Analogues of nucleosides: Synthesis of chiral pyrrolidin-2-ones or pyrrolidines-bearing nucleobases

Article abstract of DOI:10.1007/s00706-014-1251-4

Novel analogues of nucleosides tethered on a chiral pyrrolidin-2-one were prepared, but the imide functionality was unstable under basic conditions. Thus, the carbonyl group was removed from pyrrolidin-2-one, and nucleoside analogues bearing a pyrrolidine ring were synthesized, which were unaffected under basic conditions. A nucleoside dimer was also obtained, bearing a carbamate linkage between two units.

Full text of DOI:10.1007/s00706-014-1251-4

Monatsh Chem
DOI 10.1007/s00706-014-1251-4
ORIGINAL PAPER
Analogues of nucleosides: synthesis of chiral pyrrolidin-2-ones
or pyrrolidines-bearing nucleobases
•
Gianluca Martelli Antonella Monsignori
•
•
Mario Orena Samuele Rinaldi
Received: 27 March 2014 / Accepted: 12 May 2014
Ó Springer-Verlag Wien 2014
Abstract Novel analogues of nucleosides tethered on a
chiral pyrrolidin-2-one were prepared, but the imide
functionality was unstable under basic conditions. Thus,
the carbonyl group was removed from pyrrolidin-2-one,
and nucleoside analogues bearing a pyrrolidine ring were
synthesized, which were unaffected under basic conditions.
A nucleoside dimer was also obtained, bearing a carbamate
linkage between two units.
were further introduced, in which the replacement of the
oxygen atom by a methylene group resulted in higher
metabolic and chemical stability, often leading to lower
cytotoxicity and increased bioavailability [21–23].
Results and discussion
In recent years, starting from functionalized trans-3,4-
disubstituted pyrrolidin-2-ones 1a, 1b (Fig. 1), we prepared
a lot of conformationally restricted amino acids analogues
[24, 25] and monomers of foldamers [26]. Moreover, the
2-oxo functionality was easily reduced leading to the cor-
responding pyrrolidines, whose five-membered ring is
isosteric with the carbohydrate unit of nucleosides. This
gave rise to interesting biological properties, like improved
resistance against enzymes and increased hydrolytic sta-
bility [27, 28].
Keywords Imides ꢀ Pyrrolidin-2-ones ꢀ Pyrrolidines ꢀ
Carbamate linkage ꢀ Nucleosides
Introduction
The use of nucleic acids in therapeutics [1, 2] and bio- and
nanotechnologies [3] is troubled by denaturation and/or
biodegradation [4], whereas introduction of non-natural
nucleosides may afford improved in vivo half-life [5],
better structural stability [6–11], or novel interacting
groups [12–15]. Thus, a large number of these latter
compounds have been prepared to seek out new antiviral
and anticancer agents. Among them, 20,30-dideoxynucleo-
sides (ddNs) were the most effective therapeutic agents
against human immunodeficiency virus (HIV) and hepatitis
B virus (HBV) [16, 17], together with 30-azido-20,30-dide-
oxythymidine (AZT) [18], 20,30-dideoxyinosine (DDI) [19],
and 20,30-dideoxycytidine (DDC) [20]. Moreover, since the
glycosidic linkage of these compounds can undergo both
acidic and enzymatic hydrolysis, carbocyclic derivatives
Thus, we devised that pyrrolidin-2-ones 1a, 1b [24]
could be useful starting material in order to prepare
monomeric structures bearing nucleobases and/or to build
nanostructured systems. In fact, according to our previous
work, we planned to insert these monomers into foldamers
already synthesized in our laboratory, directed to obtaining
novel structures able to recognize nucleic acid portions or
to generate strong interchain interactions.
At first, the chiral inducer at nitrogen atom was removed
from compound 1a to give 2, and subsequent acylation at
N-1 with chloroacetyl chloride afforded the imide 3 in
moderate yield. This latter, by reaction with thymine or
adenine in presence of NaI and K2CO3, afforded in low
yield derivatives 4 and 5, respectively (Scheme 1).
Although we planned to insert these products into already
prepared foldameric structures, we were disappointed by
their low stability. In fact, cleavage of the imido group
G. Martelli ꢀ A. Monsignori ꢀ M. Orena ꢀ S. Rinaldi (&)
`
Di.S.V.A. – Chemistry, Universita Politecnica delle Marche,
Via Brecce Bianche, 60131 Ancona, Italy
e-mail: s.rinaldi@univpm.it
123

G. Martelli et al.
largely occurred even under mild conditions required for
ester hydrolysis, thus making problematic the use of these
compounds for the synthesis of either diagnostic or nano-
structured foldamers stable in biological environment.
Thus, we turned our attention toward pyrrolidin-2-one
1b, and we devised it could be easily converted in few
steps into diprotected nucleoside analogues 6, tethered on a
pyrrolidine ring (Fig. 2). In fact, compound 1b was fully
reduced with LiAlH4 in refluxing THF, to give in good
yield the corresponding amino alcohol 7 that, by reaction
with Boc2O, was in turn converted into its Boc derivative
8. Subsequent benzoylation afforded derivative 9 and
eventual removal of the chiral phenylethyl group, by
reaction with 1-chloroethylchlorocarbonate, led in good
yield to diprotected 3,4-trans-disubstituted pyrrolidine 10,
used in the next step without any further purification
(Scheme 2).
acid 14, precursor of guanylacetic acid, arose from
2-amino-6-benzyloxypurine [31] (Fig. 3).
The preparation of monomers is outlined in Scheme 3.
The reaction of pyrrolidine derivative 10 with the substi-
tuted acetic acids 11–14, carried out in presence of EDC,
gave in moderate yield the four orthogonally protected
analogues of natural nucleosides 6a–6d. Having in hands
compounds 6a–6d, we directed our efforts toward the
synthesis of dimer 15, in which a carbamate linkage was
chosen in place of a phosphate group in order to obtain a
stable structure between two 6a units. At first, removal of
Boc-protecting group from compound 6a, on treatment
with trifluoroacetic acid (TFA) in DCM, gave the corre-
sponding ammonium salt 15 in good yield. On the other
hand, cleavage of the benzoyl group of 6a, carried out with
polymer-supported hydroxide anion in methanol [32], led
to the hydroxymethyl derivative 16 that in turn reacted with
phosgene in dry THF leading to the corresponding chlo-
rocarbonate 17 (Scheme 4). Eventually, dimer 18, bearing
a carbamate bridge highly resistant against hydrolytic
cleavage, was obtained in moderate yield by reaction of
compounds 15 and 17 in dichloromethane in presence of
TEA at room temperature (Scheme 5).
Then, with the aim of synthesizing monomers 6, we
prepared acetic acid derivatives 11–14 bearing a nucleo-
base (thymine, cytosine, adenine, and guanine) according
to literature methods. Thus, (thymin-1-yl)acetic acid 11
and the Cbz-cytosine derivative 12 were prepared starting
from thymine and Cbz-substituted cytosine [29], respec-
tively. Adenylacetic acid 13 was prepared starting from
adenine [30], and (2-amino-6-benzyloxypurin-9-yl)acetic
Conclusion
In summary, with the aim of obtaining novel compounds
able to give self-assembly into well-ordered nanoscale
systems, useful for therapy and diagnostics, we prepared
novel nucleoside analogues 6a–6d, tethered on a pyrroli-
dine ring isosteric of deoxyribose. In addition, the
orthogonally protected dinucleoside analogue 18, display-
ing two units linked through a carbamate bridge—a
Fig. 1 trans-3,4-Disubstituted pyrrolidin-2-ones used as starting
materials
Scheme 1
123

Analogues of nucleosides
functional group inert in a biological environment—was
prepared in moderate overall yield. The incorporation of
these analogues into foldamers, directed toward buildup of
nanostructured systems, is currently under investigation,
and results will be reported in due course.
were distilled from sodium-benzophenone, calcium
hydride, sodium, and phosphorus pentoxide, respectively,
under an argon atmosphere. Compounds 1a, 1b, and 2 were
obtained according to Ref. [24]. Compounds 11–14 were
1
obtained according to Ref. [29–31]. All H NMR and 13C
NMR spectra were found to be identical with the ones
described in the cited references.
Experimental
(3S,4R)-Methyl 4-(t-butoxycarbonylamino)-1-
(2-chloroacetyl)-5-oxopyrrolidine-3-carboxylate
1H and 13C NMR spectra were determined on a Varian
(3, C13H19ClN2O6)
1
Gemini 200 spectrometer at 200 and 50 MHz for H and
To 776 mg of compound 2 (3 mmol) [24] dissolved in
12 cm3 dry THF at -78 °C under argon atmosphere,
2.07 cm3 n-BuLi (1.6 M solution in hexane) was added.
After 30 min, 0.33 cm3 chloroacetyl chloride (4.2 mmol)
dissolved in 6 cm3 dry THF was slowly dropped, and the
reaction was stirred for 1 h. Then, the mixture was poured in
water and extracted with ethyl acetate (3 9 75 cm3) and the
organic layer was washed with brine and dried (Na2SO4).
Volatiles were removed, and the residue was purified by
silica gel chromatography (cyclohexane:ethyl acetate 70:30)
to give 692 mg (69 %) 3. Colorless oil; 1H NMR (200 MHz,
CDCl3): d = 1.43 (s, 9H), 3.53 (m, 1H), 3.67 (dd, 1H,
J = 9.6, 11.5 Hz), 3.78 (s, 3H), 4.22 (dd, 1H, J = 9.6,
11.5 Hz), 4.35 (dd, 1H, J = 7.2, 10.2 Hz), 4.70 (s, 2H), 5.31
(d, 1H, J = 7.2 Hz, NH) ppm; 13C NMR (50 MHz, CDCl3):
d = 28.6, 41.1, 43.4, 52.6, 52.9, 56.3, 81.3, 155.8, 166.4,
167.9, 171.1 ppm; ESI–MS: m/z = 357.1 ([M ? Na]?);
[a]2D0 = -21.6° cm2 g-1 (c = 0.5, CHCl3).
13C, respectively, and on a Varian MR 400 spectrometer at
400 and 100 MHz, respectively, in CDCl3 unless otherwise
reported. Chemical shifts are given as ppm from tetra-
methylsilane, and J values are given in Hertz. Optical
rotations, [a]D, were recorded at room temperature on a
Perkin-Elmer Model 241 polarimeter at the sodium D line
(concentration in g/100 cm3). LC electrospray ionization
mass spectra were obtained with a Finnigan Navigator LC/
MS single-quadrupole mass spectrometer, cone voltage
25 V and capillary voltage 3.5 kV, injecting samples dis-
solved in methanol. Elemental analyses were performed
with a Carlo Erba CHN Elemental Analyzer, and their
results were found to be in good agreement ( 0.3 %) with
the calculated values. Column chromatography was per-
formed using Kieselgel 60 Merck (230–400 mesh ASTM).
Tetrahydrofuran, dichloromethane, methanol, and DMF
General procedure for preparation of compounds
4 and 5
To a solution of 335 mg compound 3 (1 mmol) in 3 cm3
dry DMF, 252 mg thymine (2 mmol) or 270 mg adenine
(2 mmol), 691 mg dry K2CO3 (5 mmol), and 75 mg NaI
Fig. 2 Nucleosides analogues prepared from pyrrolidin-2-one 1b
Scheme 2
123

G. Martelli et al.
(0.5 mmol) were added at once. After 4 h at rt, the mixture
was poured in water and extracted with ethyl acetate
(3 9 25 cm3), and combined organic layers were washed
with brine and dried (Na2SO4). The solvent was removed
under reduced pressure and the residue was purified by
silica gel chromatography (cyclohexane : ethyl acetate
80:20) to give compound 4 or 5.
Fig. 3 Nucleobase-substituted acetic acids 11–14
Scheme 3
Scheme 4
123

Analogues of nucleosides
solution of LiAlH4 in dry THF (32 mmol) was added under
inert atmosphere, and the mixture was refluxed for 3 h.
Then, 10 cm3 methanol and 60 cm3 aqueous saturated
NH4Cl solution were sequentially added, and the mixture
was extracted with ethyl acetate (3 9 200 cm3). The
organic layer was dried (Na2SO4), filtered, and then
concentrated under reduced pressure. Purification of the
residue by silica gel chromatography (ethyl acetate:meth-
Scheme 5
1
anol 8:2) gave 1.55 g (88 %) 7. Colorless oil; H NMR
(200 MHz, CDCl3): d = 1.38 (d, J = 7.2 Hz, 3H),
1.90–2.04 (m, 1H), 2.21 (dd, J = 4.2, 5.8 Hz, 1H),
2.41–2.59 (m, 4H, 1H ? OH ? NH2), 2.64–2.85 (m,
2H), 3.33 (q, J = 7.2 Hz, 1H), 3.28–3.38 (m, 1H), 3.69
(d, J = 6.8 Hz, 2H), 7.24–7.35 (m, 5ArH) ppm; 13C NMR
(50 MHz, CDCl3): d = 16.3, 41.0, 41.6, 49.8, 60.7, 61.9,
126.8, 127.9, 128.7, 139.3, 170.4 ppm; ESI–MS: m/z =
221.2 ([M ? H]?), 243.2 ([M ? Na]?); [a]2D0
=
-32.3° cm2 g-1 (c = 0.5, CHCl3).
(3R,4S,10S)-3-(t-Butoxycarbonylamino)-4-
(hydroxymethyl)-1-(1-phenylethyl)pyrrolidine
(8, C18H28N2O3)
(3S,4R)-Methyl 4-(t-butoxycarbonylamino)-1-[2-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]-5-
oxopyrrolidine-3-carboxylate (4, C18H24N4O8)
To 1.48 g of compound 7 (6.7 mmol) and 1.1 cm3 TEA
(8 mmol) in 14 cm3 methanol, 1.6 g Boc2O (7.4 mmol)
was added, and the reaction was stirred for 2 h at room
temperature. After removal of methanol under reduced
pressure, 10 cm3 water and 100 cm3 ethyl acetate were
added, the organic layer was separated, and the aqueous
phase was extracted with ethyl acetate (2 9 100 cm3). The
combined organic layers were dried (Na2SO4), filtered, and
concentrated to give 1.98 g (92 %) of compound 8.
According to the above-reported general procedure, 178 mg
1
(42 %) 4 was obtained. Colorless viscous oil; H NMR
(200 MHz, CDCl3): d = 1.45 (s, 9H), 1.92 (s, 3H), 3.55 (m,
1H), 3.68 (m, 1H), 3.79 (s, 3H), 4.17 (dd, 1H, J = 9.2,
11.7 Hz), 4.41 (dd, 1H, J = 7.3, 9.9 Hz), 4.95 (ABq, 2H,
J = 7.4 Hz), 5.39 (d, 1H, J = 6.9 Hz, NH), 6.88 (s, 1H),
8.58 (br s, 1H, NH) ppm; 13C NMR (50 MHz, CDCl3):
d = 28.2, 41.9, 43.2, 52.4, 52.7, 56.4, 81.0, 140.5, 151.0,
155.1, 164.3, 167.4, 171.3, 171.7 ppm; ESI–MS: m/
1
Colorless oil; H NMR (200 MHz, CDCl3): d = 3.49 (d,
J = 6.6 Hz, 3H), 1.42 (s, 9H), 2.03–2.19 (m, 2H),
2.55–2.77 (m, 3H), 3.17 (q, J = 6.6 Hz, 1H), 3.54 (d,
J = 5.9 Hz, 2H), 3.56 (br s, 1H, OH), 3.79–3.92 (m, 1H),
5.13 (d, J = 7.3 Hz, 1H, NH), 7.18–7.34 (m, 5ArH) ppm;
13C NMR (50 MHz, CDCl3): d = 22.5, 28.4, 53.9, 55.2,
58.5, 64.5, 65.1, 80.0, 127.0, 127.2, 128.4, 144.3,
156.1 ppm; ESI–MS: m/z = 321.2 ([M ? H]?), 343.2
([M ? Na]?); [a]D20 = -37.6° cm2 g-1 (c = 0.5, CHCl3).
z = 447.2
(c = 0.5, CHCl3).
([M ? Na]?);
[a]2D0 = -16.4° cm2 g-1
(3S,4R)-Methyl 1-[2-(6-amino-9H-purin-9-yl)acetyl]-4-
(t-butoxycarbonylamino)-5-oxopyrrolidine-3-carboxylate
(5, C18H23N7O6)
According to the above-reported general procedure, 148 mg
1
(34 %) 5 was obtained. Colorless viscous oil; H NMR
t-Butyl [(3R,4S)-4-(hydroxymethyl)-1-[(S)-1-phenyl
(200 MHz, CDCl3): d = 1.44 (s, 9H), 3.56 (m, 1H), 3.65
(m, 1H), 3.76 (s, 3H), 4.17 (m, 1H), 4.42 (m, 1H), 5.56 (m,
2H), 5.85 (br s, 1H, NH), 6.07 (br s, 2H, NH), 7.80 (s, 1H),
8.30 (s, 1H) ppm; 13C NMR (50 MHz, CDCl3): d = 28.4,
29.8, 41.8, 43.4, 48.0, 52.9, 56.6, 81.3, 119.1, 141.3, 150.4,
153.2, 155.8, 167.1, 171.6 ppm; ESI–MS: m/z = 434.2
([M ? H]?), 456.2 ([M ? Na]?); [a]D20 = -12.3° cm2 g-1
(c = 0.5, CHCl3).
ethyl]pyrrolidin-3-yl]carbamate (9, C25H32N2O4)
To a solution containing 1.98 g of compound 8 (6.2 mmol)
in 13 cm3 dry dichloromethane, 72 mg DMAP (0.6 mmol)
was added at room temperature, followed by 0.79 cm3
benzoyl chloride (6.8 mmol). After stirring for 1 h, 20 cm3
water and 50 cm3 ethyl acetate were added, the organic
layer was separated, and the aqueous phase was extracted
with ethyl acetate (2 9 50 cm3). The combined organic
layers were washed with 10 cm3 of a saturated NaHCO3
solution, dried (Na2SO4), filtered, concentrated, and the
residue was purified by silica gel chromatography (cyclo-
hexane:ethyl acetate 9:1) to give 2.26 g (86 %) of
[(3S,4R)-4-Amino-1-[(S)-1-phenylethyl]pyrrolidin-3-
yl]methanol (7, C13H20N2O)
To a solution containing 2.90 g of compound 1b [24]
(8.0 mmol) in 20 cm3 dry THF at rt, 16 cm3 of 2 M
123

G. Martelli et al.
compound 9. Low-melting white solid; 1H NMR
(200 MHz, CDCl3): d = 1.35 (d, J = 6.6 Hz, 3H), 1.43
(s, 9H), 2.10–2.27 (m, 1H), 2.29–2.44 (m, 1H), 2.67–2.84
(m, 3H), 3.22 (q, J = 6.6 Hz, 1H), 4.01–4.16 (m, 1H), 4.34
(dd, J = 1.9, 6.3 Hz, 2H), 5.00 (d, J = 7.3 Hz, 1H, NH),
7.18–7.33 (m, 4ArH), 7.35–7.59 (m, 4ArH), 7.99–8.06 (m,
2ArH) ppm; 13C NMR (50 MHz, CDCl3): d = 22.6, 28.3,
45.3, 52.7, 54.8, 64.8, 65.8, 79.2, 126.9, 128.2, 128.3,
128.4, 129.5, 132.8, 144.4, 155.2, 166.3 ppm; ESI–MS: m/z
solvents under reduced pressure, the residue was chro-
matographed on silica gel (ethyl acetate:methanol 9:1) to
give the products 6a–6d.
[(3S,4R)-4-(t-Butoxycarbonylamino)-1-[2-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]pyrrolidin-3-
yl]methyl benzoate (6a, C24H30N4O7)
According to the above-reported general procedure,
730 mg (75 %) of compound 6a was obtained by reaction
of 10 with acid 11 [29]. White solid; m.p.: 138–139 °C; 1H
NMR (200 MHz, CDCl3, mixture of rotamers): d = 1.42
(s, 9H), 1.88 (s, 3H), 2.45–2.62 (m, 1H, 55 %), 2.63–2.78
(m, 1H, 45 %), 3.21–3.53 (m, 2H), 3.75–4.51 (m, 7H), 5.65
(d, J = 7.1 Hz, 1H), 7.00 (s, 1Het-H), 7.35–7.47 (m,
2ArH), 7.48–7.56 (m, 1ArH), 7.93–8.04 (m, 2ArH), 9.80
(s, 1H, 45 %), 9.83 (s, 1H, 55 %) ppm; 13C NMR
(50 MHz, CDCl3, mixture of rotamers): d = 12.2, 28.3,
41.9, 44.5, 47.4, 48.4, 48.6, 50.8, 51.1, 52.8, 63.7, 63.8,
80.2, 110.7, 128.4, 129.6, 130.1, 133.3, 140.9, 151.2,
155.3, 155.4, 164.1, 165.0, 166.2, 166.3 ppm; ESI–MS: m/
= 425.2 ([M ? H]?), 447.2 ([M ? Na]?); [a]2D0
=
-19.2° cm2 g-1 (c = 0.5, CHCl3).
[(3S,4R)-4-(t-Butoxycarbonylamino)pyrrolidin-3-
yl]methyl benzoate (10, C17H24N2O4)
To
a
solution containing 2.26 g of compound
9
(5.3 mmol) in 16 cm3 dry dichloromethane at 0 °C,
1.17 cm3 1-chloroethyl chloroformate (10.8 mmol) was
added, and after 30 min, the solvent was removed under
reduced pressure. The residue was dissolved in 10 cm3
methanol, and the solution was refluxed for 40 min.
After removal of the volatiles, the residue was dissolved
in 15 cm3 AcOEt, and the organic layer washed with
10 cm3 of a saturated solution of Na2CO3. The aqueous
phase was extracted with additional 50 cm3 AcOEt and,
after separation, the organic layers were dried (Na2SO4)
and evaporated under reduced pressure. Eventually, the
residue was purified by silica gel chromatography (ethyl
acetate:methanol 8:2), to give 1.45 g (85 %) of com-
pound 10. Colorless oil; 1H NMR (200 MHz, CDCl3):
d = 1.40 (s, 9H), 2.35–2.49 (m, 1H), 2.75–2.94 (m, 2H),
3.19–3.41 (m, 2H), 3.94–4.09 (m, 1H), 4.31 (dd,
J = 7.0, 11.2 Hz, 1H), 4.41 (dd, J = 6.0, 11.2 Hz,
1H), 4.81 (br s, 1H, NH), 5.39 (d, J = 6.2 Hz, 1H,
NH), 7.32–7.45 (m, 2ArH), 7.47–7.58 (m, 1ArH),
7.95–8.04 (m, 2ArH) ppm; 13C NMR (50 MHz, CDCl3):
d = 28.3, 45.8, 48.7, 52.9, 54.3, 65.2, 79.6, 128.3, 129.5,
133.0, 155.4, 166.3 ppm; ESI–MS: m/z = 321.2
([M ? H]?), 343.2 ([M ? Na]?); [a]D20 = -42.6° cm2 g-1
(c = 0.5, CHCl3).
z = 509.2
(c = 0.5, CHCl3).
([M ? Na]?);
[a]2D0 = -38.7° cm2 g-1
[(3S,4R)-1-[2-[4-(Benzyloxycarbonylamino)-2-oxo-
pyrimidin-1(2H)-yl]acetyl]-4-(t-butoxycarbonylamino)-
pyrrolidin-3-yl]methyl benzoate
(6b, C31H35N5O8)
According to the above-reported general procedure,
764 mg of compound 6b (63 %) was obtained by reaction
1
of 10 with acid 12 [29]. White solid; m.p.: 67–68 °C; H
NMR (200 MHz, CDCl3, mixture of rotamers): d = 1.42
(s, 9H), 2.45–2.71 (m, 1H), 3.22–4.39 (m, 8H), 4.47 (dd,
J = 5.0, 11.0 Hz, 1H), 4.88 (br s, 1H, NH), 5.19 (s, 2H),
5.45 (br s, 1H, NH), 7.19–7.64 (m, 8ArH ? 2Het-H),
7.95–8.05 (m, 2ArH) ppm; 13C NMR (50 MHz, CDCl3,
mixture of rotamers): d = 28.4, 38.3, 44.5, 44.7, 46.8,
47.6, 50.9, 51.3, 52.2, 52.3, 63.8, 64.0, 67.9, 68.1, 80.4,
80.7, 95.3, 127.2, 128.1, 128.3, 128.4, 128.5, 128.6, 128.8,
129.6, 130.1, 130.8, 133.5, 135.2, 149.6, 152.8, 153.1,
155.7, 155.9, 163.5, 164.9, 165.2, 166.2, 169.2 ppm; ESI–
MS: m/z = 628.3 ([M ? Na]?); [a]2D0 = -56.7° cm2 g-1
(c = 0.5, CHCl3).
General procedure for preparation of compounds
6a–6d
[(3S,4R)-1-[2-[6-(Benzyloxycarbonylamino)-9H-purin-9-
yl]acetyl]-4-(t-butoxycarbonylamino)pyrrolidin-3-yl]-
methyl benzoate (6c, C32H35N7O7)
To a solution containing 640 mg of compound 10
(2 mmol) in 4 cm3 dry dichloromethane and 1 cm3 dry
DMF, acids 11-14 (2.2 mmol) were added, followed by
0.14 cm3 TEA (1 mmol) and 460 mg EDC (2.4 mmol).
After stirring for 12 h at room temperature, 8 cm3 0.1 M
HCl and 20 cm3 ethyl acetate were added, the organic layer
was separated and the extraction with ethyl acetate was
repeated twice (2 9 40 cm3). The combined organic layers
were washed with a 10 cm3 of a saturated solution of
NaHCO3 and dried (Na2SO4). After removal of the
According to the above-reported general procedure,
630 mg (50 %) of compound 6c was obtained by reaction
1
of 10 with acid 13 [30]. White solid; m.p.: 81–82 °C; H
NMR (200 MHz, CDCl3, mixture of rotamers): d = 1.42
(s, 9H, 45 %), 1.44 (s, 9H, 55 %) 2.51–2.66 (m, 1H, 55 %),
2.69 (m, 1H, 45 %), 3.27–3.47 (m, 1H), 3.48–3.65 (m, 1H),
3.79 (s, 2H, 55 %), 3.82–4.03 (m, 1H), 4.05–4.25 (m, 1H),
4.26–4.37 (m, 1H), 4.42 (dd, J = 5.3, 11.2 Hz, 1H), 4.53
123

Analogues of nucleosides
(dd, J = 4.4, 11.2 Hz, 1H), 4.92 (br s, 1H, NH), 4.96 (s,
2H, 45 %), 5.27 (s, 2H, 55 %), 5.29 (s, 2H, 45 %),
7.28–7.64 (m, 8ArH ? 1Het-H), 7.96–8.06 (m, 2ArH),
8.15 (br s, 1H, NH), 8.69 (s, 1Het-H, 55 %), 8.72 (s, 1Het-
H, 45 %) ppm; 13C NMR (50 MHz, CDCl3, mixture of
rotamers): d = 28.3, 44.1, 44.5, 44.6, 44.7, 47.5, 51.0,
51.1, 53.0, 51.1, 53.0, 63.6, 63.7, 67.8, 80.5, 121.0, 128.5,
128.6, 129.5, 129.6, 133.3, 133.4, 135.5, 143.2, 144.2,
149.3, 151.0, 151.5, 152.8, 153.0, 155.1, 155.2, 164.0,
t-Butyl [(3R,4S)-4-(hydroxymethyl)-1-[2-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]pyrrolidin-3-
yl]carbamate (16, C17H26N4O6)
A solution of 486 mg of compound 6a (1 mmol) in 2 cm3
methanol was added at rt under argon atmosphere to a
suspension of 0.5 g Amberlite IRA 900 in the hydroxide
form [32] in 2 cm3 methanol. After stirring for 6 h, the
resin was filtered off and washed with 15 cm3 methanol.
The solvent was evaporated under reduced pressure, and
the residue was washed with 10 cm3 ethyl ether to afford
270 mg (70 %) of compound 16. White solid; m.p.:
67–68 °C; 1H NMR (200 MHz,CDCl3, mixture of rota-
mers): d = 1.46 (s, 9H, 55 %), 1.49 (s, 9H, 45 %), 1.86 (s,
3H, 55 %), 1.88 (s, 3H, 45 %), 2.18–2.31 (m, 1H, 55 %),
2.33–2.44 (m, 1H, 45 %), 3.18–3.41 (m, 1H), 3.46–4.12
(m, 6H), 4.52 (s, 2H, 55 %), 4.56 (s, 2H, 45 %), 7.31 (s,
1Het-H) ppm; ESI–MS: m/z = 405.2 ([M ? Na]?);
[a]2D0 = ?27.4° cm2 g-1 (c = 0.5, CHCl3).
166.2 ppm;
ESI–MS:
m/z = 652.3
([M ? Na]?);
[a]2D0 = -26.3° cm2 g-1 (c = 0.5, CHCl3).
[(3S,4R)-1-[2-(2-Amino-6-benzyloxy-9H-purin-9-yl)-
acetyl]-4-(t-butoxycarbonylamino)pyrrolidin-3-yl]-
methyl benzoate (6d, C31H35N7O6)
According to the above-reported general procedure,
614 mg (51 %) of compound 6d was obtained by reaction
1
of 10 with acid 14 [31]. Yellow solid; m.p.: 57–58 °C; H
NMR (200 MHz, CDCl3, mixture of rotamers): d = 1.39
(s, 9H, 35 %), 1.41 (s, 9H, 65 %), 2.41–2.68 (m, 1H),
3.22–3.49 (m, 2H), 3.73–3.89 (m, 2H), 4.02–4.18 (m, 1H),
4.24 (dd, J = 6.2, 11.5 Hz, 1H), 4.41 (dd, J = 4.9,
11.5 Hz, 1H), 4.67 (s, 2H, 65 %), 4.73 (s, 2H, 35 %),
5.03 (br s, NH2, 65 %), 5.14 (br s, 1H, NH), 5.44 (br s, 2H,
NH2, 35 %), 5.59 (s, 2H, 35 %), 5.51 (s, 2H, 65 %),
7.21–7.62 (m, 8ArH), 7.67 (s, 1Het-H), 7.94–8.05 (m,
2ArH) ppm; 13C NMR (50 MHz, CDCl3, mixture of
rotamers): d = 28.3, 44.1, 44.4, 50.7, 50.9, 52.7, 63.7,
68.0, 80.1, 114.7, 127.1, 127.9, 128.1, 128.2, 128.3, 128.4,
129.5, 130.1, 133.2, 133.3, 136.4, 140.2, 140.3, 154.1,
155.2, 155.3, 159.2, 160.9, 164.6, 164.7, 166.2, 166.3 ppm;
[(3S,4R)-4-(t-Butoxycarbonylamino)-1-[2-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]pyrrolidin-3-
yl]methyl carbonochloridate (17, C18H25ClN4O7)
To a solution containing 246 mg 16 (0.64 mmol) in 3 cm3
dry THF under argon atmosphere at rt, 0.67 cm3 of 20 %
phosgene solution in toluene (1.28 mmol) was added. After
stirring for 5 h, organics were evaporated at low temper-
ature under reduced pressure, to give directly 201 mg 17
(70 %). Low-melting white solid; 1H NMR (400 MHz,
CDCl3, mixture of rotamers): d = 1.46 (s, 9H), 1.93 (s,
3H), 2.45–2.60 (m, 1H, 50 %), 2.60–2.75 (m, 1H, 50 %),
3.30 (dd, J = 8.4 Hz, 4.0 Hz, 1H), 3.43–3.54 (m, 1H),
3.87–4.02 (m, 2H), 4.02–4.40 (m, 3H), 4.40–4.61 (m, 2H),
4.69–4.83 (m, NH, 50 %), 4.87–4.99 (m, NH, 50 %), 7.15
(s, 1Het-H, 50 %), 7.29 (s, 1Het-H, 50 %), 8.47 (s, 1H,
NH, 50 %), 8.52 (s, 1H, NH, 50 %) ppm; ESI–MS: m/
ESI–MS:
m/z = 624.3
([M ? Na]?);
[a]2D0 = -
43.6° cm2 g-1 (c = 0.5, CHCl3).
z = 467.9
(c = 0.5, CHCl3).
([M ? Na]?);
[a]2D0 = -21.5° cm2 g-1
(3R,4S)-4-[(Benzoyloxy)methyl]-1-[2-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]pyrrolidin-
3-aminium 2,2,2-trifluoroacetate (15, C21H23F3N4O7)
To a solution containing 190 mg of compound 6a
(0.4 mmol) in 3 cm3 dry dichloromethane, 1.5 cm3 TFA
was slowly added and the clear solution was stirred for 1 h
at rt. After removal of organics under reduced pressure, the
residue was co-evaporated with ethyl ether (3 9 5 cm3) to
give the compound 196 mg (98 %) of compound 15.
Colorless oil, used in the next step without any further
purification. 1H NMR (400 MHz, CD3OD, mixture of
rotamers): d = 1.87 (s, 3H), 2.80–2.93 (m, 1H, 40 %),
2.93–3.05 (m, 1H, 60 %), 3.53–3.81 (m, 2H), 3.86–4.18
(m, 4H), 4.40–4.68 (m, 3H), 7.30 (s, 1Het-H), 7.48–7.56
(m, 2ArH), 7.61–7.68 (m, 1ArH), 8.03–8.10 (m, 2ArH)
ppm; ESI–MS: m/z = 387.3 ([M-CF3COO]?), 409.2 ([M-
CF3COOH ? Na]?); [a]D20 = ?54.2° cm2 g-1 (c = 0.5,
CHCl3).
[(3S,4R)-4-[[[[(3S,4R)-4-(t-Butoxycarbonylamino)-1-
[2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)acetyl]pyrrolidin-3-yl)methoxy)carbonyl)amino)-1-
[2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)acetyl]pyrrolidin-3-yl]methyl benzoate
(18, C37H46N8O12)
To a solution containing 196 mg of compound 15
(0.39 mmol) in 1.5 cm3 dry dichloromethane, 0.11 cm3
TEA (0.8 mmol) and then 179 mg of compound 17
(0.4 mmol) were added, and the mixture was stirred for
12 h at room temperature. Then, 1 cm3 of 1 M HCl was
added, and the mixture was extracted with ethyl acetate
(3 9 10 cm3). After drying (Na2SO4) and removal of the
solvents, the residue was purified by precipitation from
dichloromethane to give 149 mg (48 %) of compound 18.
1
Low-melting spongy solid; H NMR (400 MHz, CD3OD,
123

G. Martelli et al.
8. Zhang X, Lee I, Berdis AJ (2004) Org Biomol Chem 2:1703
9. Zhang X, Motea E, Lee I, Berdis AJ (2010) Biochemistry
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(2004) Curr Med Chem 11:2585
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mixture of rotamers): d = 1.44 (s, 9H, 40 %), 1.45 (s, 9H,
60 %), 1.88 (s, 6H), 2.20–2.30 (m, 1H, 40 %), 2.32–2.44
(m, 1H, 60 %), 2.61–2.72 (m, 1H, 60 %), 2.72–2.83 (m,
1H, 40 %), 3.28–3.36 (m, 2H), 3.44–3.85 (m, 6H),
3.88–4.13 (m, 2H ? 2H, 40 %), 4.37 (dd, J = 7.0,
7.8 Hz, 2H, 60 %), 4.39–4.69 (m, 6H), 7.30–7.32 (m,
2Het-H), 7.48–7.52 (m, 2ArH), 7.59–7.67 (m, 1ArH),
8.01–8.08 (m, 2ArH) ppm; 13C NMR (100 MHz, CD3OD,
mixture of rotamers): d = 12.2, 12.3, 12.4, 28.3, 28.4,
41.2, 41.4, 42.0, 44.4, 44.5, 44.7, 48.3, 48.4, 48.6, 48.8,
50.8, 51.0, 51.2, 52.5, 52.7, 52.9, 61.2, 61.4, 63.7, 63.8,
80.3, 110.7, 110.8, 111.0, 128.4, 128.5, 129.6, 129.7,
130.2, 133.2, 140.8, 140.9, 151.0, 151.2, 151.3, 155.3,
155.4, 155.8, 156.0, 164.0, 164.1, 164.9, 166.1, 166.2,
12. Morrow JR, Iranzo O (2004) Curr Opin Chem Biol 8:192
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166.4 ppm;
ESI–MS:
m/z = 817.3
([M ? Na]?);
[a]2D0 = -32.6° cm2 g-1 (c = 0.3, CHCl3).
Acknowledgments We thank Nanodream, s.r.l. (Jesi, Ancona,
Italy) for financial support and use of Varian MR-400 NMR
spectrometer.
´
ˇ ˇ
´ ´
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