Synthesis of new acadesine (AICA-riboside) analogues having acyclic D-ribityl or 4-hydroxybutyl chains in place of the ribose

Article abstract of DOI:10.3390/molecules18089420

The antiviral activity of certain acyclic nucleosides drew our attention to the fact that the replacement of the furanose ring by an alkyl group bearing hydroxyl(s) could be a useful structural modification to modulate the biological properties of those nucleosides. Herein, we report on the synthesis of some novel acadesine analogues, where the ribose moiety is mimicked by a D-ribityl or by a hydroxybutyl chain.

Full text of DOI:10.3390/molecules18089420

Molecules 2013, 18, 9420-9431; doi:10.3390/molecules18089420
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of New Acadesine (AICA-riboside) Analogues
Having Acyclic D-Ribityl or 4-Hydroxybutyl Chains in Place
of the Ribose
Stefano D’Errico 1, Giorgia Oliviero 1,*, Nicola Borbone 1, Jussara Amato 1, Vincenzo Piccialli 2,
Michela Varra 1, Luciano Mayol 1 and Gennaro Piccialli 1
1
Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Via D. Montesano 49,
Napoli 80131, Italy; E-Mails: stefano.derrico@unina.it (S.D.); nicola.borbone@unina.it (N.B.);
jussara.amato@unina.it (J.A.); varra@unina.it (M.P.); mayoll@unina.it (L.M.);
picciall@unina.it (G.P.)
2
Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II, Via Cintia 21,
Napoli 80126, Italy; E-Mail: vinpicci@unina.it
* Author to whom correspondence should be addressed; E-Mail: golivier@unina.it;
Tel.: +39-081-679-896.
Received: 27 May 2013; in revised form: 19 July 2013 / Accepted: 1 August 2013 /
Published: 6 August 2013
Abstract: The antiviral activity of certain acyclic nucleosides drew our attention to the fact
that the replacement of the furanose ring by an alkyl group bearing hydroxyl(s) could be a
useful structural modification to modulate the biological properties of those nucleosides.
Herein, we report on the synthesis of some novel acadesine analogues, where the ribose
moiety is mimicked by a D-ribityl or by a hydroxybutyl chain.
Keywords: AICAR; ZMP; acadesine; AMPK; AMPK activation; imidazole nucleosides;
nucleoside analogues; modified nucleosides; acyclic nucleosides; acyclic nucleotides
1. Introduction
A complete understanding of the interactions of the complex metabolic network and of its
numerous and in some cases unclear regulator mechanisms still today constitutes a challenge for many
researchers in the biological and biomedical fields. This is especially significant if it refers to cells that
are affected by diseases and survive with certain modified metabolic pathways. The nucleosides, and

Molecules 2013, 18
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their structurally related biomolecules have a very important role in the metabolism acting as synthetic
precursors and regulatory agents and being involved in signal transduction. They can be agonists or
antagonists of central enzymes in normal or altered metabolic pathways and can be useful tools to
demonstrate or affect metabolic rewiring. In most cases they have become important drugs [1–6]. In
summary they are molecules that stimulate intense research aimed at the development of new structural
analogues possessing potential regulatory or pharmacological activities [7–11].
In this context, 5-aminoimidazole-4-carboxamide riboside (acadesine or AICAR, Figure 1) has
central role, acting as both a purine biosynthetic precursor and as a modulator of a very high number of
biological properties. AICAR, after its 5'-phosphorylation to ZMP, is involved in important metabolic
pathways through the activation of the AMP-activated protein kinase (AMPK). In the cells AICAR is
phosphorylated to ZMP (Figure 1) that is a mimic of adenosine 5'-monophosphate (AMP) [12,13]. The
direct binding of ZMP to an allosteric site of AMPK causes its phosphorylation and activation by a
cellular kinase, resulting in a series of important metabolic events, including the inhibition of the basal
and insulin-stimulated glucose uptake [14,15], the inhibition of lipid synthesis and the activation of
certain ATP-generating processes such as glycolysis and fatty acid oxidation [16]. In the treatment of
ischemia, the cardio-protective effect of AICAR has been attributed to the stimulation of the release of
extracellular adenosine levels as well as to the activation of AMPK [13,17].
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 [18,19]. In particular, AICAR has been revealed to also be
an antagonist of the protein Hsp90, a chaperone that regulates the correct interaction between
proteins [20]. In such tumors Hsp90 is over-expressed, promoting aberrant cell survival and
reproduction even in hostile environments [21].
Recently, it has been 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 [22]. By using a mouse model of malignant hyperthermia having a
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 [22].
Nevertheless, AICAR is far from being a good drug lead because it has a short half-life in cells and
is not strictly specific for the AMPK enzyme; furthermore, it suffers from a number of side effects: it
increases uric acid production and favors lactic acidosis [15]. In light of the fact that AICAR can enter

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into AMPK-dependent or –independent processes [23–25], the design and synthesis of novel AICAR
derivatives/analogues could be useful to better understand how the related metabolic pathways work
and how to obtain new drug candidates.
The discovery of the antiviral activity of acyclovir and the acyclic nucleoside phosphonates [6,26,27]
has emphasized that the replacement of the furanose ring by an alkyl group bearing hydroxyl(s) could
be an interesting structural modification to induce new properties in the biological activity of the
nucleosides. In most cases, the activity of these sugar-modified nucleosides has been attributed to the
conformational freedom adopted by the alkyl chain that could, in principle, promote the recognition by
the nucleoside related enzymes and prevent the development of viral resistance [27]. Furthermore, the
conformational flexibility of acyclic nucleosides and nucleotides could also influence their base-pairing
properties. In fact, Van Aerschot et al., with the aim of discovering universal nucleoside analogues,
have inserted novel acyclic nucleoside derivatives in oligonucleotide strains, evaluating their hybridizing
properties [28].
In recent years, we have focused our attention on the preparation of new base- and sugar-modified
nucleosides and nucleotides both by classic solution chemistry and, more recently, by a solid-phase
approach [29–32], enlarging the collection of new potential antimetabolites. Herein, we report on the
synthesis of a small set of 5-aminoimidazole-4-carboxamides (AICAs) carrying D-ribityl or
4-hydroxybutyl chains at the N1-imidazole position, as well as 5-hydroxypentyl chains at the 4N
carboxamide position.
2. Results and Discussion
The preparation of imidazole nucleoside analogues having non-glycosidic linkages has usually been
accomplished by the condensation of 2-amino-2-cyanoacetate with a suitable chiral or achiral
aminoalcohol in the presence of triethylorthoformate, obtaining compounds that show potent
adenosine deaminase activities [33]. Other authors have reported on the synthesis of 1-(4-O-methyl-2-
deoxy-D-ribityl)-5-amino-4-carboxamide imidazole and its derivatives coupling the imidazole portion
with a fully protected and activated polyhydroxyalkyl chain by SN2 displacement [28].
In accordance with Hirota’s procedure [34], in the first part of our work we attempted the
preparation of derivative 5 (Scheme 1), in which a ribityl chain replaces the ribose moiety, by the
direct reductive cleavage of the C1'-O4' bond of AICAR and of 2',3'-O-isopropylidene AICAR.
However, this approach failed, resulting only in complex reaction mixtures in both cases. Therefore,
we designed a new synthetic route, on the assumption that the construction of this kind of open-ribose
AICAR could be performed starting from a suitable purine nucleoside, from which the 5-amino-4-
carboxamide moiety could be obtained through the degradation of the purine ring. In the last years we
have reported some synthetic strategies to obtain AICAR, ZMP and their 4N-alkyl derivatives, through
the purine ring degradation of suitable inosine or 5'-phosphate inosine precursors, carrying a strong
electron-withdrawing group (2,4-dinitrophenyl, DNP) at the N1 of the purine ring [29–32,35].
Considering the instability of the DNP group under the reductive conditions necessary to open the
ribose, its introduction at the N1 base position was performed after the reductive cleavage of the
C1'-O4' bond of the 2',3'-O-isopropylideneinosine [34] by reaction with DIBAL-H in dry THF that

Molecules 2013, 18
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furnished the ribitylinosine 1 in 68% yield. The hydroxyls of 1 were then protected by acetylation,
yielding compound 2 (Scheme 1).
Scheme 1. Synthesis of compounds 5 and 8.
Reagents and conditions: (i) DIBAL-H, THF, 24 h, r.t.; (ii) Ac2O, Py, 16 h, r.t.; (iii) K2CO3,
2,4-dinitro-chlorobenzene (DNClB), DMF, 3 h, 80 °C; (iv) EDA, DMF, 50 °C, 16 h; (v) 10% TFA,
0 °C then r.t.; (vi) 5-amino-pentan-1-ol, DMF, 50 °C, 16 h; (vii) 5 M NaOH, EtOH, reflux, 5 h.
Compound 2 was then reacted with 2,4-dinitrochlorobenzene (DNClB) in the presence of K2CO3 to
give the intermediate 3. This reaction served to activate the C2 position of the purine, rendering it
susceptible to nucleophilic attack by the amines, which induces the cleavage of the N3-C2 purine
bond. In particular, we have previously demonstrated that when a 1,ω-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 is composed of two or three methylene groups, AICAR is formed in a high yield [35].
Therefore, compound 3 was treated with a solution of ethylendiamine (EDA) in DMF and compound 4
was obtained (71% yield) by purine-ring opening/degradation and the concomitant deacetylation of the
4'–5' hydroxyl groups of the ribityl moiety. Finally, the isopropylidene group removal on 4 was
performed by 10% trifluoroacetic acid (TFA) treatment, affording the N1-ribityl AICA 5 quantitatively.
In order to obtain further AICAR derivatives we probed the reactivity of 3 with the
5-amino-pentan-1-ol. As expected this reaction, following a mechanism that starts with the scission of
the N3-C2 bond and then proceeds with the reclosure of the purine ring, furnished the
N1-hydroxyalkyl-inosine 6 (85% yield). During this reaction a concomitant deacetylation of the
hydroxyl groups at 4' and 5' ribityl moiety was observed.

Molecules 2013, 18
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It is well known that N1-alkylated inosines are susceptible to purine ring opening/degradation when
treated with alkali [36], affording the corresponding 4N-alkyl AICARs [31,32,35]. In accordance with
these data, 6 was refluxed with 5M NaOH solution in ethanol, giving a good yield (80%) of 7. The
reaction was followed by UV spectrophotometry, because TLC monitoring was difficult to perform.
After 5 h the disappearance of the purine band at λmax 249 nm (pH = 7) and the concomitant
appearance of the imidazole band at λmax 268 nm (pH = 7) confirmed the end of the reaction. The
isopropylidene group from 7 was removed as for 4, providing 8 in an almost quantitative yield.
The success of reactions 3→6→7 furnishes the possibility of modulating the length of the
hydroxyalkylic chains bound at the N1 (hypoxanthine) or 4N (imidazole-carboxamide) positions,
opening the way to the preparation of novel 1-hydroxyalkyl-9-(D-ribityl)-hypoxanthines and
5-amino-1-(D-ribityl)-N-(hydroxyalkyl)-imidazole-4-carboxamides, respectively.
In the second part of our synthetic work we planned to tune a synthetic procedure to obtain AICA
derivatives bearing hydroxyalkyl chains at the AICA N1 and at the 4N positions [37,38]. To achieve
this goal, we identified 6-chloropurine as a useful precursor on which to introduce the suitable
4-hydroxybutyl chain, followed by its transformation into the AICA derivative 10. Specifically, in a
previous paper we demonstrated that 6-chloropurine could be readily transformed into 9 with a good
yield by the following sequence of reaction: base-mediated alkylation on N9, acidic hydrolysis of
chlorine, acetylation of the primary hydroxyl and reaction of the resulting hypoxanthine derivative
with DNClB (Scheme 2) [39]. As for 3, the treatment of 9 with EDA in DMF produced the
N1-hydroxybutyl AICA 10 in 69% yield.
Scheme 2. Synthesis of compounds 10 and 12.
Reagents and conditions: (i) EDA, DMF, 50 °C, 16 h; (ii) 5-amino-pentan-1-ol, DMF, 50 °C, 16 h; (iii) 5 M
NaOH, EtOH, reflux, 5 h.
Alternatively, the treatment of 9 with 5-amino-pentan-1-ol produced 11 in a 78% yield, which,
unexpectedly, still retained the acetyl group, as detected by spectroscopic analyses. This fact was not
detrimental for the completion of the synthesis. In fact, as for 6, by refluxing 11 with 5 M NaOH
solution in ethanol, 12 was obtained (in 75% yield), after the purine ring degradation and hydrolysis of
the acetyl group.
The synthesis of compound 12 opened the way to the preparation of novel AICA analogues,
combining sugar alterations with base modifications on the natural skeleton of the AICA riboside.

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3. Experimental
General Methods
All the reagents were obtained from commercial sources (Sigma-Aldrich, Milano, Italy) and were
used without further purification. 1H (400 MHz) and 13C (100 MHz) spectra were acquired on a Varian
Mercury Plus 400 MHz in CD3OD or CDCl3. Chemical shifts are reported in parts per million (δ)
relative to the residual solvent signals: CD2HOD 3.31 and CHCl3 7.27 for 1H-NMR; CD3OD 49.0 and
1
CDCl3 77.0 for 13C-NMR. H-NMR chemical shifts were assigned by 2D NMR experiments. The
abbreviations s, bs, d, bd, dd and m stand for singlet, broad singlet, doublet, broad doublet, doublet of
doublets and multiplet, respectively. HPLC analyses and purifications were carried out on a Jasco
UP-2075 Plus pump equipped with a Jasco UV-2075 Plus UV detector using a 4.8 × 150 mm C-18
reverse-phase column (particle size 5 µm) eluted with a linear gradient of CH3CN in H2O (from 0 to
100% in 60 min, flow 1.3 mL min−1). UV spectra were recorded on a Jasco V-530 UV
spectrophotometer at pH = 7. High-resolution MS spectra were recorded on a Bruker APEX II FT-ICR
mass spectrometer using electrospray ionization (ESI) technique in positive mode. Optical rotations
were determined on a Jasco polarimeter using a 1 dm cell at 25 °C; concentrations are in g/100 mL. 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, Vimodrone (MI), Italy). Analytical
TLC analyses were performed using F254 silica gel plates (0.2 mm thick, Merck). TLC spots were
detected under UV light (254 nm).
9-(2',3'-O-Isopropylidene-4',5'-di-O-acetyl-D-ribityl)hypoxanthine (2). Compound 1 (200 mg,
0.64 mmol) [34] was dissolved in a solution of Ac2O in pyridine (4.0 mL, 4:6, v/v) and the mixture
was stirred at room temperature for 16 h (TLC monitoring: CHCl3/MeOH, 9:1). The solvents were
removed under reduced pressure to afford compound 2 that was used for the next reaction step without
1
further purification. Amorphous white solid (99%, 252 mg); H-NMR (CD3OD) δ 8.06 (s, 1H, 2-H),
8.04 (s, 1H, 8-H), 5.26–5.18 (m, 1H, 4'-H), 4.66 (dd, J = 12.3, 2.3 Hz, 1H, 5'-Ha), 4.63–4.57 (m, 1H,
2'-H), 4.51–4.43 (complex signal, 2H, 3'-H and 1'-Ha), 4.22 (dd, J = 13.9, 10.4 Hz, 1H, 1'-Hb), 4.14
(dd, J = 12.3, 5.2 Hz, 1H, 5'-Hb), 2.12 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 1.54 (s, 3H,
13
isopropylidene), 1.30 (s, 3H, isopropylidene); C-NMR (CDCl3) δ 170.7, 170.0, 158.5, 149.0, 145.1,
141.5, 123.4, 110.1, 75.4, 74.1, 68.9, 63.0, 44.0, 27.9, 25.4, 21.1, 20.8; m/z 417.1390 (HRESIMS)
([M+Na]+, C17H22N4NaO7 requires 417.1386). IR (neat) νmax 2,854, 1,740, 1,686, 1,219, 1,069 cm−1.
UV (MeOH) λmax 268 nm.
1-(2,4-Dinitrophenyl)-9-(2',3'-O-Isopropylidene-4',5'-di-O-acetyl-D-ribityl)hypoxanthine (3). A mixture
of 2 (150 mg, 0.38 mmol), DNClB (453 mg, 1.5 mmol), and K2CO3 (207 mg, 1.5 mmol) was
suspended in anhydrous DMF (5.0 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 3, consisting
of a 1:1 mixture of atropisomers at the N(1)-phenyl bond. Pale yellow amorphous solid (95%,
202 mg); 1H-NMR (CDCl3) δ 9.02 (d, J = 2.3 Hz, 1H, H-3 DNP), 9.00 (d, J = 2.3 Hz, 1H, H-3 DNP),

Molecules 2013, 18
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8.68 (bd, J = 8.5 Hz, 2H, 2 × H-5 DNP), 8.29 (bs, 1H, 2-H), 8.23 (bs, 1H, 2-H), 8.10 (bs, 1H, 8-H),
8.08 (bs, 1H, 8-H), 7.91–7.80 (m, 2H, 2 × H-6 DNP), 5.24–5.16 (m, 2H, 4'-H), 4.69 (dd, J = 12.4, 2.2
Hz, 1H, 5'-Ha), 4.64–4.52 (complex signal, 4H, 2 × 1'-Ha, 2 × 2'-H), 4.50–4.42 (m, 2H, 2 × 3'-H),
4.22–4.10 (complex signal, 4H, 2 × 1'-Hb, 2 × 5'-Hb), 2.18 (s, 3H, CH3CO), 2.15 (s, 3H, CH3CO), 2.11
(s, 6H, 2 × CH3CO), 1.58 (s, 3H, CH3), 1.56 (s, 3H, CH3); 1.37 (s, 3H, CH3), 1.34 (s, 3H, CH3);
13C-NMR (CDCl3) δ 170.7, 169.9, 154.2, 148.1, 147.2, 146.1, 145.7, 141.2, 135.3, 132.1, 129.0, 124.5,
121.2, 110.4, 75.2, 74.1, 68.8, 62.7, 44.5, 27.8, 25.3, 21.0, 20.7; m/z 583.1406 (HRESIMS) ([M+Na]+,
C23H24N6NaO11, requires 583.1401). IR (neat) νmax 3,445, 3,346, 3,099, 1,739, 1,546, 1,341, 1,220,
1,072 cm−1. UV (MeOH) λmax 244 nm.
5-Amino-1-(2',3'-O-Isopropylidene-D-ribityl)-1-H-imidazole-4-carboxamide (4). Compound 3 (100 mg,
0.18 mmol) was dissolved in DMF (2.0 mL) and then EDA (0.24 mL, 3.6 mmol) was added. The
mixture was stirred at 50 °C for 16 h (TLC monitoring: CHCl3/MeOH, 8:2) 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. Pale yellow amorphous solid (71%, 38 mg);
1H-NMR (CD3OD) δ 7.20 (s, 1H, 2-H), 4.46 (m, 1H, 2'-H), 4.42–4.34 (m, 1H, 1'-Ha), 4.22–4.13
(m, 1H, 3'-H), 3.98 (dd, J = 13.6, 10.5 Hz, 1H, 1'-Hb), 3.82–3.70 (complex signal, 2H, 4'-H and 5'-Ha),
3.61 (dd, J = 11.2, 5.1 Hz, 5'-Hb), 1.49 (s, 3H, isopropylidene), 1.30 (s, 3H, isopropylidene); 13C-NMR
(CD3OD) δ 167.2, 145.7, 133.1, 113.1, 110.4, 77.7, 77.4, 70.9, 65.1, 45.5, 28.2, 25.5; m/z 323.1328
(HRESIMS) ([M+Na]+, C12H20N4NaO5, requires 323.1331). IR (neat) νmax 3,428, 2,961, 1,665, 1,591,
1,262, 1,031, 801 cm−1. UV (MeOH) λmax 265 nm.
5-Amino-1-(D-ribityl)-1-H-imidazole-4-carboxamide (5). Compound 4 (20 mg, 0.066 mmol) was
dissolved in 1.0 mL of a solution of TFA in H2O (1:9, v/v) at 0 °C. After 15 min the cold bath was
removed and the mixture was shaken at room temperature for additional 2 h (TLC monitoring:
CHCl3/MeOH, 7:3). The solvents were evaporated under reduced pressure and the crude was purified
by HPLC (see General methods, tR = 17.7 min) to afford pure 5. Oil (99%, 17 mg). [α]D −9.7 (c = 0.2,
1
CH3OH). H-NMR (CD3OD) δ 7.21 (s, 1H, 2-H), 4.14–3.95 (complex signal, 3H, 1-Ha,b and 2'-H),
3.80–3.70 (complex signal, 2H, 5'-Ha and 3'-H), 3.67–3.60 (dd, J = 10.7, 5.3 Hz, 1H, 5'-Hb), 3.50–3.45
(m, 1H, 4'-H); 13C-NMR (CD3OD) δ 167.3, 146.2, 133.4, 112.9, 74.4, 73.6, 72.8, 64.4, 47.2; m/z
283.1021 (HRESIMS) ([M+Na]+, C9H16N4NaO5, requires 283.1018). IR (neat) νmax 3,341, 2,917,
1,635, 1,588, 1,265, 1,029, 796 cm−1. UV (MeOH) λmax 265 nm.
1-(5-Hydroxypentyl)-9-(2',3'-O-Isopropylidene-D-ribityl)hypoxanthine (6). Compound 3 (100 mg,
0.18 mmol) was dissolved in DMF (2.0 mL) and then 5-aminopentan-1-ol (186 mg, 1.8 mmol) was
added. The mixture was stirred at 50 °C for 16 h (TLC monitoring: CHCl3/MeOH, 8:2) and then the
solvent was 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 6. Oil (85%, 61 mg). 1H-NMR
(CD3OD) δ 8.27 (s, 1H, 2-H), 8.07 (s, 1H, 8-H), 4.72 (dd, J = 14.0, 2.4 Hz, 1H, 1'-Ha), 4.61–4.54 (m,
1H, 2'-H), 4.29 (dd, J = 14.0, 10.7 Hz, 1H, 1'-Hb), 4.26–4.19 (m, 1H, 3'-H), 4.08 (t, J = 7.3 Hz, 2H,
CH2N), 3.85–3.77 (complex signal, 2H, 4'-H, 5'-Ha), 3.64 (dd, J = 11.5, 5.6 Hz, 1H, 5'-Hb), 3.54 (t,
J = 6.4 Hz, 2H, CH2O), 1.83–1.71 (m, 2H, CH2), 1.62–1.51 (m, 2H, CH2), 1.48 (s, 3H, CH3),

Molecules 2013, 18
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13
1.46–1.36 (m, 2H, CH2), 1.26 (s, 3H, CH3); C-NMR (CD3OD) δ 158.2, 149.4, 149.2, 143.1, 124.2,
110.6, 77.3, 77.0, 70.9, 65.3, 62.5, 47.8, 45.8, 33.1, 30.5, 28.4, 25.7, 23.9; m/z 419.1911 (HRESIMS)
([M+Na]+, C18H28N4NaO6, requires 419.1907). IR (neat) νmax 3,345, 2,939, 1,682, 1,547, 1,366, 1,215,
1,070 cm−1. UV (MeOH) λmax 250 nm.
5-Amino-1-(2',3'-O-Isopropylidene-D-ribityl)-N-(5-hydroxypentyl)-1-H-imidazole-4-carboxamide (7).
Compound 6 (50 mg, 0.13 mmol) was dissolved in EtOH (2.0 mL) and then a 5 M solution of NaOH
(1.4 mL) was added. The mixture was refluxed and the reaction was monitored by UV
spectrophotometry. After 5 h the disappearance of the purine band at λmax 249 nm and the concomitant
appearance of the imidazole band at λmax 268 nm confirmed the end of the reaction. The reaction was
quenched with 1.4 mL of a 5 M solution of NH4Cl and the solvents were removed under reduced
pressure. The crude was adsorbed on silica gel and applied on a silica gel column eluted with
increasing amounts of MeOH in CHCl3 (from 0 to 10%) affording pure 7. Oil (80%, 40 mg). 1H-NMR
(CD3OD) δ 7.21 (s, 1H, 2-H), 4.50–4.42 (m, 1H, 2'-H), 4.42–4.34 (dd, J = 14.7, 1.9 Hz, 1H, 1'-Ha),
4.20–4.14 (m, 1H, 3'-H), 3.98 (dd, J = 14.7, 10.3 Hz, 1H, 1'-Hb), 3.79 (dd, J = 11.3, 2.6 Hz, 1H, 5'-Ha),
3.76–3.70 (m, 1H, 4'-H), 3.65–3.59 (dd, J = 11.3, 5.3 Hz, 1H, 5'-Hb), 3.56 (t, J = 6.5 Hz, 2H, CH2O),
3.32 (t, J = 7.0 Hz, 2H, CH2N, partially covered by solvent signal), 1.66–1.52 (m, 4H, 2 × CH2), 1.49
13
(s, 3H, CH2), 1.48–1.39 (m, 2H, CH2), 1.30 (s, 3H, CH3); C-NMR (CD3OD) δ 166.9, 144.8, 133.0,
113.8, 110.4, 77.7, 77.3, 71.0, 65.2, 62.8, 45.5, 39.6, 33.4, 30.7, 28.2, 25.5, 24.3; m/z 409.2066
(HRESIMS) ([M+Na]+, C17H30N4NaO6, requires 409.2063). IR (neat) νmax 3,324, 2,928, 1,621, 1,554,
1,245, 1,218, 1,067 cm−1. UV (MeOH) λmax 265 nm.
5-Amino-1-(D-ribityl)-N-(5-hydroxypentyl)-1-H-imidazole-4-carboxamide (8). Compound 7 (20 mg,
0.052 mmol) was dissolved in 1.0 mL of a solution of TFA in H2O (1:9, v/v) at 0 °C. After 15 min the
cold bath was removed and the mixture was shaken at room temperature for additional 2 h (TLC
monitoring: CHCl3/MeOH, 7:3). The solvents were removed under reduced pressure and the crude was
adsorbed on silica gel and applied on a silica gel column eluted with increasing amounts of MeOH in
1
CHCl3 (from 0 to 20%) affording pure 8. Oil (99%, 18 mg). [α]D −33.6 (c = 0.1, CH3OH). H-NMR
(CD3OD) δ 7.19 (s, 1H, 2-H), 4.14–3.92 (complex signal, 3H, 1'-Ha,b and 2'-H), 3.79–3.69 (complex
signal, 2H, 5'-Ha and 3'-H), 3.67–3.59 (dd, J = 10.7, 5.4 Hz, 5'-Hb), 3.59–3.52 (t, J = 6.5 Hz, 2H,
CH2OH), 3.50–3.43 (m, 1H, 4'-H), 3.32 (t, J = 7.0 Hz, 2H, CH2N, partially covered by solvent signal),
1.66–1.52 (m, 4H, 2 × CH2), 1.49–1.38 (m, 2H, CH2); 13C-NMR (CD3OD) δ 166.8, 145.3, 133.3,
113.6, 74.4, 73.6, 72.9, 64.4, 62.8, 47.1, 39.6, 33.3, 30.7, 24.2; m/z 369.1753 (HRESIMS) ([M+Na]+,
C14H26N4NaO6, requires 369.1750). IR (neat) νmax 3,341, 2,923, 1,621, 1,566, 1,253, 1,051 cm−1.
UV (MeOH) λmax 265 nm.
5-Amino-1-(4-hydroxybutyl)-1-H-imidazole-4-carboxamide (10). Compound 9 [39] (50 mg, 0.12
mmol) was dissolved in DMF (1.0 mL) and then EDA (0.16 mL, 2.4 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 10. Amorphous solid (69%, 16 mg).
1H-NMR (CD3OD) δ 7.19 (s, 1H, 2-H), 3.89 (t, J = 7.1 Hz, 2H, CH2N), 3.58 (t, J = 6.2 Hz, 2H, CH2O),

Molecules 2013, 18
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13
1.87–1.74 (m, 2H, CH2), 1.60–1.47 (m, 2H, CH2); C-NMR (CD3OD) δ 167.3, 145.5, 132.1, 112.9,
62.3, 44.3, 30.3, 27.1; m/z 221.1017 (HRESIMS) ([M+Na]+, C8H14N4NaO2, requires 221.1014).
IR (neat) νmax 3,308, 1,632, 1,555, 1,262, 1,081 cm−1. UV (MeOH) λmax 268 nm.
9-(4-Acetoxybutyl)-1-(5-hydroxypentyl)hypoxanthine (11). Compound 9 (50 mg, 0.12mmol) was
dissolved in DMF (1.0 mL) and then 5-aminopentan-1-ol (124 mg, 1.2 mmol) was added. The mixture
was stirred at 50 °C for 16 h (TLC monitoring: CH2Cl2/MeOH, 9:1) and then the solvent was removed
under reduced pressure. The crude was applied on a silica gel column eluted with increasing amounts
of MeOH in CH2Cl2 (from 0 to 10%) to afford pure 11. Oil (78%, 31 mg). 1H-NMR (CD3OD) δ 8.31
(s, 1H, 2-H), 8.07 (s, 1H, 8-H), 4.27 (t, J = 7.1 Hz, 2H, CH2OAc), 4.14–4.06 (complex signal, 4H,
2 × CH2N), 3.56 (t, J = 6.4 Hz, 2H, CH2O), 2.01 (s, 3H, CH3), 1.99–1.91 (m, 2H, CH2), 1.86–1.75
13
(m, 2H, CH2), 1.71–1.54 (complex signal, 4H, 2 × CH2); C-NMR (CD3OD) δ 172.2, 158.3, 149.4
(2C), 142.4, 124.6, 64.8, 62.6, 47.8, 44.6, 33.1, 30.5, 27.8, 26.7, 23.9, 20.7; m/z 359.1692 (HRESIMS)
([M+Na]+, C16H24N4NaO4, requires 359.1695). IR (neat) νmax 3,434, 2,923, 1,679, 1,575, 1,259, 1,031,
796 cm−1. UV (MeOH) λmax 249 nm.
5-Amino-1-(4-hydroxybutyl)-N-(5-hydroxypentyl)-1-H-imidazole-4-carboxamide (12). Compound 11
(20 mg, 0.059 mmol) was dissolved in EtOH (1.0 mL) and then 0.6 mL of a 5 M solution of NaOH
were added. The mixture was refluxed and the reaction was monitored by UV spectrophotometry.
After 5 h the disappearance of the purine band at λmax 249 nm and the concomitant appearance of the
imidazole band at λmax 268 nm confirmed the end of the reaction. The reaction was quenched with
0.6 mL of a 5 M solution of NH4Cl and the solvents were removed under reduced pressure. The crude
was purified by HPLC (tR = 25.8 min, see General Methods) affording pure 12. Oil (75%, 13 mg).
1H-NMR (CD3OD) δ 7.20 (s, 1H, 2-H), 3.9 (t, J = 7.2 Hz, 2H, CH2NC=C), 3.6 (t, J = 6.4 Hz, 2H,
CH2O), 3.50 (t, J = 6.5 Hz, 2H, CH2O), 3.31 (CH2NC=O, covered by solvent signal), 1.87–1.76 (m,
13
2H, CH2), 1.65–1.49 (m, 6H, 3 × CH2), 1.48–1.39 (m, 2H, CH2); C-NMR (CD3OD) δ 166.9, 144.6,
132.0, 113.4, 62.8, 62.3, 44.2, 39.5, 33.3, 30.7, 30.3, 27.1, 24.3; m/z 307.1749 (HRESIMS) ([M+Na]+,
C13H24N4NaO3, requires 307.1746). IR (neat) νmax 3,324, 1,621, 1,564 cm−1. UV (MeOH) λmax 268 nm.
4. Conclusions
We have here reported useful synthetic procedures to introduce alternative moieties (D-ribityl or
4-hydroxybutyl) into the ribose portion of the AICA-riboside. The obtained AICAR analogues have
the imidazole (AICA) without modifications (compounds 5 and 10) or bear the 5-hydroxypentyl chain
on a carboxamide function (compounds 8 and 12). We believe that these synthetic pathways could
enlarge the toolbox of the reactions operating on the AICA riboside (acadesine) and could furnish new
“tuneable” AICAR analogues in terms of their molecular size, flexibility and hydrogen bond formation
for their interactions with metabolic enzymes. Works are in progress to evaluate their activity into
AMPK-dependent and –independent processes in order to better understand the metabolic pathways
involving AICAR and ZMP.

Molecules 2013, 18
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Acknowledgments
This work was financially supported by “Progetto FARO 2011” (Finanziamento per l′Avvio di
Ricerche Originali). We are grateful to Luisa Cuorvo for her technical assistance, to CSIAS (Centro di
Servizio Interdipartimentale di Analisi Strutturale) for NMR facilities and to Brunella Pinto for her
contribution to the preparation of this manuscript.
Conflict of Interest
The authors declare no conflict of interest.
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