Synthesis of hydrazino-peptide nucleic acid monomers and dimers as new PNA backbone building blocks

Article abstract of DOI:10.1016/j.tet.2007.02.102

We describe the synthesis of new hydrazinoPNA (hydPNA) monomers and new hydPNA-containing dimers. For the hydPNA monomers, the primary terminal amino group of the aminoethylglycine unit of classical aegPNA is replaced by a hydrazine moiety. An appropriate

Full text of DOI:10.1016/j.tet.2007.02.102

Tetrahedron 63 (2007) 4108–4119
Synthesis of hydrazino-peptide nucleic acid monomers
and dimers as new PNA backbone building blocks
Paolangelo Cerea,a Clelia Giannini,a Sergio Dall’Angelo,a Emanuela Licandro,a,b,
Stefano Maioranaa,b and Rosangela Marchellic
*
aDipartimento di Chimica Organica e Industriale, Universitaꢀ degli Studi di Milano, Via Venezian 21, I-20133 Milan, Italy
bCentro Interdisciplinare Studi Bio-molecolari e Applicazioni Industriali (CISI), Via Fantoli 16/15, I-20138 Milan, Italy
cDipartimento di Chimica Organica ed Industriale, Viale G. P. Usberti 17/a, Campus Universitario, Universitaꢀ di Parma,
I-43100 Parma, Italy
Received 14 December 2006; revised 8 February 2007; accepted 22 February 2007
Available online 25 February 2007
Abstract—We describe the synthesis of new hydrazinoPNA (hydPNA) monomers and new hydPNA-containing dimers. For the hydPNA
monomers, the primary terminal amino group of the aminoethylglycine unit of classical aegPNA is replaced by a hydrazine moiety. An
appropriate choice of two orthogonal protecting groups on the two hydrazine nitrogen atoms makes it possible to drive their coupling with
other monomers selectively on one or the other nitrogen atom, thus obtaining two different types of PNA dimers. These dimers represent new
building blocks that can be used to generate novel PNA oligomers.
Ó 2007 Published by Elsevier Ltd.
1. Introduction
PNA is a very interesting and potentially useful tool in mo-
lecular biology, and may have both diagnostic and therapeu-
tic applications.7–9
Peptide nucleic acid (PNA) is an artificial DNA mimic intro-
duced by Nielsen in 1991,1,2 which has a pseudopeptide
backbone replacing the sugar-phosphate chain. The back-
bone is made of N-(2-aminoethyl)glycine units linked in
a polyamide structure, and the purine (A, G) and pyrimidine
nucleobases (C, T) are linked to the a-nitrogen atom of the
amino acid unit through methylene carbonyl residues. In
this way, the repeating unit consists of six atoms, exactly as
in DNA and RNA.
Within the framework of a research project aimed at design-
ing and synthesising new PNA monomers, we have recently
prepared some organometallic conjugates (Fig. 1) in which
aFischer-typechromiumcarbene,10,11 atricarbonylchromium-
arene moiety12 and one or more ferrocenyl unit13,14 have
been introduced as potential spectroscopic and electrochemi-
cal probes. All of these new bio-organometallic conjugates
have interesting spectroscopic and electrochemical proper-
ties,15 and are therefore potentially useful for diagnostic
purposes.16
B
O
O
N
N
H
Despite these interesting and useful properties, a number of
drawbacks justify the search for new PNA mimics that over-
come the existing limited water solubility,17 tendency to
self-aggregation and poor cell permeability.18
n
PNA
B = nucleobase
Although its backbone structure is completely different from
that of natural nucleic acids, N-(2-aminoethyl)glycine PNA
(aegPNA) has high binding affinity and specificity3–6 for the
complementary single strands of DNA (ssDNA) and RNA,
which is at least partially attributable to the absence of elec-
trostatic repulsion between the neutral molecule of PNA and
the polyanionic target DNA and RNA. For these reasons,
Another quite important aspect is the presence of molecular
constraints within the backbone that can influence the effi-
ciency and specificity of PNA–DNA binding. A number of
studies have been carried out in an attempt to increase the
binding affinity of PNA to complementary DNA and RNA
by modifying the rigidity of the PNA backbone, for example,
Nielsen et al.19 have reported that the flexibility of a PNA
backbone bearing a tertiary amine instead of the tertiary
amide reduces DNA binding affinity, whereas Appella
* Corresponding author. E-mail: emanuela.licandro@unimi.it
0040–4020/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.tet.2007.02.102

P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
4109
B
B
B
O
O
O
H
H
N
O
O
O
R
β
N
(OC)5M
N
PG
N
N
N
N
N
H
OMe
O
N
H
NHAr
ArCr(CO)3
N
H
O
H
O
M = Cr, W
B
A
B
Oligomer of N -hydPNA
O
PG
N
B
B
N
H
NHAr
O
O
O
O
N
N
N
N
O
Fc
NH2
NH2
Fc = ferrocenyl
B = nucleobase, PG = protecting group
B
Oligomer of N -hydPNA
Figure 1. Organometallic conjugates of PNA monomers.
B = nucleobase
et al.20 have recently found that a PNA decamer in which the
secondary amide bond is replaced by a more flexible second-
ary amine has the same binding affinity for the complemen-
tary DNA strand as the unmodified oligomer.
Figure 3.
In principle, hydPNA monomers could be oligomerised as
such in order to obtain type A and B homo-oligomers of hy-
dPNA (Fig. 3), or could be inserted as co-monomers in
aegPNA oligomers. In both cases, two different oligomerisa-
tion sites should be available: the terminal (Na) and the inter-
nal (Nb) nitrogen atoms of the hydrazine moiety. The
appropriate choice of the two protecting groups (PG1 and
PG2 in Fig. 2) would assure the possibility of driving the
growth of the PNA oligomer chain on the terminal (Na) or in-
ternal (Nb) nitrogen atom. In the first case, type A homo-olig-
omers would beobtained with a seven-atom repetitiveunit; in
the second case, type B oligomers would be formed with
a six-atom repetitive unit and pendant amino groups (Fig. 3).
Marchelli et al. showed that a PNA oligomer containing
three modified monomers with chiral lysines (a chiral box)
is excellent at discriminating mismatched and matched tar-
gets; moreover, the chiral box allowed the formation of the
anti-parallel PNA–DNA duplex, whereas the parallel
PNA–DNA duplex failed to form.21,22 The molecular basis
of this selectivity was described in detail by solving the crys-
tal structure of the PNA–DNA duplex.23
These results indicate that a chiral constraint in the middle of
a PNA sequence greatly affects direction selectivity in DNA
complexation. The effect of the substitution of the a- or g-
carbon of the PNA backbone,24 and its stereochemistry,25
has more recently been studied in detail. The relevance of
these studies is well exemplified by the fact that some of
these modified PNAs have been used in cell systems, such
as tumoral or stem cells,26,27 or in advanced molecular diag-
nostics.28,29
The presence of the additional nitrogen atom should give
hydPNA oligomers particular chemico-physical characteris-
tics, such as a reduced loss of entropy in the duplex
formation due to the highly expected rigidity of the PNA
oligomers of Na-hydPNA and, perhaps, increased water sol-
ubility for both oligomers due to the presence of the hydro-
philic amino groups. Moreover, the presence of the amino
groups in both A and B should make it possible to obtain
poly-cationic PNA oligomers whose binding affinity for
complementary ssDNA should be enhanced.
However, as no conclusive data are yet available concerning
the importance of other molecular constraints in the PNA
backbone, research in this field still seems to be useful.
In order to achieve and verify these objectives, we synthes-
ised new hydPNA monomers and looked for the best condi-
tions in which to couple hydPNA and aegPNA, which
required the formation of a hydrazide bond in view of the
aim to use the resulting dimers as building blocks to make
PNA oligomers.
In an attempt to contribute towards solving some of the above
problems, we have designed new PNA monomers; the hydr-
azinoPNA30 of general formula 1 (hydPNA, Fig. 2), in which
the terminal amino group of aegPNA is replaced by a hydr-
azine moiety that can be protected by two orthogonal protect-
ing groups at the nitrogen atoms.
We here describe the optimised synthetic methods for pre-
paring new hydPNA monomers and new hydPNA-contain-
ing dimers.
B
B
O
(PG2)PG1
HN
O
O
O
N
N
H2N
OR
N
OR
PG2(PG1)
1, hydPNA
2. Results and discussion
aegPNA
As stated above, the synthesis of oligomers A and B requires
appropriately designed hydPNA monomers.
PG1, PG2 = orthogonal protecting groups
B = nucleobase
In particular, obtaining type A oligomers requires the use of
Nb-protected hydPNA monomers in which the unprotected
Figure 2. Aminoethylglycine- and hydrazinoPNA (aeg- and hydPNA)
monomers.

4110
P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
B
O
O
H
N
H
N
O
O
H
N
+
(Boc)Cbz
N
OCH3
B
N
HO
(Boc)Cbz
N
OCH3
Boc(Cbz)
Boc(Cbz)
8, (9)
2-7
10-12
Cbz
Cbz
NH
NH
O
N
N
N
NH
O
N
3, 6, 11: B =
2, 5, 10: B =
4, 7, 12: B =
N
O
N
N
Scheme 1. Retrosynthetic scheme for hydPNA monomers.
terminal amino group (Na) is available for coupling with
a carboxylic acid function of a second monomer, whereas
obtaining type B oligomers requires the preparation of
monomers in which the internal nitrogen atom (Nb) is free
for coupling. This strategy based on the use of orthogonal
protecting groups led us to synthesise the new compounds
Na-Cbz,Nb-Boc 2–4, and Na-Boc,Nb-Cbz 5–7, substituted
with the three nucleobases thymine, cytosine and adenine
(Scheme 1).
group using Dess–Martin periodinane. The subsequent chain
elongation to give the hydrazinoPNA backbone 8 was
achieved in high yield by means of the reductive amination
of aldehyde 16 with theglycine methyl ester using NaBH3CN
and ZnCl2.32
Compounds 15, 16 and 8 are new, and are completely char-
acterised by means of spectroscopic data.
All of the compounds shown in Scheme 2 were obtained in
high yield; no column chromatography purification was nec-
essary for compounds 14 and 15 and so the whole sequence
was completed within one week.
Using the Boc strategy for oligomerisation (i.e., deprotecting
the Boc amino group and growing the oligomer chain upon
it), type B oligomers (Fig. 3) should be obtainable from
monomers 2–4 and type A oligomers from monomers 5–7.
Similarly, the hydPNA backbone 9 was synthesised follow-
ing the strategy shown in Scheme 3.
Retrosynthetic analysis of compounds 2–7 led us to synthe-
sise the two backbones 8 and 9 from which to obtain the
target new hydPNA monomers 2–7 through the coupling
of nucleobases 10–12 (Scheme 1).
Cbz
Cbz
a
b
H
N
Boc
N
N
N
H
OH
H2N
OH
71%
80%
H2N
OH
The first backbone 8 (with the Cbz group on the terminal
nitrogen atom and the Boc group on the internal nitrogen
atom) was prepared following the synthetic sequence shown
in Scheme 2.
17
18
13
88%
c
Boc
Boc
O
Cbz
N
a
b
H
N
H
N
H
N
Cbz
d
N
N
Boc
N
H
OH
H2N
OH
>98%
93%
Boc
N
OCH3
H2N
OH
N
H
O
54%
Cbz
13
14
15
9
19
c
90%
Scheme 3. Reagents: (a) Cbz-Cl, Et3N, CH2Cl2; (b) (Boc)2O, Et3N, THF;
(c) (i) Dess–Martin periodinane, CH2Cl2; (ii) Na2S2O3, NaHCO3, H2O,
MTBE; (d) H2NCH2CO2CH3$HCl, MeOH, NaBH3CN, CH3COOH.
Boc
N
O
H
d
Boc
N
Cbz
N
OCH3
N
H
This time, the first step was to protect the internal nitrogen
atom of compound 13 with the Cbz group by reacting 13
with Cbz-Cl, followed by protecting the terminal amino
group (Na) with Boc. Oxidation of the obtained alcohol 18
with Dess–Martin periodinane gave the expected aldehyde
19, which was converted into the backbone 9 by means of
reductive amination.
43%
O
NH
Cbz
16
8
Scheme 2. Reagents: (a) (Boc)2O, EtOH; (b) Cbz-Cl, aq NaOH, CH2Cl2; (c)
(i) Dess–Martin periodinane, CH2Cl2; (ii) Na2S2O3, NaHCO3, H2O, MTBE;
(d) H2NCH2CO2CH3$HCl, Et3N, ZnCl2, NaBH3CN, MeOH.
The commercially available N-2-hydroxyethylhydrazine 13
was chosen as the starting compound. The internal nitrogen
atom was protected to give the Boc derivative 14,31 and
protecting the external amino group with Cbz-Cl gave the or-
thogonally diprotected compound 15. The primary alcoholic
function of 15 was then oxidised in high yield to the formyl
Compounds 18 and 9 are new and are completely character-
ised by means of spectroscopic data, whereas 1733 and 1934
have been previously described. Unlike compounds 14 and
15, the analogous compounds 17 and 18 required column
chromatography purification.

P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
4111
O
N
At this point, all that was necessary to obtain the target hy-
O
N
H3C
O
H3C
O
dPNA monomers 2–7 was to introduce the nucleobases
onto the glycine nitrogen atom of 8 and 9, which was
achieved in good yield using standard conditions for cou-
pling the nucleobases with aegPNA.35 In particular, 8 and
9 were coupled with the 1-carboxymethyl derivatives of
nucleobases 10–12 in the presence of 3,4-dihydro-3-hydroxy-
4-oxo-1,2,3-benzotriazine (DhbtOH), N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide hydrochloride (EDC$HCl)
and diisopropylethylamine (DIEA) (Scheme 4).
NH
NH
O
O
O
O
H
N
N
N
N
H
OCH3
Cbz
N
Boc
20, hydPNA-aegPNA
O
The new PNA monomers 2–7, the first target of our project,
constitute the building blocks necessary for the construction
of hydPNA oligomers. They can be easily and efficiently ob-
tained using the synthetic methodologies shown in Schemes
2–4.
H3C
NH
O
N
O
Cbz
N
O
OCH3
Boc
N
N
N
H
N
H
O
The next step was to set up and optimise the coupling condi-
tions between the hydPNA and aegPNA monomers to give
the new dimers 20–22, which have either a classical amide
(compound 20) or hydrazide-type bond (compounds 21
and 22) (Fig. 4). This work was much less simple than ex-
pected.
O
N
O
NH
H3C
21, aegPNA-N hydPNA
O
O
N
O
In order to synthesise these dimers, it was necessary to find
the appropriate conditions for methyl ester hydrolysis and
Boc deprotection in the hydPNA monomers.
H3C
O
H3C
O
NH
NH
O
O
N
O
O
The ester group in compounds 2 and 5 was hydrolysed very
efficiently and easily by means of a reaction with aq potas-
sium hydroxide to give the corresponding acids 23 and 24
in high yields (Scheme 5).
Boc
N
N
N
N
H
OCH3
HN
Cbz
22, aegPNA-N hydPNA
Figure 4. New PNA dimers.
The two new hydPNA acids 23 and 24 are stable, and are
completely characterised by means of spectroscopic data.
Similarly, in a preliminary study aimed at finding the appro-
priate conditions for selectively deprotecting the Na- or Nb-
Boc groups in hydPNA, we found that this could be easily
and efficiently achieved using trifluoroacetic acid in di-
chloromethane. In this way, compounds 2, 3, 5 and 6 gave
monomers 25–28, with a free amino group suitable for the
following coupling steps (Scheme 6).
As we were able to deprotect the ester function and Na-Boc
or Nb-Boc selectively, we set up the conditions to synthesise
the target dimers 20–22 between aegPNA and hydPNA. The
monomer 23 was reacted with the aegPNA monomer 29 as
shown in Scheme 7, and the new dimer 20 was isolated after
purification on column chromatography in 92% yield. The
B
O
Cbz
Cbz
O
O
O
H
N
HN
B
+
HN
N
N
OCH3
OH
N
OCH3
Boc
Boc
8
10-12
2: B = Thymine
75%
3: B = Cytosine (Cbz) 71%
4: B = Adenine (Cbz) 44%
B
O
Boc
Boc
O
O
O
H
HN
N
HN
N
+
B
N
OCH3
N
OCH3
OH
Cbz
Cbz
9
10-12
5: B = Thymine
82%
6: B = Cytosine (Cbz) 92%
7: B = Adenine (Cbz) 70%
Scheme 4. Reagents and conditions: DhbtOH, DIEA, EDC$HCl, DMF, 16–48 h, rt.

4112
P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
O
N
O
T
T
H3C
O
H3C
O
O
O
NH
NH
O
O
H
N
+
N
N
O
N
O
Cbz
N
OH
H2N
OCH3 TFA
Cbz
HN
Cbz
HN
Boc
O
O
95%
N
N
23
29
N
OCH3
N
OH
92%
Boc
Boc
2
23
T
T
O
O
O
O
H
N
O
N
O
N
N
N
H3C
H3C
Cbz
N
N
H
OCH3
NH
NH
Boc
O
O
20
O
O
Boc
Boc
O
O
T = thymine
Scheme 7. Reagents: DCC, PFP, DIEA, DMF.
95%
HN
N
HN
N
N
OH
N
OCH3
Cbz
Cbz
5
24
Scheme 5. Reagents: (i) 2 M aq KOH; (ii) 1 M aq HCl.
After verifying the good reactivity of the free carboxylic acid
group in hydPNA monomer 23 (Scheme 7), we checked the
reactivity of the hydrazine moiety at the positions of Na-Boc
(compound 5) and Nb-Boc (compound 2), which were de-
protected as shown in Scheme 6 and reacted with Boc-
aegPNA-COOH 32 (Scheme 9) to afford dimers 21 and 22
in 65 and 56% yield, respectively.
B
B
O
O
Cbz
HN
Cbz
O
O
N
HN
N
N
OCH3
N
OCH3 TFA
77%
H
Boc
2: B = T
25: B = T
The reaction conditions shown in Scheme 9 are the result of
a study carried out in order to optimise the formation of the
hydrazide bond. The unprotected amino groups in both hy-
dPNA 25 and 27 (Nb and Na, respectively) showed unexpect-
edly less reactivity than the corresponding amino group of
aegPNA and so their reaction with the carboxylic function
of aegPNA 32 under the conditions shown in Scheme 7
only gave 5% of the corresponding dimers after 30 h. On
the contrary, by coupling 25 and 27 with 32 in the presence
of EDC$HCl, DhbtOH and DIPEA (Scheme 9), we could
isolate the target dimers 22 and 21 in satisfactory yields, al-
though this required long reaction times (24–48 h). The new
dimers, 21 and 22, are stable crystalline compounds, and
have been completely characterised.
3: B = C(Cbz)
26: B = C(Cbz)
B
B
O
O
Boc
HN
O
O
N
H2N
N
80,70%
N
OCH3
N
OCH3 TFA
Cbz
Cbz
5: B = T
6: B = C(Cbz)
27: B = T
28: B = C(Cbz)
Scheme 6. Reagents: TFA, CH2Cl2.
use of HBTU as the condensing agent instead of DCC and
pentafluorophenol (PFP) led to a lower yield (76%).
The new dimer 20 could be deprotected at the ester function
by means of LiOH in methanol to give acid 30 in 50% yield,
and at the Nb-Boc site by means of 20% TFA in CH2Cl2 to
give 31 in 98% yield (Scheme 8).
3. Conclusions
We here describe the synthetic methodologies for obtaining
new PNA monomers with a modified backbone (hydPNA
T
T
O
O
O
O
H
N
N
N
Cbz
N
N
H
OH
a
T
T
Boc
50 %
O
O
30
O
O
H
N
N
N
Cbz
N
N
H
OCH3
T
T
b
O
O
Boc
> 98%
O
O
H
20
N
N
N
Cbz
N
H
N
H
OCH3
TFA
31
Scheme 8. Reagents: (a) (i) LiOH, MeOH; (ii) 0.5 M aq KHSO4; (b) TFA, CH2Cl2.

P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
4113
T
T
T
O
O
O
.TFA
H2N
Cbz
N
O
O
O
Boc
N
N
Boc
N
OCH3
+
N
H
N
H
OH
N
OCH3
N
H
N
65%
O
Cbz
O
32
27
21
T
T
T
T
T
.TFA
O
O
O
O
O
O
O
O
H
N
Boc
N
N
Boc
N
N
N
N
H
OH
Cbz
N
OCH3
N
H
OCH3
+
56%
H
HN
Cbz
32
25
22
Scheme 9. Reagents: EDC$HCl, DhbtOH, DIPEA, dry DMF.
€
2–7) in which the primary terminal amino group of the
aminoethylglycine unit of classical aegPNA is replaced by
a hydrazine moiety. We have shown that an appropriate
choice of the two orthogonal protecting groups on the hydr-
azine nitrogen atoms makes it possible to drive their coupling
with other monomers on one or the other nitrogen atom, thus
obtaining two different types of new PNA dimer (21 and 22).
Melting points were obtained by Buchi Melting Point B-540
and (dec) indicates decomposition. Mass spectra were re-
corded using a Thermo Finnigan LCQ Advantage; high-reso-
lution mass spectra were recorded using a Bruker Daltonics
ICR-FTMS APEX II.
4.1.1. Methyl-N-[2-(Na-benzyloxycarbonyl-Nb-tert-bu-
toxycarbonyl-hydrazino)ethyl]-N-[(thymin-1-yl)acetyl]-
glycinate (2). To a solution of 8 (394.5 mg, 1.0 mmol) in
dry DMF (9 mL) under vigorous stirring, 10 (209 mg,
1.1 mmol), DhbtOH (186 mg, 1.1 mmol) and DIEA
(200 mL, 1.2 mmol) were added. The mixture was cooled to
0 ꢀC in an ice bath and EDC$HCl (228 mg, 1.2 mmol) was
added portionwise in 30 min. The mixture was allowed to
warm to rt. Thereaction was stirred for 22 h while monitoring
by TLC (eluent: ethyl acetate/petroleum ether 8:2, Rf ¼0.24).
The solvent was removed in vacuo and the residue was
partitioned between water (15 mL) and ethyl acetate
(35 mL). The organic layer was washed with 0.1 M aq
KHSO4 (2ꢁ10 mL), water (2ꢁ10 mL), satd aq NaHCO3
(4ꢁ20 mL) and brine (1ꢁ10 mL). The aqueous phases
were combined and extracted with ethyl acetate (3ꢁ50 mL).
All of the monomers and dimers described above are new
building blocks that can be used to generate the novel
PNA oligomers, whose binding affinity for complementary
DNA strands is currently being investigated.
4. Experimental
4.1. General
All reagents were obtained from commercial suppliers and
used without further purification. Dry DMF and dry metha-
nol over molecular sieves were obtained from Fluka. THF
was dried over sodium/benzophenone. Dess–Martin period-
inane was prepared according to the literature.36 Thymine-1-
acetic acid was obtained from Aldrich. Compounds 11 and
12 were prepared according to the literature.37,38
The organic phases were dried over Na2SO4, filtered and the
filtrate was concentrated to dryness to afford 475 mg of
crude product as a dark orange oil. Column chromatography
on silica gel (gradient from ethyl acetate/petroleum ether 9:1
to ethyl acetate/methanol 7:3) afforded 2 (427 mg, 75%) as
Unless otherwise specified, all of the reactions were per-
formed in an inert atmosphere under dry conditions.
1
a white solid: mp¼89–94 ꢀC (dec); H NMR (300 MHz,
The IR spectra were recorded on a Perkin–Elmer FTIR
1725X1. 1H and 13C NMR were recorded on Bruker AC200,
AC300 and AMX300 machines, and the chemical shifts (d)
are reported in parts per million relative to solvent peak.
Many of the compounds described below, in particular 20–
22, 30 and 31, showed many rotamers and consequently
CDCl3) (two rotamers): d 9.26 (br s, 1H), 7.83 (s, 1H),
7.39–7.35 (m, 5H), 6.99 (ma.) and 6.95 (mi.) (s, 1H), 5.14
(ma.) and 5.11 (mi.) (s, 2H), 4.52 (ma.) and 4.38 (mi.) (s,
2H), 4.18 (mi.) and 4.07 (ma.) (s, 2H), 3.68 (s, 3H), 3.76
(m, 2H), 3.61 (m, 2H), 1.85 (s, 3H), 1.34 (s, 9H); 13C
NMR (75 MHz, CDCl3): d 169.5, 169.3, 167.2, 164.2,
155.1, 151.1, 141.3, 135.7, 128.4, 110.5, 81.5, 67.5, 52.3,
48.2, 47.6, 47.5, 46.0, 27.9, 12.2; mass spectrum ESI m/z
570.2143 (C25H33N5O9+Na requires 570.2176); IR (film,
CCl4, cmꢂ1): 3403, 1754, 1695, 1369, 1110, 1068, 629.
1
had complex H NMR patterns. Some of the signals in the
1H NMR are therefore described as major (ma.) and minor
(mi.) components, wherever possible; however, in many
cases, some minor signals were obscured by the major signal
of another proton. In such cases, only the major signal is re-
ported or a range containing two or more CH2. In some
cases, the 13C spectrum shows many peaks that cannot be
distinguished in a small range: ov. means ‘overlapped’.
4.1.2. Methyl-N-[2-(Na-benzyloxycarbonyl-Nb-tert-bu-
toxycarbonyl-hydrazino)ethyl]-N-{[4-N-(benzyloxycar-
bonyl)cytosin-1-yl]acetyl}glycinate (3). To a solution of 11

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P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
(135 mg, 0.46 mmol) and 8 (158 mg, 0.41 mmol) in dry
DMF (2 mL), DhbtOH (75 mg, 0.46 mmol) and DIEA
(80 mL, 0.46 mmol) were added under stirring. The mixture
was cooled to 0 ꢀC in an ice bath and then EDC$HCl (88 mg,
0.46 mmol) was added portionwise. The mixture was al-
lowed to warm to rt. The pH was adjusted to 9 with DIEA.
The reaction was stirred overnight. The solvent was removed
in vacuo and the residue, a yellow oil, was partitioned be-
tween water (7.5 mL) and ethyl acetate (17 mL). The or-
ganic layer was washed with 0.1 M aq KHSO4 (2ꢁ5 mL),
water (2ꢁ6 mL), satd aq NaHCO3 (4ꢁ11 mL) and brine
(1ꢁ5 mL). All the aqueous phases were combined and
extracted with ethyl acetate (2ꢁ25 mL). These organic
phases were combined with the first and dried over
Na2SO4. After filtration, the filtrate was concentrated
in vacuo to afford 352 mg of crude product as a pale yellow
solid. Column chromatography on silica gel (ethyl acetate/
petroleum ether 9:1) afforded 3 (196.3 mg, Rf ¼0.14, 71%)
as a white solid: mp¼128.6–130.9 ꢀC (dec); 1H NMR
(200 MHz, CDCl3) (two rotamers): d 8.36 (br s, 1H), 7.60
(d, J¼7 Hz, 1H), 7.48–7.33 (m, 10H), 7.23 (d, J¼7 Hz,
1H), 5.21 (ma.) and 5.14 (mi.) (s, 4H), 4.75 (ma.) and 4.55
(mi.) (s, 2H), 4.18 (mi.) and 4.07 (ma.) (s, 2H), 3.69 (m,
2H), 3.72 (mi.) and 3.62 (ma.) (s, 3H), 3.61 (m, 2H), 1.31
(br s, 9H); 13C NMR (75 MHz, CDCl3): d 169.8 (mi.) and
169.4 (ma.), 167.7 (mi.) and 166.9 (ma.), 162.2, 155.7,
155.2, 152.8, 150.2, 136.2, 127.6–128.2, 95.2, 81.7, 67.5,
52.6 (mi.) and 52.2 (ma.), 49.7, 48.2, 47.1, 46.3, 27.9;
mass spectrum ESI m/z 689.2511 (C32H38N6O10+Na re-
quires 689.2547); IR (Nujol, cmꢂ1): 3250, 3055, 1754,
1670, 1266, 740–699.
4.1.4. Methyl-N-[2-(Nb-benzyloxycarbonyl-Na-tert-bu-
toxycarbonyl-hydrazino)ethyl]-N-[(thymin-1-yl)acetyl]-
glycinate (5). To a solution of 10 (574 mg, 3.12 mmol) and 9
(1080 mg, 2.83 mmol) in dry DMF (7 mL), DhbtOH
(508 mg, 3.11 mmol) and DIEA (532 mL, 3.12 mmol) were
added. The mixture was cooled to 0 ꢀC in an ice bath and
then EDC$HCl (596 mg, 3.12 mmol) was added portionwise
with vigorous stirring. The mixture was allowed to warm to
rt. The pH was adjusted to basic conditions with DIEA. The
reaction was stirred for 16 h. The solvent was removed in va-
cuo and the residue was partitioned between water (45 mL)
and ethyl acetate (90 mL). The organic layer was washed
with 0.1 M aq KHSO4 (2ꢁ30 mL), water (2ꢁ30 mL), satd
aq NaHCO3 (2ꢁ30 mL) and brine (1ꢁ25 mL). The organic
phase was dried over Na2SO4, filtered and the filtrate was
concentrated in vacuo to afford 1.6 g of crude product as
a pale yellow oil.
Column chromatography on silica gel (ethyl acetate/petro-
leum ether 9:1) afforded 5 (1.28 g, Rf¼0.14, 82%) as a white
solid: mp¼120 ꢀC (dec); 1H NMR (300 MHz, CDCl3) (mix-
ture of rotamers): d 8.99 (s, 1H), 7.37–7.27 (m, 5H), 7.07–
6.98 (m, 1H), 5.17–5.14 (m, 2H), 4.6–3.9 (m, 4H), 3.79–
3.65 (m, 7H), 1.90–1.86 (m, 3H), 1.47–1.39 (m, 9H); 13C
NMR (75 MHz, CDCl3): d 169.5, 167.5, 164.2, 156.0,
155.0, 151.0, 141.3 and 140.7, 135.7, 129.0–127.1, 110.7,
81.0, 68.0, 52.3, 49.1, 47.7, 47.4, 46.0, 28.0, 12.2; mass spec-
trum ESI m/z 570.2149 (C25H33N5O9+Na requires
570.2173); IR (film, CH2Cl2, cmꢂ1): 3306, 2918, 1679,
1471, 1415, 1368, 1252, 1214, 1161, 1082, 1048, 1021,
756, 698.
4.1.3. Methyl-N-[2-(Na-benzyloxycarbonyl-Nb-tert-bu-
toxycarbonyl-hydrazino)ethyl]-N-{[6-N-(benzyloxycar-
bonyl)adenin-9-yl]acetyl}glycinate (4). To a solution of 12
(151 mg, 0.46 mmol) and 8 (160 mg, 0.42 mmol) in dry
DMF (1.9 mL), DhbtOH (75 mg, 0.46 mmol) and DIEA
(81 mL, 0.46 mmol) were added. The mixture was cooled
to 0 ꢀC in an ice bath and then EDC$HCl (89 mg,
0.47 mmol) was added portionwise in 1 h. The mixture
was allowed to warm to rt. The pH was brought to 8.5 with
DIEA. The reaction was stirred for 21 h at rt. The solvent
was removed in vacuo and the residue was partitioned be-
tween water (18 mL) and ethyl acetate (48 mL). The organic
layer was washed with 0.1 M aq KHSO4 (2ꢁ15 mL), water
(2ꢁ15 mL), satd aq NaHCO3 (2ꢁ30 mL) and brine
(1ꢁ15 mL). All the aqueous phases were combined and ex-
tracted with ethyl acetate (2ꢁ100 mL). These organic phases
were combined with the first and dried over Na2SO4. After
filtration, the filtrate was concentrated to dryness to afford
271 mg of crude product as a pale yellow solid. Column
chromatography on silica gel (ethyl acetate) afforded 4
(87 mg, Rf¼0.35, 44%) as a white solid: mp¼170 ꢀC
4.1.5. Methyl-N-[2-(Nb-benzyloxycarbonyl-Na-tert-bu-
toxycarbonyl-hydrazino)ethyl]-N-{[4-N-(benzyloxycar-
bonyl)cytosin-1-yl]acetyl}glycinate (6). To a solution of 11
(365.4 mg, 1.25 mmol) and 9 (434.5 mg, 1.14 mmol) in dry
DMF (4 mL), DhbtOH (204.5 mg, 1.25 mmol) and DIEA
(218 mL, 1.25 mmol) were added. The mixture was cooled
to 0 ꢀC in an ice bath and then EDC$HCl (239.6 mg,
1.25 mmol) was added portionwise with vigorous stirring.
The mixture was allowed to warm to rt. The pH was adjusted
to basic conditions with DIEA. The reaction was stirred
overnight. The solvent was removed in vacuo and the residue
was partitioned between water (17 mL) and ethyl acetate
(36 mL). The organic layer was washed with 0.1 M aq
KHSO4 (2ꢁ12 mL), water (2ꢁ12 mL), satd aq NaHCO3
(3ꢁ25 mL) and brine (1ꢁ12 mL). All the aqueous phases
were combined and extracted with ethyl acetate
(2ꢁ50 mL). These organic phases were combined with the
first and dried over Na2SO4. After filtration, the filtrate
was concentrated in vacuo to afford 943 mg of crude product
as a pale yellow solid. Column chromatography on silica gel
(gradient from ethyl acetate/petroleum ether 9:1 to pure eth-
yl acetate) afforded 6 (699 mg, Rf¼0.17, 92%) as a white
solid: mp¼125–135 ꢀC (dec); 1H NMR (300 MHz, CDCl3)
(mixture of rotamers): d 8.32 (br s, 1H), 7.61 (d, J¼5 Hz,
1H), 7.33–7.23 (br m, 10H), 7.18 (d, J¼5 Hz, 1H), 5.17–
5.10 (two br s, 4H), 4.76–4.52 (m, 2H), 4.29–3.96 (m,
2H), 3.77–3.62 (m, 7H), 1.37 (br s, 9H); 13C NMR
(75 MHz, CDCl3) (rotamers): d 169.3, 167.1, 162.73, 156,
155.5, 152.3, 150.17 (mi.) and 149.64 (ma.), 135.02 (2C),
128.5–127.6, 94.8, 81.4, 67.6 (2C), 52.5 (mi.) and 52.0
(ma.), (some of the signals of the minor rotamer are obscured
1
(dec); H NMR (300 MHz, CDCl3) (two rotamers): d 9.61
(s, 1H), 8.70 (s, 1H), 8.32 (s, 1H), 8.04 (s, 1H), 7.42–7.29
(m, 10H), 5.27–5.19 (m, 4H), 5.05 (s, 2H), 4.13 (s, 2H),
3.79 (m, 2H), 3.70 (s, 3H), 3.69 (m, 2H), 1.40 (br s, 9H);
13C NMR (75 MHz, CDCl3): d 168.5, 165.5, 156.6, 156.2,
152.6, 150.5, 150.3, 148.9, 144.7, 135.1–134.3, 128.1–
127.9, 120.7, 81.4, 67.7, 52.4, 53.0, 46.7, 43.5, 28.0; mass
spectrum ESI m/z 713.2628 (C33H38N8O9+Na requires
713.26594; IR (Nujol, cmꢂ1): 3192, 1748, 1672, 1616,
1589, 1212, 1158, 1049, 1027, 997, 743, 698.

P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
4115
by the major signals) 49.5, 49.2, 48.4, 47.7, 47.3, 47.0, 45.8,
28.0 (ma.) and 26.8 (mi.); mass spectrum ESI m/z 689.2536
(C32H38N6O10+Na requires 689.2547); IR (film, CH2Cl2,
cmꢂ1): 3276, 1808, 1748, 1666, 1628, 1561, 1500, 1456,
1411, 1369, 1212, 1063, 789, 746, 699.
382.1978); IR (thin film, CCl4, cmꢂ1): 3392, 2980, 1747,
1718, 1438, 1394, 1368, 1155, 1108, 1068.
4.1.8. Methyl-N-[2-(Nb-benzyloxycarbonyl-Na-tert-bu-
toxycarbonyl-hydrazino)ethyl]glycinate (9). A solution
of 19 (610 mg, 1.98 mmol) and glycine methyl ester hydro-
chloride (207 mg, 1.65 mmol) in dry MeOH (18 mL) was
stirred at 0 ꢀC in an ice bath for 15 min. NaBH3CN
(62 mg, 0.99 mmol) and AcOH (112 mL, 1.98 mmol) were
added. The mixture was allowed to warm to rt and stirred
for 3.5 h. The solvent was removed in vacuo and satd aq
NaHCO3 (20 mL) was added. The solution was extracted
three times with ethyl acetate (3ꢁ60 mL). The combined or-
ganic phases were washed with brine, dried over Na2SO4 and
concentrated in vacuo to afford 749 mg of crude product as
a yellow oil. Column chromatography on silica gel (ethyl
acetate/petroleum ether 8:2) afforded 9 (408 mg, Rf ¼0.2,
4.1.6. Methyl-N-[2-(Nb-benzyloxycarbonyl-Na-tert-bu-
toxycarbonyl-hydrazino)ethyl]-N-{[6-N-(benzyloxycar-
bonyl)adenin-9-yl]acetyl}glycinate (7). To a solution of 12
(159 mg, 0.48 mmol) and 9 (168 mg, 0.44 mmol) in dry
DMF (2 mL), DhbtOH (79 mg, 0.48 mmol) and DIEA
(85 mL, 0.49 mmol) were added. The mixture was cooled to
0 ꢀC in an ice bath and then EDC$HCl (93 mg, 0.49 mmol)
was added portionwise with vigorous stirring. The mixture
was allowed to warm to rt. The pH was adjusted to basic con-
ditions with DIEA (pH¼9). The reaction was stirred for 48 h
at rt. The solvent was removed in vacuo and the residue was
partitioned between water (8 mL) and ethyl acetate (18 mL).
The organic layer was washed with 0.1 M aq KHSO4
(2ꢁ6 mL), water (2ꢁ6 mL), satd aq NaHCO3 (4ꢁ12 mL)
and brine (1ꢁ6 mL). All the aqueous phases were combined
and extracted with ethyl acetate (2ꢁ30 mL). These organic
phases were combined with the first and dried over
Na2SO4. After filtration, the filtrate was dried in vacuo to af-
ford 390 mg of crude product as a pale yellow solid. Column
chromatography on silica gel (ethyl acetate) afforded 7
(212 mg, Rf ¼0.2, 70%) as a white solid: mp¼114 ꢀC
(dec); 1H NMR (300 MHz, CDCl3) (many rotamers):
d 9.77 (br s, 0.8H), 9.32 (br s, 0.2H), 8.62–8.46 (m, 1H), 7.99–
7.89 (m, 1H), 7.38–7.06 (m, 10H), 5.05–4.89 (m, 4H), 4.26–
3.68 (m, 11H), 1.42 (br s, 9H); 13C NMR (75 MHz, CDCl3):
d 150.8, 149.5, 135.7, 135.2, 128.6–127.8, 121.2, 110.7, 81.7,
68.2, 67.7, 52.9, 52.2, 49.3, 47.54, 46.9, 46.5, 45.8, 44.0,
43.2, 28.1, 27.0 (some signals are obscured); mass spectrum
ESI m/z 713.2630 (C33H38N8O9+Na requires 713.2659); IR
(film, CH2Cl2, cmꢂ1): 3250, 2977, 1747, 1672, 1615, 1589,
1541, 1466, 1412, 1368, 1321, 1287, 1212, 1157, 1083,
993, 895, 800, 738, 699, 642.
1
54%) as a pale yellow oil; H NMR (300 MHz, CDCl3):
d 7.34–7.26 (m, 5H), 5.15 (s, 2H), 3.70 (s, 3H), 3.64
(br t, J¼5.1 Hz, 2H), 3.41 (s, 2H), 2.80 (br t, J¼5.1 Hz,
2H), 1.40 (br s, 9H); 13C NMR (75 MHz, CDCl3): d 172.5,
156.3, 156.0, 135.7, 129.0–127.1, 81.0, 67.5, 51.4, 49.7,
49.0, 46.0, 27.8; mass spectrum ESI m/z 382.1955
(C18H27N3O6+H requires 382.1978); IR (neat film, cmꢂ1):
3317, 2977, 1736, 1455, 1412, 1367, 1218, 1152, 756, 699.
4.1.9. Nb-(tert-Butoxycarbonyl)-Nb-(2-hydroxyethyl)hy-
drazide (14). To a solution of 13 (5.00 g, 65.8 mmol) in
dry ethanol (49 mL) at 0 ꢀC, a solution of (Boc)2O
(14.34 g, 65.8 mmol), previously dissolved in dry ethanol
(39 mL), was added dropwise (30 min). The mixture was al-
lowed to warm to rt and stirred for 26 h. The solvent was re-
moved in vacuo to afford 14 (11.58 g, >98%) as a pale
yellow oil; 1H NMR (200 MHz, CDCl3): d 3.78 (t,
J¼4.9 Hz, 2H), 3.53 (t, J¼4.9 Hz, 2H), 1.45 (s, 9H); 13C
NMR (75 MHz, CDCl3): d 156.9, 80.6, 60.8, 51.7, 28.1;
mass spectrum ESI m/z 199.1049 (C7H16N2O3+Na requires
199.1059); IR (neat film, cmꢂ1): 3335, 2977, 1690, 1402,
1368, 1252, 1170, 1059, 871, 769.
4.1.7. Methyl-N-[2-(Na-benzyloxycarbonyl-Nb-tert-bu-
toxycarbonyl-hydrazino)ethyl]glycinate (8). To a solution
of glycine methyl ester hydrochloride (7.14 g, 57.1 mmol)
in dry methanol (465 mL), DIEA (9.95 mL, 57.1 mmol)
and 16 (16.00 g, 51.9 mmol) were added under stirring.
Then a freshly prepared solution of NaBH3CN (3.91 g,
62.3 mmol) and ZnCl2 (4.25 g, 31.2 mmol) in dry methanol
(382 mL) was added dropwise to the reaction mixture. The
mixture was stirred for 25 h while monitoring by TLC (elu-
ent ethyl acetate/petroleum ether 7:3, Rf¼0.20), and the
solvent was removed in vacuo. The residue was dissolved
in ethyl acetate and the organic phase was washed with satd
aq NaHCO3 and the aqueous layer was extracted four times
with ethyl acetate. The combined organic phases were
washed with brine and dried over Na2SO4. After filtration,
the filtrate was concentrated in vacuo to afford 17.77 g of
crude product as a yellow oil. Flash chromatography on sil-
ica gel (ethyl acetate/petroleum ether 8:2) afforded 8 (8.51 g,
4.1.10. Na-(Benzyloxycarbonyl)-Nb-(tert-butoxycar-
bonyl)-Nb-(2-hydroxyethyl)hydrazide (15). To a solution
of NaOH (2.63 g, 65.8 mmol) in water (68 mL), CH2Cl2
(68 mL) was added and the mixture was cooled to 0 ꢀC in
an ice bath. Then 14 (11.58 g, 65.8 mmol) was added.
Cbz-Cl (9.3 mL, 65.8 mmol) was added dropwise. The mix-
ture was allowed to warm to rt and stirred for 20 h. After this
time, the stirring was stopped, and the organic layer was col-
lected and washed with water and a 20% citric acid aq solu-
tion. The organic layer was dried over Na2SO4 and filtered.
The filtrate was collected and the solvent was removed in va-
cuo to afford 15 (19.01 g, 93%) as a pale yellow oil; 1H NMR
(300 MHz, CDCl3): d 7.36–7.32 (m, 5H), 6.90 (br s, 1H),
5.18 (s, 2H), 3.70 (t, J¼4.4 Hz, 2H), 3.58 (t, J¼4.4 Hz,
2H), 1.42 (br s, 9H); 13C NMR (75 MHz, CDCl3): d 157.8,
155.2, 135.4, 129.2–127.7, 81.6, 67.6, 59.2, 53.0, 27.9;
mass spectrum ESI m/z 333.1418 (C15H22N2O5+Na requires
333.1426); IR (neat film, cmꢂ1): 3294, 2978, 1713, 1499,
1456, 1396, 1369, 1256, 1216, 1164, 1066, 864, 753, 699.
1
43%) as a bright yellow oil; H NMR (300 MHz, CDCl3):
d 7.38–7.34 (m, 5H), 7.12 (br s, 1H), 5.18 (s, 2H), 3.73 (s,
3H), 3.61 (br t, 2H), 3.41 (s, 2H), 2.82 (t, J¼5.9 Hz, 2H),
1.43 (br s, 9H); 13C NMR (75 MHz, CDCl3): d 172.6, 156.7,
155.3, 135.8, 128.4, 81.6, 67.4, 52.1, 48.7, 47.5, 46.1, 28.0;
mass spectrum ESI m/z 382.1953 (C18H27N3O6+H requires
4.1.11. Na-(Benzyloxycarbonyl)-Nb-(tert-butoxycar-
bonyl)-Nb-(formylmethyl)hydrazide (16). To a solution
of 15 (19.00 g, 61.3 mmol) in water-saturated CH2Cl2

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P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
(530 mL), Dess–Martin periodinane (54.60 g, 129 mmol)
was added under stirring. The white suspension was stirred
for 50 min at rt. Then, methyl tert-butyl ether (MTBE)
(190 mL) and a satd aq NaHCO3 (190 mL) containing
Na2S2O3 (106.5 g, 674 mmol) were added to the mixture,
and stirred vigorously for 25 min. The stirring was stopped
and the two layers were separated. The aqueous layer was
extracted twice with CH2Cl2 (240 mL). The combined or-
ganic phases were washed with satd aq NaHCO3 (60 mL),
water (2ꢁ80 mL) and brine (2ꢁ60 mL). The organic layer
was dried over Na2SO4 and concentrated to dryness to afford
16 (17.00 g, Rf¼0.28 in hexane/ethyl acetate 1:1, 90%) as
a yellow oil. The product obtained in this way was usually
sufficiently pure to be used in the next step. Pure product
and an analytically pure sample were obtained by column
chromatography on silica gel (gradient from hexane/ethyl
acetate 6:4 to hexane/ethyl acetate 1:1); 1H NMR
(300 MHz, CDCl3): d 9.67 (br s, 1H), 7.35 (m, 5H), 6.84
(s, 1H), 5.17 (s, 2H), 4.30 (br s, 2H), 1.42 (br s, 9H); 13C
NMR (75 MHz, CDCl3): d 197.8, 157.8, 155.2, 135.4,
128.5–128.2, 81.6, 67.7, 59.5, 27.9; mass spectrum ESI
m/z 331.1263 (C15H20N2O5+Na requires 331.1270); IR (neat
film, cmꢂ1): 3305, 2979, 1732, 1500, 1456, 1370, 1260,
1221, 1152, 1067, 1029, 989, 904, 855, 745, 699.
ESI m/z 333.1419 (C15H22N2O5+Na requires 333.1426);
IR (neat film, cmꢂ1): 3299, 2978, 1713, 1499, 1455, 1413,
1368, 1288, 1254, 1214, 1162, 1065, 755, 698.
4.1.14. Nb-(Benzyloxycarbonyl)-Na-(tert-butoxycar-
bonyl)-Nb-(formylmethyl)hydrazide (19). To a solution
of 18 (1.06 g, 3.42 mmol) in water-saturated CH2Cl2
(22 mL), Dess–Martin periodinane (3.05 g, 7.19 mmol)
was added under stirring. The white suspension was stirred
for 2.5 h at rt. Then, methyl tert-butyl ether (MTBE)
(11 mL) and a satd aq NaHCO3 (11 mL) containing
Na2S2O3 (5.95 g, 37.6 mmol) were added to the mixture
and stirred vigorously for 30 min. The stirring was stopped
and the two layers were separated. The aqueous layer was
extracted three times with CH2Cl2. The combined organic
phases were washed with satd aq NaHCO3, twice with water
and twice with brine. The organic phase was dried over
Na2SO4 and concentrated in vacuo to afford 1.02 g of crude
product as a yellow oil. Column chromatography on silica
gel (gradient from petroleum ether/ethyl acetate 6:4 to
pure ethyl acetate) afforded 19 (933 mg, 88%, Rf ¼0.33 in
1
petroleum ether/ethyl acetate 1:1) as a pale yellow oil; H
NMR (300 MHz, CDCl3): d 9.68 (br s, 1H), 7.34–7.26 (m,
5H), 5.17 (s, 2H), 4.34 (br s, 2H), 1.42 (br s, 9H); 13C
NMR (75 MHz, CDCl3): d 191.4, 156.3, 156.0, 135.0,
129.0–127.0, 82.0, 68.3, 55.2, 28.0; mass spectrum EI m/z
308 (M)+. IR (neat film, cmꢂ1): 3307, 2980, 1713, 1497,
1456, 1412, 1369, 1255, 1157, 1069, 1031, 738, 699.
4.1.12. Nb-(Benzyloxycarbonyl)-Nb-(2-hydroxyethyl)hy-
drazide (17). To a solution of 13 (6.37 g, 90% by weight,
75 mmol) in CH2Cl2 (100 mL) at 0 ꢀC, Et3N (10.4 mL,
75 mmol) was added. Under vigorous stirring, Cbz-Cl
(10.6 mL, 75 mmol) was added dropwise. The resulting
mixture was allowed to warm to rt and then stirred for
24 h. The mixture was concentrated in vacuo to give
22.70 g of a pale yellow oil. The crude product was sus-
pended in ethyl acetate and filtered on a 3 cm silica gel
bed, isolating the first fraction containing the unreacted
Cbz-Cl and eluting the product with ethyl acetate afforded
4.1.15. Methyl-N-[-2-[2-(Na-benzyloxycarbonyl-Nb-tert-
butoxycarbonyl-hydrazino)ethyl]-N-[(thymin-1-yl)ace-
tyl]glycyl]aminoethyl-N-[(thymin-1-yl)acetyl]glycinate
([Na-Cbz-Nb-Boc-hyd(T)PNA]-[aeg(T)PNA-COOCH3],
20). Compound 23 (100 mg, 0.19 mmol) was dissolved in
dry DMF (2.0 mL) and a solution of pentafluorophenol
(38 mg, 0.21 mmol) in dry DMF (1 mL) was added. The
solution was cooled to 0 ꢀC in an ice bath. DCC (47 mg,
0.23 mmol) was added under vigorous stirring. The solu-
tion was stirred for 1 h at 0 ꢀC and, then, for 5 h at rt.
The mixture was filtered in an inert atmosphere to remove
the DCU and the white solid was washed twice with dry
DMF (2ꢁ1 mL). The filtrate was collected. In another
round-bottomed flask, 29 (77 mg, 0.19 mmol) was
dissolved in dry DMF (4 mL) and DIEA (53 mL,
0.31 mmol), and stirred for 1 min. This mixture was then
added to the previous filtrate containing the pentafluorophe-
nol ester and stirred for 24 h. The solvent was concentrated
in vacuo to obtain 276 mg of crude product. Column chro-
matography on silica gel (ethyl acetate/methanol 8:2)
afforded 20 (139 mg, Rf¼0.13, 91%) as a white solid, no
melting was observed; at 160 ꢀC, it became a yellow rubber
(dec); 1H NMR (300 MHz, DMSO-d6) (mixture of ro-
tamers): d 8.30 (m, 2H), 7.39–7.23 (m, 7H), 5.11 (s, 2H),
4.68–4.06 (m, 12H), 4.02–3.86 (m, 2H), 3.71–3.43 (m,
5H), 1.70–1.67 (m, 6H), 1.36–1.32 (m, 9H); 13C NMR
(75 MHz, DMSO-d6): d 170.0, 169.5, 168.5, 167.8, 164.3
(2C q. ov.), 156.3, 156.0, 151.0 (2 q. ov.), 142.0 (2 ]CH
ov.), 136.3, 128.8–128.3 (5CH arom. ov.), 108.8 (2 ]C
q. ov.), 79.7, 65.7, 51.7, 49.5–46.7 (7CH2 ov.), 38.0, 27.4,
11.7 (2CH3 ov.); mass spectrum ESI m/z 836.3192
(C36H47N9O13+Na requires 836.3191); IR (Nujol, cmꢂ1):
3481, 1674, 1541, 1337, 1208, 1136, 1082, 1048, 1021,
837, 797, 719.
1
17 (12.52 g, Rf ¼0.19, 80%) as a colourless oil; H NMR
(300 MHz, CDCl3): d 7.4–7.3 (m, 5H), 5.15 (s, 2H), 4.19
(br s, 2H), 3.80 (t, J¼4.5 Hz, 2H), 3.60 (t, J¼4.5 Hz, 2H);
13C NMR (75 MHz, CDCl3): d 157.5, 136.0, 128.3–126.9,
67.5, 60.3, 51.9; mass spectrum ESI m/z 233.0891
(C10H14N2O3+Na requires 233.0902); IR (neat film,
cmꢂ1): 3412, 3344, 3055, 2948, 1696, 1626, 1455, 1416,
1359, 1266, 1217, 1126, 1060, 910, 735, 700.
4.1.13. Nb-(Benzyloxycarbonyl)-Na-(tert-butoxycar-
bonyl)-Nb-(2-hydroxyethyl)hydrazide (18). To a solution
of 17 (1.025 g, 4.88 mmol) in dry THF (15 mL), Et3N
(680 mL, 4.9 mmol) was added dropwise and the mixture
was cooled to 0 ꢀC in an ice bath. (Boc)2O was added under
vigorous stirring and the mixture was allowed to warm to rt.
After stirring for 3 h, the mixture was concentrated to dry-
ness. The residue was partitioned between water and ethyl
acetate. The aqueous layer was extracted three times with
ethyl acetate. The combined organic phases were dried
over Na2SO4 and concentrated to dryness to afford 1.46 g
of the crude product as a pale yellow oil. Column chromato-
graphy on silica gel (ethyl acetate/petroleum ether 1:1) af-
forded 18 (1.08 g, Rf ¼0.22, 71.3%) as a pale yellow oil;
1H NMR (300 MHz, CDCl3): d 7.33–7.28 (m, 5H), 6.50
(br s, 1H), 5.16 (s, 2H), 3.70 (br s, 2H), 3.60 (br s, 2H),
1.42 (br s, 9H); 13C NMR (CDCl3): d 156.3, 156.0, 135.6,
129.1–127.1, 82.0, 67.9, 58.8, 52.8, 27.9; mass spectrum

P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
4117
4.1.16. [Boc-aeg(T)PNA] [Nb-Cbz-hyd(T)PNA-
COOCH3] (21). Compound 32 (301 mg, 0.78 mmol) was
dissolved in dry DMF (2 mL) and then 27 (400 mg,
0.71 mmol) was added. DhbtOH (128 mg, 0.78 mmol) and
DIEA (268 mL, 202 mg, 1.57 mmol) were added under vig-
orous stirring. The reaction mixture was then cooled to 0 ꢀC
in an ice bath, and EDC$HCl (150 mg, 0.78 mmol) was
added portionwise. The mixture was allowed to warm to rt
and the pH was adjusted to 9 with DIEA. The mixture was
stirred for 85 h. The reaction mixture was then concentrated
to dryness, and dissolved in water (20 mL) and ethyl acetate
(30 mL). The aqueous layer was extracted with ethyl acetate
(4ꢁ15 mL). The organic phases were collected and washed
with 0.1 M aq KHSO4 (2ꢁ80 mL), water (2ꢁ60 mL), satd
aq NaHCO3 (3ꢁ60 mL) and brine (1ꢁ60 mL). All of the
aqueous phases were combined and extracted with ethyl ace-
tate (2ꢁ80 mL). These organic phases were combined with
the first and dried over Na2SO4. The organic phase was fil-
tered and concentrated to dryness in vacuo to afford
426 mg of the crude product. Column chromatography on
silica gel (ethyl acetate/MeOH 8:2) afforded 21 (376 mg,
110.6 (2 ]C q. ov.), 79.9, 67.7, 55.1, 51.0–46.5 (7CH2
ov.), 38.2, 28.3, 13.4–12.1 (2CH3 ov.); mass spectrum ESI
m/z 836.3169 (C36H47F3N9O13+Na requires 836.3185); IR
(KBr, cmꢂ1): 3467, 3066, 1666, 1473, 1421, 1385, 1251,
1218, 1172, 1092, 1044, 964, 819, 784, 734, 698, 564, 462.
4.1.18. N-[2-(Na-Benzyloxycarbonyl-Nb-tert-butoxycar-
bonyl-hydrazino)ethyl]-N-[(thymin-1-yl)acetyl]glycine
(23). Compound 2 (1.00 g, 1.83 mmol) was dissolved in 2 M
KOH aq (30 mL) and the solution was stirred for 1 h at rt.
The pH was then adjusted to 2–3 with 1 M aq HCl. A white
precipitate was observed. The mixture was extracted with
CH2Cl2 (3ꢁ70 mL), and the organic phases were combined
and dried over Na2SO4, filtered and the filtrate concentrated
to dryness to afford 23 (927 mg, 95%) as a white solid:
1
mp¼147 ꢀC (dec); H NMR (300 MHz, CDCl3) (two ro-
tamers): d 9.80 (br s, 1H), 9.30 (br s, 1H), 7.39–7.28 (m,
5H), 7.08 (ma.) and 7.05 (mi.) (s, 1H), 5.18 (s, 2H), 4.15
(s, 2H), 4.11 (s, 2H), 3.66 (m, 2H), 3.63 (m, 2H), 1.92 (s,
3H), 1.37 (br s, 9H); 13C NMR (75 MHz, CDCl3): d 171.6,
168.3, 167.8, 164.5, 155.1, 151.4, 141.7, 135.7, 128.4,
110.5, 81.5, 67.5, 48.2, 47.9, 47.5, 46.2, 27.9, 12.0; mass
spectrum FAB m/z 556 (M+Na)+; IR (Nujol, cmꢂ1): 3515,
3263, 1714, 1694, 1669, 1412, 1252, 1215, 1163, 1082,
1045, 1025, 963, 906, 841, 783, 756, 736, 698.
1
Rf¼0.29, 65%) as a white solid: mp¼95–100 ꢀC (dec); H
NMR (300 MHz, CDCl3) (mixture of rotamers): d 7.40–
7.24 (m, 5H), 7.05–6.95 (m, 2H), 5.22–5.07 (m, 2H),
4.52–3.77 (m, 8H), 3.73–3.13 (m, 11H), 1.85–1.82 (m,
6H), 1.42 (br s, 9H); 13C NMR (50 MHz, CDCl3) (some ro-
tamers not completely attributed are present): d 169.7,
168.1–167.2 (3C q. ov.), 165.6–156.3 (4C q. ov.), 151.5
(2C q. ov.), 141.6–141.2 (2 ]CH ov.), 135.6, 128.4 (5CH
arom. ov.), 110.6 (2 ]C q. ov.), 79.8, 68.3, 53.4 (mi.) and
52.3 (ma.), 49.5–46.6 (7CH2 ov.), 38.5, 28.4, 12.2 (2CH3
ov.); mass spectrum ESI m/z 836.3181 (C36H47F3N9O13+Na
requires 836.3185); IR (Nujol, cmꢂ1): 3416, 1712, 1505,
1263, 1166, 721.
4.1.19. N-[2-(Nb-Benzyloxycarbonyl-Na-tert-butoxycar-
bonyl-hydrazino)ethyl]-N-[(thymin-1-yl)acetyl]glycine
(24). Compound 5 (200 mg, 0.37 mmol) was dissolved in
2 M aq KOH (10 mL) and the solution was stirred for 1 h
at rt. The pH was then adjusted to 2–3 with 1 M aq HCl. A
white precipitate was observed. The mixture was extracted
with CH2Cl2 (3ꢁ20 mL), and the organic phases were com-
bined and dried over Na2SO4, filtered and the filtrate concen-
trated to dryness to afford 24 (185 mg, 95%) as a white solid:
4.1.17. [Boc-aeg(T)PNA] [Na-Cbz-hyd(T)PNA-
COOCH3] (22). Compounds 32 (230 mg, 0.60 mmol) and
25 (304 mg, 0.54 mmol) were dissolved in dry DMF
(1.5 mL) with stirring. DhbtOH (97 mg, 0.60 mmol) and
DIEA (204 mL, 154 mg, 1.19 mmol) were added. The mix-
ture was then cooled to 0 ꢀC in an ice bath. EDC$HCl
(114 mg, 0.60 mmol) was added portionwise. The reaction
was allowed to warm to rt and the pH was adjusted to 9
with DIEA (140 mL). The mixture was stirred for 24 h and
then concentrated to dryness in vacuo. The residue was dis-
solved in water (20 mL) and ethyl acetate (30 mL). The or-
ganic layer was washed with 0.1 M aq KHSO4 (2ꢁ15 mL),
water (2ꢁ15 mL), satd aq NaHCO3 (2ꢁ20 mL) and brine
(2ꢁ15 mL). All of the aqueous phases were combined and
extracted with ethyl acetate (3ꢁ20 mL). These organic
phases were combined with the first and dried over
Na2SO4. The organic phase was filtered and concentrated
to dryness to afford 324 mg of the crude product as a yellow
solid. Column chromatography on silica gel (ethyl acetate/
MeOH 8:2) afforded 22 (254 mg, Rf¼0.28, 56%) as a white
solid: mp¼100–120 ꢀC (dec); 1H NMR (300 MHz, CDCl3)
(mixture of rotamers): d 7.36–7.32 (m, 5H), 7.14–6.90 (m,
2H), 5.18 (ma.) and 5.17 (mi.) (m, 2H), 4.51–3.80 (m,
8H), 3.80–3.00 (m, 11H), 1.92–1.83 (m, 6H), 1.42 (br s,
9H); 13C NMR (50 MHz, CDCl3) (some rotamers not com-
pletely assigned are present): d 171.4, 169.6–167.3 (3C q.
ov.), 165.5–164.7 (4C q. ov.), 151.5 (2C q. ov.), 143.1–
139.5 (2 ]CH ov.), 135.5, 130.1–126.9 (5CH arom. ov.),
mp¼103–110 ꢀC (dec); H NMR (300 MHz, CDCl3) (two
1
rotamers): d 8.99 (br s, 1H), 7.36 (m, 5H), 7.07 (ma.) and
7.02 (mi.) (s, 1H), 5.14 (s, 2H), 4.49 (s, 2H), 4.13 (s, 2H),
3.89 (m, 2H), 3.60 (m, 2H), 1.85 (s, 3H), 1.44 (s, 9H); 13C
NMR (75 MHz, CDCl3): d 172.2, 167.5, 164.2, 156.3,
156.0, 151.0, 141.0 and 142.0, 135.9, 128.1–129, 110.7,
81.0, 68.1, 49.0, 48.5, 48.4, 46.2, 28.3, 12.3; mass spectrum
ESI m/z 556 (M+Na)+; IR (thin film, CH2Cl2, cmꢂ1): 3474,
3264, 2614, 1694, 1681, 1471, 1368, 1286, 1217, 1085,
1049, 1021, 967, 787, 756.
4.1.20. Methyl-N-[2-(Na-benzyloxycarbonyl-hydr-
azino)ethyl]-N-[(thymin-1-yl)acetyl]glycinate trifluoro-
acetic salt (25). Compound 2 (1.70 g, 3.11 mmol) was
dissolved in
a solution of TFA (7.09 g, 4.8 mL,
62.16 mmol) at 40% by weight in CH2Cl2 (8 mL) under vig-
orous stirring. Gas formation was observed. The reaction was
stirred overnight. The solution was concentrated in vacuo to
an orange oil. The oil was stirred in diethyl ether for 1 h at rt
and a white precipitatewas obtained. Centrifugation afforded
1
25 (1.35 g, 77%) as a white solid: mp¼115 ꢀC (dec); H
NMR (300 MHz, DMSO-d6) (mixture of rotamers): d 9.90
(br s, 1H), 7.38–7.33 (m, 6H), 5.13 (ma.) and 5.09 (mi.) (s,
2H), 4.69 (mi.) and 4.67 (ma.) (m, 2H), 4.50–4.25 (m, 2H),
4.13 (m, 2H), 3.72 (m, 2H), 3.62 (br s, 3H), 1.76 (s, 3H);
13C NMR (75 MHz, DMSO-d6) (mixture of rotamers):
d 170.0, 167.8, 164.8, 164.5, 151.4, 142.4, 135.5, 128.8–
128.2, 108.5, 66.1, 49.5, 48.2, 47.1, 46.6, 46.0, 12.2; mass

4118
P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
spectrum ESI m/z 448 (MꢂCF3COOH+H)+; IR (Nujol,
7.42–7.29 (m, 10H), 7.24 (br d, 1H), 5.23–5.16 (m, 4H),
4.59–4.00 (m, 6H), 3.78–3.66 (m, 5H); 13C NMR
(75 MHz, CD3OD): d 169.4, 165.2, 164.7, 159.1, 158.4,
154.3, 151.8, 148.4, 137.1, 129.6–128.7, 96.9 (ma.) and
95.9 (mi.), 69.3, 68.7, 52.8, 51.9, 47.8, 47.2, 46.5; mass spec-
trum ESI m/z 589.2016 (C29H31F3N6O10-CF3COOH+Na re-
quires 589.2023); IR (Nujol, cmꢂ1) 3341, 1754, 1675, 1566,
1504, 1454, 1208, 1128, 841, 800, 722, 699.
cmꢂ1): 3200, 1675, 1216.
4.1.21. Methyl-N-[2-(Na-benzyloxycarbonyl-hydr-
azino)ethyl]-N-{[4-N-(benzyloxycarbonyl)cytosin-1-yl]-
acetyl}glycinate trifluoroacetic salt (26). Compound 3
(100 mg, 0.15 mmol) was dissolved in a solution of TFA
(342 mg, 220 mL, 3.0 mmol) at 20% by weight in CH2Cl2
(1.5 mL) under vigorous stirring. Gas formation was ob-
served. The reaction was stirred overnight. The solution
was concentrated in vacuo to an orange oil. The oil was dis-
solved in CH2Cl2 and concentrated in vacuo. This operation
was repeated several times in order to obtain a bright yellow
foam. Column chromatography on silica gel (ethyl acetate/
MeOH 9:1) afforded 26 (70 mg, 69%) as a white solid:
mp¼110 ꢀC (dec); 1H NMR (300 MHz, CD3OD) (many ro-
tamers): d 7.91 (br s, 1H), 7.41–7.31 (m, 10H), 7.13 (br s,
1H), 5.22 (mi.) and 5.15 (ma.) (s, 4H), 4.69 (mi.) and 4.67
(ma.) (m, 2H), 4.50–4.25 (m, 2H), 4.13 (m, 2H), 3.72 (m,
2H), 3.62 (br s, 3H); 13C NMR (75 MHz, CD3OD) (many ro-
tamers): d 170.0, 169.1, 167.8, 163.0, 162.8, 152.7, 151.5,
151.2, 135.9, 135.3, 128.2–127.5 95.2, 95.2, 67.7, 67.2,
52.1, 49.3, 47.3, 46.7, 46.2, 45.5, 43.9, 42.0; mass spectrum
ESI m/z 589.2016 (C27H30N6O8+Na requires 589.2023)+;
IR (Nujol, cmꢂ1): 3474, 1731, 1663, 1640, 1456, 1374,
1201, 1140, 1028, 909, 838, 800, 722, 698.
4.1.24. N-[-2-[2-(Na-Benzyloxycarbonil-Nb-tert-butoxy-
carbonyl-hydrazino)ethyl]-N-[(thymin-1-yl)acetyl]gly-
cyl]aminoethyl-N-[(thymin-1-yl)acetyl]glycine ([Na-
Cbz-Nb-Boc-hyd(T)PNA] [aeg(T)PNA-COOH], 30).
Compound 20 (132 mg, 0.16 mmol) was dissolved in meth-
anol (7 mL), and a solution of LiOH (13 mg, 0.31 mmol) in
methanol (2.5 mL) was added. The mixture was stirred for
24 h and then another portion of LiOH (13 mg,
0.31 mmol) in methanol (1 mL) was added. After a further
5 h, the solvent was concentrated in vacuo. The residue
was dissolved in water (a few drops) and 1 M aq KHSO4
was added dropwise to adjust the pH to 2. The solution
was extracted 10 times with ethyl acetate, and the solvent
was concentrated in vacuo to afford 30 (65 mg, 50%) as
a white solid: no melting was observed; at 180 ꢀC, it became
brown; 1H NMR (300 MHz, CD3OD): d 7.38–7.33 (m, 7H),
5.18 (ma.) and 5.16 (mi) (s, 2H), 4.70–3.95 (m, 8H), 3.75–
3.40 (m, 8H), 1.85 (m, 6H), 1.40 (br s, 9H); 13C NMR
(75 MHz, CD3OD): d 173.4, 167.4–156.0 (7C q. ov.),
152.0 (2C q. ov.), 144.4, 143.6, 134.1, 129.5–129.1 (5CH
arom. ov.), 111.0 (2C q. ov.), 83.1, 68.4, 50.0–46.7 (8CH2
ov.), 28.2 (3CH3 ov.), 11.8 (2CH3 ov.); mass spectrum ESI
m/z 844.2871 (C35H45N9O13-H+2Na requires 844.2854);
IR (Nujol, cmꢂ1): 3436, 1675, 1256, 1205, 1141.
4.1.22. Methyl-N-[2-(Nb-benzyloxycarbonyl-hydr-
azino)ethyl]-N-[(thymin-1-yl)acetyl]glycinate trifluoro-
acetic salt (27). Compound 5 (100 mg, 0.18 mmol) was
dissolved in
a solution of TFA (416 mg, 270 mL,
3.65 mmol) at 40% by weight in CH2Cl2 (470 mL). Gas for-
mation was observed. The reaction was stirred for 16 h at rt.
The solution was concentrated in vacuo. The solid was
washed three times with Et2O and separated by centrifuga-
tion. The residue was dried in vacuo to afford 27 (83 mg,
80%) as a white solid: mp¼130 ꢀC (dec); 1H NMR
(300 MHz, CDCl3) (two rotamers): d 8.99 (m, 1H), 7.35–
6.75 (m, 6H), 5.15 (s, 2H), 4.75–3.50 (m, 11H), 1.95 (ma.)
and 1.90 (mi.) (s, 3H); 13C NMR (75 MHz, CDCl3):
d 172.0, 164.6, 164.2, 156.0, 151.0, 141.5 and 141.0, 135.5,
129.0–127.7, 110.8, 68.0, 52.3, 49.1, 48.1, 47.3, 46.6, 12.1;
mass spectrum ESI m/z 448.3 (MꢂCF3COOH+H)+; IR
(film, CH2Cl2, cmꢂ1): 3208, 3037, 2962, 2921, 2846, 1745,
1680, 1470, 1439, 1419, 1358, 1260, 1212, 1167, 1085,
1020, 967, 906, 799, 756, 739, 701.
4.1.25. Methyl-N-[-2-[2-(Na-benzyloxycarbonyl-hydrazi-
no)ethyl]-N-[(thymin-1-yl)acetyl]glycyl]aminoethyl-N-
[(thymin-1-yl)acetyl]glycinate trifluoroacetic salt ([Na-
Cbz-hyd(T)PNA] [aeg(T)PNA-COOCH3] trifluoroacetic
salt, 31). Compound 20 (143 mg, 0.18 mmol) was dissolved
in a freshly prepared solution of trifluoroacetic acid (270 mL,
3.52 mmol) at 20% by weight in CH2Cl2 (1.2 mL). The mix-
ture was stirred overnight. The solvent was evaporated at re-
duced pressure to afford a pale yellow oil. The residue was
repeatedly diluted with CH2Cl2 and concentrated until 31
was obtained (143 mg, >98%) as a white foam: mp¼190 ꢀC
(dec); 1H NMR (300 MHz, CD3OD) (mixture of rotamers):
d 7.45–7.17 (m, 7H), 5.37–4.87 (m, 4H), 4.72–4.10 (m, 4H),
3.99–3.19 (m, 11H), 1.84–1.76 (m, 6H); 13C NMR (75 MHz,
CD3OD) (some not completely attributed rotamers): d 172.2,
172.0, 169.8 (2C q. ov.), 166.9 (2C q. ov.), 159.3, 153.1 (2C
q. ov.), 144.2–143.8 (2 ]CH ov.), 137.9, 137.4, 129.6–128.8
(5CH arom. ov.), 111.1–110.7 (2C q. ov.), 68.6 (mi.) and
68.0 (ma.), 53.3, 50.0–47.3 (7CH2 ov.), 38.0, 12.3 (2CH3
ov.); mass spectrum ESI m/z 736.2653 (C33H40F3N9O13-
CH3COOH+Na requires 736.2667); IR (Nujol, cmꢂ1):
3433, 1674, 1201, 1140, 1025, 797, 719.
4.1.23. Methyl-N-[2-(Nb-benzyloxycarbonyl-hydr-
azino)ethyl]-N-{[4-N-(benzyloxycarbonyl)cytosin-1-yl]-
acetyl}glycinate trifluoroacetic salt (28). Compound 6
(100 mg, 0.15 mmol) was dissolved in a solution of TFA
(1.026 g, 670 mL, 3.00 mmol) at 20% by weight in CH2Cl2
(3.1 mL) under vigorous stirring. Gas formation was ob-
served. The reaction was stirred overnight. The solution
was concentrated in vacuo to a pale yellow oil. The oil
was dissolved in few drops of CH2Cl2 and treated with petro-
leum ether. A white precipitate was observed. The precipi-
tate was dried in vacuo to afford 102 mg of crude product
as a pale yellow solid foam. Column chromatography on
silica gel (ethyl acetate/methanol 9:1) afforded 28 (70 mg,
69%) as a white solid: no melting was observed; at
120 ꢀC, it became a brown rubber (dec); 1H NMR
(300 MHz, CD3OD) (two rotamers): d 7.74 (br d, 1H),
Acknowledgements
The authors gratefully acknowledge joint financial support
from the Ministero dell’Istruzione, dell’Universitaꢀ e della
Ricerca Scientifica (MIUR), Rome and from the University

P. Cerea et al. / Tetrahedron 63 (2007) 4108–4119
4119
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