Bio-inspired dual-selective BCL-2/c-MYC G-quadruplex binders: Design, synthesis, and anticancer activity of drug-like imidazo[2,1-i]purine derivatives

Article abstract of DOI:10.1021/acs.jmedchem.9b00262

In the search for new drug-like selective G-quadruplex binders, a bioinspired design focused on the use of nucleobases as synthons in a multicomponent reaction was herein proved to be viable and successful. Hence, a new class of multifunctionalized imidaz

Full text of DOI:10.1021/acs.jmedchem.9b00262

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Article
Bio-Inspired Dual Selective BCL-2/c-MYC G-
Quadruplex Binders: Design, Synthesis and Anticancer
Activity of Drug-like Imidazo[2,1-i]purine Derivatives.
Sveva Pelliccia, Jussara Amato, Domenica Capasso, Sonia Di Gaetano,
Alberto Massarotti, Marialuisa Piccolo, Carlo Irace, Gian Cesare Tron, Bruno
Pagano, Antonio Randazzo, Ettore Novellino, and Mariateresa Giustiniano
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00262 • Publication Date (Web): 26 Jun 2019
Downloaded from http://pubs.acs.org on June 27, 2019
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Bio-Inspired Dual Selective BCL-2/c-MYC G-Quadruplex Binders: Design,
Synthesis and Anticancer Activity of Drug-like Imidazo[2,1-i]purine Derivatives.
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Sveva Pelliccia,†,# Jussara Amato,†,# Domenica Capasso,† Sonia Di Gaetano,‡ Alberto Massarotti,§ Marialuisa
Piccolo,† Carlo Irace,† Gian Cesare Tron,§ Bruno Pagano,† Antonio Randazzo,† Ettore Novellino,† and
Mariateresa Giustiniano*,†.
† Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano 49, 80131, Naples, Italy
‡ Institute of Biostructures and Bioimaging, CNR, Via Mezzocannone 16, 80134, Naples, Italy
§ Department of Pharmaceutical Sciences, University of Piemonte Orientale, Largo Donegani 2, 28100
Novara, Italy
KEYWORDS. G-Quadruplex; BCL2; c-MYC; Groebke-Blackburn-Bienaymé Reaction; Anticancer Drugs
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Abstract
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In the search for new drug-like selective G-quadruplex binders, a bio-inspired design focused on the use of
nucleobases as synthons in a multicomponent reaction has been herein proved to be viable and successful.
Hence, a new class of multi-functionalized imidazo[2,1-i]purine derivatives, easily synthesized with a
convergent approach, allowed for the identification of the first dual BCL2/c-MYC gene promoter G-
quadruplex ligand. Biophysical studies involving CD melting experiments, MST measurements, NMR
titrations, and computational docking calculations, as well as biological investigations including cytotoxicity
and apoptotic assays, and qPCR and Western Blot analyses, have been carried out to assess the potency and
to characterize the binding mode of the newly identified lead compound. The absence of toxicity towards
normal cells, together with the small molecular weight (≅ 500), the water solubility, the ease of
functionalization, and the selectivity profile are promising and desirable features to develop G-quadruplex
binders as safe and effective anticancer agents.
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Introduction
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Since the early observations that guanine-rich nucleic acids can adopt highly ordered secondary structures,1
biological relevance and therapeutic value of non-canonical DNA secondary structures have been both very
active and attractive fields in the drug discovery arena. Among them, G-quadruplexes (G4s) stand for the
most investigated ones and have gained the status of clinically validated targets in oncology, with Quarfloxin
as first-in-class compound reaching Phase II clinical trials,2 but failing to proceed for bioavailability issues.
The G4s are a heterogeneous class of structural topologies composed of hydrogen-bonding arrangements of
four guanine bases, namely the G-tetrads, which are the G4s’ core-motif, or, quoting S. Neidle “the cement
that holds G-quadruplex structures together”. 3 In the late 80’ pioneering works by Henderson4 and Sundquist
and Klug,5 sparked an interest in the involvement of G4s in telomeric processes6-9 and in potential therapeutic
applications. A few years later, cardinal work of the Hurley and Neidle labs provided the first G4 ligands that
effectively inhibited telomerase and induced cancer cell death by restoring the activation of the apoptotic
pathways.10-12
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G4-forming motifs have also been identified in oncogene promoter regions,13-15 bringing to light the
therapeutic potential for targeted gene regulation at the transcriptional level.16 The most studied oncogene
promoter G4s include c-MYC,17 BCL-2,18-19 h-RAS,20 k-RAS,21 and c-KIT.22-23 Soon after the leading works
addressing the identification and the biological relevance of gene promoters’ G4 binders, it became apparent
that the indirect targeting of a protein overexpressed in tumor cells, by the direct targeting of its coding gene
could have some key advantages compared to proteins,24 such as fewer copies of target, lower probability of
resistance and mutations, and possibility to target genes even when their products proved to be undruggable
as in the case of c-MYC. Nevertheless, important challenges are associated with the development of potent
and selective G4 binders, with the poor availability of promoter G4s’ structural information being one the
most limiting issues. Albeit selective ligands rationale design is most desirable,25 and some successful
applications of computational techniques have been reported, different G4s’ selective ligands also arose from
the application of fragment-based screening,26 and dynamic combinatorial libraries.27-29 Thanks to these
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approaches, a good number of ligands with different selectivity profiles have been reported so far, the most
being c-MYC G4 selective binders.30-36 Other reported chemotypes able to selectively bind gene promoter
G4s over duplex and sometimes also over telomeric G4s, include, but are not limited to, indolo[3,2-
b]quinolines (human telomeric, c-KIT2 and Hsp90A G4s),37 indenopyrimidine derivatives (c-KIT and c-MYC
G4s),38 indolylmethyleneindanone scaffolds (c-KIT and c-MYC G4s),39 piperidinyl-amines (c-KIT and c-
MYC G4s),40 synthetic derivatives of two natural compounds, berberine and palmatine (c-KIT and c-MYC
G4s),41 and furopyridazinones (BCL-2 G4),42 tri-substituted isoalloxazines (c-KIT1 and c-KIT2 G4s),43 and
bis-indole carboxamides (c-KIT2 and c-MYC G4s).44 These important findings demonstrated that, in
particular for the end-stacker ligands, selectivity for the G4s over the duplex DNA, could be achieved
increasing both the number of side chains substituents and ligand size, so that π-π interactions with the larger
surface area of a terminal G-tetrad could be maximized. But, clearly, these structural requirements – higher
molecular weights and increased cationic charges – lower the drug-likeness of the G4 ligands. In the search
for new drug-like molecular entities able to selectively target gene promoter G4s, here we propose a bio-
inspired approach focusing on the use of nucleobases as chemical platform to get three different new classes
of compounds: 6-alkyl-3-(alkylamino)-2arylimidazo[1,2-c]pyrimidin-5(6H)-ones (1), 1-alkyl-8-
(alkylamino)-7-aryl-1H-imidazo[2,1-b]purin-4(5H)-ones (2), and N-alkyl-3-alkyl-8-aryl-3H-imidazo[2,1-
i]purin-7-amines (3) when cytosine, guanine, and adenine were used as starting materials, respectively
(Figure 1).
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Figure 1. Bio-inspired key scaffolds 1, 2, and 3 (nucleobases’ structural cores are highlighted in blue).
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The underlying concept aims to create libraries of new molecular scaffolds exploiting the chemical nature of
nucleobases, which are intrinsically able to accept and donate hydrogen bonds and to make efficient π-
stacking interactions with each other and hence potentially, with the target nucleic acids sequences. We
speculated that harnessing the structural features of the nucleobases would create a library with desirable
G4s’ binding properties. Moreover, the presence of a high nitrogen/carbon ratio in the imidazo[2,1-i]purin-
7-amine molecular frame (which means, potentially, formation of more hydrogen bonds), and the possibility
to functionalize this scaffold with a range of cationic side chains, would offer the possibility to fine tune
selectivity towards different G4 topologies.
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Results and Discussion
Selection of the lead compound. In order to test our hypothesis, we synthesized compounds 4-6 (Figure 2),
representative of the use of three different nucleobases as synthons (cytosine, guanine, and adenine,
respectively), and functionalized with a 2-ethylmorpholino moiety in order to add a basic amino group,
which, in its protonated form, could provide an ionic interaction with the negatively charged DNA
phosphates. Compounds 4-6 were hence preliminarily screened for their ability to stabilize G4s in a circular
dichroism (CD) melting assay.45
Figure 2. Bio-inspired potential leads 4, 5, and 6.
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In particular, three G4 -forming sequences from the oncogene promoter regions of BCL-2 (BCL2-G4), c-KIT
(c-KIT1-G4) and c-MYC (c-MYC-G4), as well as a 23-mer truncation of human telomeric DNA sequence
(Tel23-G4) were used in these experiments. Moreover, a 20-mer hairpin-duplex-forming sequence (Hairpin
20-mer) was also used in order to evaluate the G4s’ over duplex selectivity of the compounds.
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The overall topology adopted by each investigated DNA sequence was first verified by CD measurements
(Supporting Information, Figure S1). c-KIT1 and c-MYC sequences adopted parallel G4 conformations,
showing the characteristic positive band at 262 nm and negative band at 240 nm in their CD spectra. The
presence of an additional band at 290 nm in the CD spectrum of BCL-2 clearly indicates the formation of a
mixed parallel/antiparallel conformation. On the other hand, Tel23 sequence showed a CD spectrum having
two positive bands at 290 and 270 nm and a weak negative band at 240 nm, consistent with the formation of
a hybrid [3+1] G4 folding topology. As mentioned above, we also took into consideration a hairpin-duplex-
forming sequence showing a positive band at 270 nm and a negative one at around 230 nm in the CD
spectrum, which are characteristic values of duplex formation. Next, CD studies were performed to explore
the potential of 4-6 to alter the native folding topology of the investigated G4s. Upon addition of an excess
of ligands (10 molar equivalents relative to the DNA), no relevant variations of DNA chiroptical signals were
observed, suggesting an overall preservation of the DNA structure in the presence of 4-6 (Supporting
Information, Figure S1). Then, the DNA stabilizing properties of compounds 4-6 were evaluated by CD-
melting experiments measuring the ligand-induced change in the melting temperature (ΔTm) either of G4 and
duplex structures (Supporting Information, Figure S2). Results of these experiments clearly revealed that 4
and 5 did not increase significantly the stability of any G4 as well as of duplex DNA (Figure 3). On the other
hand, compound 6 turned out to be able to induce a noticeable increase of the melting temperatures of the
three gene promoter G4s (BCL2 G4: ΔTm = 9.2 ±0.5 °C; c-MYC G4: ΔTm = 7.2 ±0.5 °C; c-KIT1 G4: ΔTm =
5.2 ±0.5 °C), suggesting a good affinity of 6 for the parallel G4 conformation adopted by them. Conversely
compound 6 did not increase significantly the thermal stabilities of the antiparallel telomeric G4 (Tel23 G4:
ΔTm = 2.4 ±0.5 °C), as well as of the hairpin-duplex DNA (Hairpin 20-mer: ΔTm = 1.9 ±0.5 °C) giving a
glimpse of its G4’s over duplex selectivity, and also providing a first evidence of its ability to discriminate
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among different G4 topologies. In light of these preliminary results, compound 6 was selected as our lead
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compound and a library of 16 analogues was synthesized and tested in both biophysical and biological assays.
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Figure 3. Variation of the melting temperature (ΔTm) of different G4-forming sequences and of a hairpin-
duplex-forming sequence induced by compounds 4-6. Errors are ±0.5 °C.
Synthesis of the focused library. Compounds 4-21 were synthesized in two steps, namely a Groebke-
Blackburn-Bienaymé 3-component reaction (GBB-3CR)46 and a subsequent SN2 with 2- and 3-aminoalkyl
halides (Scheme 1). The GBB-3CR is a multicomponent reaction involving a 2-amino-aza-heterocycle, a
benzaldehyde and an isocyanide. Herein we used nucleobases47 such as cytosine, guanine and adenine as
starting materials, six different substituted benzaldehydes, and tert-butylisocyanide. By performing the GBB-
3CR in DMSO as the solvent, using ZrCl4 as Lewis acid catalyst, at 70 °C overnight, compounds A-G (GBB-
3CR adducts) were obtained in 11–50 % yields. Their subsequent alkylation was performed in DMF, using
Cs2CO3 as base, at 100 °C, overnight, to give compounds 4-21 with yields ranging from 22 to 84%. While
variously substituted aldehydes and different 2- and 3-aminoalkyl halides have been selected as starting
materials, tert-butyl isocyanide was chosen as a highly nucleophilic component of the GBB-3CR.
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Compd
6
R1
R2
Yield
4-N(CH3)2
56%
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8
4-N(CH3)2
4-N(CH3)2
63%
84%
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4-N(CH3)2
4-Cl
56%
37%
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4-Cl
4-Cl
2-F
63%
59%
42%
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2-F
2-F
69%
47%
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2-OR2
2-OR2
31%
35%
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2-OR2
22%
68%
4-morpholine
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4-morpholine
4-morpholine
67%
48%
Scheme 1. Synthesis of compounds 4-21.
According to Scheme 2, compound 9 was deprotected with hydrazine hydrate, to give the free amino group
in compound 22, and the latter was further derivatized with S-ethylthiopseudourea in order to get the
guanidino functional group (23).48
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Scheme 2. Synthesis of compounds 22-23.
Circular dichroism studies. With the aim of evaluating the interaction of compounds 7-8, 10-23 with G4
structures and their selectivity for G4 over duplex DNA, CD melting experiments were performed using the
same DNA sequences already used for testing compounds 4-6. Results of such experiments are reported in
Table 1 (and Supporting Information, Figures S3-S4).
Starting from lead compound 6, when the ethylmorpholino chain (-R2, Scheme 1) was substituted with
ethylpyrrolidine (7) or N,N-dimethylpropylamine (8), an increase of thermal stabilization of G4s was
observed even at the expenses of their selectivity. In fact, compounds 7 and 8 showed to be not able to
discriminate G4 over duplex DNA (Table 1). On the other hand, the functionalization of the alkyl chain with
a primary amino group (22) or a guanidino unit (23) led to a dramatic drop in potency. In addition, while the
replacement of the 4-N,N-dimethylamino group of the aryl ring of lead compound 6 (R1, Scheme 1) with a
halogen atom as a 4-Cl (10) led to a lower Tm variation , the concomitant presence of aminopyrrolidine (11)
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and N,N-dimethylpropylamine (12), induced an increased G4 stabilization and a better selectivity towards
BCL2 G4 and c-MYC G4 over the other investigated G4s and duplex DNA (Table 1).
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Table 1. Melting temperature variations of G4 and hairpin-duplex DNAs upon addition
of 10 molar equivalents of compounds 7-8 and 10-23 measured by CD melting
experiments.a
Compound
BCL2-G4
c-MYC-G4
c-KIT1-G4
Tel23-G4 Hairpin-20-mer
7
+ 13.4
+ 16.9
+ 2.1
+ 4.5
+6.7
+ 19.5
> + 25.0b
+ 1.6
+ 7.3
+ 11.9
+ 1.1
+ 2.3
+ 2.8
+ 1.0
+ 2.1
+ 2.6
+ 0.8
+ 4.5
+ 4.5
+ 1.9
+ 2.9
+ 5.1
+ 1.4
+ 2.1
+ 4.8
+ 7.2
+ 0.3
+ 2.3
+ 2.4
+ 0.7
+ 1.1
+ 1.2
+ 0.1
+ 4.9
+ 5.7
+ 1.0
+ 2.2
+ 3.8
- 0.1
+ 7.5
N.D.c
- 0.5
8
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20
21
22
23
+ 6.4
+ 1.9
- 0.5
+ 12.8
+ 1.9
+ 2.6
+ 5.0
+ 8.2
+ 1.9
+ 7.1
+ 7.8
+ 4.9
+ 9.1
+ 10.1
+ 1.7
+ 2.8
+ 0.5
+ 0.3
+ 0.2
+ 1.2
+ 2.2
+ 3.5
+ 1.5
+ 6.0
+ 9.7
+ 0.5
+ 0.2
+ 5.8
+ 11.5
+ 1.9
+ 8.4
> + 25.0b
+ 2.1
+ 8.7
+ 9.5
+ 1.1
+ 2.5
+ 0.6
aTm represents the difference in melting temperature [Tm = Tm (DNA + 10 ligand equivalents) - Tm (DNA)]. The
Tm values of DNAs alone are: BCL2= 61.5  0.5 °C; c-MYC = 78.4  0.5 °C c-KIT1= 54.7  0.5 °C; Tel23= 54.5 
b
0.5 °C; Hairpin-20-mer = 62.3  0.5 °C. These compounds increase significantly the thermal stability of the G4;
however Tm values are not accurately determinable (Tm >100 °C). cTm value is not determinable due to the presence
of multiple transitions in the melting curve.
When the halogen atom on the aryl ring was a 2-F group as in compounds 13-15, the ethylmorpholino chain
(13) provided weak interactions, while ethylpyrrolidine (14) and N,N-dimethylpropylamine (15) were able
to induce a higher thermal stabilization of both BCL2 and c-MYC G4s, while showing poor binding to c-KIT1
and Tel23 G4s, and absolutely no binding to the duplex DNA (Table 1). We also investigated the effect of a
double aminoalkyl chain as in compounds 16-18, with the aim to check if the presence of two cationic
charges, as present in many G4 binding ligands, could improve affinity of the lead compound 6. When the
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scaffold was decorated with two ethylmorpholino chains (16), a very weak stabilization of both G4s and
duplex DNA structures was observed, while unfortunately the ethylpyrrolidine (17) and N,N-
dimethylpropylamine (18) chains induced an indiscriminate binding to both G4s and duplex DNA (Table 1).
Finally, compounds 19-21 showed that the effect of a bulkier substituent on the aryl ring such as the 4-
morpholino group, was detrimental, since it led to a loss of selectivity for G4s over duplex DNA (Table 1).
In view of the above results, compounds 12, 15, and 19 were selected as the best performing ones due to their
high selectivity over duplex DNA and preference for the BCL2 and c-MYC G4 architectures over others.
Therefore, a more in-depth biological and biophysical characterization of the interaction properties of 12, 15,
and 19 with BCL2 and c-MYC G4s was carried out.
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Evaluation of the cytotoxic activity of the selected compounds. The simultaneous targeting of two non-
canonical DNA secondary structures, in particular c-MYC G4 and BCL2 i‐Motif, has been shown as a
synergistic and valid approach to lower mRNA levels of anti-apoptotic proteins and overcome aberrant
pathways in cancer cells.49 Encouraged by these previous findings, and with the aim to evaluate the cellular
effects of compounds 12, 15, and 19 upon interaction with the gene promoter BCL2 and c-MYC G4s, further
biological studies were performed. Within the framework of preliminary screening in vitro, the bioactivity
profile of the compounds was first evaluated on the human leukemia cell line Jurkat, where Bcl2 is known
to play a critical role in the regulation of cell survival.50 To this aim, at the outset the cells were incubated in
the presence of the ligands for 48 h at concentrations ranging from 5 to 50 µM. The results depicted in Figure
4A - here reported grouping data into a single indicator defined as "cell survival index" combining
mitochondrial metabolic activity and cell counting - show a remarkable dose-dependent cytotoxic activity
for compound 12 on Jurkat cells, with a cell survival index that drops below 25% to the highest tested
concentrations. In the same experimental condition, the bioactivities related to compounds 15 and 19 were
yet significant but less effective in blocking cell growth and proliferation. Indeed, compound 15 induced a
decrease of cell survival of about 60% at 50 µM, while compound 19 showed only a weak cytotoxic activity.
Therefore, we next investigated more thoroughly the bioactivity profile of compound 12 by using a selected
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panel of human cancer cells of different histological origin. Concentration-effect curves in Figure 4B suggest
compound 12 as basically inactive on human breast adenocarcinoma cells (MCF-7 cell line), as well as on
colorectal and melanoma cells (HCT-116 and A375, respectively). This selectivity of action for compound
12 is highlighted by the calculation of IC50 values (data reported in Figure 4D), indicating a marked
antiproliferative effect on Jurkat cells with an IC50 in the low micromolar range (17 µM) and no interference
with the growth of the other tumor cells tested - an interesting outcome for further future development of this
compound. Accordingly, under the same conditions, evidences emerging from bioscreen on non-malignant
cells (Figure 4C), as human keratinocytes (HaCaT) and human dermal fibroblast (HDF), only show a weak
cytotoxicity at concentrations in vitro higher than 50 µM.
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Figure 4. (A) Cell survival index, evaluated by the MTT assay and the analysis of the live/dead cell ratio,
for Jurkat cells following 48 h of incubation with the indicated concentration (5, 10, 25 and 50 µM) of
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compounds 12, 15 and 19. Data are expressed as percentage of untreated cells and are reported as mean of 3
independent experiments ± SEM (n = 18). ***p<0.001 vs. control (untreated cells). (B) Cell survival index
for human cancer MCF-7, HCT-116, A375 and Jurkat cells, and (C) for human non-cancer HaCaT and HDF
cells, after treatments for 48 h with a range of concentrations (5 → 100 µM) of compound 12, as indicated
in the legends. *p<0.05 vs. control (untreated cells); ***p<0.001 vs. control (untreated cells). (D) IC50 values
(M) of compound 12 in the indicated cancer and non-cancer cell lines after 48 h of incubation in vitro. IC50
values are reported as mean ± SEM (n=18).
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To determine whether 12, 15, and 19 were able to induce apoptotic cell death, Jurkat cells were treated with
the compounds for 48 h, and the apoptosis analysis was performed with annexin V-FITC/PI double staining
by flow cytometry analysis.51 Consistent with bioscreen in vitro, the assay showed that the entire population
of cells (99%) treated with compound 12 was in the late apoptotic state (Figure 5B), while for compound 15
the 68% of cells were found to be in advanced apoptosis and 13% in early apoptosis (Figure 5C) with respect
to the control (Figure 5A). In contrast, when cells were treated with compound 19, only slight percentages, 9
and 8%, were found in the advanced and in the early apoptotic stage, respectively (Figure 5D).
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Figure 5. Apoptosis analysis with annexin V-FITC/PI double staining on Jurkat cells. Jurkat were treated
with 25 M molecules for 48 h. (A) Control (vehicle treated cells); (B) 12; (C) 15; (D) 19 treated cells. Lower
left quadrant: viable cells; upper left: necrotic cells; upper right: advanced apoptotic cells, low right: early
apoptotic cells. This picture is representative of two independent experiments.
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Downregulation of Bcl2 and c-Myc mRNAs, and effects on protein expression. To determine the effect
of 12, 15, and 19 on BCL2 and c-MYC gene transcription, quantitative analysis of mRNA was carried out by
means of real-time qPCR analyses (Figure S5). Upon treatment of Jurkat cells with 25 M of selected
compounds for 24 h, the cells were lysed to obtain the total RNA. qPCR analysis showed a reduction of about
66 and 56 % of c-MYC expression when cells were treated with compounds 12 and 15, respectively.
Differently, 19 induced a decrease of the expression level of c-MYC of only 27%. In the same manner, BCL2
expression resulted to be inhibited of 67 and 43% after 12 and 15 treatments, respectively. In parallel, to
confirm that the observed decrease in mRNA levels effectively leads to a decrease in Bcl-2 and c-Myc protein
content, Western blot analysis was performed on cellular extracts by using anti-Bcl-2 and anti-c-Myc
antibodies (Figure 6). Accordingly, immunoblot experiments revealed compound 12 able to decrease of about
50 and 40% c-Myc and Bcl-2 levels, respectively. In line with qPCR analysis, compounds 15 and 19 were
less effective but still able to significantly decrease c-Myc and Bcl-2 protein content under the same
experimental conditions.
Based on the results obtained from cytotoxicity assays, on the ability to induce apoptotic death in Jurkat cells,
to down-regulate Bcl2 and c-Myc mRNAs, as well as to decrease both c-Myc and Bcl-2 protein expression,
compound 12 was selected as the most promising of the library and engaged in further biophysical studies in
order to better characterize its activity towards BCL2 and c-MYC G4s.
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Figure 6. (A) Western blot analysis showing the effects of compounds 12, 15 and 19 in Jurkat cells on the
expression of c-Myc and Bcl-2 proteins following 24 h of incubation at 25 µM. The shown blots are
representative of three independent experiments and are cropped from different parts of the same gel, as
explicit by using clear delineation with dividing lines and white space. (B) After chemiluminescence, the
bands resulting from cell extracts were quantified by densitometric analysis and plotted in bar graphs as
percentage of controls (100% of protein content), as indicated. Shown are the average ± SEM values of three
independent experiments. The anti-β-actin antibody was used to standardize the amount of proteins in each
lane. *** p<0.001 vs. control untreated cells.
Microscale Thermophoresis experiments. The binding affinity of 12 for BCL2 and c-MYC G4s was
measured by means of microscale thermophoresis (MST).52 MST is a rapid and easy methodology to quantify
interactions between small molecules and nucleic acids in solution.53 In MST experiment, one of the
interacting partners must be fluorescent (either intrinsically fluorescent or a fluorophore is conjugated to a
non-fluorescent target). The binding of a ligand to the investigated molecule changes the thermophoretic
behavior of the target. This effect can be used to evaluate equilibrium constants, such as the dissociation
constant Kd. To this purpose, serial dilutions of 12 were prepared, mixed with a constant concentration of
Cy5-labeled oligonucleotides (BCL2 and c-MYC), loaded into capillaries and analyzed by MST. Experiments
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were run by using 5% final DMSO, in order to better solubilize the ligand. The results of these experiments
(Figure 7A, B) showed that compound 12 was able to bind both BCL2 and c-MYC G4s. with estimated Kd
values in the micromolar range [Kd= 26 (±7) µM and 60 (±20) µM for c-MYC and BCL2, respectively].
Control experiments were also performed using c-KIT1, telomeric and hairpin 20-mer sequences. Results of
such experiments (Figure S6) clearly show no interaction of compound 12 with both telomeric and hairpin
20-mer DNAs. On the other hand, compound 12 turned out to be able to interact also with c-KIT1 G4
(estimated Kd= 51 (±10) µM, Figure S6C), thus confirming a ligand binding specificity towards parallel G4s.
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Figure 7. MST measurements on the interaction of compound 12 with (A) BCL2 and (B) c-MYC G4s. (Top)
Time traces recorded by incubating increasing concentrations of 12 with the labeled G4s and (bottom) the
corresponding binding curves.
NMR spectroscopy titration experiments. In order to get information about the binding mode of compound
12 to BCL2 G4 and c-MYC G4, 1H-NMR titration experiments were performed.45 According to the literature,
the BCL2 and c-MYC sequences form, under the experimental conditions used, a single G4 conformation
characterized by 12 well-resolved imino proton peaks, corresponding to the 12 guanines involved in the three
G-tetrad planes (Figure 8). 19, 54 The investigated oligonucleotides were then titrated with 12 up to a 5:1
ligand/G4 ratio. In both cases, upon addition of increasing amounts of ligand to the G4 solutions, gradual
chemical shift changes for some DNA proton signals were observed. However, the imino and the aromatic
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regions of the spectra were affected with a different extent for the two G4s. Some signals of the compound
were also detected in the spectra. These peaks only grew in intensity, without showing any significant change
in chemical shift values by increasing ligand concentration, clearly suggesting a rapid binding process on the
NMR timescale. To probe the structural origin of the interaction between 12 and the two G4 structures, the
chemical shift variations (Δδ) of BCL2 G4 and c-MYC G4 were calculated (Supporting Information, Table
S1).
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As far as BCL2 G4 is concerned, the largest perturbations were observed for the imino protons belonging the
3’ G-tetrad (G3, G7 and G19), as well as for some of those of the middle G-tetrad (G2 and G8). Conversely,
the imino protons of the 5’ G-tetrad (G1−G9−G17−G21) were not affected at all. Furthermore, ligand
interaction with BCL2 G4 (Figure 8A) also significantly affected the aromatic proton of C4 located in the
lateral loop adjacent to the 3’-tetrad, as well as the aromatic protons of G3 and G7.
On the other hand, upon addition of compound 12 to the c-MYC G4 solution the imino signals belonging to
the G-tetrad on the 5’-side (G7−G11−G16−G20) as well as of those belonging to the 3’-face
(G9−G13−G18−G22) were strongly affected, while the remaining imino protons of the middle tetrad
(G8−G12−G17−G21) turned out to be slightly less affected (Figure 8B). Concerning the 5’-face of c-MYC
G4, it is more accessible for ligand stacking than 3’ one and the only 5’-flanking residue mainly affected by
the binding turned out to be the A6. At the 3’-side, the G4 NMR structure reveals that A25 folds back to form
a base pair with T23, thus covering the external 3’ G-tetrad, along with A24 that stacks on the T23:A25 base
pair. However, upon addition of 12, the aromatic protons of T23, A24, and A25 show a strong perturbation,
thus suggesting that such bases were not able to prevent the binding and that the ligand could stack on the 3’
G-tetrad displacing the architecture of 3’-flanking bases.
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Figure 8. H NMR spectra of (A) BCL2 and (B) c-MYC G4s titrated with compound 12. The ligand
equivalents are shown on the left of the spectra.
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Computational docking studies. To visualize the potential binding mode of 12 we performed a docking
study. Regarding the c-MYC G4 sequence, we selected the three NMR models with the following PDB codes:
1XAV, 2L7V, and 5W77. The first is formed by 20 conformations of a monomeric parallel-stranded G4,52
the second is formed by 10 different conformations55 and is complexed with a quinoline ligand, while the
latter is formed by 15 different conformations56 and is in complex with a benzofuran ligand. As concerns the
BCL2 G4 sequence, an NMR structure (PDB code: 2F8U) has been taken into account.19 This structure is a
monomer characterized by 10 different conformations. All the above-described experimental structures were
prepared and refined. Then, we performed a molecular docking study, using software FRED57-58 to identify
a potential binding mode of 12 against 45 and 15 conformations of c-MYC G4 and BCL2 G4 respectively
(Supporting Information, Tables S2-S3). Based on the interaction energy predicted by FRED, the best
complex of compound 12 and c-MYC and BCL2 G4S were selected.
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In the complexes with c-MYC G4, compounds 12 appeared to prefer the 5’-end position (Figure 9A, C) by
π-π stacking interactions with G5 and G16, while NH group is involved in a hydrogen bond interaction with
G11. Moreover, a putative hydrogen bond between a protonated tertiary amine and G7 could be present.
Compound 12 showed a very similar binding orientation compared to a quindoline structure already
crystalized with c-MYC G4 (PDB code: 2L7V). Docking results indicate a preference for 5’-side of
compound 12, this could be a consequence of the conformation of T23:A25 in the considered c-MYC G4
structures that prevent 12 to bind near the 3’ G-tetrad. This result is in contrast with the NMR titration spectra
showing that compound 12 is able to displace the 3’-flanking bases and stack also on the 3’ G-tetrad. As
regards the complexes of BCL2 G4 (Figure 9B, D), compound 12 assumed a different binding mode. In
accordance with spectral NMR data showing the largest perturbations for the imino protons belonging the 3’
G-tetrad (G3, G7 and G19), and the middle G-tetrad (G2 and G8), the docking results indicate that 12 is
located near C4, G7 and G8, but with an orientation that does not allow π-π stacking interactions, while NH
group gives hydrogen bond interactions with the phosphate link between G8 and G9 and also a putative
hydrogen bond between a protonated tertiary amine and phosphate group of G7 could be present. From both
NMR and molecular docking studies it is possible to elucidate the following structure-activity relationship:
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in the interaction with c-MYC G4, the heteroaromatic imidazo[2,1-i]purinic scaffold forms -stacking
interactions with guanines 5 and 16 at the 5’-end, and, as evident from NMR spectra, with the 3’-flanking
bases. Two hydrogen bonds are formed between the esocyclic NH of 12 and the guanine 11, and the
protonated tertiary amine and the guanine 7, respectively. Hydrogen bonds between the NH group of 12 and
the phosphate group linking guanines 8 and 9, and between the protonated tertiary amine and the guanine 7’s
phosphate group, represent key interactions in the binding to BCL2 G4.
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Figure 9. Best docked poses of 12 against c-MYC (A, C, E) and BCL2 G4s (B, D, F) structures, respectively.
The DNA of c-MYC (PDB id: 2L7V) and BCL2 (PDB id: 2F8U) are shown as orange and green cartoons,
respectively. The best 12 poses are represented as cyan carbon sticks. Coordinating K+ ions are reported as
violet spheres, while hydrogen bond interactions are indicated as dashed yellow lines. In the 2D depiction of
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Conclusions
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Starting from three bio-inspired scaffolds (4, 5, and 6) synthesized through an atom-economical and
convergent approach, namely a Groebke-Blackburn-Bienaymé three-component reaction, and a post-
condensation SN2, a new potential hit compound 6 has been selected by means of CD melting experiments
performed on different G4 topologies. Accordingly, a focused library of 16 analogues has been synthesized
and the binding efficiency towards different gene promoter G4s, as well as towards telomeric G4 and duplex
DNA, has been evaluated. As a result, three new lead compounds, 12, 15 and 19 have been selected as the
best performing ones, both in terms of potency and selectivity towards BCL2 and c-MYC G4s, over the other
G4 topologies screened and a duplex model. In the context of preclinical studies in vitro, bioscreens on a
selected panel of human cancer and non-cancer cells, as well as apoptotic induction assays, have been carried
out to explore the bioactivity profile of the lead compounds, allowing us to select compound 12 as the most
interesting one. Indeed, 12 exhibited a selective antiproliferative action towards Jurkat human T
lymphoblastoid cells, without showing significant cytotoxicity towards normal cells such as skin-derived
cells. Furthermore, a down-regulation at both mRNA and protein levels has been observed through qPCR
and Western Blot analyses when cells were treated with the selected compounds. Even if these correlations
do not rule out additional mechanisms contributing to the observed cellular responses, they are usually taken
as a good validation of on-target (G4) effects. This feature, along with other important properties, such as
small molecular weight (≅ 500), water-solubility (as hydrochloride salts), and ease of functionalization,
account for the drug-likeness of the newly identified G4s’ binders. Finally, in order to better characterize the
BCL2 and c-MYC G4s ligand binding profile, the dissociation constants Kd have been measured as in a low
micromolar range via MST experiments, and the binding modes have been studied by both NMR titrations
and computational docking calculations providing precious information for a future lead optimization.
Altogether, the obtained data suggest that, albeit with a slight loss in potency if compared with less- or non-
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selective G4 binders, the modulation of the selectivity towards different gene promoters’G4 could be feasible
and biological relevant. In conclusion, a new class of bio-inspired drug-like dual selective BCL2/c-MYC G4
binders has been identified as new potential anticancer agents. Based on both the herein reported biophysical
and biological data and the poor availability in literature of selective BCL2-G4 ligands, our lead 12 represents
a promising advancement towards the pathway-specific targeting ꢀ of cancer cell lines by means of gene
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promoters’ G4 binders. Moreover, the reported approach, and the results obtained, provided evidence that
capitalizing Nature’s resources, such as nucleobases59-60, as key structural elements could provide new
molecular entities able to induce a fine-tuning of the selectivity towards different gene-promoter G4s and
ergo safer anticancer drugs.
Experimental Section
Chemistry. General methods. Commercially available reagents and solvents were used without further
purification. Dichloromethane was dried by distillation from P2O5 and stored over activated molecular sieves
(4 Å). When necessary the reactions were performed in oven-dried glassware under a positive pressure of
dry nitrogen. Melting points were determined in open glass capillaries and are uncorrected. All the
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compounds were characterized by IR. H and C APT NMR spectra were recorded on a 400 MHz. High-
resolution ESI-MS spectra were performed on a Thermo LTQ Orbitrap XL mass spectrometer. The spectra
were recorded by infusion into the ESI source using MeOH as the solvent. Chemical shifts (δ) are reported
in part per million (ppm) relative to the residual solvent peak. Column chromatography was performed on
silica gel (70–230 mesh ASTM) using the reported eluents. Thin layer chromatography (TLC) was carried
out on 5 x 20 cm plates with a layer thickness of 0.25 mm (Silica gel 60 F254). When necessary they were
developed with KMnO4. Compounds 12, 15 and 19 were analyzed by analytical reversed-phase Ultra High
Performance Liquid Chromatography (RP-UHPLC) (Shimadzu Nexera liquid chromatograph LC- 30AD)
equipped with a C18-bonded column (Phenomenex Kinetex, 150 mm × 4.6 mm, 5 μm, 100 Å), using isocratic
10% MeCN (0.1% TFA) in water (0.1% TFA) over 5 min, and gradient 10% to 90% elution of MeCN (0.1%
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TFA) in water (0.1% TFA) over 15 min, with a flow rate of 1 mL/min and UV detection at 220 nm by a diode
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array UV−Vis detector. The purity of compounds was confirmed as ≥ 95%.
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General procedure for the GBB-3CR (A-G). The nucleobase (adenine, guanine, or cytosine) (1 mmol, 1
eq.), the substituted benzaldehyde (1 mmol, 1 eq.) and the isocyanide (1 mmol, 1 eq.) were one-pot mixed in
DMSO (2.5 mL, 0.4 M); the catalyst, ZrCl4 (0.1 mmol, 0.1 eq.) was added and the reaction was stirred at 100
°C for 20 hours. The formation of the GBB-3CR adduct was monitored by TLC (typically
dichloromethane/methanol 9:1), and the crude material was purified by column chromatography.
3-(tert-butylamino)-2-(4-(dimethylamino)phenyl)imidazo[1,2-c]pyrimidin-5(6H)-one (A). The crude
material was purified by column chromatography (DCM/MeOH 95:5) to give the product as beige solid (88
mg, 27% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.20 (br s, -NH), 7.92 (d, J= 7.2 Hz, 2H), 7.07-7.05 (m,
1H), 6.70-6.68 (m, 2H), 6.41-6.39 (m, 1H), 6.45 (br s, -NH), 2.90 (s, 6H), 0.95 (s, 9H). HRMS (ESI) m/z
Calcd for C18H24N5O+: 326.1976; Found: 326.1976 [M+H]+.
8-(tert-butylamino)-7-(4-chlorophenyl)-1H-imidazo[2,1-b]purin-4(5H)-one (B). The crude material was
purified by column chromatography (DCM/MeOH 95:5) to give the product as amorphous solid (39 mg,
11% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.21 (br s, -NH), 8.18 (s, 1H), 8.06 (d, J= 8.8 Hz, 2H), 7.37
(d, J= 8.8 Hz, 2H), 4.11 (br s, -NH), 1.03 (s, 9H). HRMS (ESI) m/z Calcd for C17H18ClN6O+: 357.1226;
Found: 357.1229 [M+H]+.
N-(tert-butyl)-8-(4-(dimethylamino)phenyl)-3H-imidazo[2,1-i]purin-7-amine (C). The crude material
was purified by column chromatography (DCM/MeOH 96:4) to give the product as yellowish solid (59 mg,
17% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.20 (s, 1H), 8.03 (d, J= 8.4 Hz, 2H), 6.75 (d, J=
+
8.4 Hz, 2H), 4.61 (br s, -NH), 2.93 (s, 6H), 1.03 (s, 9H). HRMS (ESI) m/z Calcd for C19H24N7 : 350.2088;
Found: 350.2087 [M+H]+.
N-(tert-butyl)-8-(4-chlorophenyl)-3H-imidazo[2,1-i]purin-7-amine (D). The crude material was purified
by column chromatography (DCM/MeOH 97:3) to give the product as light orange solid (112 mg, 33%
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yield). 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.23-8.20 (m, 3H), 7.47 (d, J= 8.8 Hz, 2H), 4.79 (br
5
6
+
s, -NH), 1.01 (s, 9H). HRMS (ESI) m/z Calcd for C17H18ClN6 : 341.1276; Found: 341.1273 [M+H]+.
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N-(tert-butyl)-8-(2-fluorophenyl)-3H-imidazo[2,1-i]purin-7-amine (E). The crude material was purified
by column chromatography (DCM/MeOH 95:5) to give the product as yellowish solid (162 mg, 50% yield).
1H NMR (400 MHz, DMSO-d6) δ 9.02 (br s, -NH), 7.93 (s, 1H), 7.75-7.72 (m, 1H), 7.40-7.35 (m, 1H), 7.29-
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+
7.26 (m, 2H), 7.19-7.14 (m, 1H), 5.25 (br s, -NH), 0.95 (s, 9H). HRMS (ESI) m/z Calcd for C17H18FN6 :
325.1572; Found: 325.1569 [M+H]+.
2-(7-(tert-butylamino)-3H-imidazo[2,1-i]purin-8-yl)phenol (F). The crude material was purified by
column chromatography (DCM/MeOH 95:5) to give the product as yellowish solid (116 mg, 36% yield). 1H
NMR (400 MHz, DMSO-d6) δ 13.83 (br s, -NH), 11.74 (br s, -OH), 9.06 (s, 1H), 8.28 (s, 1H), 8.17-8.06 (m,
1H), 7.25-7.18 (m, 1H), 6.93-6.89 (m, 2H), 5.62 (br s, -NH), 1.04 (s, 9H). HRMS (ESI) m/z Calcd for
C17H19N6O+: 323.1615; Found: 323.1613 [M+H]+.
N-(tert-butyl)-8-(4-morpholinophenyl)-3H-imidazo[2,1-i]purin-7-amine (G). The crude material was
purified by column chromatography (DCM/MeOH 96:4) to give the product as white solid (74 mg, 19%
1
yield). H NMR (400 MHz, DMSO-d6) δ 13.70 (br s, -NH), 8.97 (s, 1H), 8.21 (s, 1H), 8.10-8.05 (m, 2H),
6.97 (d, J= 8.4 Hz, 2H), 4.68 (br s, -NH), 3.75-3.73 (m, 4H), 3.17-3.14 (m, 4H), 1.03 (s, 9H). HRMS (ESI)
m/z Calcd for C21H26N7O+: 392.2194; Found: 392.2188 [M+H]+.
General procedure for the alkylation of GBB-3CR products. The GBB-3CR intermediate (A-G) (0.1
mmol, 1 eq.) was dissolved in DMF (0.33 mL, 0.3 M), the aminoalkyl halide (0.3 mmol, 3 eq.) and Cesium
carbonate (0.5 mmol, 5 eq.) were added and the reaction was stirred overnight at 60 °C. The formation of the
alkylated GBB-3CR product was monitored by TLC (typically dichloromethane/methanol 95:5 or 90:10),
and the crude material was purified by column chromatography.
3-(tert-butylamino)-2-(4-(dimethylamino)phenyl)-6-(2-morpholinoethyl)imidazo[1,2-c]pyrimidin-
5(6H)-one (4). The crude material was purified by column chromatography (DCM/MeOH 97:3) to give the
product as reddish sticky (25 mg, 57% yield). 1H NMR (400 MHz, CD3OD) δ 7.93 (d, J= 8.0 Hz, 2H), 7.33
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(br d, 1H), 6.69 (d, J= 8.0 Hz, 2H), 6.48 (br d, 1H), 4.48 (br s, 1H), 3.98-3.95 (m, 2H), 3.54-3.49 (m, 4H),
4
5
6
+
2.89 (s, 6H), 2.59-2.56 (m, 2H), 2.48-2.40 (m, 4H), 0.95 (s, 9H). HRMS (ESI) m/z Calcd for C24H35N6O2 :
7
8
9
439.2816; Found: 439.2812 [M+H]+.
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8-(tert-butylamino)-7-(4-chlorophenyl)-1-(2-morpholinoethyl)-1H-imidazo[2,1-b]purin-4(5H)-one (5).
The crude material was purified by column chromatography (DCM/MeOH 90:10) to give the product as
yellowish solid (13 mg, 28% yield). 1H NMR (400 MHz, CD3OD) δ 8.06 (s, 1H), 7.96 (br d, AA’XX’, 2H),
7.36 (br d, AA’XX’, 2H), 4.60-4.57 (m, 2H), 3.64-3.61 (m, 4H), 2.85-2.82 (m, 2H), 2.52-2.50 (m, 4H), 1.05
+
(s, 9H). HRMS (ESI) m/z Calcd for C23H29ClN7O2 : 470.2066; Found: 470.2067 [M+H]+.
N-(tert-butyl)-8-(4-(dimethylamino)phenyl)-3-(2-morpholinoethyl)-3H-imidazo[2,1-i]purin-7-amine
(6). The crude material was purified by column chromatography (DCM/MeOH 95:5) to give the product as
yellow solid (26 mg, 56% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.23 (s, 1H), 8.03 (d, J= 8.8
Hz, 2H), 6.75 (d, J= 8.8 Hz, 2H), 4.66-4.63 (m, 3H), 3.46-3.44 (m, 4H), 2.93 (s, 6H), 2.92-2.89 (m, 2H),
2.50-2.48 (m, 4H), 1.03 (s, 9H). HRMS (ESI) m/z Calcd for C25H35N8O+: 463.2929; Found: 463.2922
[M+H]+.
N-(tert-butyl)-8-(4-(dimethylamino)phenyl)-3-(2-(pyrrolidin-1-yl)ethyl)-3H-imidazo[2,1-i]purin-7-
amine (7). The crude material was purified by column chromatography (DCM/MeOH 90:10) to give the
product as reddish amorphous solid (28 mg, 63% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.24
(s, 1H), 8.04 (d, J= 8.8 Hz, 2H), 6.75 (d, J= 8.8 Hz, 2H), 4.64 (br s, -NH), 4.62-4.60 (m, 2H), 3.06-3.04 (m,
+
2H), 2.93 (s, 6H), 2.56-2.53 (m, 4H), 1.65-1.61 (m, 4H), 1.04 (s, 9H). HRMS (ESI) m/z Calcd for C25H35N8 :
447.2980; Found: 447.2976 [M+H]+.
N-(tert-butyl)-8-(4-(dimethylamino)phenyl)-3-(3-(dimethylamino)propyl)-3H-imidazo[2,1-i]purin-7-
amine (8). The crude material was purified by column chromatography (DCM/MeOH 90:10) to give the
product as reddish sticky solid (36.5 mg, 84% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.20 (s,
1H), 8.04 (d, J= 8.4 Hz, 2H), 6.74 (d, J= 8.8 Hz, 2H), 4.64 (br s, -NH), 4.53-4.50 (m, 2H), 2.93 (s, 6H), 2.34-
+
2.31 (m, 2H), 2.20 (s, 6H), 2.17-2.15 (m, 2H), 1.03 (s, 9H). HRMS (ESI) m/z Calcd for C24H35N8 : 435.2980;
Found: 435.2975 [M+H]+.
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N-(tert-butyl)-8-(4-chlorophenyl)-3-(2-morpholinoethyl)-3H-imidazo[2,1-i]purin-7-amine (10). The
crude material was purified by column chromatography (DCM/MeOH 97:3) to give the product as yellowish
solid (17 mg, 37% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.27 (s, 1H), 8.21 (d, J= 8.8 Hz,
2H), 7.48 (d, J= 8.0 Hz, 2H), 4.82 (br s, -NH), 4.65 (m, 2H), 3.45-3.43 (m, 4H), 2.91-2.88 (m, 2H), 2.48-2.43
(m, 4H), 1.03 (s, 9H). HRMS (ESI) m/z Calcd for C23H29ClN7O+: 454.2117; Found: 454.2113 [M+H]+.
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N-(tert-butyl)-8-(4-chlorophenyl)-3-(2-(pyrrolidin-1-yl)ethyl)-3H-imidazo[2,1-i]purin-7-amine
(11).
The crude material was purified by column chromatography (DCM/MeOH 90:10) to give the product as light
orange solid (28 mg, 63.5% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.27 (s, 1H), 8.22 (d, J=
8.0 Hz, 2H), 7.48 (d, J= 7.6 Hz, 2H), 4.82 (br s, -NH), 4.65-4.62 (m, 2H), 3.05-3.03 (m, 2H), 2.53-2.50 (m,
+
4H), 1.64-1.60 (m, 4H), 1.03 (s, 9H). HRMS (ESI) m/z Calcd for C23H29ClN7 : 438.2168; Found: 438.2166
[M+H]+.
N-(tert-butyl)-8-(4-chlorophenyl)-3-(3-(dimethylamino)propyl)-3H-imidazo[2,1-i]purin-7-amine (12).
The crude material was purified by column chromatography (DCM/MeOH 90:10) to give the product as
yellowish solid (25 mg, 59% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.24-8.22 (m, 3H), 7.48
(d, J= 8.4 Hz, 2H), 4.83 (br s, -NH), 4.54-4.51 (m, 2H), 2.30-2.27 (m, 2H), 2.16 (s, 6H), 2.18-2.13 (m, 2H),
1.03 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 148.3, 135.9, 134.3, 133.9, 132.2, 129.7, 128.5, 124.6, 113.7,
+
56.3, 56.2, 45.5, 45.4, 30.3, 28.2. HRMS (ESI) m/z Calcd for C22H29ClN7 : 426.2168; Found: 426.2164
[M+H]+. tR (min): 11.6.
N-(tert-butyl)-8-(2-fluorophenyl)-3-(2-morpholinoethyl)-3H-imidazo[2,1-i]purin-7-amine (13). The
crude material was purified by column chromatography (DCM/MeOH 97:3) to give the product as yellowish
solid (18 mg, 42% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.28 (s, 1H), 7.81-7.77 (m, 1H),
7.46-7.40 (m, 1H), 7.32-7.29 (m, 2H), 4.64-4.61 (m, 2H), 4.20 (br d, -NH), 3.43-3.41 (m, 4H), 2.89-2.86 (m,
2H), 2.45-2.41 (m, 4H), 0.91 (s, 9H). HRMS (ESI) m/z Calcd for C23H29FN7O+: 438.2413; Found: 438.2408
[M+H]+.
N-(tert-butyl)-8-(2-fluorophenyl)-3-(2-(pyrrolidin-1-yl)ethyl)-3H-imidazo[2,1-i]purin-7-amine
(14).
The crude material was purified by column chromatography (DCM/MeOH 90:10) to give the product as
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reddish solid (29mg, 69% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.28 (s, 1H), 7.83-7.79 (m,
1H), 7.46-7.41 (m, 1H), 7.33-7.28 (m, 2H), 4.62-4.59 (m, 2H), 4.19 (br s, -NH), 2.52-2.48 (m, 6H), 1.63-
6
7
8
9
+
1.61 (m, 4H), 0.91 (s, 9H). HRMS (ESI) m/z Calcd for C23H29FN7 : 422.2463; Found: 422.2458 [M+H]+.
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N-(tert-butyl)-3-(3-(dimethylamino)propyl)-8-(2-fluorophenyl)-3H-imidazo[2,1-i]purin-7-amine (15).
The crude material was purified by column chromatography (DCM/MeOH 90:10) to give the product as
amorphous solid (19 mg, 47% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.24 (s, 1H), 7.83-7.79
(m, 1H), 7.46-7.42 (m, 1H), 7.31-7.28 (m, 2H), 4.51-4.48 (m, 2H), 4.19 (br s, -NH), 2.45-2.23 (m, 2H), 2.16-
2.13 (m, 2H), 2.13 (s, 6H), 0.91 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 160.0 (d, JC-F = 245.5 Hz), 148.1,
134.3, 133.5 (d, JC-F = 2.1 Hz), 132.2 (d, JC-F = 3.5 Hz), 130.4 (d, JC-F = 8.1 Hz), 125.7, 124.8 (d, JC-F = 3.2
Hz), 123.1 (d, JC-F = 14.1 Hz), 116.2 (d, JC-F = 21.8 Hz), 113.7, 56.1, 55.7, 45.5, 45.4, 30.0, 28.1. HRMS
+
(ESI) m/z Calcd for C22H29FN7 : 410.2463; Found: 410.2460 [M+H]+. tR (min): 10.8.
N-(tert-butyl)-8-(2-(2-morpholinoethoxy)phenyl)-3-(2-morpholinoethyl)-3H-imidazo[2,1-i]purin-7-
amine (16). The crude material was purified by column chromatography (DCM/MeOH 95:5) to give the
product as amorphous solid (17 mg, 31.5% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.26 (s,
1H), 7.68 (d, J= 7.6 Hz, 1H), 7.36-7.32 (m, 1H), 7.16 (d, J= 8.4 Hz, 1H), 7.10-7.06 (m, 1H), 4.64-4.61 (m,
2H), 4.21 (br s, -NH), 4.15-4.13 (m, 2H), 3.51-3.43 (m, 8H), 2.89-2.86 (m, 2H), 2.62-2.60 (m, 2H), 2.48-
+
2.44 (m, 4H), 2.35-2-34 (m, 4H), 0.90 (s, 9H). HRMS (ESI) m/z Calcd for C29H41N8O3 : 549.3297; Found:
549.3297 [M+H]+.
N-(tert-butyl)-8-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-3-(2-(pyrrolidin-1-yl)ethyl)-3H-imidazo[2,1-
i]purin-7-amine (17). The crude material was purified by column chromatography (DCM/MeOH 90:10) to
give the product as yellowish solid (18 mg, 35% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.27
(s, 1H), 7.70 (dd, J= 7.6, 1.6 Hz, 1H), 7.37-7.33 (m, 1H), 7.16-7.06 (m, 2H), 4.63-4.60 (m, 2H), 4.34 (br s, -
NH), 4.15-4.13 (m, 2H), 3.07-3.04 (m, 2H), 2.80-2.78 (m, 2H), 2.55-2.49 (m, 8H), 1.67-1.62 (m, 8H), 0.88
(s, 9H). HRMS (ESI) m/z Calcd for C29H41N8O+: 517.3398; Found: 517.3393 [M+H]+.
N-(tert-butyl)-8-(2-(3-(dimethylamino)propoxy)phenyl)-3-(3-(dimethylamino)propyl)-3H-
imidazo[2,1-i]purin-7-amine (18). The crude material was purified by column chromatography
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