Facile solid-phase synthesis of AICAR 5-monophosphate (ZMP) and its 4-N-Alkyl derivatives

Article abstract of DOI:10.1002/ejoc.200901271

We report herein a facile, solid-phase synthesis of 5-aminol-β-D- ribofuranosylimidazole-4-carboxamide-5'-monophosphate (ZMP), a biosynthetic precursor of purine nucleotides, as well as a small collection of its 4-N-alkyl derivatives. The very difficult, direct, chemical phosphorylation of 5-amino-lβ-D-ribofuranosylimidazole-4-carboxamide (AICAR) was circumvented by installing a suitable, fully protected, phos phate group on the 5'-position of A-l-(2,4-dinitrophenyl)inosine, connected to the solid support through the 2',3'-positions, prior to the purine degradation, which led to the 5amino-imidazole-4-ca:rboxamide moiety. A plausible reaction mechanism for the formation of the ZMP imidazole was also reported.

Full text of DOI:10.1002/ejoc.200901271

FULL PAPER
DOI: 10.1002/ejoc.200901271
Facile Solid-Phase Synthesis of AICAR 5Ј-Monophosphate (ZMP) and Its
4-N-Alkyl Derivatives
Giorgia Oliviero,[a] Stefano D’Errico,[a] Nicola Borbone,[a] Jussara Amato,[a]
Vincenzo Piccialli,[b] Gennaro Piccialli,*[a] and Luciano Mayol[a]
Keywords: ZMP / Nucleotides / Solid-phase synthesis / Phosphorylation
We report herein a facile, solid-phase synthesis of 5-amino-
1-β- -ribofuranosylimidazole-4-carboxamide-5Ј-monophos-
phate (ZMP), a biosynthetic precursor of purine nucleotides,
as well as a small collection of its 4-N-alkyl derivatives. The
very difficult, direct, chemical phosphorylation of 5-amino-1-
phate group on the 5Ј-position of N-1-(2,4-dinitrophenyl)-
inosine, connected to the solid support through the 2Ј,3Ј-posi-
tions, prior to the purine degradation, which led to the 5-
amino-imidazole-4-carboxamide moiety. A plausible reaction
mechanism for the formation of the ZMP imidazole was also
reported.
D
β-D-ribofuranosylimidazole-4-carboxamide (AICAR) was cir-
cumvented by installing a suitable, fully protected, phos-
vated protein kinase (AMPK)[4] without altering the cellu-
lar levels of ATP, ADP or AMP.[5] The activation of AMPK
has been reported to be associated with a variety of benefi-
cial effects on the cardiovascular system, including an in-
crease of the regional blood flow and local adenosine con-
centration, inhibition of neutrophil activation, suppression
of platelet activation, prevention of intracoronary thrombo-
sis, prevention of oxidant-induced injury and potentiation
of the protective mechanisms induced by myocardial pre-
conditioning.[3] Furthermore, recent studies have suggested
that AMPK is a mediator of glucose transport in skeletal
muscles[6] and that its activation strongly inhibits basal and
insulin-stimulated glucose uptake, lipogenesis, glucose oxi-
dation as well as lactate production in fat cells.[7] In this
context, ZMP is expected to possess an activity higher than
that of AICAR toward AMPK activation, and both have
been indicated as promising prodrugs. Consequently, the
production of ZMP and new ZMP derivatives is an appeal-
ing objective in the field of medicinal chemistry.
Introduction
Nucleosides and nucleotides exhibit a broad spectrum of
biological activities including antiviral, anticancer, antibac-
terial and antiparasitic activity, which generally result from
their ability to inhibit specific enzymes. Both naturally oc-
curring and synthetic nucleosides and nucleotides are of
value in antiviral chemotherapy, with nucleoside mimics sig-
nificantly contributing to the arsenal of agents for the treat-
ment of diseases ranging from chickenpox to acquired im-
munodeficiency syndrome (AIDS). Imidazole nucleosides
and nucleotides closely related to their purine counterparts
have, to a large extent, been neglected, possibly because
they are not constituents of nucleic acids. However, it is
well established that imidazole nucleotides are important
intermediates in de novo purine nucleotide biosynthesis.[1]
In addition, they are also produced during histidine biosyn-
thesis and are associated with histamine metabolism.[2]
In recent years, particular attention has been paid to 5-
amino-1-β--ribofuranosylimidazole-4-carboxamide (AICAR,
Figure 1) and its phosphorylated derivative, 5-amino-imid-
azole-4-carboxamide ribotide (ZMP, Figure 1), a central in-
termediate in the de novo biosynthesis of purine nucleo-
tides.[3] AICAR is phosphorylated in cells by adenosine kin-
ase to ZMP, which mimics 5Ј-adenosine monophosphate
(AMP) and activates 5Ј-adenosine monophosphate-acti-
[a] Dipartimento di Chimica delle Sostanze Naturali, Università
degli Studi di Napoli ”Federico II”,
Via D. Montesano 49, 80131 Napoli, Italy
Fax: +39-081-678552
E-mail: picciall@unina.it
Figure 1. AICAR and ZMP (AICA ribotide).
[b] Dipartimento di Chimica Organica e Biochimica, Università
degli Studi di Napoli ”Federico II”,
As experienced by others, we found the direct chemical
preparation of ZMP from AICAR to be very difficult. It
has been reported[8] that only adenosine kinase efficiently
Via Cynthia 4, 80126, Napoli, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901271.
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G. Piccialli et al.
FULL PAPER
catalyses the phosphorylation of AICAR to ZMP. The first
Our synthetic approach used the inosine-binding solid
chemical synthesis of ZMP appeared in a paper published support 3 (0.72 mmol/g, Scheme 2) as a key intermediate.
in 1961.[9] In this work 1-methoxymethyl-2Ј,3Ј-isopropylid- This was prepared, in almost quantitative yield, by the reac-
eninosine 5Ј-di-p-nitrophenyl phosphate was converted into tion of the 4-(hydroxymethyl)-2Ј,3Ј-benzylidene-5Ј-TBDPS-
ZMP by the degradation of the purine with sodium hydrox- inosine (2, Scheme 2) with the commercially available poly-
ide and the enzymatic removal of the p-nitrophenyl groups styrene monomethoxytrityl chloride resin (MMTCl resin,
with a yield of 11–16%. In an alternative synthetic pro- 1.3 mmol/g). In this way, inosine was bound to the trityl
cedure, which used as the starting material the methyl 5- resin by a linker having two acid-labile groups (a benzyl
aminoimidazole-4-carboxylate ribonucleoside, a 40–50% trityl ether and a 2Ј,3Ј-acetal), which are selectively cleav-
yield of ZMP was obtained.[10] In addition, to the best of able by treatment with anhydrous and aqueous acid, respec-
our knowledge, only two 4-N-alkyl AICA-ribotides have tively. Intermediate 2 was, in turn, prepared in 85% yield
been synthesised so far, both showing inhibition of inosine- by the reaction of 5Ј-protected inosine (1, Scheme 2) with
monophosphate cyclohydrolase, an enzyme involved in the 4-(hydroxymethyl)benzaldehyde dimethyl acetal in the pres-
de novo purine biosynthetic pathway.[11] These 4-N-alkyl ri- ence of catalytic amounts of TsOH. This reaction proved
botides were obtained in low to moderate yields by the 5Ј- highly diastereoselective giving the stereoisomer possessing
phosphorylation of the corresponding 4-N-alkyl ribosides. the (R) configuration at the benzylidene acetal carbon (de
We report herein a fast and high-yielding, solid-phase ap- Ͼ 96%), as ascertained by a 2D-NOESY experiment, which
proach for the preparation of ZMP and 4-N-alkyl AICA- showed intense correlations between the acetal proton and
ribotides as well as evidence on the mechanism of conver- the ribose 2Ј-H and 3Ј-H protons.
sion of the purine of inosine into the imidazole ring of
ZMP.
Results and Discussion
In previous studies we had shown that AICAR and its
4-N-alkyl derivatives (Scheme 1) can be easily obtained
starting from N-1-(2,4-dinitrophenyl)inosine and N-1-alkyl-
inosines, respectively.[12] In particular, the introduction of
the strongly electron–withdrawing, 2,4-dinitrophenyl group
on N-1 of inosine increases the electrophilicity of the purine
Scheme 2. Reagents and conditions: (i) 4-(hydroxymethyl)benzalde-
hyde dimethyl acetal, p-toluenesulfonic acid (TsOH), DCM, 7 h,
reflux; (ii) MMTCl resin, pyridine, DMAP, 24 h, room temp.
C-2, allowing its reaction with amino nucleophiles. There-
fore, the treatment of the activated inosine with ethylenedi-
amine (EDA) quantitatively gave AICAR, whereas its reac-
tion with alkylamines furnished N-1-alkylinosines, which
The key step of the whole synthetic pathway (Scheme 3)
could, in turn, be converted into 4-N-alkyl AICARs by proved to be the phosphorylation of the 5Ј-OH group. In
NaOH-mediated, purine degradation.[13]
particular, the choice of suitable protecting groups on the
These precedents provided the basis for this paper, phosphate moiety was found to be crucial in order to obtain
wherein we describe the synthesis of ZMP and a collection ZMP and its 4-N-alkyl derivatives in high yields and purity.
of its 4-N-alkyl derivatives by the introduction of a suitable Thus, the desilylation of support 3 in the presence of NH4F
phosphorylation step into the synthetic routes described gave support 4, which furnished the fully 5Ј-protected inos-
above.
ine phosphate 5 upon treatment with bis-cyanoethyl-N,NЈ-
Scheme 1. Synthetic pathways leading to AICAR, N-1-alkylinosines and 4-N-alkyl AICARs.
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Solid-Phase Synthesis of AICAR 5Ј-Monophosphate
diisopropylphosphoramidite [(iPr)2NP(OCE)2], in the pres- employ an alternative phosphorylating reagent having base-
ence of tetrazole, followed by phosphate oxidation with tert- stable protecting groups. We focused our attention on the
butyl hydroperoxide (tBuOOH). The yield (95% from 4) of commercially available bis-trimethylsilylethoxy-N,NЈ-di-
the phosphorylation step was evaluated by detaching inos- isopropylphosphoramidite[14] [(iPr)2NP(OTMSEt)2], in
ine 5Ј-monophosphate (IMP) by treatment of support 5 which the TMSEt groups can be removed by fluoride or
with concentrated aqueous ammonia followed by acid. trifluoroacetic acid treatment. This phosphorylating rea-
Compound 5, suspended in dry DMF, was then treated gent has largely been employed to phosphorylate sugars
with 2,4-dinitrochlorobenzene (DNCB) in the presence of and peptides[15] but, to the best of our knowledge, it has
K2CO3 to give support 6. In an attempt to obtain ZMP never been used with nucleosides. According to the new
through a one-pot procedure, support 6 was treated with strategy, the reaction of 4 with (iPr)2NP(OTMSEt)2 in
EDA, a reagent that could both degrade the purine to THF, in the presence of tetrazole, afforded support 9 in
AICA by acting as a nucleophile on C-2 as well as deprotect high yield after the usual phosphate oxidation with
the phosphate by acting as a base capable of promoting the tBuOOH (released IMP, 95% yield). Support 9 was then
removal of the 2-cyanoethyl protecting groups by β-elimi- converted into support 10 as described above for support 5.
nation, to give, in principle, resin 7. Disappointingly, the Pleasingly, the treatment of 10 with EDA in DMF, followed
acidic treatment of a weighed amount of the resulting resin by the acidic cleavage of the nucleotidic material from 11,
1
gave a complex mixture of products, from which ZMP was gave ZMP in an overall 86% yield (from 3). H and 31P
isolated in less than 10% yield. Likewise, the reaction of 6 NMR and UV data were in full agreement with those ob-
with alkylamines failed to give the expected N-1-alkylinos- tained from the commercially available, authentic sample.
ine supports 8. We hypothesise that a partial or complete We note that the treatment of support 11 with TFA, fol-
deprotection of the phosphate took place by a reaction with lowed by washing with H2O, allowed for the release of the
either the diamine or the alkylamine, which caused the pro- nucleotidic material from the resin as well as the deprotec-
cess to take a different course. These results prompted us to tion of the phosphate group.
Scheme 3. Reagents and conditions: (i) NH4F, MeOH, reflux, 15 h; (ii) a) (iPr)2NP(OCE)2 or (iPr)2NP(OTMSEt)2, tetrazole, room temp.,
15 h; b) tBuOOH, THF, room temp., 2.5 h; (iii) concentrated NH4OH, 15 h, 55 °C; (iv) TFA (2% in DCM), room temp., 20 min, followed
by washing with H2O; (v) DNCB, K2CO3, DMF, 80 °C, 3 h; (vi) R2-NH2, DMF, 50 °C, 8 h; (vii) EDA or 1,3-diaminopropane, DMF,
50 °C, 8 h; (viii) NaOH (2  in EtOH), reflux, 5 h.
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G. Piccialli et al.
FULL PAPER
The stability of TMSEt groups under basic conditions Table 1. Products and reaction yields.
opened the way to the synthesis of 4-N-alkyl AICA-ribotide
derivatives 15c–g. In particular, we explored the possibility
of alkylating the N-1 position of the inosine followed by
opening of the purine in the presence of the fully protected
phosphate group. Thus, the reaction of 10 with some alk-
ylamines (R2-NH2, c–g, Table 1) furnished the N-1-alkylin-
osine supports 12c–g. Next, supports 12c–g were incubated
in a ethanolic NaOH to give supports 14c–g. The TFA
treatment of 14c–g furnished 4-N-alkyl AICA-ribotide de-
rivatives 15c–g in 74–77% yield (Table 1), the structures of
which were confirmed by spectroscopic data. On the other
Entry R2–NH2
Yield of
Yield of 13 Yield of 15
ZMP [%][a]
[%][a]
[%][a]
1
2
3
4
5
6
7
H2N–(CH2)2–NH2
86
85
17
8
n.f.
n.f.
n.f.
n.f.
n.f.
69
77
83
83
84
n.f.
n.f.
75
74
76
77
77
H2N–(CH2)3–NH2
H2N–(CH2)4–NH2
H2N–(CH2)5–NH2
H2N–(CH2)6–NH2
OH–(CH2)5–NH2
H3C–(CH2)3–NH2
[a] Overall yield starting from support 3; n.f.: not formed.
hand, the acid treatment of supports 12c–g provided, in ble for the purine degradation by an intramolecular attack
high yields (Table 1), N-1-alkylinosine-5Ј-phosphates 13c–g on the formamidine carbon. This hypothesis prompted us
as well. The use of 5-hydroxypentylamine (f) and bu- to test the reaction of 10 with a set of linear α,ω-diaminoal-
tylamine (g) allowed us to enlarge the chemical diversity kanes of different chain length [NH2-(CH2)n-NH2, n = 2–6].
attainable through the above synthetic routes and prove the Interestingly, we found that if the diamine was comprised of
compatibility of the chemistry with a hydroxyl-bearing side two or three methylene groups, ZMP was formed
chain on N-1.
(Scheme 4). When longer diamines (four to six methylene
Finally, we studied the reaction of N-1-(2,4-dinitrophen- groups) were used, purine reclosure was observed with the
ylinosine) support 10 with some linear α,ω-diaminoalkanes. formation of N-1-ω-aminoalkylinosines (12c–e, Scheme 5).
In particular, we wanted to shed light on the diverse reactiv- These findings suggested that if the diamine is short, the
ity of 10, which leads to N-1-alkylinosines or ZMP when terminal amino group in 16 can attack the formamidine
reacted with alkylamines or EDA, respectively.
carbon forming the cyclic orthoamide 17, which then un-
In our previous studies on the opening-reclosure of the dergoes degradation by the attack of a second diamine mo-
purine of inosine,[16] we demonstrated that a formamidine lecule at the orthoamide carbon to afford AICAR deriva-
intermediate (16, Scheme 4) was formed upon the attack of tive 18 and orthoamides 20. ZMP was eventually obtained
amino nucleophiles on C-2. We hypothesised that a similar through the attack of an additional diamine on the dini-
intermediate was formed upon the attack of EDA on 10 trophenyl ipso carbon with the concomitant formation of
and that the free, terminal, amino group could be responsi- N-substituted 2,4-dinitroanilines 19. On the other hand, a
Scheme 4. A mechanistic hypothesis that explains the formation of ZMP by the reaction of 10 with shorter α,ω-diaminoalkanes.
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Solid-Phase Synthesis of AICAR 5Ј-Monophosphate
Scheme 5. A mechanistic hypothesis for the formation of N-1-(ω-aminoalkyl)inosines by the reaction of 10 with longer α,ω-diamino-
alkanes.
sources and were used without further purification. The reactions
on solid phase were performed using glass columns [10 mm dia-
meter, 100 mm length, with fused-in sintered glass-disc of porosity
3 (bore of plug: 2.5 mm)], which were shaken in an orbital shaker.
High-temperature reactions were performed in round-bottomed
flasks. Column chromatography was performed on Merck silica gel
(Kieselgel 60, 0.063–0.200 mm); preparative thin-layer chromatog-
longer diamine possesses increased conformational free-
dom, and the alternative route (Scheme 5), leading to 12c–
e, can operate, wherein the N linked to the amidine carbon
in 16 participates in the reclosure of the purine by attacking
the carbonyl group and causing dinitroaniline displace-
ment. In summary, with a too-long diamine (n = 4–6), the
formation of cyclic orthoamides 17 is disfavoured because
an unfavourable seven- to nine-membered ring would be in-
volved. On the contrary, this step can easily take place when
raphy was performed on TLC plates (Merck, silica gel 60, F254
,
0.5 mm thick). 1H and 13C NMR spectra were acquired with a
Varian Mercury Plus 400 MHz or a Varian Unity Inova 500 MHz
the formation of a five-/six-membered ring (n = 2,3) is in- instrument using D2O, CD3OD or [D6]acetone as the solvent.
Chemical shifts were reported in parts per million (δ) relative to
the residual solvent signal [1H: 4.80 (HDO), 3.31 (CD2HOD) and
2.09 ([D5H]acetone) ppm; 13C: 49.0 (CD3OD) ppm]. 31P NMR
were performed with a Varian Unity Inova 500 MHz instrument
using 85% H3PO4 as an external standard (δ = 0 ppm). IR spectra
were recorded with a Jasco FT-IR 430 spectrometer. UV spectra
were recorded with a Jasco V-530 UV spectrophotometer. Mass
spectra were recorded with an Applied Biosystems API 2000 mass
spectrometer using electron spray ionisation (ESI) in negative
mode. RP-HPLC analyses and purifications were carried out with
a Jasco UP-2075 Plus pump, equipped with a Jasco UV-2075 Plus
volved, leading to AICAR through the purine system de-
gradation. These hypothesised mechanisms were supported
by the isolation of formamides 21 from the hydrolysis of
cyclic orthoamides 20 and 2,4-dinitroaniline 22 or N-substi-
tuted 2,4-dinitroanilines 19 from 16, depending on the
length of the diamine employed (see Schemes 4 and 5).
Conclusions
In conclusion, we have described an efficient, solid-phase
synthesis of ZMP, 4-N-alkyl AICA-ribotides and 5Ј-phos-
phate N-1-alkylinosines. The main goal of the synthesis, the
installation of the phosphate at the 5Ј position of AICAR,
has been successfully achieved by postponing the degrada-
tion of the purine until after the phosphorylation step. The
latter has been performed using (iPr)2NP(OTMSEt)2, where
the TMSEt protecting groups are stable under basic condi-
tions and removable by mild acidic treatment. In addition,
a plausible hypothesis for the formation of the 5-amino-
imidazole-4-carboxamide ring and N-1-alkylinosines has
been given by studying the reaction of N-1-(2,4-dini-
trophenyl)inosine 5Ј-phosphate with a set of linear α,ω-di-
aminoalkanes.
UV detector and
a
C-18, reverse-phase column (5 µm,
4.8ϫ150 mm), eluted with a linear gradient of CH3CN in 0.1 
triethylammonium hydrogen carbonate buffer (TEAB, pH = 7.0).
System A: from 0 to 20% CH3CN over 90 min, flow rate: 1.6 mL/
min; System B: from 0 to 100% CH3CN over 90 min, flow rate:
1.6 mL/min).
5Ј-O-TBDPS-2Ј,3Ј-O-[4-Hydroxymethyl-(1R)-benzyliden]inosine
(2): A mixture of 1 (1.0 g, 3.7 mmol), TsOH (0.19 g, 0.99 mmol)
and 4-(hydroxymethyl)benzaldehyde dimethyl acetal (9.9 mmol,
1.8 g) was suspended in dry DCM (15 mL), and the solution was
refluxed for 7 h. The reaction was monitored by TLC (AcOEt/
MeOH = 95:5). After the mixture was cooled, the solvent was re-
moved under reduced pressure, and the residue was applied to a
silica gel column and eluted with increasing amounts of MeOH in
AcOEt (from 0 to 5%) to afford pure 2 (1.6 g, 70%) as an amorph-
ous, white solid. IR (neat): ν = 3500 (broad, OH), 1690 (carbonyl)
˜
1
cm–1. H NMR (400 MHz, CD3OD): δ = 8.20 (s, 1 H, 8-H or 2-
Experimental Section
H), 7.80 (s, 1 H, 8-H or 2-H), 7.61–7.24 (complex signals, 14 H,
aromatic of TBDPS), 6.35 (d, J = 1.7 Hz, 1 H, 1Ј-H), 6.01 (s, 1 H,
CH-benzylidene), 5.52 (dd, J = 6.6, 2.0 Hz, 1 H, 2Ј-H), 5.13 (dd, J
= 6.7, 3.0 Hz, 1 H, 3Ј-H), 4.65 (s, 2 H, CH2-benzylidene), 4.54 (m,
1 H, 4Ј-H), 3.87 (dd, J = 11.2, 5.1 Hz, 1 H, 5Ј-Ha), 3.81 (dd, J =
General Methods: 4-Methoxytrityl chloride resin (MMTCl resin,
1% divinylbenzene, 200–400 mesh, 1.3 mmol/g loading) was pur-
chased from CBL Patras, Greece. Anhydrous solvents were used for
all reactions. All the other reagents were obtained from commercial
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G. Piccialli et al.
11.2, 5.1 Hz, 1 H, 5Ј-Hb), 0.99 (s, 9 H, tBu) ppm. 13C NMR relevant portions of the plate were then scratched from the plates
FULL PAPER
(100 MHz, CD3OD, assignments were made by an HSQC experi-
ment): δ = 20.0 (C-Si) 27.3 (3 CH3, tBu), 64.8 (CH2Ph), 65.5 (C-
5Ј), 83.8 (C-4Ј), 86.4 (C-2Ј), 89.1 (C-3Ј), 91.9 (C-1Ј), 108.8 (CH
acetal), 126.2 (C-5), 127.9 (C-2 and C-6 benzylidene), 128.2 (C-3
and C-5 benzylidene), 128.7, 128.8, 130.9, 131.0, 134.0, 134.3, 135.7
(aromatic), 136.7 (C-1 benzylidene), 136.7 (aromatic), 141.3 (C-8),
144.7 (C-4 benzylidene), 146.5 (C-2), 149.2 (C-4), 158.8 (C-6) ppm.
ESI-MS: m/z = 625 [M + H]+.
and eluted with CHCl3/MeOH (6:4) to give 19a and 21a or 19b
and 21b.
N-(2,4-Dinitrophenyl)-1,2-diaminoethane (19a): Amorphous yellow
1
solid. H NMR (400 MHz, [D6]acetone): δ = 9.03 (d, J = 2.7 Hz,
1 H, 3-H phenyl), 8.34 (dd, J = 9.6, 2.7 Hz, 1 H, 5-H phenyl), 7.38
(d, J = 9.6 Hz, 1 H, 6-H phenyl), 3.81 (m, 2 H, CH2NH), 3.62 (t,
J = 5.9 Hz, 2 H, CH2NH2) ppm. ESI-MS: m/z = 227 [M + H]+.
N-(2,4-Dinitrophenyl)-1,3-diaminopropane (19b): Amorphous yel-
low solid. 1H NMR (400 MHz, [D6]acetone): δ = 9.02 (d, J =
2.7 Hz, 1 H, 3-H phenyl), 8.33 (dd, J = 9.5, 2.7 Hz, 1 H, 5-H
phenyl), 7.31 (d, J = 9.5 Hz, 1 H, 6-H phenyl), 3.72 (m, 2 H,
CH2NH), 3.41 (t, J = 5.7 Hz, 2 H, CH2NH2) ppm. 2.09 (CH2,
obscured by the residual solvent signal); ESI-MS: m/z = 241 [M +
H]+.
N-(2-Aminoethyl)formamide (21a): Oil. 1H NMR (500 MHz,
CD3OD): δ = 8.13 (s, 1 H, formamide), 3.41 (t, J = 6.1 Hz, 2 H,
CH2NH), 2.91 (t, J = 6.1 Hz, 2 H, CH2NH2) ppm. ESI-MS: m/z
= 89 [M + H]+.
Loading of 2 onto MMTCl Resin–Solid Support 3: A mixture of 2
(1.0 g, 1.6 mmol) and DMAP (0.23 mmol, 0.028 g), coevaporated
with dry pyridine (3ϫ1.5 mL) and dissolved in dry pyridine
(6 mL), was added to the MMTCl resin (0.9 g, 1.1 mmol), and the
mixture was gently shaken for 24 h at room temp. The obtained
support 3 (inosine loading of 0.72 mmol/g) was filtered and washed
with DCM (3ϫ5 mL), DCM/MeOH (1:1, 3ϫ5 mL) and MeOH
(3ϫ5 mL) and finally dried under reduced pressure. The reaction
yield (96%) was calculated both by measuring the weight increase
of the support and analysing and quantitating the nucleoside mate-
rial detached from the solid support by treatment with 2% TFA in
DCM (8 min, room temp.).
N-(2-Aminopropyl)formamide (21b): Oil. 1H NMR (400 MHz,
CD3OD): δ = 8.05 (s, 1 H, formamide), 3.31 (CH2NH, obscured
by the residual solvent signal), 2.69 (t, J = 6.1 Hz, 2 H, CH2NH2),
1.68 (m, 2 H, CH2) ppm. ESI-MS: m/z = 103 [M + H]+.
ZMP: Solid support 3 (0.50 g, 0.36 mmol) was desilylated by treat-
ment with NH4F according to a reported procedure.[17] Support 4
(0.22 g, 0.37 mmol), previously swollen in dry THF, was treated
with (iPr)2NP(OTMSEt)2 (1.6 g, 4.4 mmol) and tetrazole (0.48 g,
6.6 mmol) in dry THF/DCM (9:1, 4.5 mL) and shaken for 15 h at
room temp. After the resin was filtered, the solid support was
washed with THF (3ϫ5 mL), THF/MeOH (1:1, 3ϫ5 mL) and
MeOH (3ϫ5 mL) and finally dried in vacuo. The resin was treated
with tBuOOH (5.5  in decane, 2.2 mL, 12.4 mmol) in THF
(4.5 mL) for 2.5 h at room temp. and then washed with THF
(3ϫ5 mL), THF/MeOH (1:1, 3ϫ5 mL) and MeOH (3ϫ5 mL) to
give support 9 with a 0.67 mmol/g inosine-5Ј-monophosphate load-
ing. The reaction yield (95%) was evaluated by detaching inosine-
5Ј-monophosphate by treating the resin with 2% TFA in DCM
for 20 min at room temp., washing it with H2O and collecting the
filtrates.
13c–g: Support 10 (0.10 g, 0.059 mmol), obtained as described
above for the synthesis of ZMP, swollen in DMF, was incubated
with the appropriate amine (c–g, 5.0 mmol, Table 1) in DMF
(1.5 mL) under shaking for 8 h at 50 °C. After the resin was filtered
and washed with DMF (3ϫ5 mL), DMF/MeOH (1:1, 3ϫ5 mL)
and MeOH (3ϫ5 mL), supports 12c–g were dried under reduced
pressure. The yields of N-1-alkylinosine 5Ј-monophosphates 13c–g
(69–84% from 3, Table 1) were calculated by UV quantitation of
the products obtained after HPLC purification (System B, see the
General Methods) of the crude nucleotidic material released from
a weighed amount of resin 12c–g by treating it with 2% TFA in
DCM for 20 min at room temp., washing it with H2O and collect-
ing the filtrates. In particular, 19 mg of 13c, 21 mg of 13d, 23 mg
1
of 13e, 23 mg of 13f and 22 mg of 13g were obtained. H and 31P
To the solid support 9 (0.30 g, 0.2 mmol), suspended in dry DMF
(6 mL), K2CO3 (0.14 g, 1.0 mmol) and DNCB (0.30 g, 1.0 mmol)
were added, and the reaction was maintained at 80 °C for 3 h. After
the resin was filtered, the support was washed with DMF
(3ϫ5 mL), DMF/H2O (1:1, 3ϫ5 mL), H2O (3ϫ5 mL), H2O/
MeOH (1:1, 3ϫ5 mL) and MeOH (3ϫ5 mL) and then dried under
reduced pressure to give support 10.
NMR, UV, IR and MS data confirmed the purity and the structure
of the products (see below).
15c–g: Supports 12c–g (0.10 g, 0.060–0.062 mmol) were suspended
in ethanolic NaOH (2.0  in EtOH, 2.0 mL) for 5 h at reflux. After
the resin was cooled, it was filtered, washed with EtOH (3ϫ5 mL),
EtOH/H2O (1:1, 3ϫ5 mL), H2O (3ϫ5 mL) and MeOH (3ϫ5 mL)
and finally dried under reduced pressure, giving supports 14c–g.
The yields of 4-N-alkyl AICA ribotides 15c–g (74–77%, from 3)
were calculated by UV quantitation of the products obtained after
HPLC purification (System B, see the General Methods) of the
crude nucleotidic materials released from a weighed amount of
resin 14c–g by treating it with 2% TFA in DCM for 20 min at room
temp., washing it with H2O and collecting the filtrate. In particular,
21 mg of 15c, 21 mg of 15d, 23 mg of 15e, 22 mg of 15f and 21 mg
of 15g were obtained. 31P NMR, UV, IR and MS data confirmed
the purity and the structure of the products (see below).
Support 10 (0.10 g, 0.059 mmol) was incubated with EDA (0.30 g,
5.0 mmol, a, Table 1) or 1,3-diaminopropane (0.37 g, 5.0 mmol, b,
Table 1) in DMF (4.5 mL) under shaking for 8 h at 50 °C. After
the resin was filtered and washed with DMF (3ϫ5 mL), DMF/
MeOH (1:1, 3ϫ5 mL) and MeOH (3ϫ5 mL), support 11 was
dried under reduced pressure. Cleavage of the nucleotidic material
was performed by treating the resin with 2% TFA in DCM for
20 min at room temp., washing it with H2O and collecting the fil-
trates. The crude material, purified according to the procedure re-
ported in the General Methods (System A), furnished 18 mg (86%
from 3) of pure ZMP, which showed chromatographic behaviour
and spectroscopic data identical to those exhibited by an authentic
specimen. The washings of resin 11 from the reaction of support
10 with EDA or 1,3-diaminopropane, collected as described above,
were dried under reduced pressure. The crude material was purified
on TLC plates using CHCl3/MeOH (7:3) as the solvent system. The
bands were detected either by direct exposure to UV light (254 nm)
or by treatment of a narrow strip of the plates with ninhydrin. The
1-(4-Aminobutyl)inosine 5Ј-Monophosphate (13c): Amorphous so-
lid. IR (neat): ν = 3600–2600 (broad, OHs and NH ), 1681 (strong,
˜
2
C=O), 1069, 973 (both strong, phosphate) cm–1. 1H NMR
(500 MHz, D2O): δ = 8.51 (s, 1 H, 8-H or 2-H), 8.39 (s, 1 H, 8-H
or 2-H), 6.10 (d, J = 5.8 Hz, 1 H, 1Ј-H), 4.80 (2Ј-H, obscured by
the residual solvent signal), 4.52 (m, 1 H, 3Ј-H), 4.40 (m, 1 H, 4Ј-
H), 4.19 (t, J = 6.9 Hz, 2 H, CH2N), 4.05 (m, 2 H, 5Јa,b-H), 3.04
(t, J = 7.4 Hz, 2 H, CH2NH2), 1.89 (m, 2 H, CH2), 1.72 (m, 2 H,
1522
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 1517–1524

Solid-Phase Synthesis of AICAR 5Ј-Monophosphate
1
CH2) ppm. 31P NMR (202 MHz, D2O): δ = 3.81 (s) ppm. UV cm–1. H NMR (400 MHz, D2O): δ = 7.51 (s, 1 H, 2-H), 5.64 (d,
(H2O): λmax = 250 nm; ESI-MS: m/z = 418 [M – H]–.
J = 7.0 Hz, 1 H, 1Ј-H), 4.66 (m, 1 H, 2Ј-H), 4.44–4.39 (m, 1 H, 3Ј-
H), 4.30 (m, 1 H, 4Ј-H), 4.06–3.97 (m, 2 H, 5Јa,b-H), 3.34 (t, J =
6.1 Hz, 2 H, CH2N), 2.99 (t, J = 7.3 Hz, 2 H, CH2NH2), 1.69 (m,
2 H, CH2), 1.61 (m, 2 H, CH2), 1.42 (m, 2 H, CH2) ppm. 31P NMR
(202 MHz, D2O): δ = 2.81 (s) ppm. UV (H2O): λmax = 267 nm;
ESI-MS: m/z = 422 [M – H]–.
1-(5-Aminopentyl)inosine 5Ј-Monophosphate (13d): Amorphous so-
lid. IR (neat): ν = 3600–2600 (broad, OHs and NH ), 1682 (strong,
˜
2
C=O), 1058 and 941 (both strong, phosphate) cm–1. 1H NMR
(500 MHz, D2O): δ = 8.45 (s, 1 H, 8-H or 2-H), 8.37 (s, 1 H, 8-H
or 2-H), 6.10 (d, J = 5.8 Hz, 1 H, 1Ј-H), 4.80 (2Ј-H, obscured by
the residual solvent signal), 4.50 (m, 1 H, 3Ј-H), 4.37 (m, 1 H, 4Ј-
5-Amino-1-(β-D-ribofuranosyl)imidazole-4-[N-(6-aminohexyl)]carb-
H), 4.15 (t, J = 6.9 Hz, 2 H, CH2N), 4.13–4.06 (m, 2 H, 5Јa,b-H), oxamide 5Ј-Monophosphate (15e): Amorphous solid. IR (neat): ν =
˜
2.98 (t, J = 7.5 Hz, 2 H, CH2NH2), 1.83 (m, 2 H, CH2), 1.70 (m, 3600–2600 (broad, OHs and NH2s), 1681 (strong, C=O), 1627
2 H, CH2), 1.42 (m, 2 H, CH2) ppm. 31P NMR (202 MHz, D2O): (strong, amide II band), 1132 and 991 (both strong, phosphate)
1
δ = 2.15 (s) ppm. UV (H2O): λmax = 250 nm; ESI-MS: m/z = 432
cm–1. H NMR (400 MHz, D2O): δ = 7.52 (s, 1 H, 2-H), 5.65 (d,
J = 6.8 Hz, 1 H, 1Ј-H), 4.63 (m, 1 H, 2Ј-H), 4.43–4.39 (m, 1 H, 3Ј-
H), 4.31 (m, 1 H, 4Ј-H), 4.09–4.04 (m, 2 H, 5Јa,b-H), 3.34 (br. t, 2
H, CH2N), 3.19 (q, J = 7.3 Hz, CH2 triethylammonium), 2.98 (t, J
= 7.2 Hz, 2 H, CH2NH2), 1.70–1.54 (m, 4 H, 2 CH2), 1.46–1.35
(m, 4 H, 2 CH2), 1.27 (t, J = 7.3 Hz, CH3 triethylammonium) ppm.
[M – H]–.
1-(6-Aminohexyl)inosine 5Ј-Monophosphate (13e): Amorphous so-
lid. IR (neat): ν = 3600–2600 (broad, OHs and NH ), 1671 (strong,
˜
2
C=O), 1175 and 1126 (both strong, phosphate) cm–1. 1H NMR
(400 MHz, D2O): δ = 8.42 (s, 1 H, 8-H or 2-H), 8.37 (s, 1 H, 8-H
or 2-H), 6.10 (d, J = 5.9 Hz, 1 H, 1Ј-H), 4.80 (2Ј-H, obscured by
the residual solvent signal), 4.49 (m, 1 H, 3Ј-H), 4.37 (m, 1 H, 4Ј-
H), 4.16–4.09 (complex signal, 4 H, CH2N, 5Јa,b-H), 3.18 (q, J =
7.3 Hz, CH2 triethylammonium), 2.96 (t, J = 7.7 Hz, 2 H,
CH2NH2), 1.79 (m, 2 H, CH2), 1.64 (m, 2 H, CH2), 1.39 (m, 4 H, 2
CH2), 1.27 (t, J = 7.3 Hz, CH3 triethylammonium) ppm. 31P NMR
(202 MHz, D2O): δ = 1.26 (s) ppm. UV (H2O): λmax = 250 nm;
ESI-MS: m/z = 446 [M – H]–.
31P NMR (202 MHz, D2O): δ = 2.93 (s) ppm. UV (H2O): λmax
268 nm; ESI-MS: m/z = 436 [M – H]–.
=
5-Amino-1-(β-
D-ribofuranosyl)imidazole-4-[N-(5-hydroxypentyl)]-
carboxamide 5Ј-Monophosphate (15f): Amorphous solid. IR (neat):
ν = 3600–2600 (broad, OHs and NH ), 1683 (strong, C=O), 1635
˜
2
(strong, amide II band), 1133 and 991 (both strong, phosphate)
1
cm–1. H NMR (500 MHz, D2O): δ = 7.52 (s, 1 H, 2-H), 5.66 (d,
J = 7.01 Hz, 1 H, 1Ј-H), 4.65 (m, 1 H, 2Ј-H), 4.42 (m, 1 H, 3Ј-H),
4.32 (m, 1 H, 4Ј-H), 4.06 (m, 2 H, 5Јa,b-H), 3.61 (t, J = 6.5 Hz, 2
H, CH2O), 3.34 (t, J = 6.4 Hz, 2 H, CH2N), 3.20 (q, J = 7.3 Hz,
CH2 triethylammonium), 1.59 (m, 4 H, 2 CH2), 1.41 (m, 2 H, CH2),
1.28 (t, J = 7.3 Hz, CH3 triethylammonium) ppm. 31P NMR
(202 MHz, D2O): δ = 3.54 (s) ppm. UV (H2O): λmax = 266 nm;
ESI-MS: m/z = 423 [M – H]–.
1-(5-Hydroxypentyl)inosine 5Ј-Monophosphate (13f): Amorphous
solid. IR (neat): ν = 3600–2600 (broad, OHs), 1678 (strong, C=O),
˜
1
1201 and 956 (both strong, phosphate) cm–1. H NMR (400 MHz,
D2O): δ = 8.44 (s, 1 H, 8-H or 2-H), 8.39 (s, 1 H, 8-H or 2-H), 6.12
(d, J = 5.8 Hz, 1 H, 1Ј-H), 4.75 (m, 1 H, 2Ј-H), 4.49 (m, 1 H, 3Ј-
H), 4.38 (m, 1 H, 4Ј-H), 4.17–4.09 (complex signal, 4 H, CH2N
and 5Јa,b-H), 3.58 (t, J = 6.5 Hz, 3 H, CH2O), 3.19 (q, J = 7.3 Hz,
CH2 triethylammonium), 1.81 (m, 2 H, CH2), 1.58 (m, 2 H, CH2),
1.39 (m, 2 H, CH2), 1.27 (t, J = 7.3 Hz, CH3 triethylammonium)
ppm. 31P NMR (202 MHz, D2O): δ = 1.25 (s) ppm. UV (H2O):
λmax = 250 nm; ESI-MS: m/z = 433 [M – H]–.
5-Amino-1-(β-D-ribofuranosyl)imidazole-4-(N-butyl)carboxamide 5Ј-
Monophosphate (15g): Amorphous solid. IR (neat): ν = 3600–2600
˜
(broad, OHs and NH2), 1624 (strong, C=O), 1559 (strong, amide
1
II band), 1081 and 982 (both strong, phosphate) cm–1. H NMR
(400 MHz, D2O): δ = 7.51 (s, 1 H, 2-H), 5.65 (d, J = 6.8 Hz, 1 H,
1Ј-H), 4.63 (m, 1 H, 2Ј-H), 4.41 (m, 1 H, 3Ј-H), 4.30 (m, 1 H, 4Ј-
H), 4.05 (m, 2 H, 5Јa,b-H), 3.32 (br. t, 2 H, CH2N), 3.19 (q, J =
7.3 Hz, CH2 triethylammonium), 1.54 (m, 2 H, CH2), 1.35 (m, 2
H, CH2), 1.27 (t, J = 7.3 Hz, CH3 triethylammonium), 0.90 (t, J =
7.2 Hz, 3 H, CH3) ppm. 31P NMR (202 MHz, D2O): δ = 2.78 (s)
ppm. UV (H2O): λmax = 266 nm; ESI-MS: m/z = 393 [M – H]–.
1-Butylinosine 5Ј-Monophosphate (13g): Amorphous solid. IR
(neat): ν = 3600–2600 (broad, OHs), 1683 (strong, C=O), 1057 and
˜
1
945 (both strong, phosphate) cm–1. H NMR (400 MHz, D2O): δ
= 8.38 (s, 1 H, 8-H or 2-H), 8.43 (s, 1 H, 8-H or 2-H), 6.11 (d, J
= 5.9 Hz, 1 H, 1Ј-H), 4.75 (m, 1 H, 2Ј-H), 4.49 (m, 1 H, 3Ј-H),
4.37 (m, 1 H, 4Ј-H), 4.15–4.08 (complex signal, 4 H, CH2N and
5Јa,b-H), 3.18 (q, J = 7.3 Hz, CH2 triethylammonium), 1.75 (m, 2
H, CH2), 1.34 (m, 2 H, CH2), 1.26 (t, J = 7.3 Hz, CH3 triethylam-
monium), 0.91 (t, J = 7.4 Hz, 3 H, CH3) ppm. 31P NMR
(202 MHz, D2O): δ = 1.33 (s) ppm. UV (H2O): λmax = 250 nm;
ESI-MS: m/z = 403 [M – H]–.
Supporting Information (see also the footnote on the first page of
this article): 1H and 13C NMR spectra of 2, 1H and 31P NMR
spectra of 13c–g and 15c–g, and 1H NMR spectra of 19a,b and
21a,b.
5-Amino-1-(β-D-ribofuranosyl)imidazole-4-[N-(4-aminobutyl)]carb-
Acknowledgments
oxamide 5Ј-Monophosphate (15c): Amorphous solid. IR (neat): ν =
˜
3600–2600 (broad, OHs and NH2s), 1626 (strong, C=O), 1558
We are grateful to the Italian Ministero dell’Università e della
Ricerca Scientifica e Tecnologica (MURST) (PRIN 2007), for fin-
ancial support in favour of this investigation. We are also grateful
to the Centro di Servizio Interdipartimentale di Analisi Strumen-
tale of the University of Napoli Federico II for NMR facilities. The
authors are thankful to Dr Luisa Cuorvo for technical assistance.
(strong, amide II band), 1082 and 975 (both strong, phosphate)
1
cm–1. H NMR (500 MHz, D2O): δ = 7.52 (s, 1 H, 2-H), 5.65 (d,
J = 6.8 Hz, 1 H, 1Ј-H), 4.66 (m, 1 H, 2Ј-H), 4.45–4.39 (m, 1 H, 3Ј-
H), 4.31 (m, 1 H, 4Ј-H), 4.10–3.99 (m, 2 H, 5Јa,b-H), 3.38 (m, 2 H,
CH2N), 3.03 (br. t, J = 7.6 Hz, 2 H, CH2NH2), 1.77–1.61 (m, 4 H,
2 CH2) ppm. 31P NMR (202 MHz, D2O): δ = 4.38 (s) ppm. UV
(H2O): λmax = 268 nm; ESI-MS: m/z = 408 [M – H]–.
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˜
3600–2600 (broad, OHs and NH2s), 1626 (strong, C=O), 1563
(strong, amide II band), 1082 and 974 (both strong, phosphate)
Eur. J. Org. Chem. 2010, 1517–1524
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1523

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Received: November 5, 2009
Published Online: January 28, 2010
1524
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 1517–1524