Synthesis of triazole-functionalized 2-DOS analogues and their evaluation as A-site binders

Article abstract of DOI:10.1016/j.bmcl.2013.12.125

Aminoglycoside-antibiotics represent important tools for studying the biological functions of RNA. An orthogonal protection strategy applied on 2-deoxystreptamine (2-DOS) revealed a series of key intermediates that enable its regioselective functionalization. Our approach allowed the construction of selected representatives of triazole-containing analogues with diverse molecular frameworks for biological evaluation regarding their binding and antibacterial potencies.

Full text of DOI:10.1016/j.bmcl.2013.12.125

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1122–1126
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of triazole-functionalized 2-DOS analogues and their
evaluation as A-site binders
Panoula Anastasopoulou, Georgia Kythreoti, Tilemachos Cosmidis, Constantina Pyrkotis,
⇑
Victoria R. Nahmias, Dionisios Vourloumis
Chemical Biology Laboratories, National Center for Scientific Research ‘Demokritos’, Agia Paraskevi Attikis, GR 15310, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminoglycoside-antibiotics represent important tools for studying the biological functions of RNA. An
orthogonal protection strategy applied on 2-deoxystreptamine (2-DOS) revealed a series of key interme-
diates that enable its regioselective functionalization. Our approach allowed the construction of selected
representatives of triazole-containing analogues with diverse molecular frameworks for biological eval-
uation regarding their binding and antibacterial potencies.
Received 10 December 2013
Revised 30 December 2013
Accepted 31 December 2013
Available online 9 January 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Aminoglycosides
Ribosomal A-site
RNA recognition
2-Deoxystreptamine
Orthogonal protection strategy
Our rapidly expanding knowledge of the key biological roles
that RNA plays has fueled a growing interest in exploiting RNA
as a drug target. The decoding site of the bacterial ribosome, or
A-site, an internal loop within the 16S rRNA, is the molecular target
for natural aminoglycoside antibiotics (Fig. 1).1–7 Most of these
natural products embody in their structures a highly conserved
diaminocyclohexitol, 2-deoxystreptamine (2-DOS, Fig. 1), whose
essential role for binding to RNA5,8–12 involves not only direct
hydrogen bonding interactions but also the precise orientation of
its peripheral functionalities for increased binding affinity. Thus,
classification of 2-DOS as a ‘privileged’ RNA-targeting chemical
entity for the development of novel antiviral and antimicrobial
pharmaceuticals is highly substantiated. Some of the most studied
sub-families of aminoglycoside antibiotics are presented in
Figure 1, including the 4-monosubstituted neamine (neomycin
A), the 4,6-disubstitution pattern present in the kanamycin class,
the 4,5-disubstituted neomycin series and the 1,4,6-trisubstituted
semi-synthetic amikacin.13 The observed variety in their substitu-
tion pattern stimulated several efforts towards the synthesis and
utilization of an orthogonally protected 2-DOS intermediate,
enabling the obvious opportunity to construct analogues of the
natural products through a unified approach from a common
precursor.14–18 Herein we would like to present our findings on
that subject, resulting in the synthesis of diverse representative
Figure 1. Structures of the 4,5-disubstituted neomycin
4-monosubstituted neamine (neomycin A), 2-deoxystreptamine (2-DOS), 4,
6-disubstituted kanamycin B and 1,4,6-trisubstituted semi-synthetic amikacin.
B and paromomycin,
⇑
Corresponding author. Tel.: +30 210 650 3624; fax: +30 210 651 1766.
E-mail address: vourloumis@chem.demokritos.gr (D. Vourloumis).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.125

P. Anastasopoulou et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1122–1126
1123
examples of mono-, di- and tri-substituted triazole-containing
2-DOS analogues for further biological evaluation. Our approach
was based on existing literature precedent19–21 and our experience
in this field.22–25 Triazoles were selected primarily due to their pre-
vious success in similar systems, as the functionality that dramat-
ically increases binding affinities for the ribosomal decoding site,
when placed in the area occupied by position-4 substituents
(Fig. 1 numbering).26 Consequently, adjacent positioning (3 and
5), amplified H-bonding potential and overall rigidity modifica-
tions of the final structures were rationally targeted.
Our synthetic approach was initiated from optically pure alco-
hol 1.25 This intermediate was obtained from neamine, exploiting
to our advantage the chiral substitution pattern of 2-DOS in the
natural products, and thus avoiding chiral auxiliary- or enzymati-
cally-induced resolution strategies. Formation of oxazolidinone 2
under basic conditions, followed by activation of the cyclic carba-
mate towards hydrolysis through the introduction of a Boc-group,
resulted, after basic treatment with LiOH, to the isolation of 2-DOS
analogue 3, with obvious orthogonal diversification of the amino-
protecting groups (Scheme 1).
ketal, diol 5. Finally, the two hydroxyls at positions 5 and 6
(Scheme 1 numbering) were differentiated, as before for 2, by the
formation of a cyclic-carbamate, producing orthogonally protected
2-DOS 6.
The same alcohol could be synthesized by an alternative ap-
proach, initiated by etherification of 1 with MOMCl and selective
cleavage of the cyclic ketal, to provide diol 7 in good yield. Subse-
quently, intramolecular oxazolidinone formation under basic con-
ditions furnished alcohol 8. Hydrogenolysis of the benzyl
carbamate and transformation of the liberated amine to the corre-
sponding azide (triflic azide, CuII catalysis),27 furnished compound
9. Finally the desired alcohol 6 resulted in 70% yield from the effi-
cient introduction of a PMB-group to the more acidic oxazolidi-
none NH after treatment of alcohol 9 with stoichiometric NaH
(1 equiv) and PMB-Br (3 equiv) in THF at 45 °C for 4 h. The trans-
formations presented in Scheme 1 along with their products, ad-
vanced intermediates
6 and 9, embody all the required
characteristics of an orthogonality concept, allowing the efficient
regioselective modification of 2-DOS, as it will be presented for
representative examples in the following schemes.
When alcohol 1 was subjected to stronger hydrolyzing condi-
tions, only the amine at position 3 was liberated from the corre-
sponding benzyl carbamate through the neighboring group
participation of OH-4, allowing its differential protection as azide
4.27 Protection of the free hydroxyl in 4 with MOM-Cl, followed
by the introduction of a PMB-group to the acidic oxazolidinone
NH (NaH, PMB-Cl) furnished, after selective cleavage of the cyclic
The desired triazole functionality was firstly introduced at posi-
tion 3 (Scheme 2 numbering) of advanced intermediate 9 by its
reaction with propargyl alcohol under standard ‘click chemistry’
conditions,28 furnishing 10. Cleavage of the MOM-protection fol-
lowed by basic hydrolysis of the oxazolidinone moiety resulted
in the formation of 3-mono-substituted product 12 in excellent
overall yield. Hydrolysis of the oxazolidinone ring in 10 liberated
the amine at position 1, allowing the introduction of additional
functionalization. (S)-4-Amino-2-hydroxybutyric acid was selected
for that purpose, since it represents the side-chain of amikacin as
well as a potentially rich source of hydrogen-bonding interactions
with the biological target. Reaction of its succinate derivative 1329
(Supporting information) with the corresponding amine furnished,
after removal of the acid-sensitive protecting groups, 1,3-di-substi-
tuted product 15. Similar chemical transformations (hydrolysis of
the oxazolidinone, coupling of the amine with the activated ester,
reduction of the azide and cleavage of the MOM-ether) resulted in
the isolation of 1-mono-substituted analogue 14 for direct compar-
ison with 12 and 15. Compound 14 was also efficiently obtained
Scheme 1. Reagents and conditions: (a) NaH (4.0 or 7.0 equiv), DMF (0.06 or 0.1 M),
0.5–2 h, 0 °C or 23 °C, 50% for 2 and 51% for 8; (b) Boc2O (1.25 equiv), Et3N
(1.25 equiv), 4-DMAP (0.2 equiv), THF (0.01 M), 16 h, 23 °C, 91%; (c) aq LiOH
(0.5 M)/dioxane (1:3), 16 h, 23 °C, 64%; (d) aq LiOH (0.5 M)/dioxane (1:2), 4 h, 70 °C,
91%; (e) triflic azide (3.0 or 5.0 equiv), K2CO3 (1.5 equiv) or Et3N (1.5 equiv),
CuSO4ꢀ5H2O (0.1 equiv), MeOH/H2O (5:1), 18 h, 23 °C, quantitative for 4 and 60%
over 2 steps for 9; (f) MOMCl (10.0 equiv), DIPEA (12.0–14.0 equiv), TBAI
(0.1 equiv), CH2Cl2 (0.1 or 0.5 M), 16 h, 0 ? 23 °C, 95–96%; (g) PMBCl (3.0 equiv),
NaH (4.0 equiv), TBAI (0.1 equiv), DMF (0.24 M), 10 h, 0 ? 23 °C; (h) AcOH/H2O
(4:1), 1.5–3 h, 23 °C, 60% over 2 steps for 5 and 88% for 7; (i) NaH (3.0 equiv), DMF
(0.02 M), 0.5 h, 0 °C, quantitative; (j) H2, Pd/C (10% moles), MeOH (0.15 M), 3 h,
23 °C, 92%. MOMCl = Chloromethyl-methyl ether; DIPEA = N,N-Diisopropyl-ethyl-
amine; 4-DMAP = 4-dimethylamino pyridine; TBAI = Tetra-n-butyl ammonium
iodide; AcOH = Acetic acid; DMF = Dimethylformamide; Tf = Trifluoromethane sul-
fonyl; PMB = 4-Methoxybenzyl; THF = Tetrahydrofuran.
Scheme 2. Reagents and conditions: (a) propargyl alcohol (2.0 equiv), CuSO4ꢀ5H2O
(0.3 equiv), sodium ascorbate (0.5 equiv), EtOH/H2O (2:1), 12 h, 23 °C, 96%; (b) 1 M
HCl/EtOAc or 1 M HCl/MeOH (1:1), 12 h, 23 °C, 97% for 11, or 77% over 2 steps for
14, or 94% for 15; (c) aq LiOH (0.5 M)/dioxane (1:1), 2.5–12 h, 23 °C, 77% for 12; (d)
13 (2.1 equiv), satd NaHCO3 (1 mL/mmol), dioxane/H2O (3:1), 12 h, 23 °C; (e) Me3P
(1 M in THF, 5.0 equiv), NaOH (0.05–1.3 equiv), THF (0.1 M), 12 h, 0 ? 23 °C, 92%.

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P. Anastasopoulou et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1122–1126
from 3 after a 3-steps, hydrogenolysis—coupling—acidic deprotec-
tion, sequence.
reactivity, the desired triazole was in this case pre-formed as bro-
mide 29 (Supporting information) and introduced to the free 4-hy-
droxyl under basic conditions. Oxidative conditions (CAN)
universally cleaved all PMB-protecting groups. Substitution at po-
sition 1 resulted from nucleophilic opening of the oxazolidinone
with ethanolamine furnishing the corresponding urea. Hydrogena-
tion of the azide moiety completed the synthesis of tri-substituted
analogue 31.
Evaluation of the binding specificity of our analogues for the
ribosomal decoding site was performed, as previously described,30
by an RNA fluorescence assay, based on enhancement of the
fluorescence emission of 2-aminopurine (2AP) attached instead
of the flexible adenine A1492 of the model oligonucleotide (Table 1,
RNA Specific).31 The activity of the compounds as functional
Similar analogues, albeit of increased rigidity, were constructed
as presented in Scheme 3. Specifically, replacement of the benzyl
carbamates in 1 for azides resulted in the formation of diazido
alcohol 16 in 72% overall yield. Alkylation of the 4-OH in 16 with
propargyl bromide proceeded with simultaneous cyclization to
the corresponding triazole 17.30 Cleavage of the ketal protecting
group with aqueous AcOH, followed by Staudinger reduction of
the azide furnished tricyclic analogue 18 in excellent yield. Addi-
tional substitution at position 1 was introduced, either through
activated ester 13 (as shown before in the syntheses of 14 and
15), or by standard amide coupling (EDC) of N-protected amino
acid 19, yielding, after removal of the acid-labile protecting groups
with HCl, analogues 20 and 21.
bacterial-translation inhibitors was evaluated in
a coupled
Introduction of triazole-containing moieties at positions 4 or 5
of 2-DOS was performed as presented in Scheme 4. Thus, advanced
intermediate 6 was alkylated with propargyl bromide under basic
conditions (NaHMDS) furnishing, after liberation of the 4-hydroxyl,
in vitro transcription-translation assay (IVT, Table 1).32 The syn-
thetic analogues were also evaluated regarding their antibacterial
activity against Gram positive Staphylococcus aureus (ATCC
29213) as well as Gram negative Pseudomonas aeruginosa (ATCC
27853) and Escherichia coli (ATCC 25922) strains (Table 1).
alcohol 22. A second alkylation step at position 4 with
a-bromo
amide 23 (Supporting information), followed by removal of the
PMB-protection, resulted in the formation of 24. Reduction of the
azide and hydrolysis of the oxazolidinone prepared the resulting
diamino-alkyne for its reaction with azide 25 under CuI catalysis28
furnishing final analogue 26.
Based on the obtained results we speculate that the overall pro-
file of the synthetic analogues represent a composite of various fac-
tors. Some of the more important ones dictating their behaviour
include: (a) H-bonding and/or p-stacking potential, (b) availability
Similarly, alkylation of 6 with bromide 27 (Supporting informa-
tion) produced after removal of the MOM-protection under acidic
conditions, alcohol 28. Introduction of an alkynyl-moiety on the
latter would, as shown before for 17, lead to an immediate cycliza-
tion to the corresponding tricyclic system. To avoid this unwanted
Scheme 4. Reagents and conditions: (a) Propargyl bromide (3.0 equiv), NaHMDS
(1.2 equiv), TBAI (0.1 equiv), THF (0.1 M), 12 h, ꢁ4 ? 23 °C; (b) HCl (1.4 M in 1:1
MeOH/CHCl3), 12 h, 23 °C, 52% over 2 steps; (c) 23 or 27 or 29 (1.5–2.5 equiv),
NaHMDS (1.1 equiv), THF (0.05–0.1 M), 20 min. to 3 h, ꢁ4 ? 23 °C, 83–96%; (d) CAN
(5.0 equiv), CH3CN/H2O (9:1), 1–4 h, 23 °C, 75% over 2 steps for 24; (e) Me3P (1 M in
THF, 3.0 equiv), aq NaOH (1 M, 1.3 equiv), THF (0.04 M), 12 h, 0 ? 23 °C; (f) aq LiOH
(0.5 M)/dioxane (1:1), 12 h, 23 °C; (g) 25 (2.0 equiv), CuSO4ꢀ5H2O (0.3 equiv),
sodium ascorbate (0.5 equiv), EtOH/H2O (2:1), 12 h, 23 °C, 46% over 3 steps for 26;
(h) concd HCl (cat.), MeOH (0.05 M), 0.5 h, 50 °C, quantitative; (i) ethanolamine
(10 equiv), satd NaHCO3 (1.5 equiv), dioxane (0.03 M), 12 h, 23 °C, 72%; (j) H2, Pd/C
(10% mol), MeOH (0.1 M), 4 h, 23 °C, 51%. NaHMDS = sodium bis(trimethyl-
silyl)amide; CAN = cerium(IV) ammonium nitrate.
Scheme 3. Reagents and conditions: (a) H2, Pd/C (10% mol), MeOH (0.1 M), 12 h,
23 °C; (b) triflic azide (10 equiv), Et3N (20 equiv), CuSO4ꢀ5H2O (0.05 equiv), MeOH/
H2O (1.5:1), 16 h, 23 °C, 72% over 2 steps; (c) propargyl bromide (80% wt. in toluene,
1.1 equiv), NaH (5.0 equiv), TBAI (0.1 equiv), toluene (0.07 M), 12 h, 0 ? 23 °C, 88%;
(d) AcOH/H2O (4:1), 12 h, 23 °C; (e) Me3P (1 M in THF, 5.0 equiv), aq NaOH (0.1 M,
0.05 equiv), THF (0.1 M), 12 h, 0 ? 23 °C, 96% over 2 steps for 18; (f) 19 (3.0 equiv),
EDC (3.0 equiv), DIPEA (4.0 equiv), 4-DMAP (1.1 equiv), CH3CN (0.3 M), 12 h,
0 ? 23 °C, 68% over 2 steps; (g); 13 (2.1 equiv), satd NaHCO3 (1 mL/mmol),
dioxane/H2O (3:1), 12 h, 23 °C; (h) HCl (4 M)/EtOAc (1:1), 12 h, 23 °C, 79% for 20
and 73% for 21 over 3 steps. EDCl = N-(3-dimethylaminopropyl)-N0-ethylcarbo-
diimide hydrochloride.

P. Anastasopoulou et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1122–1126
1125
Table 1
Binding affinities and biological activities of synthetic analogues
Entry
Compounda
RNA Specificb (EC50
,
lM)
IVTc (IC50
,
lM)
Escherichia coli (MIC)d
Staphylococcus aureus (MIC)d
Pseudomonas aeruginosa (MIC)d
1
2
3
4
5
6
7
8
12
14
15
18
20
21
26
31
No activity
8.7
No activity
468
475
66.5
317
55.5
104
730
22.6
>700
>840
>1000
283
>870
>820
>1000
235
88
52.5
60
>565
109
102
129
78
>700
>840
>1000
283
>870
>820
>1000
157
53
22.4e
0.017e
3
62
a
b
c
All final products were quantitated by NMR spectroscopy, utilizing an internal standard (DiMethylFuran, DMFu).33
RNA-ligand EC50 values were determined by decoding-site RNA fluorescence assay (average of 3 replicate experiments per compound, 10%).
Bacterial in vitro transcription/translation (IVT) IC50 values were determined by coupled transcription-translation assay using E. coli extract (2 replicate experiments, in
duplicate, per compound, 14%).
d
Concentrations are reported in lg/mL.
Small efficacy in fluorescence intensity increase effect.
e
of the two amino-groups at positions 1 and 3 to participate into
‘neamine-like’ interactions with the A-site (with C1407–G1494
and the non-Watson–Crick pair U1406–U1495),12 docking the
compounds into a preferred conformation, (c) binding orientation
adapted within the binding cavity, (d) existence of alternative bio-
logical targets (different topologies of binding on the ribosome,
other RNAs, other proteins).
chemistry protocols, by harnessing the molecular diversity accessi-
ble to synthetic chemistry. Thus, novel specific RNA binders could
be discovered within a new molecular framework, not hampered
by complexity.
Acknowledgments
Specifically, for analogues 12 and 15 we observe the absence of
binding affinity for the bacterial decoding site. Apparently, posi-
tioning of the triazole-functionality in a perpendicular orientation
to the diaminocyclohexitol, along with the unavailability of N-3 to
participate in H-bonding, diminish their potential to participate in
effective interactions, rendering these compounds incapable to as-
sume the required binding orientation. Removal of the triazole
moiety in 14 re-establishes its binding ability. Thus, according to
our previous discussion, the observed inhibition in protein produc-
tion (IVT for the di-substituted 15) as well as their potential to in-
hibit the growth of Gram positive bacterial strains (for 12, 14 and
15) are the results of an alternative, unknown at this point, mech-
anism of action.
When the triazole is forced to adapt a more planar orientation
with respect to the 2-DOS ring, 18, 20 and 21 regain their binding
affinity for the A-site, in an analogous manner to their H-bonding
potential. This effect is also directly translated into biological activ-
ity (IVT and MICs). Small variations in the absolute values of their
potencies, comparing either analogues within the same series (12
to 15 or 18 to 20 and 21) or different series (12, 15 vs. 18, 20
and 21, respectively), appear to be directed by the extent of their
H-bonding potential and/or differential mechanisms of action
and binding orientations.
This research was supported by Marie Curie Actions, Excellence
Teams (EXT) Grants, Contract number MEXT-CT-2006-039149. P.A.
thanks the ‘Leonidas Zervas’ Foundation for financial support (2011
Organic Synthesis Award). G.K. and D.V. acknowledge support by
the Action ‘Supporting Postdoctoral Researchers’ Grant LS9_1057
of the Operational Program ‘Education and Lifelong Learning’.
Supplementary data
Supplementary data (experimental procedures and NMR spec-
tra of all intermediates and final products along with titration
curves) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmcl.2013.12.125.
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