picolinonitrile, fragment 17: acroyl chloride (0.12 mL, 1.48 × 10− mol
)
0
−3
was added to a solution of(Z)-6-cyano-N -hydroxypicolinimidamide (0.2 g, 3.7
×
10 mol) in 15 mL
of chloroform. The reaction mixture was stirred at room temperature for 3 h, then quenched with a
saturated solution of NaHCO and extracted with chloroform (3 20 mL). The organic phase was dried
under vacuum and the intermediate obtained was directly used for the cyclization step. The crude was
×
3
−
3
◦
dissolved in 25 mL of 1,4-dioxane, treat with K CO (0.335 g, 2.4 × 10 mol) and heated at 100 C for
2
3
6
h. The reaction mixture was cooled to r.t. and the solvent was dried under vacuum. The crude solid
was treat with chloroform in order to dissolve only the product. The organic phase was then dried
1
◦
under vacuum (white solid, 0.292 g, yield 80%). H-NMR (300 MHz, CDCl , 25 C, TMS):
δ
= 8.5 (dd,
J(H, H) = 8.0 Hz; 3J(H, H) = 1.1 Hz, 1H), 8.16 (t, 3J(H, H) = 7.9 Hz, 1H), 7.97 (dd, 3J(H, H) = 7.9 Hz;
J(H, H) = 1.1 Hz, 1H), 6.95 (dd, 3J(H, H) = 17.7 Hz; 3J(H, H) = 10.7 Hz, 1H), 6.81 (dd, 3J(H, H) = 17.7 Hz;
3
3
3
3
2
13
J(H, H) = 1.1 Hz, 1H), 6.21 (dd, 3J(H, H) = 10.7 Hz; 3J(H, H) = 1.1 Hz, 1H). C-NMR (75 MHz, CDCl3,
◦
5 C, TMS):
δ = 175.6, 167.1, 147.9, 138.3, 134.5, 130.0, 129.9, 126.1, 120.0, 116.3; elemental analysis calcd
(
%) for C H N O: C, 60.60; H, 3.05; N, 28.27; found C, 60.62; H, 3.01; N, 28.27.
10 6 4
General procedure A for the synthesis of the amidoxime derivatives 18
, 27, 28, 29, 32, 35 and 36.
A mixture of hydroxylamine hydrochloride (0.68 mmol) and of Na CO (0.34 mmol) in water (10 mL)
2
3
was added dropwise in 45 min to a solution containing the nitrile derivatives (0.33 mmol) in EtOH

Molecules 2018, 23, 1874
12 of 18
(
30 mL). The reaction mixture was stirred at r.t. for 1 h. The precipitated white solid was filtered and
dried. All the carbonitrile fragment were converted in the corresponding amidoxime derivatives with
0
high yields (Z)-N -hydroxy-6-(5-methyl-1,2,4-oxadiazol-3-yl)picolinimidamide, fragment 18: white
1
solid, yield 89%. H-NMR (300 MHz, DMSO-d ):
(
δ
= 10.13 (s, 1 H), 8.06–8.02 (m, 3H), 5.87 (s, 2H), 2.71
= 177.9, 167.3, 150.5, 148.7, 144.5, 138.2, 123.3, 121.5, 12.1;
elemental analysis calcd (%) for C H N O : C, 49.31; H, 4.14; N, 31.95; found C, 49.38; H, 4.12; N, 31.91.
6
s, 3H). 13C-NMR (75 MHz, DMSO-d6):
δ
9
9
5
2
General cyclization protocol B for the synthesis of the 1,2,4-oxadiazole derivatives 19
33 and 34. The opportune carboxylic acid (1.46 mmol) and 1,1 -carbonyldiimidazole (CDI, 1.46 mmol)
,
20
,
22
,
24
,
25
,
0
26,
were dissolved in DMF (10 mL) and stirred for 30 min at r.t.. After this period, the correspondent
amidoxime derivatives (1.46 mmol) was added and the reaction mixture was stirred at r.t. overnight.
◦
CDI (1.46 mmol) was further added and the reaction mixture was heated at 150 C for 6 h. After cooling
down, the resulting solution was poured into water to induce the precipitation of a solid, which was
filtered and characterised as pure product. 5-(6-Bromopyridin-2-yl)-3-(pyridin-2-yl)-1,2,4-oxadiazole,
1
fragment 19: white solid, yield 53%. H-NMR (300 MHz, DMSO-d ):
δ = 8.81 (d, 3J(H, H) = 4.0 Hz; 1H),
6
8
7
.38 (dd, 3J(H, H) = 7.4 Hz; 3J(H, H) = 0.9 Hz, 1H), 8.2 (d, 3J(H, H) = 7.4 Hz; 1H), 8.12–8.00 (m, 3H),
.68–7.63 (m, 1H). 13C-NMR (75 MHz, DMSO-d6):
δ
= 173.3, 168.5, 150.4, 145.4, 143.3, 141.8, 141.3, 137.8,
1
32.1, 126.4, 124.0, 123.5; elemental analysis calcd (%) for C H BrN O: C, 47.55; H, 2.33; N, 18.48; found
12 7 4
C, 47.58; H, 2.31; N, 18.45.
6
-(5-(6-Bromopyridin-2-yl)-1,2,4-oxadiazol-3-yl)picolinonitrile, fragment 20: general method B. White solid,
1
yield 57%. H-NMR (300 MHz, DMSO-d ):
δ
= 8.48 (d, 3J(H, H) = 9.0 Hz; 1 H), 8.38 (t, 3J(H, H) =
6
7
.4 Hz, 1H), 8.32 (d, 3J(H, H) = 9 Hz; 1H), 8.23 (d, 3J(H, H) = 7.4 Hz; 1H), 8.08 (t, 3J(H, H) = 7.7 Hz, 1
13
H), 7.99 (d, 3J(H, H) = 7.7 Hz; 1H). C-NMR (75 MHz, DMSO-d6):
δ = 173.8, 167.5, 146.9, 143.2, 141.9,
1
41.1, 139.9, 133.4, 132.1, 130.9, 127.1, 124.1, 116.7; elemental analysis calcd (%) for C H BrN O: C,
13
6
5
4
7.59; H, 1.84; N, 21.34; found C, 48.01; H, 1.83; N, 21.36.
-(6-Azidopyridin-2-yl)-3-(pyridin-2-yl)-1,2,4-oxadiazole, fragment 21
5
:
0.108 g of sodium azide
−
3
−3
(
1.66 × 10 mol) was added to a solution of (19) (0.05 g 1.66
×
10
mol) in 2 mL of
◦
dimethylformammide. The reaction mixture was heated at 100 C and stirred overnight. The solvent
was removed under pressure, the crude product was washed with water and filtered in order to obtain
1
◦
a pure white solid (0.038 g, yield 87%). H-NMR (300 MHz, CDCl , 25 C, TMS):
δ
= 8.87 (d, 3J(H, H) =
3
4
.8 Hz, 1H), 8.28 (d, 3J(H, H) = 7.9 Hz, 1H), 8.19 (dd, 3J(H, H) = 7.6 Hz; 3J(H, H) = 0.7 Hz, 1H), 7.94–7.85
13
(m, 2H), 7.52–7.47 (m, 1H), 7.01 (dd, 3J(H, H) = 8.0 Hz; 3J(H, H) = 0.7 Hz, 1H). C-NMR (75 MHz,
◦
CDCl , 25 C, TMS):
δ = 174.3, 168.8, 155.5, 150.4, 146.0, 142.4, 139.7, 137.0, 126.1, 123.7, 120.7, 117.6;
3
elemental analysis calcd (%) for C H N O: C, 54.34; H, 2.66; N, 36.97; found C, 54.32; H, 2.65; N, 37.01.
12
7
7
6
4
3
(
1
-(5-(Pyridin-2-yl)-1,2,4-oxadiazol-3-yl)picolinonitrile, fragment 22: general method B. White solid, yield
1
5%. H-NMR (300 MHz, DMSO-d ):
δ = 8.9 (d, 3J(H, H) = 4.2 Hz; 1H), 8.5 (dd, 3J(H, H) = 7.7 Hz;
6
J(H, H) = 1.1 Hz, 1H), 8.4–8.27 (m, 3H), 8.15 (dt, 3J(H, H) = 7.7 Hz; 3J(H, H) = 1.6 Hz 1H), 7.79–7.74
= 171.1, 167.3, 150.7, 146.9, 142.5, 140.0, 138.2, 133.3, 131.1,
27.7, 127.1, 124.7, 116.9; elemental analysis calcd (%) for C H N O: C, 62.65; H, 2.83; N, 28.10; found
m, 1H). 13C-NMR (75 MHz, DMSO-d6):
δ
13
7
5
C, 62.63; H, 2.81; N, 28.14.
.
6
2
(
-(3-(6-Cyanopyridin-2-yl)-1,2,4-oxadiazol-5-yl)picolinic acid 2HCl, fragment 23: to a solution of fragment
−
4
4
(0.100 g, 3.25
×
10
mol) in THF/H O (2:1, 6 mL), were added 0.07 g of LiOH H O
2 2
−
3
1.62 × 10 mol). The reaction mixture was stirred for 4 h at r.t., after this period, the solution
was acidified with chloridric acid (1 mL) and the organic solvent removed under pressure. The product
precipitate was filtered and washed with water in order to obtain a pure white solid (0.087 g, yield
1
9
1
1
1%). H-NMR (300 MHz, DMSO-d ):
δ
= 8.6–8.5 (m, 1H), 8.41–8.35 (m, 4H), 8.32–8.26 (m, 1H), 8.0 (bs,
= 174.6, 167.9, 165.3, 151.1, 149.5, 146.8, 144.4, 140.1,
33.3, 128.0, 125.9, 116.8; elemental analysis calcd (%) for C H Cl N O : C, 45.92; H, 2.48; N, 19.13;
6
H), 7.9 (bs, 1H). 13C-NMR (75 MHz, DMSO-d6):
δ
14
9
2
5
3
found C, 44.88; H, 2.46; N, 19.16.

Molecules 2018, 23, 1874
13 of 18
Methyl 6-(3-(6-cyanopyridin-2-yl)-1,2,4-oxadiazol-5-yl)picolinate, fragment 24: general method B. White
1
solid, yield 46%. H-NMR (300 MHz, DMSO-d ):
8
δ = 8.60 (dd, 3J(H, H) = 6.0 Hz; 3J(H, H) = 2.9 Hz; 1H),
6
13
.51 (dd, 3J(H, H) = 7.6 Hz; 3J(H, H) = 1.0 Hz; 1H), 8.38–8.29 (m, 4H), 3.98 (s, 3H). C-NMR (75 MHz,
DMSO-d6):
δ = 174.4, 167.3, 164.3, 148.3, 146.7, 142.7, 140.1, 140.0, 133.3, 131.1, 128.2, 127.8, 127.1 116.9,
5
2.8; elemental analysis calcd (%) for C H N O : C, 58.63; H, 2.95; N, 22.79; found C, 58.69; H, 2.93;
15 9 5 3
N, 22.82.
.
6
-(5-(6-Methoxypyridin-2-yl)-1,2,4-oxadiazol-3-yl)picolinonitrile 2HCl, fragment 25: general method B.
1
White solid, yield 19%. H-NMR (300 MHz, DMSO-d ):
δ = 8.55 (d, 3J(H, H) = 3.9 Hz; 1H), 8.37–8.32
6
(m, 2H), 8.25–8.22 (m, 2H), 7.96 (bs, 1H), 7.87 (bs, 1H), 7.67 (dd, 3J(H, H) = 8.8 Hz; 3J(H, H) = 2.8 Hz;
1H), 3.96 (s, 3H). Elemental analysis calcd (%) for C H Cl N O : C, 47.75; H, 3.15; N, 19.89; found C,
14 11 2 5 2
4
7.72; H, 3.18; N, 19.85.
6
6
-(5-(Quinolin-2-yl)-1,2,4-oxadiazol-3-yl)picolinonitrile, fragment 26: general method B. White solid, yield
1
0%. H-NMR (300 MHz, DMSO-d ):
δ
= 8.73 (d, 3J(H, H) = 8.4 Hz; 1H), 8.53 (d, 3J(H, H) = 7.8 Hz,
6
1
H), 8.43 (d, 3J(H, H) = 7.8 Hz; 1H), 8.36 (t, 3J(H, H) = 7.8 Hz; 1H), 8.29–8.26 (m, 2H), 8.17 (d, 3J(H,
H) = 8.0 Hz; 1H), 7.96 (t, 3J(H, H) = 7.1 Hz; 1H), 7.82 (t, 3J(H, H) = 7.4 Hz; 1H). 13C-NMR (75 MHz,
DMSO-d6): = 175.2, 167.5, 147.4, 147.0, 142.6, 139.9, 138.4, 133.4, 131.2, 130.1, 129.7, 129.1, 128.9, 127.1,
20.6, 116.8; elemental analysis calcd (%) for C H N O: C, 68.22; H, 3.03; N, 23.40; found C, 68.18; H,
δ
1
3
17
9
5
.05; N, 23.38.
0
(
Z)-6-(5-(Furan-2-yl)-1,2,4-oxadiazol-3-yl)-N -hydroxypicolinimidamide, fragment 27: general method A.
1
White solid, yield 90%. H-NMR (300 MHz, DMSO-d ):
δ = 10.15 (s, 1H), 8.20 (d, 3J(H, H) = 0.7 Hz;
6
1
1
1
H), 8.14 (q, 3J(H, H) = 4.4 Hz, 1H), 8.11–7.96 (m, 2H), 7.73 (d, 3J(H, H) = 7.8 Hz; 1H), 6.91–6.74 (m,
H), 5.89 (s, 2H). 13C-NMR (75 MHz, DMSO-d6):
= 167.7, 167.6, 150.6, 148.8, 148.6, 144.1, 138.9, 138.3,
23.7, 121.8, 117.9, 113.2; elemental analysis calcd (%) for C H N O : C, 53.14; H, 3.34; N, 25.82; found
δ
12
9
5
3
C, 53.20; H, 3.36; N, 25.80.
0
(
Z)-N -Hydroxy-6-(5-(pyridin-2-yl)-1,2,4-oxadiazol-3-yl)picolinimidamide, fragment 28: general method
1
A. White solid, yield 86%. H-NMR (300 MHz, DMSO-d ):
δ
= 10.26 (s, 1H), 8.99–8.97 (m, 1H), 8.48
d, 3J(H, H) = 6.8 Hz; 1H), 8.33–8.23 (m, 2H), 8.20 (d, 3J(H, H) = 1.28 Hz; 1H), 8.18 (s, 1H), 7.89–7.84
m, 1H), 6.02 (s, 2H). 13C-NMR (75 MHz, DMSO-d6):
= 174.7, 168.1, 150.6, 148.8, 144.3, 142.7, 140.1,
38.3, 138.2, 127.6, 124.6, 123.6, 121.8; elemental analysis calcd (%) for C H N O : C, 55.32; H, 3.57;
6
(
(
1
δ
13
10
6
2
N, 29.77; found C, 55.28; H, 3.56; N, 29.82.
0
(
Z)-N -Hydroxy-6-(5-(quinolin-2-yl)-1,2,4-oxadiazol-3-yl)picolinimidamide, fragment 29: general method A.
1
White solid, yield 92%. H-NMR (300 MHz, DMSO-d ):
δ = 10.09 (s, 1H), 8.24 (d, 3J(H, H) = 8.5 Hz;
6
1
H), 8.45 (d, 3J(H, H) = 8.5 Hz, 1H), 8.27–8.25 (m, 2H), 8.18 (d, 3J(H, H) = 8.2 Hz; 1H), 8.11 (d, 3J(H, H)
=
=
4.1 Hz; 2H), 7.96 (dt, 3J(H, H) = 8.0 Hz, 3J(H, H) = 1.4 Hz, 1H), 7.82 (dt, 3J(H, H) = 8.0 Hz, 3J(H, H)
1.4 Hz, 1H), 5.89 (s, 2H). 13C-NMR (75 MHz, DMSO-d6):
δ
= 174.8, 168.3, 150.8, 148.9, 147.3, 144.3,
142.8, 138.4, 138.3, 131.2, 129.7, 129.1, 128.9, 128.3, 123.7, 121.8, 120.6; elemental analysis calcd (%) for
C H N O : C, 61.44; H, 3.64; N, 25.29; found C, 61.49; H, 3.62; N, 25.25.
17
12
6
2
Compound 30 has been synthesized according to the published procedures [42].
0
(
Z)-N -Hydroxy-6-(5-(6-(phenylethynyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)picolinimidamide, fragment 31
:
−
6
−6
10 mol) were added to a suspension
Pd(Ph P) Cl (0.005 g, 7.1
of 19 (0.1 g 3.3
×
10 mol) and CuI (0.0006 g, 3.15
×
3
2
2
−
10 mol) in 5 mL of THF/Et N (1:1). The solution was stirred for fiftheen min
3
4
×
−
3
at r.t.. After the addition of ethynylbenzene (0.128 mL, 1.16
overnight, at 50 C, under nitrogen atmosphere. After this period, the solvent was removed under
vacuum. The resulting crude was treat with 20 mL of water and then extracted with three portions of
×
10 mol), the mixture was stirred
◦
chloroform (3
×
20 mL). The organic phase was dried on Na SO , filtered and removed under reduced
2 4
pressure. The crude product was purified by column chromatography and the product was obtained
as a yellow solid (0.067 g, yield 63%). H-NMR (300 MHz, CDCl , 25 C, TMS):
1
◦
δ = 8.85 (d, 3J(H, H) =
3

Molecules 2018, 23, 1874
14 of 18
4.0 Hz, 1H), 8.37 (d, 3J(H, H) = 7.0 Hz, 1H), 8.29 (d, 3J(H, H) = 7.8 Hz, 1H), 7.95 (d, 3J(H, H) = 7.8 Hz,
1
H), 7.93–7.87 (m, 1H), 7.75 (dd, 3J(H, H) = 7.8 Hz, 3J(H, H) = 0.8 Hz, 1H), 7.66–7.63 (m, 2H), 7.49–7.45
m, 1H), 7.42–7.37 (m, 3H). 13C-NMR (75 MHz, CDCl , 25 C, TMS):
◦
δ
= 174.4, 168.8, 150.4, 146.0, 144.4,
43.7, 137.5, 137.9, 132.1, 130.1, 129.3, 128.3, 125.6, 123.4, 123.2, 121.6, 91.0, 87.6; elemental analysis calcd
%) for C H N O : C, 65.96; H, 3.69; N, 21.98; found C, 66.00; H, 3.66; N, 21.97.
(
3
1
(
21
14
6
2
0
Methyl (Z)-6-(3-(6-(N -hydroxycarbamimidoyl)pyridin-2-yl)-1,2,4-oxadiazol-5-yl)picolinate, fragment 32
:
1
general method A. White solid, yield 88%. H-NMR (300 MHz, DMSO-d ):
δ = 10.16 (bs, 1H), 8.60 (dd,
6
3
(
1
J(H, H) = 6.2 Hz; 3J(H, H) = 2.7 Hz; 1H), 8.38–8.32 (m, 2H), 8.25–8.21 (m, 1H), 8.11–8.08 (m, 2H), 5.91
s, 2H), 3.97 (s, 3H). 13C-NMR (75 MHz, DMSO-d6):
= 174.1, 168.2, 164.4, 150.7, 148.8, 148.3, 144.2,
42.9,139.9, 138.3, 128.1, 127.8, 123.7, 121.9, 52.8; elemental analysis calcd (%) for C H N O : C, 52.94;
δ
15
12
6
4
H, 3.55; N, 24.70; found C, 52.91; H, 3.54; N, 24.74.
4
.2.3. Synthesis of the Fragment Family Shown in Figure 2c
Synthesis of intermediate 48 49: The alkyne ( or , 1.24 mmol) was dissolved in tert-butanol
–
6
7
(
(
10 mL) and added to a water solution (10 ml) of N,N-Dimethyl-N-(3-azidopropyl)amine hydrochloride
226 mg, 1.37 mmol), copper (II) sulphate pentahydrate (31 mg, 10% mol) and sodium ascorbate (246 mg,
1.24 mmol). The resulting mixture was stirred for 24 h, at r.t. The organic solvent was removed under
vacuum and the residue was diluted with saturated NaHCO (10 mL) and extracted with DCM
3
(
3 × 20 mL). The combined organic phases were dried over Na SO and the solvent was removed
2
4
under reduced pressure. The crude was purified by flash chromatography (CHCl /MeOH 95:5).
3
Methyl 6-(1-(3-(dimethylamino)propyl)-1H-1,2,3-triazol-4-yl)picolinate (48): colourless liquid, yield = 90%.
1
◦
H-NMR (300 MHz, CDCl , 25 C, TMS)
δ
(ppm) 8.39 (d, J = 7.9 Hz, 1H), 8.36 (s, 1H), 8.06 (d, J = 7.7 Hz
,
3
1
(
H), 7.95 (t, J = 7.8 Hz, 1H), 4.53 (t, J = 7.0 Hz, 2H), 4.02 (s, 3H), 2.34 (t, J = 6.8 Hz, 2H), 2.25 (s, 6H), 2.14
quint, J = 6.9 Hz, 2H). 13C-NMR (75 MHz, CDCl , 25 C, TMS)
◦
δ
(ppm) 165.4, 150.7, 147.5, 147.3, 137.7,
3
1
23.9, 123.2, 122.9, 55.7, 52.6, 48.2, 45.2, 28.0.
Methyl 3-(1-(3-(dimethylamino)propyl)-1H-1,2,3-triazol-4-yl)benzoate (49): colourless liquid, yield = 95%.
1
◦
H-NMR (300 MHz, CDCl , 25 C, TMS)
δ
(ppm) 8.42 (s, 1H), 8.11 (d, J = 7.8 Hz 1H), 7.99 (d, J = 7.8 Hz,
3
1
(
H), 7.89 (s, 1H), 7.51 (t, J = 7.8 Hz, 1H), 4.49 (t, J = 6.9 Hz, 2H), 3.94 (s, 3H), 2.31 (t, J = 6.7 Hz, 2H), 2.24
s, 6H), 2.11 (quint, J = 6.8 Hz, 2H). 13C-NMR (75 MHz, CDCl , 25 C, TMS)
◦
δ (ppm) 166.7, 146.6, 131.0,
3
1
30.6, 129.9, 128.92, 128.88, 126.6, 120.3, 55.6, 52.1, 48.1, 45.2, 28.0.
Synthesis of intermediates 50 51: The corresponding methyl ester 48–49 (1.20 mmol) and
–
potassium carbonate (248 mg, 1.80 mmol) were dissolved in 1:1 aqueous THF (10 mL). The solution was
stirred at reflux 24 h. Hence, 1% HCl (10 mL) was added until cease of gas evolution and the solvent
was removed under reduced pressure. The crude was dissolved in isopropanol (5 mL) and filtered,
these steps were repeated three times. The filtrates were combined and the solvent was removed under
reduced pressure.
3
-(4-(6-Carboxypyridin-2-yl)-1H-1,2,3-triazol-1-yl)-N,N-dimethylpropan-1-aminium chloride (50): white
1
◦
solid, yield = 98%. H-NMR (300 MHz, DMSO-d , 25 C, TMS)
δ
(ppm) 8.81 (s, 1H), 8.25 (d, J = 7.7
6
Hz, 1H), 8.09 (t, J = 7.8 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 4.8–5.8 (bs), 4.58 (t, J = 6.7 Hz, 2H), 3.11 (t,
J = 7.8 Hz, 2H), 2.78 (s, 6H), 2.33 (quint, J = 7.8 Hz, 2H). 13C-NMR (75 MHz, DMSO-d , 25 C, TMS)
◦
δ
6
(ppm) 165.8, 149.9, 148.3, 146.7, 138.6, 124.2, 123.6, 122.5, 53.9, 47.0, 42.1, 24.6.
3
-(4-(3-Carboxyphenyl)-1H-1,2,3-triazol-1-yl)-N,N-dimethylpropan-1-aminium chloride (51): white solid,
1
◦
yield = 97%. H-NMR (300 MHz, CDCl , 25 C, TMS)
δ (ppm) 10.71 (bs, NH), 8.79 (s, 1H), 8.42 (s,
3
1
H), 8.09 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 7.8 Hz), 7.60 (t, J = 7.7 Hz, 1H), 4.55 (t, J = 6.7 Hz, 2H), 3.09
t, J = 7.6 Hz, 2H), 2.74 (s, 6H), 2.33 (quint, J = 7.2 Hz, 2H). 13C-NMR (75 MHz, CDCl , 25 C, TMS)
◦
δ
(
(
3
ppm) 167.1, 145.6, 131.5, 131.1, 129.3, 128.6, 125.8, 122.1, 53.7, 47.0, 42.0, 24.5.

Molecules 2018, 23, 1874
15 of 18
1
◦
Fragment 33: general method B. White solid, yield 57%. H-NMR (300 MHz, CDCl , 25 C, TMS):
3
δ = 8.51 (s, 1H), 8.44 (bs, 1H), 8.41 (bs, 1H), 8.39 (s, 1H), 8.2 (dd, 3J(H, H) = 7.7 Hz, 3J(H, H) = 0.9 Hz,
1
=
H), 8.01 (t, 3J(H, H) = 7.8 Hz, 1H), 7.80 (dd, 3J(H, H) = 7.7 Hz; 3J(H, H) = 0.9 Hz; 1H), 7.65 (t, 3J(H, H)
7.8 Hz, 1H), 4.55 (t, 3J(H, H) = 7.0 Hz; 2H), 2.36 (t, 3J(H, H) = 7.0 Hz; 2H) 2.26 (s, 6H) 2.16 (quin, 3J(H,
13
H) = 7.0 Hz; 2H). C-NMR (75 MHz, DMSO-d6):
31.1, 129.7, 128.0, 123.3, 123.2, 123.1, 117.8, 113.3, 55.7, 48.3, 45.2, 28.0; elemental analysis calcd (%) for
C H N O: C, 62.99; H, 5.03; N, 27.98; found C, 62.92; H, 5.05; N, 28.01.
δ = 174.8, 167.4, 151.5, 147.0, 142.8, 138.1, 134.4, 131.4,
1
21
20
8
1
◦
Fragment 34: general method B. White solid, yield 61%. H-NMR (300 MHz, CDCl , 25 C, TMS):
3
δ = 8.64 (s, 1H), 8.45 (d, 3J(H, H) = 8.0 Hz, 1H), 8.18 (dd, 3J(H, H) = 7.8 Hz, 3J(H, H) = 1.6 Hz, 2H), 8.08
(t, 3J(H, H) = 7.9 Hz, 1H), 8.01 (s, 1H), 7.87 (d, 3J(H, H) = 7.7 Hz, 1H), 7.63 (t, 3J(H, H) = 7.8 Hz, 1H),
4
2
.54 (t, 3J(H, H) = 7.0 Hz; 2H), 2.37 (t, 3J(H, H) = 7.0 Hz; 2H) 2.28 (s, 6H) 2.16 (quint, 3J(H, H) = 7.0 Hz;
13
H). C-NMR (75 MHz, DMSO-d6):
δ = 176.6, 167.4, 148.1, 146.0, 138.4, 134.4, 132.0, 130.2, 130.0, 129.7,
1
27.5, 126.3, 125.3, 123.9, 120.7, 116.4, 55.6, 48.1, 45.1, 27.8; elemental analysis calcd (%) for C H N O:
21 20 8
C, 62.99; H, 5.03; N, 27.98; found C, 62.94; H, 5.02; N, 28.00.
1
Fragment 35: general method A. White solid, yield 88%. H-NMR (300 MHz, DMSO-d ):
1
8
δ
= 9.9 (s,
H), 8.93 (s, 1H), 8.57 (bs, 1H), 8.45–8.38 (m, 2H), 8.32 (dt, 3J(H, H) = 7.8 Hz, 3J(H, H) = 0.9 Hz, 1H),
.22 (dd, 3J(H, H) = 7.7 Hz, 3J(H, H) = 1 Hz, 1H), 8.02 (dd, 3J(H, H) = 7.7 Hz, 3J(H, H) = 1 Hz, 1H), 7.73
t, 3J(H, H) = 7.8 Hz, 1H), 6.1 (s, 2H), 4.59 (t, 3J(H, H) = 7.0 Hz; 2H), 2.34 (t, 3J(H, H) = 7.0 Hz; 2H) 2.26
s, 6H) 2.14 (quin, 3J(H, H) = 7.0 Hz; 2H). 13C-NMR (75 MHz, DMSO-d6):
= 174.3, 168.3, 151.0, 150.2,
46.0, 142.7, 139.3, 134.4, 129.3, 128.5, 127.4, 125.9, 124.3, 123.5, 123.0, 55.7, 47.9, 45.1, 27.7; elemental
analysis calcd (%) for C H N O : C, 58.19; H, 5.35; N, 29.08; found C, 58.24; H, 5.38; N, 29.05.
6
(
(
δ
1
21
23
9
2
1
Fragment 36: general method A. White solid, yield 82%. H-NMR (300 MHz, DMSO-d ): δ = 10.16 (s,
6
1H), 8.85 (s, 1H), 8.68 (bs, 1H), 8.31–8.19 (m, 3H), 8.09 (d, 3J(H, H) = 4.4 Hz, 2H), 7.77 (t, 3J(H, H) =
7
.8 Hz, 1H), 5.92 (s, 2H) 4.47 (t, 3J(H, H) = 7.0 Hz; 2H), 2.32 (t, 3J(H, H) = 7.0 Hz; 2H) 2.20 (s, 6H) 2.06
13
(
quin, 3J(H, H) = 7.0 Hz; 2H). C-NMR (75 MHz, DMSO-d6):
38.6, 132.5, 130.7, 130.1, 127.5, 124.5, 124.4, 124.0, 122.7, 122.1, 55.8, 48.1, 45.2, 27.7; elemental analysis
calcd (%) for C H N O : C, 58.19; H, 5.35; N, 29.08; found C, 58.23; H, 5.36; N, 29.02.
δ = 176.0, 168.4, 151.0, 149.2, 145.3, 144.8,
1
21
23
9
2
4
.3. Biophysical and Biological Assays
4
.3.1. FRET Assay
0
For fluorescence melting curves, 6-carboxyfluorescein (FAM) 5 -end and 6-carboxy-
0
tetramethylrhodamine (TAMRA) 3 -end labelled oligonucleotides (0.25
µ
M) (Tabel S1) were folded
in lithium cacodylate buffer (10 mM, pH 7.4) and KCl 100 mM by heating at 95 C for 5 min and
◦
gradually cooling to r.t.. Where indicated, fragments were added at the final concentration of 25
µ
M or
◦
1
mM and, after stabilization at 4 C, samples were processed in a LightCycler 480 II (Roche, Milan,
Italy). Oligonucleotide melting was monitored by observing FAM emission in the temperature range of
◦
◦
3
0–95 C with 1 C/min gradient. Melting profiles were normalized as previously described [47] and
Tm was defined as the temperature corresponding to the 0.5 fraction of the normalized fluorescence.
0
0
For competition assays, 5 -FAM and 3 -TAMRA labelled LTR-III oligonucleotide and unlabelled
◦
oligonucleotides (competitors) (Tabel S1) were separately folded for 5 min at 95 C in lithium cacodylate
buffer (10 mM, pH 7.4) supplemented with KCl (100 mM). After 4 h at r.t., labelled oligonucleotide
(
3
0.25
(25
described above.
µ
M) was mixed with increasing amounts of competitor (0–32-fold excess) in the presence of
6
µ
M). Samples were processed by Light Cycler (Roche, Milan, Italy) and T were obtained as
m
4
.3.2. Circular Dichroism (CD)
For CD analysis, oligonucleotides (Table S1) were diluted to a final concentration of 1.5
lithium cacodylate buffer (10 mM, pH 7.4) and KCl 100 mM. Samples were annealed by heating
µM in

Molecules 2018, 23, 1874
16 of 18
◦
at 95 C for 5 min and gradually cooled to r.t. and, where indicated, fragments were added at the
final concentration of 15 µM. CD spectra were recorded on a Chirascan-Plus (Applied Photophysics,
Leatherhead, UK) equipped with a Peltier temperature controller using a quartz cell of 5 mm optical
path length, over a wavelength range of 230–320 nm. The reported spectrum of each sample represents
◦
the average of 2 scans at 20 C and it is baseline corrected for signal contributions due to the buffer.
2
−1
Observed ellipticities were converted to mean residue ellipticity (
ellip.). For the determination of T
with temperature increase of 5 C. T
θ
) = deg
×
cm
×
dmol (mol.
◦
m
, spectra were recorded over a temperature range of 20–90 C,
◦
m
values were calculated according to the van’t Hoff equation,
applied for a two-state transition from folded to unfolded state, assuming that the heat capacity of the
folded and unfolded states are equal [48].
4
.3.3. Taq Polymerase Stop Assay
0
◦
-32P]ATP using T4 polynucleotide kinase
The DNA primer (Table S1) was 5 -end labelled with [
γ
(
Thermo Scientific, Milan, Italy) at 37 C for 30 min and then purified with Illustra MicroSpin G-25
columns (GE Healthcare, Milan, Italy). The labelled primer (final concentration 72 nM) was annealed
to the template (final concentration 36 nM) (Table S1) in lithium cacodylate buffer (10 mM, pH 7.4)
◦
in the presence or absence of KCl 100 mM by heating at 95 C for 5 min and gradually cooling to r.t.
to allow both primer annealing and G4 folding. Where specified, 35, 36 or NDI were added at the
indicated concentrations and incubated overnight. Primer extension was performed with 2 U/reaction
◦
of AmpliTaq Gold DNA polymerase (Applied Biosystem, Carlsbad, CA, USA) at 42 C for 30 min.
Reactions were stopped by ethanol precipitation and primer extension products were separated on a
1
6% denaturing gel and finally visualized by phosphorimaging (Typhoon FLA 9000, GE Healthcare,
Milan, Italy). Markers were prepared based on Maxam & Gilbert sequencing by PCR reaction with
3
2
◦
P-labeled primer. PCR products were treated with formic acids for 5 min at 25 C and then with
◦
piperidin for 30 min at 90 C.
Supplementary Materials: The following are available online. Table S1: Oligonucleotides used in this study,
1
13
NMR Figures: H and C-NMR characterization of all compounds presented in the main text.
Author Contributions: Conceptualization, M.F., F.D. and S.N.R.; Methodology, M.T., M.F., F.D. and S.N.R.;
Validation, M.T., M.N., F.D. and S.N.R.; Formal Analysis ad investigation, M.T., A.L., V.P., M.N., E.B., F.D.;
Resources, M.F. and S.N.R.; Data Curation, M.T., E.B., F.D.; Writing-Original Draft Preparation, F.D. and
S.N.R.; Writing-Review & Editing, M.T., F.D., M.F. and S.N.R.; Supervision, M.N., F.D., M.F. and S.N.R.; Project
Administration, F.D., M.F. and S.N.R.; Funding Acquisition, M.F. and S.N.R.
Funding: This research was funded by [the Bill and Melinda Gates Foundation] grant numbers [OPP1035881,
OPP1097238] and [the European Research Council] grant number [ERC Consolidator 615879]. The APC was
funded by [the Bill and Melinda Gates Foundation].
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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