Synthesis of Triazole-Linked Analogues of c-di-GMP and Their Interactions with Diguanylate Cyclase

Article abstract of DOI:10.1021/acs.jmedchem.5b01184

Cyclic di-GMP (c-di-GMP) is a widespread second messenger that plays a key role in bacterial biofilm formation. The compound's ability to assume multiple conformations allows it to interact with a diverse set of target macromolecules. Here, we analyzed the binding mode of c-di-GMP to the allosteric inhibitory site (I-site) of diguanylate cyclases (DGCs) and compared it to the conformation adopted in the catalytic site of the EAL phosphodiesterases (PDEs). An array of novel molecules has been designed and synthesized by simplifying the native c-di-GMP structure and replacing the charged phosphodiester backbone with an isosteric nonhydrolyzable 1,2,3-triazole moiety. We developed the first neutral small molecule able to selectively target DGCs discriminating between the I-site of DGCs and the active site of PDEs; this molecule represents a novel tool for mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for future optimization studies to target DGCs in vivo.

Full text of DOI:10.1021/acs.jmedchem.5b01184

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Article
Synthesis of triazole-linked analogues of c-di-
GMP and their interactions with diguanylate cyclase
Silvia Fernicola, Ilaria Torquati, Alessandro Paiardini, Giorgio Giardina,
Giordano Rampioni, Marco Messina, Livia Leoni, Fabio Del Bello, Riccardo
Petrelli, serena rinaldo, Loredana Cappellacci, and Francesca Cutruzzolà
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01184 • Publication Date (Web): 01 Oct 2015
Downloaded from http://pubs.acs.org on October 8, 2015
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Synthesis of triazole-linked analogues of c-di-GMP and their interactions with diguanylate
cyclase
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Silvia Fernicola , Ilaria Torquati , Alessandro Paiardini , Giorgio Giardina , Giordano
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Rampioni , Marco Messina , Livia Leoni , Fabio Del Bello , Riccardo Petrelli , Serena
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Rinaldo , Loredana Cappellacci , Francesca Cutruzzolà
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Istituto PasteurꢀFondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza
University of Rome, Italy.
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School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino (MC), Italy
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Department of Biology and Biotechnology “Charles Darwin” Sapienza University of Rome, Italy.
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Department of Science, University Roma Tre, Rome, Italy.
Abstract
Cyclic diꢀGMP (cꢀdiꢀGMP) is a widespread secondꢀmessenger, that plays a key role in bacterial
biofilm formation. The compound’s ability to assume multiple conformations allows it to interact
with a diverse set of target macromolecules.
Here, we analyzed the binding mode of cꢀdiꢀGMP to the allosteric inhibitory site (Iꢀsite) of
diguanylate cyclases (DGCs), and compared it to the conformation adopted in the catalytic site of
the EAL phosphodiesterases (PDEs).
An array of novel molecules has been designed and synthesized by simplifying the native cꢀdiꢀGMP
structure and replacing the charged phosphodiester backbone with an isosteric nonꢀhydrolyzable
1
,2,3ꢀtriazole moiety.
We developed the first neutral small molecule able to selectively target DGCs discriminating
between the Iꢀsite of DGCs and the active site of PDEs; this molecule represents a novel tool for
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mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for
future optimization studies to target DGCs in vivo.
Keywords: cꢀdiꢀGMP, biofilm, Pseudomonas aeruginosa, Caulobacter crescentus, WspR, PleD,
RocR, inhibitors, copper(I)ꢀcatalyzed 1,3ꢀdipolar cycloaddition, click chemistry, Huisgen
cycloaddition, alkynes, azides, dinucleoside, heterodinucleoside, 1,2,3ꢀtriazole, purine, dimer.
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Abbreviations: DGCs, diguanylate cyclases; PDEs, phosphodiesterase; Iꢀsite, inhibitory site,
CuAAC, Copper(I)ꢀcatalyzed azideꢀalkyne cycloaddition.
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INTRODUCTION
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Since its discovery in 1987 , cyclicꢀdiꢀGMP (cꢀdiꢀGMP) has attracted growing attention and is
now acknowledged as one of the most important second messengers controlling bacterial adaptation
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strategies, such as biofilm formation, persistence, cytotoxicity and cellꢀcycle progression . The
intracellular levels of cꢀdiꢀGMP are regulated by the opposing activities of diguanylate cyclases
(
DGCs), containing GGDEF domains, and phosphodiesterases (PDEs), containing either EAL or
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HDꢀGYP domains, which synthesizes and degrade cꢀdiꢀGMP, respectively . In many cases, the
activity of GGDEF and EAL or HDꢀGYP domains is modulated by environmental/metabolic
signals or by neighbouring domains.
The receptors sensing the cellular levels of this dinucleotide, which represent the endꢀnodes of cꢀ
diꢀGMP controlled networks, are quite heterogeneous, embracing riboswitches and transcription
factors or enzymes containing PilZ domains, degenerate DGCs or PDEs, or other domains, and are
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yet to be fully structurally characterized .
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CꢀdiꢀGMP shows a very rich conformational polymorphism which accounts for its capability
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to interact with structurally unrelated binding sites , making the prediction of cꢀdiꢀGMPꢀbinding
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proteins very challenging .
As an example, cꢀdiꢀGMP forms an intercalated dimer to bind to GGDEF domains via the Iꢀsite,
a widespread (but not ubiquitous) binding site for noncompetitive product inhibition, which
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switches the enzymes to an inactive conformation
. On the other hand, the binding mode of cꢀ
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diꢀGMP to the EAL active site involves a single molecule in the open conformation
, even
though structural polymorphism at the level of one of the two glycosidic bonds has also been
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reported .
In recent years, cꢀdiꢀGMP signalling systems have emerged as promising targets to hamper
biofilm formation and virulence in many bacterial pathogens. However, since many bacterial
genomes encode multiple proteins involved in cꢀdiꢀGMP turnover and reception, we still need to
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deeply understand the mechanistic details governing its synthesis, degradation and signalling to
efficiently develop strategies targeting cꢀdiꢀGMPꢀdependent pathways.
Up to now, a number of approaches have been described aimed at inhibiting biofilm formation
by interfering with cꢀdiꢀGMP pathways; these are mainly based on the screening of smallꢀmolecule
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libraries using wholeꢀcell
, in vitro , or in silico methods, or on the rational design of
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potential inhibitors (see for an upꢀtoꢀdate review) including cꢀdiꢀGMP analogues (see Tables SIꢀ1
and SIꢀ2) that are useful tools for investigating the molecular bases of cꢀdiꢀGMP/protein
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interactions .
Among the cꢀdiꢀGMP analogues tested, the most promising compounds effective against DGCs
display very conservative modifications of the starting chemical structure of cꢀdiꢀGMP, involving
the 2’ꢀOH and the bridging oxygen in the phosphodiester linkage. The most potent so far is 2’ꢀ
deoxyꢀ2’ꢀFꢀcꢀdiGMP (2’ꢀFꢀcꢀdiꢀGMP, IC50 = 11 µM, assayed on WspR from Pseudomonas
aeruginosa, Table SIꢀ1). However, this inhibitor lacks selectivity as it also strongly inhibits PDEs
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(
IC50 = 0.7 µM, assayed on RocR from P. aeruginosa) . The relatively weak effect of these cꢀdiꢀ
GMP analogues on DGCs, as compared to PDEs, could be ascribed to the substantial entropy loss
when such compounds bind as stacked dimers on the Iꢀsite.
On the other hand, the three selective inhibitors for the PDE RocR, bearing 2’ꢀOH substitutions
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(
2’ꢀH and 2’ꢀOMe ) or phosphate linkage modifications (endoꢀSꢀcꢀdiꢀGMP ), may still be
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hydrolysed to the linear product, since the phosphodiester moiety is conserved .
The development of new compounds able to selectively inhibit DGCs without affecting the
activity of PDEs is an ambitious goal with potential biomedical applications, and thus demands a
systematic study focused on the role of the various moieties of the cꢀdiꢀGMP scaffold in controlling
its interaction with the target protein.
Therefore, in the present work we have designed and synthetized an array of cꢀdiꢀGMPꢀbased
molecules, encompassing linear derivatives with a completely novel scaffold, which have been
tested for their ability to inhibit both DGCs and PDEs. This study allowed us to identify the
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minimal structural determinants required for specifically targeting the Iꢀsite of DGCs. In particular,
identification of a linker able to hold the two guanine bases (and their derivatives) at the appropriate
distance required for selective targeting of the DGCs’ Iꢀsite makes these novel molecules potent
compounds, and possibly future therapeutic agents.
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RESULTS AND DISCUSSION
Chemical Synthesis
Three series of compounds have been designed and synthetized as linear derivatives of cꢀdiꢀ
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GMP. As was recently described , the linear analogues may present features that are more
attractive from a drug development perspective, as compared to the cyclic counterpart: i) these
compounds are in principle structurally simpler and therefore cost less to synthesize; ii) the
different subset of conformations accessible to linear compounds, as compared to cꢀdiꢀGMP, could
favour the identification of molecules able to selectively target a subset of cꢀdiꢀGMPꢀinteracting
proteins.
We designed linear cꢀdiꢀGMPꢀbased compounds starting from 5,5’ꢀtriazolylꢀdinucleosides
(Chart 1, Scaffold A), which were further simplified to hybrid dimers (i.e. linking one nucleoside
and one purine base by a triazole linker) (Chart 1, Scaffold B), and then to a third scaffold, where
the sugar was completely removed (Chart 1, Scaffold C).
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The synthesis of all the compounds was carried out using a click chemistry approach
. This
simple chemistry allowed us to replace the charged and hydrolizable phosphodiester linker of cꢀdiꢀ
GMP with the neutral 1,2,3ꢀtriazole bioisostere ring, thus preventing potential hydrolysis of the
linkage portion in a cellular background; moreover, this moiety is able to form hydrogen bonds and
π – π interactions with the amino acid side chains of the target proteins. As reported in Scheme 1,
for scaffold A the click reaction of 2ꢀNꢀisobutyrylꢀ5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀ
guanosine (4) with 2ꢀNꢀisobutyrylꢀ2’,3’ꢀOꢀisopropylideneꢀ5’ꢀOꢀpropargylꢀguanosine (8) yielded the
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1,2,3ꢀtriazole intermediate 10. Similarly, the protected 5’ꢀazidoꢀ5’ꢀdeoxyꢀguanosine 3 was reacted
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with N ꢀbenzoylꢀ2’,3’ꢀOꢀisopropylideneꢀ5’ꢀOꢀpropargylꢀadenosine (9) , to obtain the intermediate
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1. The 5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀadenosine (5) , prepared starting from 2’,3’ꢀOꢀ
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isopropylideneꢀadenosine (2)
was clicked with 8 and 9 to furnish the 1,2,3ꢀtriazole
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intermediates 12 and 13, respectively. Compound 10 was first deprotected in acidic conditions and
then in saturated ammonium hydroxide solution at 50°C, to give, after purification, the triazoleꢀ
linked dinucleoside DCI028. In contrast, the deprotection reactions of dinucleosides 11ꢀ13 were
performed first by treatment with saturated methanolic ammonia solution at room temperature,
followed by 70% formic acid aqueous solution at 40 °C, to furnish the triazoleꢀlinked dinucleosides
DCI016, DCI021, DCI015 respctively.
The 5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀguanosine (3) was synthesized starting from
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’,3’ꢀOꢀisopropylideneꢀguanosine (1) . Compound 3 was then treated with isobutyrylꢀchloride in
the presence of pyridine and trimethylsililchloride to obtain the 2ꢀNꢀisobutyrylꢀ5’ꢀazidoꢀ5’ꢀdeoxyꢀ
’,3’ꢀOꢀisopropylideneꢀguanosine (4) in 65% yield, according to the general procedure described by
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Kline et al. . 2ꢀNꢀisobutyrylꢀ2’,3’ꢀOꢀisopropylideneꢀguanosine (6) was converted into the 5’ꢀOꢀ
propargyl derivative 8 by reaction with propargyl bromide and NaH in THF at room temperature
(Scheme 1). The side product 8a (Scheme 2) was also obtained during the reaction, and
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characterized by HꢀNMR. The copper(I)ꢀcatalyzed azideꢀalkyne cycloaddition (CuAAC) reaction
(Huisgen’s cycloaddition) between 2ꢀNꢀisobutyrylꢀ2’,3’ꢀOꢀisopropylideneꢀ5’ꢀOꢀpropargylꢀ
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guanosine (8) and 2ꢀaminoꢀ6ꢀchloroꢀN ꢀ(3ꢀazidopropyl)ꢀpurine (16) yielded the triazole
intermediate 20 (Scheme 3). Compound 20 was deprotected first by a mixture of TFA/H O (3:1)
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followed by hydrolysis in basic conditions, to afford the hybride triazoleꢀlinked dimer DCI091
(Figure 1, Scaffold B).
Compound DCI059 was prepared, as shown in Scheme 4 by Huisgen’s cycloaddition of 2ꢀNꢀ
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isobutyrylꢀ5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀguanosine (4) with 2ꢀaminoꢀ6ꢀchloroꢀN ꢀ
propargylꢀpurine (19). The resulting triazole intermediate 21 was deprotected as reported for 20 to
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obtain the hybride triazoleꢀlinked DCI059. The alkyl azides 23, 25, 26 were synthetized following
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the procedure reported by Wijtmans et al.
and used to prepare the Nꢀ9 (azidoalkyl)ꢀpurine
derivatives 14-17 (Scheme SIꢀ1, Supporting information). The commercial 6ꢀchloropurine (22) was
alkylated only with alkyl azide 23, while 2ꢀaminoꢀ6ꢀchloropurine (24) was alkylated with the alkyl
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azides 23, 25, and 26, following the procedure of Lindsell et al. . The reactions led to a mixture of
Nꢀ9/Nꢀ7 isomers, with the Nꢀ9 isomers always as the major products. The Nꢀ9 isomer derivatives
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4ꢀ17 were obtained with a 9.7:1 ratio, 5.8:1 ratio, 11.5:1 ratio and 6.5:1 ratio, respectively, along
with their Nꢀ7 regioisomers 14aꢀ17a. The chemical structures of the Nꢀ9 and Nꢀ7 isomers were
assigned from spectroscopic data, on the basis of the chemical shift of the protons Hꢀ8 and ꢀCH ꢀNꢀ
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as reported by Lambertucci et al. . The HꢀNMR spectrum of the Nꢀ7 isomers always showed a
downfield shift of the Hꢀ8 and ꢀCH ꢀNꢀ protons with respect to the same protons of the Nꢀ9
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isomers.
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Alkynes 18 and 19 were prepared following the same procedure of azide 16 starting from
commercially available 6ꢀchloropurine (22) and 2ꢀaminoꢀ6ꢀchloropurine (24), respectively, and
propargyl bromide (Scheme SIꢀ1, Supporting information).
The desired Nꢀ9 isomers 18 and 19 were obtained as major products (3.8:1 and 3.5:1 ratio) along
with their Nꢀ7 regioisomers 18a and 19a, respectively. The chemical structures of Nꢀ7 or Nꢀ9
isomers were assigned as reported above.
In Scheme 5 the synthesis of dinucleobases DCI058, DCI061, DCI070, DCI072, DCI095, DCI096,
and DCI133-136 was reported (Figure 1, Scaffold C). The Huisgen’s cycloaddition between the 6ꢀ
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chloroꢀN ꢀ(3ꢀazidopropyl)ꢀpurine (14) and the 6ꢀchloroꢀN ꢀpropargylꢀpurine (18) yielded the
triazole intermediate DCI070. Then, the nucleophilic substitution of DCI070 with a saturated
isopropanolic ammonia solution at 60 °C furnished the triazoleꢀlinked dimer DCI072. The click
reactions of the azides 15-17 with the alkyne 19, resulted in the formation of triazoleꢀlinked dimers
DCI133, DCI058 and DCI095, respectively. These intermediates were treated with a mixture of
TFA/H O to afford the homodimers DCI135, DCI061 and DCI096, respectively. Furthermore,
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DCI058 (linker n = 3) was treated with two different amines, methylamine and benzylamine, to
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Effect of linear c-di-GMP-based compounds on DGC and/or PDE activity
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All the compounds synthesized in the present study (Tables 1, 2, and SIꢀ3) were tested at a final
concentration of 100 µM as possible inhibitors of PleD from Caulobacter crescentus and of RocR
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from P. aeruginosa, as representatives of DGCs and PDEs, respectively
. Following a stepwise
approach for the design of the compounds, we pursued the goal to obtain the simplest cꢀdiꢀGMP
linear analogue(s) effective against PleD and/or RocR.
Scaffold A. First, the four compounds with scaffold A (Table 1, rows 1ꢀ4) were designed to
assess the impact of nonconservative modification at the level of the linkage of the two sugar
moieties; different combinations of purine bases have also been tested. Compound DCI028
significantly affects DGC activity (63% of residual activity of PleD, at 100 µM inhibitor), in
contrast compounds DCI015 and DCI016, where one or both guanines were replaced by adenine(s),
exhibited no inhibition. Compound DCI021 was not assayed due to the interference of its CD
spectrum with that of cꢀdiꢀGMP and coꢀmigration with cꢀdiꢀGMP on the C8ꢀRP column.
The guanine moiety of cꢀdiꢀGMP is indeed crucial for promoting the formation of the
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intercalated dimer targeting the Iꢀsite of DGCs ; more specifically, the carbonyl group, the –NH at
position 1 of guanine and, to a lesser extent, the –NH moiety at position 2, which are absent in the
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adenine base, are crucial both for the formation of the intercalated dimer and for the interaction with
the RxxD motif of the Iꢀsite. The carbonyl and the amino moieties establish Hꢀbonding interactions
with the two conserved arginines and aspartic residues of the Iꢀsite, respectively, while the ꢀNH at
position 1 stabilizes the two intercalated molecules by contributing an additional intramolecular Hꢀ
bond. The driving force of cꢀdiꢀGMP binding to the Iꢀsite of DGCs is the πꢀcation interaction of the
stacked bases with a charged nitrogen atom on the arginine residue, while the cyclic phosphate
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lactone ring is not involved in polar interaction with protein residues .
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Given that the two guanines are essential to form stacking interactions with the protein in the
48
intercalated dimer , it is likely that compound DCI028 targets the Iꢀsite of PleD. This model
would explain why compounds DCI015 and DCI016 failed to inhibit PleD, as their adenine
moieties are not able to form dimers.
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In light of this result, we can speculate that replacement of the phosphodiester moiety with a
triazole ring does not negatively affect the plasticity of this compound, in terms of its capability to
mimic the intercalated dimer of cꢀdiꢀGMP.
On the other hand, lack of significant inhibition of RocR (Table 1) may indicate that the linker
employed in scaffold A compounds disrupts targeting of the catalytic site of the EAL PDEs, where
22
cꢀdiꢀGMP binds as a linear monomer . It should be mentioned that, while the binding mode of cꢀ
diꢀGMP to the Iꢀsite of DGCs favours Hꢀbonding between protein residues and the guanine
48
moieties , binding of cꢀdiꢀGMP to the EAL active site mainly involves the phosphate moieties,
22
besides the stacking interactions of the bases (see also below) .
Scaffold B. Based on these initial findings, a second series of compounds, lacking one of the two
sugar moieties, was synthetized and tested. As reported in Table 1, row 5, removal of one of the two
sugars in the sugarꢀtriazolꢀsugar motif, as in compound DCI091, decreased the inhibitory effect on
PleD observed with compound DCI028. Shortening of the linker length, as in compound DCI059
(Table 1, row 6), was not able to overcome this loss of activity. Interestingly, compound DCI091
appears to enhance the catalytic activity of RocR, though the mechanistic details of this effect have
not been pursued.
Scaffold C. A third series of compounds was then designed and synthetized in order to determine
the minimal structural determinants controlling the DGC/cꢀdiꢀGMP interaction (Table 1, rows 7ꢀ8).
The main goal was to identify the simplest linker between the two purine bases able to target the Iꢀ
site of DGCs and, possibly, to generate selectivity between this site and the catalytic site of PDEs.
The sugar moiety was completely removed and the triazoleꢀbased linker was assayed. Compound
DCI061, bearing two guanines, was found to significantly inhibit both PleD (12,5% of residual
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activity, at 100 µM inhibitor) and RocR (41% of residual activity, at 100 µM inhibitor). According
to our structural hypothesis, compound DCI072, bearing two adenines, would not be a promising
inhibitor of PleD, but could act on RocR, which demonstrates a less stringent requirement for
guanine. In agreement with this prediction, DCI072 inhibits RocR, though to a lower extent than
compound DCI061 (64% of residual activity, at 100 µM inhibitor).
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Binding of cꢀdiꢀGMP to the PDE active site mainly involves interactions with the linker moiety
of cꢀdiꢀGMP (which is then hydrolysed, see below) rather than with the nitrogenous bases;
accordingly, RocR is sensitive to both DCI061 and DCI072 but the overall inhibitory activity is
lower than that exerted by DCI061 with PleD.
The effect of compound DCI061 on both PleD and RocR was analysed in greater detail to
determine the IC50, which was found to be 17.5±1.1 µM and 66.3±1.3 µM, respectively (Figure
1
A).
As mentioned above, we speculated that compound DCI061 specifically targets the Iꢀsite of
PleD. To support this assumption, compound DCI061 was assayed on two other DGCs, from P.
aeruginosa, namely WspR, containing a canonical Iꢀsite, and YfiNHAMPꢀGGDEF insensitive to cꢀdiꢀ
49
GMP feedback inhibition (due to the presence of a degenerate Iꢀsite, ). In agreement with our
hypothesis, WspR activity was strongly reduced in the presence of DCI061 (∼20% of residual
activity, at 100 µM inhibitor), while the activity of YfiNHAMPꢀGGDEF was not affected by a large
excess of DCI061 (Figure 1B), even up to 250 µM (data not shown).
GGDEF
Literature data report the case of XCC4471
, the GGDEF domain of a DGC from
Xanthomonas campestris, which, despite the absence of a canonical Iꢀsite, is competitively inhibited
by cꢀdiꢀGMP. Structural data showed a novel partially intercalated dimeric form of cꢀdiꢀGMP in the
5
0
active site, which accounts for the competitive inhibition observed . It is likely that proteins able
to bind cꢀdiꢀGMP in such a way would also be able to bind compound DCI061, as suggested by
modelling studies (Figure SIꢀ1).
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Based on these results, the chemical scaffold of compound DCI061 was used as a starting point
to design and synthetize optimized inhibitors.
Derivatives of compound DCI061 and selective inhibition of DGCs
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As reported in Table 2, eight compounds were synthesized to improve the selectivity and,
possibly, the activity of compound DCI061. First, a series of four compounds bearing the same
linker of compound DCI061 but harbouring guanine modifications were assayed (Table 2, rows 1ꢀ
4
). Among the substitutions of the carbonyl moiety in the guanine ring assayed (Table 2, rows 1ꢀ3),
the chloride derivative (compound DCI058) is the most promising, given that it retains the
inhibitory property on PleD (IC50 = 25.5±1.2 µM, Figure 2) and, more importantly, does not
significantly inhibit RocR (96% of residual activity, at 100 µM inhibitor). The chlorine atom retains
the electrophilic character at the Cꢀ6 position, a feature which is crucial for targeting the Iꢀsite of
DGCs; on the other hand, the replacement of the carbonyls with chlorine most likely abolishes the
Hꢀbonding of the nitrogenous base with the EAL active site (see below).
Since position 6 is crucial for the interaction of guanine with both DGCs and PDEs, it is not
surprising that compounds DCI134 and DCI136, where the carbonyl moiety has been replaced by
the methylaminoꢀ or the bulky phenylmethylaminoꢀ substituents, respectively, are ineffective
against PleD and exhibit only slight activity against RocR.
It has been previously shown by crystal structure analysis that the 2ꢀamino group of the guanine
moiety is involved in the Hꢀbonding of cꢀdiꢀGMP with highly conserved residues in Iꢀsites of
29, 49
DGCs
, while it is not crucial for the stabilization of substrate binding to the EAL site (see
5
1
below). Despite this, in vitro data on the Slr1143 DGC indicated that cyclic diꢀinosinylic acid,
corresponding to the cꢀdiꢀGMP analogue lacking the 2ꢀamino group, inhibits DGC activity (IC50
8.9 µM), being more effective than cꢀdiꢀGMP itself on the same enzyme (IC50 >> 100 µM).
In light of these opposite observations, the 2ꢀamino group of compound DCI058 was removed,
=
6
leading to compound DCI070 (Table 2, row 4), which is unable to inhibit either PleD or RocR.
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These data indicate that, in agreement with structural evidence, the conservation of position 2 of
guanine is crucial for the class of analogues presented in this work. The mild inhibitory effect of
51
cyclic diꢀinosinylic acid reported in could be related to the peculiarity of the DGC analysed, in
which the feedback inhibition of cꢀdiꢀGMP presents unconventional features (IC50 >> 100 µM vs
0
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PleD IC50 = 5 µM or WspR IC50 = 49 µM, ).
Finally, as compounds DCI061 and DCI058 proved to be the most interesting molecules, they
were further modified to contain longer or shorter linker regions (Table 2, rows 5ꢀ8). However, no
inhibition of either PleD or RocR was observed, thus suggesting that the optimal linker length is
that contained in compounds DCI061 and DCI058. It is worth noting that this linker length most
closely mimics both that of the intercalated dimer of cꢀdiꢀGMP and its monomeric and linear
counterpart (as in the case of the EAL active site).
In Vivo Studies
Preliminary in vivo analyses revealed that, despite the strong repressive effect that compounds
DCI061 and DCI058 exert on DGCs in vitro, they are not effective in decreasing biofilm formation
in P. aeruginosa and E. coli (Supporting information, Figure SIꢀ2). Also the evidence that neither
DCI058 nor DCI061 alter the expression of cꢀdiꢀGMP regulated genes in P. aeruginosa cells argues
against their ability to decrease intracellular levels of cꢀdiꢀGMP in vivo (Supporting information,
Figure SIꢀ3). This lack of efficacy on bacterial cells is not surprising when considering that among
the cꢀdiꢀGMP analogues with DGC inhibitory activity published so far (see supporting
informations), none has been reported to decrease biofilm formation, which is very likely due to a
lack of permeability with these compounds.
It is not ruled out that once we will find optimized compounds able to exert their effect not only
in vitro but also in vivo, they may also target other kinds of cꢀdiꢀGMP receptors, given that cꢀdiꢀ
GMP is flexible enough to bind its partners with different conformations via only minor
15
adjustments .
Molecular Modeling
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Modeling of DCI061 into the Iꢀsite of PleD gave additional clues regarding the possible binding
mode of triazole derivatives. DCI061 is predicted to bind in the same way as the intercalated dimer
of cꢀdiꢀGMP, with two molecules bound at the interface between the DGC and D2 domains of PleD
by a multitude of specific interactions (Figure 3). The latter mimic the common baseꢀarginine
pairing motif involving O6 and N7 of the guanyl groups of cꢀdiꢀGMP and arginine residues
(Arg390, Arg359, and Arg178). The length of the linker connecting the two purine moieties appears
to be optimal to place the aromatic rings at stacking distance and provides a structural rationale to
the finding that longer/shorter linkers generally result in poor inhibition (Table 2, rows 5ꢀ8). The
obtained model is also able to explain why small electrophilic groups are the most favoured at
position 6 of the purine base. Indeed, the partial negative charges of Oꢀ or Clꢀ atoms are placed at a
suitable distance to interact with Arg178 and Arg359, and possibly with the triazole ring in an
anion/πꢀlike interaction. Amines or bulkier groups, as observed in some of the triazole derivatives
tested in this study, would disrupt such favorable interactions. Finally, the importance of an amine
group at position 2 of the ring for PleD inhibition, as assessed in this study, is justified by the
interactions of this moiety with residues Asp362, Gly174 and His177 of PleD.
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cꢀdiꢀGMP is expected to bind to RocR in a manner similar to BlrP1 and TBD1265, since the key
4
7
residues that bind cꢀdiꢀGMP are conserved among the three homologous proteins . When
21
modelled in the EAL active site of RocR using the crystal structure of BlrP1 as a template , cꢀdiꢀ
GMP appears in the correct conformation to bind to the same residues that are conserved in RocR
and BlrP1.
When docked into the active site of RocR, DCI061 adopts an extended conformation, which
again resembles the binding mode of cꢀdiꢀGMP (Figure 4A and 4B, respectively). Baseꢀarginine
and baseꢀtyrosine pairing motifs involving Arg319 and Tyr374 stabilize one of the guanyl groups of
DCI061. The latter group is further stabilized by the electrostatic interaction with Glu355, and by a
hydrogenꢀbond between the mainꢀchain of Tyr374 and the carbonyl moiety of the guanine ring. The
2
,3 Nꢀ atoms of the triazole ring of DCI061 appear at suitable distance to coordinate at least one of
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the two metallic centers of RocR (2.9Å and 3.5Å, respectively), while they seem too distant to
interact with the other centre (3.9Å and 4.8Å, respectively); this may account for the poorer
inhibitory activity of DCI061 against PDEs than against DGCs. The second guanyl moiety of
DCI061 is deeply buried into a cleft of the EAL domain of RocR, as observed in the crystal
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structure of the cꢀdiꢀGMP/BlrP1 complex . Three polar interactions take place in this case: Glu116
and His235 with the O6 of the guanyl group, and a hydrogenꢀbond between a water molecule (403)
and the amino group of the guanyl ring.
CONCLUSIONS
In the present paper a new class of cꢀdiꢀGMPꢀbased compounds able to selectively target DGCs
is reported. The idea was to simplify the complex molecule of cꢀdiꢀGMP and to substitute the
charged and hydrolyzable phosphate linker of cꢀdiꢀGMP with a neutral and stable triazoleꢀbased
linker, in order to obtain a small molecule able to discriminate between the Iꢀsite of DGCs and the
active site of PDEs.
Two compounds are reported to inhibit DGCs targeting the Iꢀsite, namely compound DCI061
and DCI058, the latter being selective for this class of enzyme (See Figure 5 for a scheme
summarizing the proposed mechanism of action). Among cꢀdiꢀGMP analogues characterized so far,
29
their effect on DGCs is comparable to those of 2’ꢀFꢀcꢀdiꢀGMP , but, contrary to the latter, they do
not significantly inhibit PDEs.
The array of compounds designed, synthesized and tested in this study revealed new information
as to the specific role of each of the guanine substituents in the two different conformations
required for binding to the Iꢀsite or to the PDE active site. These compounds also represent precious
tools for future mechanistic characterization of DGCs and PDEs. In particular, compound DCI058
will be useful for studying hybrid proteins containing both GGDEF and EAL motifs, which, in spite
of their involvement in biofilm formation and bacterial pathogenesis, are still poorly characterized
biochemically. Indeed, many of the biophysics and biochemical analyses required to study kinetic
properties, binding affinity and siteꢀdirected mutants of such proteins could take advantage of the
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capability of DCI058 to selectively target GGDEF domains without altering the catalytic activity of
EAL domains.
We demonstrated that the linker region of cꢀdiꢀGMP (and the analogues characterized herein)
does not interact directly with the Iꢀsite, but rather serves as a flexible spacer to allow the correct
stacking of the four guanines (or their chloride derivatives).
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In conclusion, these results lay the foundation for the future development of cell permeable
analogues of these compounds, based on modification of the linker region, which could be used to
hamper cꢀdiꢀGMP signalling in bacteria cells, and hence biofilm formation.
EXPERIMENTAL SECTION
Chemical Synthesis. Materials and Instrumentation
All reagents and solvents were purchased from SigmaꢀAldrich Chemical Co and were analytical
grade. Thin layer chromatography (TLC) was run on silica gel 60 F254 plates; silica gel 60 (70–230
mesh, Merck and 200ꢀ400 mesh, Merck) for column chromatography was used. Preparative thin
layer chromatography was run on silica gel GF (20 x 20 cm, 1000 ꢀ, Analtech). The final
1
13
1
compounds were characterized by HꢀNMR, CꢀNMR, MS and elemental analyses. H NMR
spectra were determined at 400 MHz with a Varian Mercury AS400 instrument. The chemical shift
values are expressed in
δ
values (ppm), and coupling constants (J) are in Hertz; TMS was used as
O.
an internal standard. The presence of all exchangeable protons was confirmed by addition of D
2
The purity of final compounds was checked using an Agilent 1100 Series instrument equipped with
Synergi PolarꢀRP 80A 4 µm analytical column (150 mm x 4.6 mm, Phenomenex, Torrance, CA).
Mobile phase consisted of a mixture of water/methanol (50:50) at a flow rate of 0.6 mL/min. Peaks
were detected by UV adsorption with a diode array detector (DAD) at 230, 254, and 280 nm. All
derivatives tested for biological activity showed ≥ 96% purity by HPLC analysis (detection at 254
nm). Mass spectra were recorded on an HP 1100 series instrument.
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6
All measurements were performed in the positive ion mode using atmospheric pressure electrospray
ionization (APIꢀESI). Elemental analyses (C, H, and N) were determined on ThermoFisher
Scientific FLASH 2000 CHNS analyzer and are within 0.4% of theoretical values.
41
5
’-Azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine (3). 2’,3’ꢀOꢀisopropylideneꢀguanosine (1)
0
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0
(1 mmol) was suspended in dry 1,4ꢀdioxane (4 mL) under nitrogen atmosphere. DPPA (2 mmol)
and DBU (3 mmol) were added dropwise and the mixture was stirred at room temperature for 4 h.
Sodium azide (5 mmol), tetrabutylammonium iodide (0.1 mmol) and 15ꢀcrownꢀ5 (0.1 mmol) were
added and the reaction mixture was refluxed for 5 h. The reaction was evaporated under reduced
pressure and the residue was purified by column chromatography on silica gel (0–2% MeOH in
1
CHCl
3
) to give compound 3 (65% yield) as a white solid. H NMR (400 MHz, DMSOꢀd
6
): δ 1.30
(
s, 3H, CH ), 1.50 (s, 3H, CH ), 3.49 (dd, J = 4.8, 13.0 Hz, 1H, Hꢀ5′a), 3.58 (dd, J = 7.6 Hz, 13.0
3 3
Hz, 1H, Hꢀ5′b), 4.24ꢀ4.17 (m, 1H, Hꢀ4’), 5.03 (dd, J = 3.3, 6.2 Hz, 1H, Hꢀ3′), 5.29 (dd, J = 2.2, 6.3
Hz, 1H, Hꢀ2′), 5.99 (d, J = 2.2 Hz, 1H, Hꢀ1′), 6.54 (brs, 2H, NH ), 7.88 (s, 1H, Hꢀ8), 10.70 (s, 1H,
[M+ H] 349.13; found 349.12.
2-N-Isobutyryl-5’-azido-5’-deoxy-2’,3’-O-isopropylidene-guanosine (4). 5’ꢀAzidoꢀ5’ꢀdeoxyꢀ
’,3’ꢀOꢀisopropylideneꢀguanosine 3 (1 mmol) was suspended in pyridine (1 mL) and the suspension
2
+
16 8 4
NH). MS (APIꢀESI): m/z calcd for C13H N O
2
was evaporated. Pyridine (3 mL) was added and the mixture was cooled at 0 °C. Trimethylsilyl
chloride (4.9 mmol) and isobutyryl chloride (5 mmol) were added dropwise and the mixture was
stirred at room temperature for 3 h. The mixture was cooled to 0 °C, water (1 mL) and ammonium
hydroxide solution (1.5 mL) were added and the solution was allowed to warm to room temperature
for 1 h. The pyridine was evaporated, water was added (3 mL) to residue, then the water phase was
extracted 3 times with chloroform. The combined organic phases were dried over Na SO and
2
4
concentrated. The crude was purified by column chromatography on silica gel (0–2% MeOH in
1
CHCl
3
) to give compound 4 (65% yield) as a white solid. H NMR (400 MHz, DMSOꢀd
6
): δ 1.11
(s, 3H, CH
3
ꢀisobutyryl), 1.13 (s, 3H, CH
3
ꢀisobutyryl), 1.25 (s, 3H, CH
3
), 1.50 (s, 3H, CH ), 2.70ꢀ
3
2
.80 (m, 1H, CHꢀisobutyryl), 3.56ꢀ3.52 (m, 2H, Hꢀ5’), 4.25ꢀ4.20 (m, 1H, Hꢀ4’), 5.15 (dd, J = 3.4,
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6.4 Hz, 1H, Hꢀ3′), 5.35 (dd, J = 2.5, 6.2 Hz, 1H, Hꢀ2′), 6.02 (d, J = 2.2 Hz, 1H, Hꢀ1′), 8.20 (s, 1H,
Hꢀ8), 11.50 (s, 1H, NHꢀisobutyryl), 12.10 (s, 1H, NH). MS (APIꢀESI): m/z calcd for C H N O
5
17
22
8
+
[
M+ H] 419.17; found 419.20.
2
-N-isobutyryl-2’,3’-O-isopropylidene-5’-O-propargyl-guanosine (8). 2ꢀNꢀisobutyrylꢀ2’,3’ꢀOꢀ
10
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4
3
isopropylideneꢀguanosine (6) (1 mmol) was dissolved in dry THF (10 mL), cooled to 0 °C before
the addition of NaH (1.79 mmol, 60% dispersion in mineral oil). The mixture was stirred for 30
minutes and propargyl bromide (1.79 mmol) was added dropwise. After 26 h of stirring at room
temperature, the reaction was evaporated to dryness and the crude was purified by column
chromatography on silica gel (0–2% MeOH in CHCl
3
) to give 8 (31% yield) as a white solid and
): δ 1.11 (s, 3H, CH ꢀisobutyryl),
1
the side product 8a (17% yield). H NMR (400 MHz, DMSOꢀd
6
3
1
.13 (s, 3H, CH ꢀisobutyryl), 1.31 (s, 3H, CH ), 1.51 (s, 3H, CH ), 2.73ꢀ2.82 (m, 1H, CHꢀ
3 3 3
isobutyryl), 3.41 (t, J = 2.4 Hz, 1H, CHꢀpropargyl), 2.50ꢀ2.65 (m, 2H, Hꢀ5’), 4.13 (d, J = 2.4 Hz,
2
5
2
H, OCH ꢀpropargyl), 4.27 (dd, J = 5.0, 8.3 Hz, 1H, Hꢀ4’), 5.07 (dd, J = 3.2, 6.2 Hz, 1H, Hꢀ3′),
.27 (dd, J = 2.4, 6.2 Hz, 1H, Hꢀ2′), 6.05 (d, J = 2.3 Hz, 1H, Hꢀ1′), 8.15 (s, 1H, Hꢀ8), 11.53 (brs,
+
1H, NHꢀisobutyryl), 12.10 (brs, 1H, NH). MS (APIꢀESI): m/z calcd for C20
H N
25 5
O
6
[M+ H]
4
32.18; found 432.21.
1
1
2
-N-isobutyryl-N -propargyl-2’,3’-O-isopropylidene-5’-O-propargyl-guanosine (8a). H NMR
(400 MHz, DMSOꢀd
6
): δ 1.03 (s, 3H, CH
3
ꢀisobutyryl), 1.04 (s, 3H, CH
3
ꢀisobutyryl), 1.29 (s, 3H,
1
CH
3
), 1.51 (s, 3H, CH
3
), 2.48 (brs, 1H, CHꢀisobutyryl), 3.24 (brs, 1H, CHꢀN ꢀpropargyl), 3.38ꢀ3.42
(
2
m, 1H, CHꢀpropargyl), 3.50ꢀ3.64 (m, 2H, Hꢀ5’), 4.10 (d, J = 2.3 Hz, 2H, OCH ꢀ propargyl), 3.26ꢀ
4
5
.32 (m, 1H, Hꢀ4’), 4.54ꢀ4.58 (m, 2H, NCH ꢀ propargyl), 4.94 (dd, J = 2.9, 6.4 Hz, 1H, Hꢀ3′), 5.30ꢀ
2
.35 (m, 1H, Hꢀ2′), 6.11 (d, J = 2.2 Hz, 1H, Hꢀ1′), 8.25 (s, 1H, Hꢀ8), 12.84 (brs, 1H, NHꢀ
+
isobutyryl). MS (APIꢀESI): m/z calcd for C23
H
27
N
5
O
6
[M+ H] 470.19; found 470.18.
6
6
N -Benzoyl-2’,3’-O-isopropylidene-5’-O-propargyl-adenosine
(9).
N ꢀBenzoylꢀ2’,3’ꢀOꢀ
3
4
isopropylideneꢀadenosine (7) (1 mmol) was dissolved in dry DMF (5 mL), cooled to 0 °C before
the addition of NaH (5 mmol, 60% dispersion in mineral oil). The mixture was stirred for 30
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
minutes and propargyl bromide (1.2 mmol) was added dropwise. After 1.5 h of stirring at 0 °C, the
reaction was quenched with ethyl acetate (1 mL) and concentrated. The residue was treated with a
mixture of CH
2
Cl
2
/H
2
O; the organic phase was separated and the aqueous phase was extracted with
SO and concentrated. The
CH Cl (3 times). The combined organic phases were dried over Na
2
2
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
crude was purified by column chromatography on silica gel (0–2% MeOH in CH
2
Cl
2
) to give
), 1.55
1
compound 9 (85% yield) as a pale solid. H NMR (400 MHz, DMSOꢀd
6
): δ 1.34 (s, 3H, CH
3
(
s, 3H, CH
propargyl), 4.28ꢀ4.44 (m, 1H, Hꢀ4’), 5.01 (dd, J = 2.7, 6.0 Hz, 1H, Hꢀ3′), 5.47 (dd, J = 2.4, 6.0 Hz,
H, Hꢀ2’), 6.29 (d, J = 2.3 Hz, 1H, Hꢀ1’), 7.54 (t, J = 7.5 Hz, 2H, benzoyl), 7.64 (t, J = 7.4 Hz, 1H,
3 2
), 3.42 (s, 1H, CHꢀpropargyl), 3.50ꢀ3.70 (m, 2H, Hꢀ5’), 4.03ꢀ4.18 (m, 2H, OCH ꢀ
1
benzoyl), 8.03 (d, J = 2.3 Hz, 2H, benzoyl), 8.60 (s, 1H, Hꢀ2), 8.77 (s, 1H, Hꢀ8), 11.22 (s, 1H, NHꢀ
+
benzoyl). MS (APIꢀESI): m/z calcd for C H N O [M+ H] 450.17; found 450.19.
23
23
5
5
General procedure for the synthesis of 1,2,3-triazole derivatives via CuACC.
4 2
To a mixture of alkyne (1 mmol) in 10 mL of H O/tBuOH (3:2) was added CuSO ·5H O (0.2
2
52
mmol), sodium ascorbate (0.04 mmol), and then azide (1.3 mmol) . The heterogeneous reaction
mixture was stirred at room temperature for 2ꢀ3 days and monitored by TLC and MS. After the
completion, the mixture was evaporated to dryness and the intermediates were used directly for
following reactions or purified by column chromatography on silica gel.
5’-Deoxy-(4-(2-N-isobutyryl-2’,3’-O-isopropylidene-guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-
yl)-2-N-isobutyryl-2’,3’-O-isopropylidene guanosine (10). The reaction was carried out according
to the general procedure for the synthesis of 1,2,3ꢀtriazole derivatives, using 2ꢀNꢀisobutyrylꢀ2’,3’ꢀ
Oꢀisopropylideneꢀ5’ꢀOꢀpropargylꢀguanosine (8) and 2ꢀNꢀisobutyrylꢀ5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀ
isopropylideneꢀguanosine (4) as starting materials. The reaction mixture was stirred at room
temperature for 2 days and evaporated to dryness. The crude was purified by column
chromatography on silica gel (CHCl
3
/MeOH 98:2) to give compound 10 (85% yield) as a white
): δ 1.09ꢀ1.14 (m, 12H, CH ꢀisobutyryl), 1.29 (s, 3H, CH ),
1
solid. H NMR (400 MHz, DMSOꢀd
6
3
3
1
.31 (s, 3H, CH ), 1.49 (s, 3H, CH ), 1.50 (s, 3H, CH ), 2.71ꢀ2.80 (m, 2H, CHꢀisobutyryl), 3.47ꢀ
3 3 3
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7
8
9
3.58 (m, 2H, Hꢀ5’), 4.18ꢀ4.26 (m, 1H, Hꢀ4’), 4.42ꢀ4.50 (m, 3H, OCH ꢀtriazolyl, Hꢀ4’), 4.55ꢀ4.70
2
(
m, 2H, Hꢀ5’), 5.01 (dd, J = 3.2, 6.2 Hz, 1H, Hꢀ3′), 5.24 (dd, J = 2.3, 6.2 Hz, 1H, Hꢀ3′), 5.27ꢀ5.34
m, 2H, Hꢀ2′), 6.02 (d, J = 2.3 Hz, 1H, Hꢀ1’), 6.13 (d, J = 1.4 Hz, 1H, Hꢀ1’), 7.90 (s, 1H, =CHꢀN),
(
8
.08 (s, 1H, Hꢀ8), 8.09 (s, 1H, Hꢀ8), 11.43 (brs, 1H, NHꢀisobutyryl), 11.51 (brs, 1H, NHꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
isobutyryl), 12.10 (brs, 2H, NH). MS (APIꢀESI): m/z calcd for C37
47
H N
13
O
11 [M+ H] 850.35;
found 850.33.
6
5
2
’-Deoxy-(4-(N -benzoyl-2’,3’-O-isopropylidene-adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-
’,3’-O-isopropylidene-guanosine (11). The reaction was carried out according to the general
6
procedure for the synthesis of 1,2,3ꢀtriazole derivatives, using N ꢀbenzoylꢀ2’,3’ꢀOꢀisopropylideneꢀ
5’ꢀOꢀpropargyl adenosine (9) and 5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀguanosine (3) as
starting materials. The reaction mixture was stirred at room temperature for 3 days and the
suspension was filtered. The solid was washed with ethanol, dried and used directly for following
reaction.
5
’-Deoxy-(4-(2-N-isobutyryl-2’,3’-O-isopropylidene-guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-
yl)-2’,3’-O-isopropylidene-adenosine (12). The reaction was carried out according to the general
procedure for the synthesis of 1,2,3ꢀtriazole derivatives, using 2ꢀNꢀisobutyrylꢀ2’,3’ꢀOꢀ
isopropylideneꢀ5’ꢀOꢀpropargylꢀguanosine (8) and 5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀ
adenosine (5) as starting materials. The reaction mixture was stirred at room temperature for 3 days
and evaporated to dryness. The crude was purified by column chromatography on silica gel
1
(
CHCl
DMSOꢀd
.29 (s, 6H, CH
3.58 (m, 2H, Hꢀ5’), 4.34ꢀ4.39 (m, 1H, Hꢀ4’), 4.44 (s, 2H, OCH
3
/MeOH 95:5) to give compound 12 (56% yield) as a white solid. H NMR (400 MHz,
6
): δ 1.10 (d, J = 1.5 Hz, 3H, CH
3
ꢀisobutyryl), 1.11 (d, J = 1.5 Hz, 3H, CH
), 2.72ꢀ2.81 (m, 1H, CHꢀisobutyryl), 3.47ꢀ
ꢀtriazolyl), 4.47ꢀ4.52 (m, 1H, Hꢀ4’),
.59ꢀ4.76 (m, 2H, Hꢀ5’), 4.96 (dd, J = 3.1, 6.1 Hz, 1H, Hꢀ3′), 5.11 (dd, J = 3.2, 6.3 Hz, 1H, Hꢀ3′),
.23 (dd, J = 2.6, 6.2 Hz, 1H, Hꢀ2′), 5.43 (dd, J = 2.3, 6.2 Hz, 1H, Hꢀ2’), 6.02 (d, J = 2.4 Hz, 1H,
3
ꢀisobutyryl),
1
3
), 1.49 (s, 3H, CH ), 1.50 (s, 3H, CH
3
3
2
4
5
Hꢀ1’), 6.20 (d, J = 2.3 Hz, 1H, Hꢀ1’), 7.35 (brs, 2H, NH ), 7.92 (s, 1H, =CHꢀN), 8.11 (s, 1H, Hꢀ2),
2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8.17 (s, 1H, Hꢀ8), 8.26 (s, 1H, Hꢀ8), 11.52 (brs, 1H, NHꢀisobutyryl), 12.10 (brs, 1H, NH). MS
+
(
APIꢀESI): m/z calcd for C H N O [M+ H] 764.31; found 764.35.
33
41 13
9
6
5
2
’-Deoxy-(4-(N -benzoyl-2’,3’-O-isopropylidene-adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-
’,3’-O-isopropylidene-adenosine (13). The reaction was carried out according to the general
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
procedure for the synthesis of 1,2,3ꢀtriazole derivatives, using N ꢀbenzoylꢀ2’,3’ꢀOꢀisopropylideneꢀ
5’ꢀOꢀpropargylꢀadenosine (9) and 5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀadenosine (5) as
starting materials. The reaction mixture was stirred at room temperature for 3 days and evaporated
3
to dryness. The crude was purified by column chromatography on a silica gel (CHCl /MeOH 95:5)
1
to give compound 13 (40% yield) as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 1.27 (s, 3H,
6
CH
3
3 3 3
), 1.31 (s, 3H, CH ), 1.48 (s, 3H, CH ), 1.53 (s, 3H, CH ), 3.50ꢀ3.64 (m, 2H, Hꢀ5’), 4.34ꢀ4.39
(
(
(
m, 1H, Hꢀ4’), 4.44 (s, 2H, OCH ꢀtriazolyl), 4.47ꢀ4.52 (m, 1H, Hꢀ4’), 4.59ꢀ4.76 (m, 2H, Hꢀ5’), 4.94
2
dd, J = 2.6, 6.1 Hz, 1H, Hꢀ3′), 5.09 (dd, J = 3.4, 6.2 Hz, 1H, Hꢀ3′), 5.38ꢀ5.43 (m, 2H, Hꢀ2’), 6.19
d, J = 2.2 Hz, 1H, Hꢀ1’), 6.24 (d, J = 2.4 Hz, 1H, Hꢀ1’), 7.35 (brs, 2H, NH ), 7.53 (t, J = 7.6 Hz,
2
2H, benzoyl), 7.63 (t, J = 7.4 Hz, 1H, benzoyl), 7.93 (s, 1H, =CHꢀN), 8.03 (d, J = 7.4 Hz, 2H,
benzoyl), 8.16 (s, 1H, Hꢀ2), 8.24 (s, 1H, Hꢀ2), 8.55 (s, 1H, Hꢀ8), 8.72 (s, 1H, Hꢀ8), 11.21 (brs, 1H,
+
NH). MS (APIꢀESI): m/z calcd for C H N O [M+ H] 782.30; found 782.31.
36
39 13
8
5
’-Deoxy-(4-(guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-guanosine (DCI028). Compound 10
1 mmol) was treated with 10 mL of formic acid aqueous solution (70%) and the reaction was
stirred at 40 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The
residue was purified by column chromatography on silica gel (CHCl /MeOH/NH 8.2:1.6:0.2) to
(
3
3
give the intermediate NꢀiBuꢀprotected as a white solid (46% yield). The intermediate NꢀiBuꢀ
protected (1 mmol) was then treated with an ammonium hydroxide solution 33% (20 mL) and the
reaction was stirred at 50 °C for 20 h, followed by evaporation and coevaporation with MeOH (3
times). The solid was treated with ethanol and diethyl ether (2 times), filtered and purified by
3 2
preparative thinꢀlayer chromatography (iPrOH/NH /H O 8:1.5:0.5) to give compound DCI028
1
(
20% yield) as a white solid. H NMR (400 MHz, DMSOꢀd
6
): δ 3.50ꢀ3.70 (m, 2H, Hꢀ5’), 3.86ꢀ3.98
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8
9
(m, 1H, Hꢀ4’), 3.99ꢀ4.06 (m, 1H, Hꢀ4’), 4.09ꢀ4.26 (m, 2H, Hꢀ5’), 4.37ꢀ4.42 (m, 1H, Hꢀ3’), 4.43ꢀ
4
5
1
7
.49 (m, 1H, Hꢀ3’), 4.52 (s, 2H, OCH ꢀtriazolyl), 4.65ꢀ4.67 (m, 1H, Hꢀ2′), 4.69ꢀ4.72 (m, 1H, Hꢀ2′),
2
.19 (brs, 1H, OH), 5.42 (brs, 2H, OH), 5.55 (d, J = 5.8 Hz, 1H, OH), 5.66 (d, J = 5.9 Hz, 1H, Hꢀ
’), 5.68 (d, J = 6.1 Hz, 1H, Hꢀ1’), 6.47 (brs, 2H, NH
2 2
ꢀguanosine), 6.48 (brs, 2H, NH ꢀguanosine),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
.78 (s, 1H, Hꢀ8), 7.83 (s, 1H, Hꢀ8), 7.97 (s, 1H, =CHꢀN), 10.44 (brs, 2H, NH). C NMR (101
MHz, DMSOꢀd ): δ 51.83, 64.15, 70.51, 70.98, 71.42, 72.86, 73.67, 82.75, 83.41, 86.78, 87.36,
6
1
17.02, 117.33, 125.03, 135.71, 136.30, 144.03, 151.57 (x2), 154.06 (x2), 157.12 (x2). MS (APIꢀ
+
ESI): m/z calcd for C23
H
27
N
13
O
9
[M+ H] 630.20; found 630.20. Elem. Anal: C, 43.88; H, 4.32; N,
2
8.92; Found: C, 43.90; H, 4.33; N, 28.90.
5’-Deoxy-(4-(adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-guanosine (DCI016). Compound 11
1 mmol) was treated with a saturated methanolic ammonia solution (50 mL) and the reaction was
stirred at room temperature overnight. After full conversion the solvent was removed under reduced
pressure and the residue was purified by preparative thinꢀlayer chromatography (CHCl /MeOH 8:2)
(
3
to give intermediate 2’,3’ꢀOꢀisopropylideneꢀprotected as a white solid (48% yield). The
intermediate 2’,3’ꢀOꢀisopropylideneꢀprotected (1 mmol) was then treated with 10 mL of formic acid
aqueous solution (70%) and the reaction was stirred at 40 °C for 2 days. After full conversion the
solvent was removed under reduced pressure followed by coevaporation with MeOH (3 times). The
3 2
residue was purified by preparative thinꢀlayer chromatography (iPrOH/NH /H O 8:1:1) to give
1
compound DCI016 (25% yield) as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 3.58ꢀ3.71 (m,
6
2
H, Hꢀ5’), 3.99ꢀ4.03 (m, 1H, Hꢀ4’), 4.10ꢀ4.21 (m, 3H, Hꢀ4’, Hꢀ5’), 4.44ꢀ4.56 (m, 4H, Hꢀ3′, Hꢀ3′,
OCH ꢀtriazolyl), 4.69ꢀ4.72 (m, 2H, Hꢀ2’, Hꢀ2’), 5.38 (s, 1H, OH), 4.45ꢀ4.49 (m, 2H, OH), 5.59 (s,
2
1
H, OH), 5.69 (d, J = 5.6 Hz, 1H, Hꢀ1’), 5.87 (d, J = 5.6 Hz, 1H, Hꢀ1’), 6.53 (brs, 2H, NH
2
ꢀ
guanosine), 7.26 (brs, 2H, NH ꢀadenosine), 7.79 (s, 1H, =CHꢀN), 7.97 (s, 1H, Hꢀ2), 8.12 (s, 1H, Hꢀ
2
1
3
8
7
1
6
), 8.29 (s, 1H, Hꢀ8), 10.47 (brs, 1H, NH). C NMR (101 MHz, DMSOꢀd ): δ 51.96, 66.25, 69.30,
0.67, 72.89, 72.95, 74.50, 84.23, 87.30, 86.78, 88.65, 117.10, 117.34, 125.10, 136.20, 136.45,
44.07, 151.40, 152.25, 154.20, 155.63, 157.12, 157.30. MS (APIꢀESI): m/z calcd for C H N O
8
2
3
27 13
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
[M+ H] 614.20; found 614.40. Elem. Anal: C, 45.03; H, 4.44; N, 28.68; found: C, 45.02; H, 4.42;
N, 28.65.
5
’-Deoxy-(4-(guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-adenosine (DCI021). Compound 12
(
1 mmol) was treated with a saturated methanolic ammonia solution (50 mL) and the reaction was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
stirred at room temperature overnight. After full conversion the solvent was removed under reduced
pressure and the crude was treated with 10 mL of formic acid aqueous solution (70%). The reaction
was stirred at 40 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times).
3 2
The residue was purified by preparative thinꢀlayer chromatography (iPrOH/NH /H O 8:1:1) to give
1
the compound DCI021 (25% yield) as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 3.53ꢀ3.66
6
(m, 2H, Hꢀ5’), 3.90ꢀ3.97 (m, 1H, Hꢀ4’), 3.97ꢀ4.04 (m, 1H, Hꢀ4’), 4.19ꢀ4.31 (m, 2H, Hꢀ5’), 4.36ꢀ
4
.53 (m, 4H, Hꢀ3′, Hꢀ3′, OCH ꢀtriazolyl), 4.62ꢀ4.69 (m, 1H, Hꢀ2’), 4.70ꢀ4.78 (m, 1H, Hꢀ2’), 5.17ꢀ
2
5
.59 (m, 4H, OH), 5.66 (d, J = 5.8 Hz, 1H, Hꢀ1’), 5.89 (d, J = 5.2 Hz, 1H, Hꢀ1’), 6.57 (brs, 2H,
NH
2 2
ꢀguanosine), 7.29 (brs, 2H, NH ꢀadenosine), 7.86 (s, 1H, =CHꢀN), 7.96 (s, 1H, Hꢀ2), 8.13 (s,
13
1H, Hꢀ8), 8.25 (s, 1H, Hꢀ8), 10.50 (brs, 1H, NH). C NMR (101 MHz, DMSOꢀd
6
): δ 51.83, 64.48,
7
1
0.36, 71.03, 72.56, 72.83, 74.52, 83.66, 85.28, 86.66, 87.43, 117.15, 117.33, 125.10, 135.88,
36.58, 144.07, 151.48, 153.73, 154.29, 155.63, 157.43, 157.55. MS (APIꢀESI): m/z calcd for
+
C
23
H
27
N
13
O
8
[M+ H] 614.21; found 614.30. Elem. Anal: C, 45.03; H, 4.44; N, 29.68; found: C,
4
5.04; H, 4.45; N, 29.65.
5’-Deoxy-(4-(adenosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-adenosine (DCI015). Compound 13
1 mmol) was treated with a saturated methanolic ammonia solution (50 mL) and the reaction was
(
stirred at room temperature overnight. After full conversion the solvent was removed under reduced
pressure and the crude was treated with 10 mL of formic acid aqueous solution (70%). The reaction
was stirred at 40 °C for 2 days, followed by evaporation and coevaporation with MeOH (3 times).
2
The residue was purified by preparative thinꢀlayer chromatography (EtOAc/AcOH/EtOH/H O
1
6
:2:2:1) to give the compound DCI015 (24% yield) as a white solid. H NMR (400 MHz, DMSOꢀ
d
6
): δ 3.57ꢀ3.70 (m, 2H, Hꢀ5’), 3.97ꢀ4.02 (m, 1H, Hꢀ4’), 4.08ꢀ4.15 (m, 1H, Hꢀ4’), 4.20ꢀ4.29 (m,
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2H, Hꢀ5’), 4.47ꢀ4.54 (m, 4H, Hꢀ3′, Hꢀ3′, OCH ꢀtriazolyl), 4.61ꢀ4.67 (m, 1H, Hꢀ2’), 4.70ꢀ4.77 (m,
2
1
H, Hꢀ2’), 5.23ꢀ5.30 (m, 1H, OH), 5.49ꢀ4.54 (m, 1H, OH), 5.54ꢀ4.58 (m, 1H, OH), 5.62ꢀ4.68 (m,
2
H, OH), 5.86ꢀ5.91 (m, 2H, Hꢀ1’, Hꢀ1’), 7.26 (brs, 2H, NH ꢀadenosine), 7.30 (brs, 2H, NH ꢀ
1
2
adenosine), 7.96 (s, 1H, =CHꢀN), 8.12 (s, 1H, Hꢀ2), 8.13 (s, 1H, Hꢀ2), 8.24 (s, 1H, Hꢀ8), 8.28 (s,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
1
6
H, Hꢀ8). C NMR (101 MHz, DMSOꢀd ): δ 52.05, 64.58, 70.58, 71.05, 71.39, 72.82, 73.50,
82.37, 83.10, 86.72, 87.26, 117.04, 117.39, 125.08, 135.88, 136.23, 144.01, 151.49 (x2), 153.84
+
(
x2), 157.56 (x2). MS (APIꢀESI): m/z calcd for C H N O [M+ H] 598.21; found 598.22. Elem.
23
27 13
7
Anal: C, 46.23; H, 4.55; N, 30.47; found: C, 46.22; H, 4.57; N, 30.45.
9
2
-Amino-6-chloro-N -(3-(4-(2-N-isobutyryl-2’,3’-O-isopropylidene-guanosin-5’-yl)-methyl-
1H-1,2,3-triazol-1-yl)propyl)-purine (20). The reaction was carried out according to the general
9
procedure for the synthesis of 1,2,3ꢀtriazole derivatives, using 2ꢀaminoꢀ6ꢀchloroꢀN ꢀ(3ꢀ
azidopropyl)ꢀpurine (16) and 2ꢀNꢀisobutyrylꢀ5’ꢀOꢀpropargylꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀ
guanosine (8) as starting materials. The reaction mixture was stirred at room temperature for 3 days
and evaporated to dryness. The crude was purified by column chromatography on a silica gel
1
(CHCl
3
/MeOH 93:7) to give 20 (53% yield) as a white solid. H NMR (400 MHz, DMSOꢀd
6
): δ
1
2
.10 (s, 3H, CH ꢀisobutyryl), 1.11 (s, 3H, CH ꢀisobutyryl), 1.30 (s, 3H, CH ), 1.49 (s, 3H, CH ),
3 3 3 3
.30ꢀ2.39 (m, 2H, CH
2
ꢀCH
ꢀNꢀpurine), 4.23ꢀ4.29 (m, 1H, Hꢀ4’), 4.36 (t, J = 7.0 Hz, 2H, CH
triazolyl), 4.50 (s, 2H, OCH ꢀCH ), 5.01ꢀ5.04 (m, 1H, Hꢀ3′), 5.23ꢀ5.26 (m, 1H, Hꢀ2′), 6.02 (d, J =
.2 Hz, 1H, Hꢀ1′), 6.89 (s, 2H, NH
2
ꢀCH
2
), 2.72ꢀ2.80 (m, 1H, CHꢀisobutyryl), 3.51ꢀ3.62 (m, 2H, Hꢀ5’), 4.05
(
t, J = 6.8 Hz, 2H, ꢀCH
2
2
ꢀNꢀ
2
2
2
2
), 8.05 (s, 1H, =CHꢀN), 8.10 (s, 2H, Hꢀ8, Hꢀ8), 11.53 (brs, 1H,
+
NHꢀisobutyryl), 12.10 (brs, 1H, NH). MS (APIꢀESI): m/z calcd for C H ClN O [M+ H] 684.22;
2
8
34
13
6
found 684.20.
9
N -(3-(4-(guanosin-5’-yl)-methyl-1H-1,2,3-triazol-1-yl)-propyl)-guanine (DCI091). Compound
2
2
0 (1 mmol) was treated with a mixture of TFA/H O (3:1, 10 mL) and the reaction was stirred at
room temperature for 24 hours, followed by evaporation and coevaporation with MeOH (3 times).
The residue was treated with an ammonium hydroxide solution 33% (20 mL) and the reaction was
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
stirred at 60 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The
solid was treated with methanol and diethyl ether (2 times), filtered and purified by preparative
3 2
thinꢀlayer chromatography (iPrOH/NH /H O 8:1.5:0.5) to give compound DCI091 (30% yield) as
1
a white solid. H NMR (400 MHz, DMSOꢀd ): δ 2.24ꢀ2.34 (m, 2H, CH ꢀCH ꢀCH ), 3.58 (dd, J =
6
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4.8, 10.6 Hz, 1H, Hꢀ5’a), 3.66 (dd, J = 3.8, 10.7 Hz, 1H, Hꢀ5’b), 3.94 (t, J = 6.6 Hz, 2H, ꢀCH
2
ꢀNꢀ
purine), 4.00ꢀ4.06 (m, 1H, Hꢀ4’), 4.29ꢀ4.42 (m, 4H, CH ꢀNꢀtriazolyl, Hꢀ2′, Hꢀ3′), 4.54 (s, 2H,
2
OCH
Hꢀ1’), 6.42 (brs, 2H, NH
H, Hꢀ8), 8.11 (s, 1H, Hꢀ8), 10.56 (brs, 2H, NH). C NMR (101 MHz, DMSOꢀd
2
ꢀCH
2
), 5.19 (d, J = 4.9 Hz, 1H, OH), 5.42 (d, J = 6.0 Hz, 1H, OH), 5.66 (d, J = 5.8 Hz, 1H,
ꢀguanosine), 6.46 (brs, 2H, NH ꢀguanine), 7.66 (s, 1H, =CHꢀN), 7.81 (s,
): δ 30.60, 47.51,
2
2
13
1
6
64.50, 70.74, 71.33, 74.05, 83.70, 87.00, 117.26, 117.32, 124.77, 135.03, 136.04, 138.02, 144.45,
+
1
5
5
51.85, 152.10, 154.22, 154.37, 157.42, 157.50. MS (APIꢀESI): m/z calcd for C H N O [M+ H]
21 25 13 6
56.20; found 556.22. Elem. Anal: C, 45.40; H, 4.54; N, 32.78; found: C, 45.42; H, 4.55; N, 32.79.
’-Deoxy-(4-(2-amino-6-chloro-9H-purin)-methyl-1H-1,2,3-triazol-1-yl)-2-N-isobutyryl-2’,3’-
O-isopropylidene-guanosine (21). The reaction was carried out according to the general procedure
9
45
for the synthesis of 1,2,3ꢀtriazole derivatives, using 2ꢀaminoꢀ6ꢀchloroꢀN ꢀpropargylꢀpurine (19)
and 2ꢀNꢀisobutyrylꢀ5’ꢀazidoꢀ5’ꢀdeoxyꢀ2’,3’ꢀOꢀisopropylideneꢀguanosine (4) as starting materials.
The reaction mixture was stirred at room temperature for 2 days and evaporated to dryness. The
crude was purified by column chromatography on a silica gel (CHCl
3
/MeOH 9:1) to give
1
compound 21 (71% yield) as a white solid. H NMR (400 MHz, DMSOꢀd
6
): δ 1.12 (dd, J = 3.6, 6.8
Hz, 6H, CH ꢀisobutyryl), 1.31 (s, 3H, CH ), 1.50 (s, 3H, CH ), 2.69ꢀ2.78 (m, 1H, CHꢀisobutyryl),
3
3
3
4
.44ꢀ4.60 (m, 2H, Hꢀ5’), 4.67 (dd, J = 4.6, 14.3 Hz, 1H, Hꢀ4’), 5.26ꢀ5.33 (m, 4H, NCH ꢀtriazolyl,
2
Hꢀ3′, Hꢀ2′), 6.12 (s, 1H, Hꢀ1’), 6.93 (s, 2H, NH
2
), 7.95 (s, 1H, =CHꢀN), 8.11 (s, 1H, Hꢀ8), 8.13 (s,
1
H, Hꢀ8), 11.44 (brs, 1H, NHꢀisobutyryl), 12.10 (brs, 1H, NH). MS (APIꢀESI): m/z calcd for
+
C
25 5
H28ClN13O [M+ H] 626.20; found 626.22.
5
’-Deoxy-(4-(9H-guanin)-methyl-1H-1,2,3-triazol-1-yl)-guanosine (DCI059). Compound 21 (1
mmol) was treated with a mixture of TFA/H O (3:1, 10 mL) and the reaction was stirred at room
2
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temperature for 20 hours, followed by evaporation and coevaporation with MeOH (3 times). The
residue was treated with an ammonium hydroxide solution 33% (20 mL) and the reaction was
stirred at 60 °C for 20 h, followed by evaporation and coevaporation with MeOH (3 times). The
solid was treated with methanol and diethyl ether (2 times), filtered and purified by preparative
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
thinꢀlayer chromatography (iPrOH/NH
3
/H
2
O 8:1.5:0.5) to give the compound 5 (30% yield) as a
): δ 4.03ꢀ4.22 (m, 2H, Hꢀ5’), 4.42ꢀ4.49 (m, 1H, Hꢀ4’),
ꢀtriazolyl), 5.40 (brs, 1H, OH), 5.56 (brs, 1H, OH),
.68 (d, J = 6.0 Hz, 1H, Hꢀ1’), 6.47 (brs, 2H, NH ꢀguanosine), 6.51 (brs, 2H, NH ꢀguanine), 7.66
1
white solid. H NMR (400 MHz, DMSOꢀd
6
4
5
.61ꢀ4.71 (m, 2H, Hꢀ2′, Hꢀ3′), 5.18 (s, 2H, NCH
2
2
2
13
(
s, 1H, =CHꢀN), 7.81 (s, 1H, Hꢀ8), 7.91 (s, 1H, Hꢀ8), 10.58 (brs, 2H, NH). C NMR (101 MHz,
DMSOꢀd ): δ 44.50, 56.38, 72.30, 75.26, 80.66, 89.54, 117.18, 117.68, 123.36, 136.58, 137.89,
45.02, 151.68, 152.17, 154.32, 154.44, 157.48, 157.63. MS (APIꢀESI): m/z calcd for C H N O
5
6
1
1
8
19 13
+
[
M+ H] 498.16; found 498.18. Elem. Anal: C, 43.46; H, 3.85; N, 36.61; found: C, 43.48; H, 3.87;
N, 36.63.
9
6-Chloro-N -(4-(6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-propyl)-purine (DCI070). The
reaction was carried out according to the general procedure for the synthesis of 1,2,3ꢀtriazole
9
9
derivatives, using 6ꢀchloroꢀN ꢀpropargylꢀpurine (18) and 6ꢀchloroꢀN -(3ꢀazidopropyl)ꢀpurine (14)
as starting materials. The reaction mixture was stirred at room temperature for 2 days and
evaporated to dryness. The crude was purified by column chromatography on a silica gel
1
(CHCl
3
/MeOH 95:5) to give the compound DCI070 (40% yield) as a white solid. H NMR (400
MHz, DMSOꢀd
6
): δ 2.38ꢀ2.46 (m, 2H, CH
2
ꢀCH
2
ꢀCH
2
2
), 4.30 (t, J = 6.9 Hz, 2H, ꢀCH ꢀNꢀpurine),
4
.38 (t, J = 7.0 Hz, 2H, CH ꢀNꢀtriazolyl), 5.59 (s, 2H, NCH ꢀC), 8.14 (s, 1H, =CHꢀN), 8.65 (s, 1H,
2
2
13
6
Hꢀ2), 8.74 (s, 1H, Hꢀ2), 8.77 (s, 1H, Hꢀ8), 8.79 (s, 1H, Hꢀ8). C NMR (101 MHz, DMSOꢀd ): δ
30.23, 43.90, 45.67, 49.73, 122.67, 123.56, 123.87, 144.67, 145.70, 145.93, 150.09, 150.56, 151.67,
+
1
51.78, 156.32, 156.87. MS (APIꢀESI): m/z calcd for C16
H13Cl
2
N
11 [M+ H] 430.07; found 430.20.
Elem. Anal: C, 44.67; H, 3.05; N, 35.81; found: C, 44.68; H, 3.07; N, 35.83.
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2
2
2
2
2
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3
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3
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3
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5
5
5
5
5
5
6
9
N -(4-(adenine)-methyl-1H-1,2,3-triazol-1-yl)propyl)-adenine (DCI072). DCI070 (1 mmol) was
treated with a saturated isopropanol ammonia solution (25 mL) and the reaction was stirred at 60 °C
overnight. After this time the suspension was filtered and the solid was treated with methanol and
1
diethyl ether (2 times), dried to give the compound DCI072 (55% yield) as a white solid. H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
400 MHz, DMSOꢀd
6 2 2 2 2
): δ 2.28ꢀ2.38 (m, 2H, CH ꢀCH ꢀCH ), 4.12 (t, J = 6.9 Hz, 2H, ꢀCH ꢀNꢀ
purine), 4.33 (t, J = 7.0 Hz, 2H, CH ꢀNꢀtriazolyl), 5.41 (s, 2H, NCH ꢀC), 7.19 (s, 2H, NH ), 7.21 (s,
2
2
2
1
3
2
H, NH ), 8.09 (s, 2H, =CHꢀN, Hꢀ2), 8.12 (s, 2H, Hꢀ8, Hꢀ2), 8.17 (s, 1H, Hꢀ8). C NMR (101
2
MHz, DMSOꢀd
6
): δ 30.15, 45.67, 46.78, 49.63, 121.75, 122.46, 122.81, 139.37, 143.46, 143.58,
+
1
17
49.97, 149.99, 152.45, 152.68, 156.56, 156.79. MS (APIꢀESI): m/z calcd for C16H N13 [M+ H]
392.18; found 392.22. Elem. Anal: C, 49.10; H, 4.38; N, 46.52; found: C, 49.11; H, 4.36; N, 46.53.
9
2
-Amino-6-chloro-N -(4-(2-amino-6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-ethyl)-purine
(
DCI133). The reaction was carried out according to the general procedure for the synthesis of
9
1
,2,3ꢀtriazole derivatives, using 2ꢀaminoꢀ6ꢀchloroꢀN ꢀpropargylꢀpurine (19) and 2ꢀaminoꢀ6ꢀchloroꢀ
9
N ꢀ(2ꢀazidoethyl)ꢀpurine (15) as starting materials. The reaction mixture was stirred at room
temperature for 5 days and evaporated to dryness. The crude was purified by column
chromatography on a silica gel (CHCl /MeOH 95:5) to give the compound DCI133 (22% yield) as
3
1
a white solid. H NMR (400 MHz, DMSOꢀd ): δ 4.49 (t, J = 5.7 Hz, 2H, ꢀCH ꢀNꢀpurine), 4.77 (t, J
6
2
=
5.7 Hz, 2H, ꢀCH
2
ꢀNꢀtriazolyl), 5.29 (s, 2H, NCH
2
ꢀC), 6.93 (s, 2H, NH
2
2
), 6.94 (s, 2H, NH ), 7.71
1
3
(d, J = 1.5 Hz, 1H, =CHꢀN), 7.99 (s, 1H, Hꢀ8), 8.10 (d, J = 1.5 Hz, 1H, Hꢀ8). C NMR (101 MHz,
DMSOꢀd
6
): δ 38.53, 42.76, 46.53, 123.3, 128.24, 130.73, 142.96, 143.67, 144.54, 149.53, 150.03,
+
1
4
54.33, 154.98, 160.41, 161.04. MS (APIꢀESI): m/z calcd for C H Cl N [M+ H] 446.08; found
1
5
13
2
13
46.10. Elem. Anal: C, 40.37; H, 2.94; N, 40.80; Found: C, 40.38; H, 2.91; N, 40.83.
9
2-Amino-6-chloro-N -(4-(2-amino-6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-propyl)-
purine (DCI058). The reaction was carried out according to the general procedure for the synthesis
9
of 1,2,3ꢀtriazole derivatives, using 2ꢀaminoꢀ6ꢀchloroꢀN ꢀpropargylꢀpurine (19) and 2ꢀaminoꢀ6ꢀ
9
chloroꢀN -(4ꢀazidopropyl)ꢀpurine (16) as starting materials. The reaction mixture was stirred at
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room temperature for 2 days and evaporated to dryness. The crude was purified by column
chromatography on a silica gel (CHCl /MeOH 95:5) to give the compound DCI058 (66% yield) as
3
1
a white solid. H NMR (400 MHz, DMSOꢀd
6
): δ 2.26ꢀ2.38 (m, 2H, CꢀCH
ꢀCH ꢀNꢀtriazolyl), 5.54 (s, 2H, NCH
2
), 8.05 (s, 1H, =CHꢀN), 8.09 (s, 1H, Hꢀ8), 8.15 (s, 1H, Hꢀ8). C NMR
2
ꢀC), 4.02 (t, J = 6.9 Hz,
2
H, ꢀCH
2
ꢀNꢀpurine), 4.38 (t, J = 7.0 Hz, 2H, CH
2
2
2
ꢀC), 6.97 (s,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
2
H, NH
2
), 6.98 (s, 2H, NH
(101 MHz, DMSOꢀd ): δ 30.05, 38.93, 41.15, 47.60, 123.84, 124.05, 124.33, 142.96, 143.68,
6
1
43.83, 150.02, 150.08, 154.57, 154.78, 160.41, 160.56. MS (APIꢀESI): m/z calcd for C H Cl N
16 15 2 13
+
[
M+ H] 460.09; found 460.20. Elem. Anal: C, 41.75; H, 3.28; N, 39.56; found: C, 41.77; H, 3.31;
N, 39.54.
2-Amino-6-chloro-N -(4-(2-amino-6-chloropurin)-methyl-1H-1,2,3-triazol-1-yl)-butyl)-purine
9
(DCI095). The reaction was carried out according to the general procedure for the synthesis of
9
1
,2,3ꢀtriazole derivatives, using 2ꢀaminoꢀ6ꢀchloroꢀN ꢀpropargylꢀpurine (19) and 2ꢀaminoꢀ6ꢀchloroꢀ
9
N ꢀ(3ꢀazidobutyl)ꢀpurine (17) as starting materials. The reaction mixture was stirred at room
temperature for 3 days and evaporated to dryness. The crude was purified by column
2 2
chromatography on a silica gel (0–5% MeOH in CH Cl ) to give the compound DCI095 (40%
1
yield) as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 1.68ꢀ1.74 (m, 4H, CꢀCH ꢀCH ꢀC), 4.01ꢀ
6
2
2
4
.06 (m, 2H, ꢀCH ꢀNꢀpurine), 4.30ꢀ4.35 (m,, 2H, ꢀCH ꢀNꢀtriazolyl), 5.31 (s, 2H, NCH ꢀC), 6.89 (s,
2
2
2
1
3
2
H, NH
2
2
), 6.93 (s, 2H, NH ), 8.02 (s, 1H, =CHꢀN), 8.10 (s, 1H, Hꢀ8), 8.14 (s, 1H, Hꢀ8). C NMR
(101 MHz, DMSOꢀd
6
): δ 26.38, 28.77, 38.93, 43.86, 49.73, 124.70, 125.03 (x2), 143.04, 144.45,
1
44.48, 150.46, 150.83, 154.23, 154.61, 159.89, 159.93. MS (APIꢀESI): m/z calcd for C17H17Cl N
2 13
+
[
M+ H] 474.11; found 474.13. Elem. Anal: C, 43.05; H, 3.61; N, 38.39; found: C, 43.02; H, 3.63;
N, 38.38.
9
N -(4-(guanine)-methyl-1H-1,2,3-triazol-1-yl)-ethyl)-guanine (DCI135). DCI133 (1 mmol) was
2
treated with a mixture of TFA/H O (3:1, 10 mL) and the reaction was stirred at room temperature
for 2 days, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated
with methanol and diethyl ether (2 times), filtered and dried to give compound DCI135 (58% yield)
27
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Journal of Medicinal Chemistry
Page 28 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 4.37 (t, J = 5.7 Hz, 2H, ꢀCH ꢀNꢀpurine), 4.73 (t,
6
2
J = 5.7 Hz, 2H, ꢀCH ꢀNꢀtriazolyl), 5.19 (s, 2H, NCH ꢀC), 6.52 (brs, 4H, NH ), 7.41 (d, J = 7.9 Hz,
2
2
2
13
1
H, =CHꢀN), 7.80 (d, J = 11.0 Hz, 1H, Hꢀ8), 7.91 (s, 1H, Hꢀ8), 10.65 (brs, 2H, NH). C NMR
(
101 MHz, DMSOꢀd
6
): δ 43.15, 44.70, 54.53, 115.25, 116.04, 123.89, 137.23, 139.03, 145.83,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
15 13 2
51.23, 151.55, 153.89, 154.20, 157.37, 157.68. MS (APIꢀESI): m/z calcd for C15H N O [M+ H]
410.15; found 410.17. Elem. Anal: C, 44.01; H, 3.69; N, 44.48; found: C, 44.02; H, 3.66; N, 44.49.
9
N -(4-(guanine)-methyl-1H-1,2,3-triazol-1-yl)-propyl)-guanine (DCI061). DCI058 (1 mmol)
2
was treated with a mixture of TFA/H O (3:1, 10 mL) and the reaction was stirred at room
temperature for 2 days, followed by evaporation and coevaporation with MeOH (3 times). The solid
was treated with methanol and diethyl ether (2 times), filtered and dried to give the compound
1
DCI061 (76% yield) as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 2.24ꢀ2.36 (m, 2H, CH ꢀ
6
2
CH ꢀCH ), 3.99 (t, J = 6.9 Hz, 2H, ꢀCH ꢀNꢀpurine), 4.35 (t, J = 7.0 Hz, 2H, ꢀCH ꢀNꢀtriazolyl), 5.24
2
2
2
2
(
s, 2H, NꢀCH
2
ꢀCH
2
), 6.58 (s, 2H, NH
2
), 6.67 (s, 2H, NH
2
), 7.96 (s, 1H, Hꢀ8), 8.05 (s, 1H, =CHꢀN),
13
8
6
.14 (s, 1H, Hꢀ8), 10.77 (brs, 1H, NH), 10.89 (brs, 1H, NH). C NMR (101 MHz, DMSOꢀd ): δ
30.12, 38.97, 41.53, 47.52, 114.05, 115.24, 124.46, 137.86, 137.96, 143.03, 151.33, 151.43, 154.76,
+
1
54.91, 156.29, 156.80. MS (APIꢀESI): m/z calcd for C H N O [M+ H] 424.16; found 424.30.
16
17 13
2
Elem. Anal: C, 45.39; H, 4.05; N, 43.01; found: C, 45.41; H, 4.08; N, 42.98.
9
N -(4-(guanine)-methyl-1H-1,2,3-triazol-1-yl)-butyl)-guanine (DCI096). DCI095 (1 mmol) was
2
treated with a mixture of TFA/H O (3:1, 10 mL) and the reaction was stirred at room temperature
for 2 days, followed by evaporation and coevaporation with MeOH (3 times). The solid was treated
with methanol and diethyl ether (2 times), filtered and dried to give the compound DCI096 (93%
1
yield) as a white solid. H NMR (400 MHz, DMSOꢀd ): δ 1.64ꢀ1.77 (m, 4H, CH ꢀCH ꢀCH ), 3.93ꢀ
6
2
2
2
4
.00 (m, 2H, ꢀCH
2
ꢀNꢀpurine), 4.29ꢀ4.36 (m, 2H, ꢀCH
2
ꢀNꢀtriazolyl), 5.22 (s, 2H, NꢀCH
2
2
ꢀCH ), 6.54
(s, 2H, NH
2
2
), 6.60 (s, 2H, NH ), 7.88 (s, 1H, Hꢀ8), 8.00 (s, 1H, =CHꢀN), 8.05 (s, 1H, Hꢀ8), 10.70
13
(
6
brs, 1H, NH), 10.80 (brs, 1H, NH). C NMR (101 MHz, DMSOꢀd ): δ 27.43, 29.06, 40.47, 44.53,
4
9.79, 115.25, 116.63, 123.83, 135.64, 136.15, 142.03, 150.65, 151.68, 154.34, 154.76, 157.29,
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
157.85. MS (APIꢀESI): m/z calcd for C H N O [M+ H] 438.18; found 438.30. Elem. Anal: C,
1
7
19 13
2
4
6.68; H, 4.38; N, 41.63; found: C, 46.67; H, 4.40; N, 41.62.
6
9
6
2
-Amino-(N -methyl)-N -(4-(2-amino-(N -methyl)-purin)-methyl-1H-1,2,3-triazol-1-yl)-
propyl)-purine (DCI134). To a solution of DCI058 (1 mmol) in ethanol (20 mL) was added
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
methylamine (33% in absolute ethanol, 10 mmol) and the reaction was stirred at 80 °C for 4 h.
After this time the suspension was filtered and the solid was treated with ethanol and diethyl ether
1
(
2 times), dried to give the compound DCI134 (68% yield) as a white solid. H NMR (400 MHz,
DMSOꢀd
CH ꢀNꢀpurine), 4.31 (t, J = 7.0 Hz, 2H, CH
NH ), 7.17 (brs, 2H, NHꢀCH ), 7.65 (s, 1H, Hꢀ8), 7.70 (s, 1H, Hꢀ8), 8.07 (s, 1H, =CHꢀN). C NMR
101 MHz, DMSOꢀd ): δ 27.24, 27.33, 30.06, 40.63, 41.42, 49.53, 118.46, 118.78, 123.75, 136.23,
6
): δ 2.22ꢀ2.31 (m, 2H, CH
2
ꢀCH
2
ꢀCH
2
), 2.85 (brs, 6H, NHꢀCH
3
), 3.93 (t, J = 6.9 Hz, 2H, ꢀ
2
2
ꢀNꢀtriazolyl), 5.22 (s, 2H, NCH
2
ꢀC), 5.86 (brs, 4H,
1
3
2
3
(
6
1
36.55, 142.79, 149.55, 150.27, 156.73, 156.91, 158.43, 158.90. MS (APIꢀESI): m/z calcd for
+
C
18
H
23
N
15 [M+ H] 450.23; found 450.23. Elem. Anal: C, 48.10; H, 5.16; N, 46.74; found: C,
4
8.08; H, 5.17; N, 46.76.
6
9
6
2-Amino-(N -benzyl)-N -(4-(2-amino-(N -benzyl)-purin)-methyl-1H-1,2,3-triazol-1-yl)propyl)-
purine (DCI136). To a solution of DCI058 (1 mmol) in ethanol (20 mL) was added benzylamine
(10 mmol) and the reaction was stirred at 80 °C for 4 h. After this time the suspension was filtered
and the solid was treated with ethanol and diethyl ether (2 times), dried to give the compound
1
DCI136 (68% yield) as a white solid. H NMR (400 MHz, DMSOꢀd
6
): δ 2.22ꢀ2.33 (m, 2H, CꢀCH
ꢀNꢀtriazolyl), 4.61 (brs,
H, ꢀCH ꢀbenzyl), 7.17 (brs, 2H, NH), 5.23 (s, 2H, NꢀCH ꢀCH ), 5.86 (brs, 4H, NH ), 7.14ꢀ7.20 (m,
2
ꢀ
2 2
C), 3.93 (t, J = 6.4 Hz, 2H, ꢀCH ꢀNꢀpurine), 4.32 (t, J = 6.8 Hz, 2H, CH
4
2
2
2
2
2
H, arom), 7.23ꢀ7.29 (m, 4H, arom), 7.29ꢀ7.33 (m, 4H, arom), 7.68 (d, J = 1.5 Hz, 1H, Hꢀ8), 7.73
13
(
d, J = 1.5 Hz, 1H, Hꢀ8), 8.07 (s, 1H, =CHꢀN). C NMR (101 MHz, DMSOꢀd
6
): δ 30.54, 41.67
(x2), 41.60 (x2), 49.63, 119.67, 119.34, 122.89, 126.89 (x6), 127.69 (x4), 130.67, 140.63 (x2),
1
43.59, 145.84, 150.46, 150.81, 157.96, 157.98, 159.43, 159.67. MS (APIꢀESI): m/z calcd for
29
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