A facile synthesis of 5′-Fluoro-5′-deoxyacadesine (5′-F-AICAR): A novel non-phosphorylable AICAR Analogue

Article abstract of DOI:10.3390/molecules171113036

The substitution of a hydroxyl group by a fluorine atom in a potential drug is an efficient reaction that can, in principle, improve its pharmacological properties. Herein, the synthesis of the novel compound 5′-fluoro- 5′-deoxyacadesine (5′-F-AICAR), a strict analogue of AICAR that cannot be 5′-phosphorylated to ZMP by cellular kinases, is reported.

Full text of DOI:10.3390/molecules171113036

Molecules 2012, 17, 13036-13044; doi:10.3390/molecules171113036
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
A Facile Synthesis of 5-Fluoro-5-deoxyacadesine (5-F-AICAR):
A Novel Non-phosphorylable AICAR Analogue
Stefano D’Errico 1, Giorgia Oliviero 1,*, Nicola Borbone 1, Jussara Amato 1, Daniele D’Alonzo 2,
Vincenzo Piccialli 2, Luciano Mayol 1 and Gennaro Piccialli 1
1
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II,
Via D. Montesano 49, 80131, Napoli, Italy
2
Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II, Via Cintia 21,
80126, Napoli, Italy
* Author to whom correspondence should be addressed; E-Mail: golivier@unina.it;
Tel.: +39-081-679-986.
Received: 29 September 2012; in revised form: 31 October 2012 / Accepted: 31 October 2012 /
Published: 2 November 2012
Abstract: The substitution of a hydroxyl group by a fluorine atom in a potential drug is an
efficient reaction that can, in principle, improve its pharmacological properties. Herein,
the synthesis of the novel compound 5′-fluoro-5′-deoxyacadesine (5′-F-AICAR), a strict
analogue of AICAR that cannot be 5′-phosphorylated to ZMP by cellular kinases, is reported.
Keywords: AICAR; ZMP; AMPK; AMPK activation; fluorinated nucleosides;
fluorination; imidazole nucleosides; nucleoside analogues; modified nucleosides
1. Introduction
New synthetic methodologies have been developed to prepare novel nucleoside and nucleotide
analogues for enlarging the collection of potential antitumoral and antiviral drugs and for improving
the pharmacological and pharmacokinetic properties of the existing ones [1–10]. Particular attention
has been paid to imidazole nucleosides and nucleotides considering that their precursors,
5-aminoimidazole-4-carboxamide riboside (acadesine or AICAR) and 5-aminoimidazole-4-carboxamide
ribotide (ZMP, Figure 1), are involved in the activation of AMP-activated protein kinase (AMPK) [11,12].
In the cells AICAR is phosphorylated to ZMP that is a mimic of AMP. The direct binding of ZMP to
an allosteric site of AMPK causes a cascade of important metabolic events, including inhibition of the

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basal and insulin-stimulated glucose uptake, lipogenesis and glucose oxidation [13]. In the treatment of
ischemia, AICAR has also been shown to be cardioprotective [14], preventing the endoplasmic
reticulum stress, the inhibition of the sodium/hydrogen exchanger and thus the rapid rise of
intracellular sodium, which normally occurs during the reperfusion [15].
Figure 1. Structures of AICAR and ZMP.
The AMPK pathway is also implicated in the regulation of cell proliferation and activation by
AICAR could result in pro-apoptotic effects [16,17]. Moreover, it was established that mutations
affecting the Ca2+ releasing channel RYR1 are associated with a broad spectrum of human disorders,
including malignant hyperthermia, central core disease and core-rod myopathy [18]. By using a mouse
model of malignant hyperthermia having mutation in the RYR1 gene, Lanner et al. have recently
demonstrated that AICAR can inhibit Ca2+ leakage through RYR1 by a mechanism independent from
AMPK activation, thus preventing heat-induced sudden death in the mutated mouse [18]. Considering
that AICAR has a short half-life in cells, does not cross efficiently the blood-brain barrier, is poorly
adsorbed by the gastrointestinal tract, increases uric acid production and the lactic acidosis [15], the
development of novel AICAR derivatives/analogues endowed with an improved pharmacological
profile is an appealing goal in the field of medicinal chemistry.
In recent years we have focused our attention on the preparation of new base- and sugar-modified
nucleosides and nucleotides [4,10,19–24] with the aim to enlarge the pool of new potential
antimetabolites and to obtain new probes to better understand how certain metabolic pathways work. It
is well known that the introduction of fluorine in a potential drug can dramatically change its chemical
behaviour, influencing its pharmacokinetic and pharmacodynamics properties; furthermore, increased
metabolic stability and a more efficient permeation of biological membranes are a consequence,
respectively, of the strength of the C-F bond and of the higher hydrophobicity of fluorine respect to
hydrogen [25–27].
In the last years several novel synthetic methodologies have been proposed for the selective
introduction of fluorine at the 2′-, 3′- or 5′-position of a nucleoside [28,29]; in some cases these
substitutions were revealed to be so important that novel anti-tumor and anti-viral drugs have been
obtained [28]. In this paper we report on the synthesis of the novel analogue 5′-fluoro-5′-deoxy-
acadesine (5′-F-AICAR, 8, Scheme 1) in which the 5′-hydroxyl group is replaced by a fluorine atom.
The latter is an isopolar mimic of the hydroxyl group, but prevents the kinase-mediated
5′-phosphorylation by cellular kinases. We hypothesized that this new derivative, which can be

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considered the most similar and non-phosphorylable AICAR analogue, could be a useful tool to
explore the wide and not completely understood physiological roles of AICAR and ZMP. Futhermore,
5′-F-AICAR may be also considered as a mimic of 5′-fluoro-5′-deoxyadenosine (5′-FDA) that has been
employed as a tool for studying the Actinomycete bacterium Strepromyces cattleya [30]. Interestingly,
some 5′-FDA derivatives were revealed to be inhibitors of the enzyme S-adenosyl-L-homocysteine
hydrolase (an enzyme involved in the replication of some viruses) thanks to their chemical inertness
toward the phosphorylation [30,31]. Hence, it would not be surprising if the novel compound
5′-F-AICAR could also work as an antiviral agent.
Scheme 1. Preparation of compound 5′-F-AICAR 8.
Reagents and conditions: (i) Acetone, DMP, p-TsOH, 2 h, r.t.; (ii) TsF, TBAF, THF, reflux, 18 h;
(iii) 0.1 M NaOH, 4 h, r.t.; (iv) 70% TFA, 0 °C then r.t.; (v) Ac2O, Py, 1 h, r.t; (vi) K2CO3,
2,4-dinitro-chlorobenzene (DNClB), DMF, 3 h, 80 °C; (vii) EDA, DMF, 50 °C, 16 h.
2. Results and Discussion
At first, we tried the selective insertion of the fluorine atom at the 5′-position of AICAR starting
from its 2′,3′-O-isopropilydene derivative, using common fluorinating agents, such as: (a) DAST,
(b) deoxofluor, (c) the Olah reagent [28] and (d) the tosyl fluoride (TsF)/tetrabutylammonium fluoride
system (TBAF) [30]. Unfortunately, all these procedures failed, resulting in the recovery of only the
unreacted starting material (reagent d) or decomposition of the nucleoside (reagents a–c). These results
thus prompted us to explore an alternative synthetic route. We hypothesized that the construction of

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the 5-aminoimidazole-4-carboxamide moiety could be performed starting from an easily fluorinable
purinic substrate, from which AICAR could be obtained through the degradation of the purine ring. In
this context, we have recently reported efficient synthetic strategies to obtain AICAR, ZMP and their
4-N-alkyl derivatives, through the purine ring degradation of suitable inosine or 5′-phosphate inosine
precursors, respectively, after the introduction of a suitable strong electron-withdrawing group
(2,4-dinitrophenyl, DNP) at the N1 position of the base moiety [20–22,24].
Accordingly, for the preparation of 5′-F-AICAR (8) we used as starting material commercially
available 6-chloronebularine (1, Scheme 1), which was readily transformed into its 2′,3′-O-
isopropylidene derivative 2 [32]. Then, the 5′-OH group of 2 was replaced by a 5′-F in a one-pot
tosylation/fluorination sequence, treating 2 with the TsF/TBAF reagent system, according to the
Schimizu’s procedure [30]. It should be noted that the presence of an electron-withdrawing atom, such
as chlorine, at the C6 position is necessary to prevent the intramolecular nucleophilic attack of N3 on
the first-formed C5′-OTs, that would led to the unwanted cyclic side product containing the N3-C5′
bond [29]. As previously observed by Ashton et al. [30], we noted (TLC monitoring) that the process
led not only to the formation of the expected fluorinated product 3a, but also to the formation of the
bis-fluorinated product 3b (Scheme 1, X = F), as a consequence of the substitution in 2 of the C6
chlorine atom by fluoride. This was in no way detrimental to the continuation of the synthetic plan
since both 3a and 3b could be used in the next step. Thus, the 3a/3b mixture was converted into the
corresponding inosine derivative 4 by reaction with a 0.1 M NaOH solution, affording 4 in a 60% yield.
In particular, 3a and 3b were revealed to be reactive to the same extent toward the hydroxyl ion, and
the displacement of chlorine and fluorine atoms at C6 could both be monitored by TLC.
Importantly, the success of this reaction was strongly dependent upon the concentration of the base
employed. In fact, concentrations of NaOH higher than 0.1 M caused the decomposition of the
nucleosidic material leading to the formation of several side-products, one of which was identified as
2′,3′-O-isopropylideneinosine.
Then, removal of the isopropylidene group in 4 with 70% trifluoroacetic acid (TFA) gave inosine
derivative 5 [33], which was acetylated with Ac2O in pyridine under standard conditions to give the
2′,3′-di-O-acetylated product 6 quantitatively. The change of the 2′,3′-protecting group was necessary
since the deprotection of 2′,3′-O-isopropylidene group in the final step led to a partial decomposition
of the 5′-fluoro-acadesine 8. Next, introduction in 6 of the strong DNP electron-withdrawing group at
the N1 position was carried out by treatment with K2CO3 and 2,4-dinitrochlorobenzene (DNClB) to
give 7 [34,35]. This process served to activate the C2 position of the purine, rendering it susceptible to
nucleophilic attack by amines, thus leading to the cleavage of the N3-C2 bond. In particular, we have
previously demonstrated that when a 1,-diaminoalkane is employed, the fate of the open intermediate
depends on the length of the alkyl chain separating the two amino groups. If the diamine comprises
two or three methylene groups, AICAR is formed as the sole product [22].
Eventually, when compound 7 was left into contact with a solution of ethylendiamine (EDA) in
DMF, 5′-F-AICAR 8 was obtained by purine-ring opening and concomitant deacetylation of the
hydroxyl groups at the 2′-3′ of the ribose moiety. During this reaction no loss of the fluorine at the 5′C
position was observed. Compound 8 was obtained in 70% yield starting from 7 and an overall 25%
yield from 1. Its structure was confirmed by 1D/2D-NMR evidence and by high-resolution mass
spectrometry (HRMS).

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3. Experimental
General
All the reagents were obtained from commercial sources (Sigma-Aldrich, city, state abbrev if USA,
country) and were used without further purification. 1H-, 13C-, and 19F-NMR spectra were acquired on
a Varian Mercury Plus 400 MHz and on a Varian Unit Inova 500 MHz in CD3OD. Chemical shifts are
1
reported in parts per million (δ) relative to the residual solvent signals: CD2HOD 3.31 for H-NMR;
1
CD2HOD 49.0 for 13C-NMR. H-NMR chemical shifts were assigned by 2D NMR experiments.
19
Trifluorotoluene was used as external standard for F-NMR experiments. The abbreviations s, bs, d,
dd, ddd and m stand for singlet, broad singlet, doublet, doublet of doublets, doublet doublet of doublets
and multiplet, respectively. UV spectra were recorded on a Jasco V-530 UV spectrophotometer.
High-resolution MS spectra were recorded on a Bruker APEX II FT-ICR mass spectrometer using
electrospray ionization (ESI) technique in positive mode. IR spectra were recorded on a Jasco FT-IR
430 spectrophotometer. Column chromatography was performed by using silica gel 60 (70-230 mesh
ASTM, Merck). Analytical TLC analyses were performed using F254 silica gel plates (0.2 mm thick,
Merck). TLC spots were detected under UV light (254 nm).
2′,3′-O-Isopropylidene-6-chloronebularine (2). Compound 2 (102 mg) was obtained by reaction of 1
(100 mg, 0.37 mmol) according to the procedure by Kappler et al. [32]. Spectroscopic data and yields
were in agreement with those reported by the authors.
5′-Fluoro-5′-deoxy-2′,3′-O-isopropylidene-6-chloronebularine (3a) and 5′-fluoro-5′-deoxy-2′,3′-O-
isopropylidene-6-fluoro-nebularine (3b). Compound 2 (102 mg, 0.31 mmol) was reacted according to
the procedure by Ashton et al. [30]. Spectroscopic data and yields were in agreement with those
reported by the authors. A mixture (86 mg) of 3a (Rf = 0.48, hexane/AcOEt, 1:1) and 3b (Rf = 0.42,
hexane/AcOEt, 1:1) were obtained; HRESIM: m/z 351.0641 (3a), 335.0938 (3b) ([M+Na]+
C13H14ClFN4NaO3, requires 351.0636 for 3a; [M+Na]+, C13H14F2N4NaO3, requires 335.0932 for 3b).
5′-Fluoro-5′-deoxy-2′,3′-O-isopropylideneinosine (4). To a solution of 3a and 3b (86 mg, 0.27 mmol)
in dioxane (5 mL) was a added 0.1 M NaOH solution (5 mL). The mixture was stirred at room
temperature for 4 h (TLC monitoring: CHCl3/MeOH, 9:1, Rf = 0.29) and then neutralized with
Dowex-50 resin (H+ form), previously washed with H2O, MeOH and then dried under vacuum. The
mixture was filtered through a glass column [10 mm diameter, 100 mm length, with fused-in sintered
glass-disc of porosity 3 (bore of plug: 2.5 mm)] and the resin was washed several times with MeOH
and then the solvents were removed under reduced pressure. The crude was applied on a silica gel
column eluted with increasing amounts of MeOH in CHCl3 (from 0 to 10%) to afford pure 4 (60%,
50 mg, 0.16 mmol) as an amorphous white solid. Spectroscopic data of 4 were in agreement with those
reported by Spitale et al. [33].
5′-Fluoro-5′-deoxyinosine (5). Compound 4 (50 mg, 0.16 mmol) was reacted according to the
procedure by Spitale et al. [33] to give 38 mg of 5. Spectroscopic data and yields were in agreement
with those reported by the authors.

Molecules 2012, 17
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5′-Fluoro-5′-deoxy-2′,3′-di-O-acetylinosine (6). Compound 5 (38 mg, 0.14 mmol) was dissolved in a
solution of Ac2O in pyridine (4:6, v/v, 2.0 mL) and the mixture was stirred at room temperature for 1 h
(TLC monitoring: CHCl3/MeOH, 9:1). The solvents were removed under reduced pressure to afford
compound 6 that was used for the next reaction step without further purification. Foam (99%, 49 mg,
0.14 mmol); 1H-NMR (400 MHz, CD3OD) δ 8.28 (bs, 1H, H-2), 7.87 (s, 1H, H-8), 6.27 (d, J = 5.1 Hz,
1H, H-1′), 5.86–5.81 (m, 1H, H-2′), 5.70–5.64 (m, 1H, H-3′), 4.84–4.63 (m, 2H, 2 × H-5′a,b), 4.53–4.40
(m, 1H, H-4′), 2.14 (s, 3H, CH3), 2.05 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ 171.3, 171.0,
158.8, 149.8, 147.9, 140.5, 125.7, 87.9, 83.2 (d, JCF = 170.8 Hz), 82.7 (d, JCF = 18.1 Hz), 74.8, 71.2,
20.4, 20.2; 19F (400 MHz) δ (126.5–126.9) (m); m/z 377.0877 (HRESIMS) ([M+Na]+, C14H15FN4NaO6
requires 377.0873). IR (neat) max 3054, 1751, 1698, 1376, 1242, 1214, 1099 cm−1. UV (MeOH)

max 250 nm.
5′-Fluoro-5′-deoxy-2′,3′-di-O-acetyl-1-(2,4-dinitrophenyl)inosine (7). A mixture of 6 (49 mg, 0.14 mmol),
DNClB (169 mg, 0.56 mmol), and K2CO3 (77 mg, 0.56 mmol) was suspended in anhydrous DMF
(3.0 mL) and stirred at 80 °C for 3 h. The reaction was monitored by TLC (CHCl3/MeOH, 95:5). After
cooling, the mixture was filtered and the solid was washed with CHCl3. The filtrates and washings,
collected and evaporated to dryness, were applied on a silica gel column eluted with increasing
amounts of MeOH in CHCl3 (from 0 to 5%) to give pure 7, consisting of a 1:1 mixture of atropisomers
at the N(1)-phenyl bond. Pale yellow amorphous solid (96%, 71 mg, 0.13 mmol); 1H-NMR (400 MHz,
CD3OD) δ 9.03 (d, J = 2.3 Hz, 2H, 2 × H-3 DNP), 8.74 (dd, J = 8.6, 2.4 Hz, 2H, 2 × H-5 DNP), 8.49
(bs, 2H, 2 × H-2), 8.28 (bs, 2H, 2 × H-8), 8.01 (d, J = 8.6 Hz, 2H, 2 x H-6 DNP), 6.33 (t, J = 4.7 Hz,
2H, 2 × H-1′), 5.92–5.87 (m, 2H, 2 × H-2′), 5.74–5.68 (m, 2H, 2 × H-3′), 4.84–4.66 (m, 4H, 2 × H-5′a,b),
4.55–4.42 (m, 2H, 2 × H-4′a,b), 2.15 (bs, 6H, CH3), 2.09 (bs, 6H, CH3); 13C-NMR (100 MHz, CDCl3) δ
171.3, 171.1, 157.0, 149.7, 149.1, 148.4, 147.4, 141.4, 136.4, 133.4, 130.2, 124.9, 122.0, 88.1, 83.1
19
(d, JCF = 171.1 Hz), 82.9 (d, JCF = 17.8 Hz), 74.9, 71.3, 20.3, 20.2; F (400 MHz) δ (127.6–126.1)
(m); m/z 543.0891 (HRESIMS) ([M+Na]+, C20H17FN6NaO10, requires 543.0888). UV (MeOH)

max 243 nm.
5′-Fluoro-5′-deoxyacadesine (5′-F-AICAR, 8). Compound 7 (71 mg, 0.13 mmol) was dissolved in
DMF (1.0 mL) and then EDA (0.17 mL, 2.6 mmol) was added. The mixture was stirred at 50 °C
for 16 h (TLC monitoring: CHCl3/MeOH, 7:3) and then the solvents were removed under reduced
pressure. The crude was applied on a silica gel column eluted with increasing amounts of MeOH in
CHCl3 (from 0 to 20%) to afford pure 8. Amorphous white solid (70%, 26 mg, 0.091 mmol); 1H-NMR
(500 MHz, CD3OD) δ 7.32 (s, 1H, H-2), 5.58 (d, J = 5.7 Hz, 1H, H-1′), 4.71 (ddd, J = 47.5, 10.8, 2.3
Hz, 1H, H-5′a), 4.61 (ddd, J = 47.5, 10.8, 2.1 Hz, 1H, H-5′b); 4.36–4.32 (m, 1H, H-2′), 4.28–4.24 (m,
13
1H, H-3′), 4.23–4.13 (m, 1H, H-4′); C-NMR (100 MHz, CDCl3) δ 164.1, 145.2, 129.7, 113.6, 89.6,
84.9 (d, JCF = 18.4 Hz), 83.8 (d, JCF = 168.2 Hz), 75.1, 71.1; 19F (400 MHz) δ (100.9–101.3) (m); m/z
283.0822 (HRESIMS) ([M+Na]+, C9H13FN4NaO4, requires 283.0819 ). IR (neat) max 3320, 1642, 1556,
1056 cm−1. UV (MeOH) max 264 nm.

Molecules 2012, 17
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4. Conclusions
In this paper we have reported a facile synthetic approach for the preparation of the novel
compound 5′-fluoro-5′-deoxyacadesine (5′-F-AICAR) starting from commercially available
6-chloro-nebularine (1). This product may be considered a novel non-phosphorylable AICAR
derivative, whose biological activity has to be explored, and that could be a useful tool to understand
the metabolic pathways involving AICAR and ZMP. The described synthetic procedure may be
extended to the preparation of novel fluorinated base modified AICAR.
Acknowledgments
We are grateful to the “Polo delle Scienze e delle Tecnologie per la Vita” (Progetto FARO 2011,
Finanziamento per l′Avvio di Ricerche Originali), for financial support. We are grateful to Luisa
Cuorvo for her technical assistance and to CSIAS (Centro di Servizio Interdipartimentale di Analisi
Strutturale) for NMR facilities.
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Sample Availability: Samples of the compounds 6–8 are available from the authors.
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