Synthesis and molecular modeling of novel dihydroxycyclopentane- carbonitrile nor-nucleosides by bromonitrile oxide 1,3-dipolar cycloaddition

Article abstract of DOI:10.1016/j.tet.2011.12.086

The regioisomeric cycloadducts of the bromonitrile oxide to the N-benzoyl-2,3-oxazanorborn-5-ene were easily prepared and elaborated into a novel class of uracil nor-nucleoside derivatives. In the key-synthetic step represented by the reductive N-O bond cleavage, an unusual double ring opening afforded the aminol intermediates containing a β-hydroxynitrile structure. By adapting known protocols, the aminols entered the linear construction of uracil rings. These novel nucleosides were found structurally similar to a potent antiviral compound, Brivudin, and molecular modeling and docking allowed to select one of the two regioisomeric structures as promising candidate for antiviral tests, due to the nice level of binding with the Thymidine Kinase, the enzyme involved in virus replication.

Full text of DOI:10.1016/j.tet.2011.12.086

Tetrahedron 68 (2012) 1845e1852
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and molecular modeling of novel dihydroxycyclopentane-carbonitrile
nor-nucleosides by bromonitrile oxide 1,3-dipolar cycloaddition
Marco Savion a, Misal Giuseppe Memeo a, Bruna Bovio a, Giovanni Grazioso b, Laura Legnani a,
,
Paolo Quadrelli a
*
a
ꢀ
Dipartimento di Chimica, Universita degli Studi di Pavia, Viale Taramelli 12, 27100 Pavia, Italy
Istituto di Chimica Farmaceutica e Tossicologica “Pietro Pratesi”, Universita degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 October 2011
Received in revised form 20 December 2011
Accepted 29 December 2011
Available online 4 January 2012
The regioisomeric cycloadducts of the bromonitrile oxide to the N-benzoyl-2,3-oxazanorborn-5-ene
were easily prepared and elaborated into a novel class of uracil nor-nucleoside derivatives. In the key-
synthetic step represented by the reductive NeO bond cleavage, an unusual double ring opening
afforded the aminol intermediates containing a b-hydroxynitrile structure. By adapting known protocols,
the aminols entered the linear construction of uracil rings. These novel nucleosides were found struc-
turally similar to a potent antiviral compound, Brivudin, and molecular modeling and docking allowed to
select one of the two regioisomeric structures as promising candidate for antiviral tests, due to the nice
level of binding with the Thymidine Kinase, the enzyme involved in virus replication.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Nor-nucleosides
Bromonitrile oxide
1,3-Dipolar cycloaddition
Molecular docking
Thymidine kinase
1. Introduction
derivatives of type 4 in quantitative yields.2 The N-benzoyl re-
moval took place easily under basic alcoholysis, followed by the
A novel approach to useful precursors for the synthesis of
isoxazoline-carbocyclic nucleosides was recently detailed starting
from the readily available N-benzoyl-2,3-oxazanorborn-5-ene 1
hydrogenolytic NeO bond cleavage to obtain the regioisomeric
aminols 6. They served as intermediates in the linear construction
of the desired heterobases in order to prepare the new nucleosidic
structures of type 7 (Scheme 2) belonging to the conformationally
locked nucleoside series.3
and introducing
a polar functional group via a condensed
carbethoxy-isoxazoline ring. These structures can be prepared
through the efficient and exo-selective 1,3-dipolar cycloaddition
reaction of carbethoxyformhydroxymoyl chloride 2 to the 2,3-
oxazanorbornene 1a affording the regioisomeric cycloadducts
3a,b in good yields (Scheme 1).1
Scheme 1.
The regioisomeric cycloadducts 3 were used as starting mate-
rials for aminol synthesis. Reduction with NaBH4 in methanol of
the carbethoxy substituents afforded the hydroxymethylene
Scheme 2.
The chemistry of nitrosocarbonyl (ReCONO) intermediates 8
represents the key strategy of the reported syntheses because
of the high dienophilic power of these fleeting intermediates
* Corresponding author. E-mail address: paolo.quadrelli@unipv.it (P. Quadrelli).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.12.086

1846
M. Savion et al. / Tetrahedron 68 (2012) 1845e1852
and the synthetic potential of their hetero DielseAlder (HDA)
cycloadducts (Scheme 3). Traditionally, nitrosocarbonyls are
generated through periodate oxidation of hydroxamic acids 9
and instantly trapped by dienes as reported by G.W. Kirby,
whose initial studies paved the way for synthetic applications
of these intermediates.4 Recently, we developed two alternative
entries to nitrosocarbonyls through the mild oxidation of nitrile
oxides 10 with N-methylmorpholine-N-oxide (NMO)5 or, al-
ternatively, the clean photolysis of 1,2,4-oxadiazole-4-
oxides 11.6
of 14a, bearing the bromine and the benzoyl groups syn to each
other, showed the isoxazoline proton H5 at
5.01 (dt, J¼8.4,
1.7 Hz), coupled with the H4 proton at
3.99 (d, J¼8.4 Hz) and
d
d
with the more deshielded of the two methylene protons at
d
2.07 through a 4J¼1.7 Hz (‘W-coupling’).1,8 The regioisomer
14b displayed an anti arrangement of the bromine and the
benzoyl groups and its 1H NMR spectrum showed the iso-
xazoline proton H5 at
H4 proton at
of the two methylene protons at
d
5.15 (dt, J¼8.0, 1.4 Hz), coupled with the
d
3.90 (d, J¼8.0 Hz) and with the more deshielded
d
2.14 through a 4J¼1.4 Hz (‘W-
coupling’). The presence of the ‘W-coupling’ in both the
cycloadducts as well as the absence of appreciable coupling
between the isoxazoline and the bridge-head protons, found as
singlets in both cases, fully support the exo structures of the
1,3-dipolar cycloadducts. This is in line with the exo-selective
addition to these dipolarophiles reported in literature for other
1,3-dipoles and for the dihydroxylation reaction.12 However,
the regiochemistry was not clearly evidenced from the spectra
or other spectroscopic experiments and only an X-ray analysis
of single crystal of 14a allowed us to unequivocally confirm the
regioisomeric structures of both the compounds. Fig. 1 reports
the ORTEP view of 14a.
Scheme 3.
The HDA reactions of nitrosocarbonyl intermediates with dienes
provide useful methods for incorporating 1,4-aminoalcohol moie-
ties into a carbocyclic framework in a diasteroselective fashion. The
2,3-oxazanorborn-5-ene structures 1a (n¼1) and the 2,3-
oxazabicyclo[2.2.2]oct-5-ene structures 1b (n¼2) are suitable for
further synthetic manipulations, which allow for a flexible in-
troduction of multifunctionality.7
Fig. 1. ORTEP plot of compound 14a with atom labeling (ellipsoid at 25% probability).
Hydrogen atom atoms are omitted for clarity with the exception of those at the C1, C4,
C5, and C6 atoms.
In the planned efforts to expand upon the versatility of 1,1,8 we
were interested in varying the substituent on the nitrile oxide in the
1,3-dipolar cycloaddition reaction, introducing not only polar and
hydrophilic groups but active functionalities suitable for di-
versification of the nucleoside structures. We wish to report here
on the studies regarding the reactivity of N-benzoyl-2,3-
oxazanorborn-5-ene 1a with the bromonitrile oxide, easily pre-
pared from glyoxylic acid,9 and the synthetic elaboration of the
regioisomeric cycloadducts to obtain valuable intermediates for
nucleoside syntheses.
In order to achieve the aminol structures, the regioisomeric
cycloadducts 14a,b were then submitted to the synthetic elabora-
tion following the well-established protocol applied in previous
studies.1,5
Alkaline hydrolysis of the cycloadducts 14a,b took place in
the presence of 1.1 equiv of NaOH in THF/H2O 1:1 as solvents,
leaving the reaction at room temperature overnight. Monitoring
the reaction by TLC allowed for the clean and complete consume
of the starting compounds and the regioisomeric hydroxyl-
amines 15a,b were obtained in 97% and 95% yields, respectively
(Scheme 5).5e
2. Results
The bromonitrile oxide 13 is generated10 in situ in an AcOEt/
NaHCO3 solution from the corresponding bromoxime 12, whose
preparation is easily conducted by treating of glyoxylic acid with
hydroxylamine hydrochloride in the presence of bromine at 5 ꢀC in
dichloromethane.11 Using the N-benzoyl-2,3-oxazanorborn-5-ene
1a as dipolarophile, the 1,3-dipolar cycloaddition reaction
smoothly occurs affording the regioisomeric compounds 14a,b in
45% and 44% yields, respectively (Scheme 4), easily isolated upon
chromatographic separation.
The structures rely upon their analytical and spectroscopic
data. The IR spectrum of hydroxylamine 15a showed a sharp
band at 3229 cmꢁ1 corresponding to the stretching of the NH
group that gave also a broad signal in the 1H NMR spectrum at
d
5.60. The isoxazoline proton H5 at
d
4.80 (d, J¼8.4 Hz) is cou-
pled with the H4 proton at
d
3.69 (d, J¼8.4 Hz). Similarly, the IR
spectrum of regioisomer 15b showed a sharp band at 3231 cmꢁ1
corresponding to the stretching of the NH group that gave also
a broad signal in the 1H NMR spectrum at
d
5.67. The isoxazoline
proton H5 at
d
4.94 (d, J¼8.2 Hz) is coupled with the H4 proton at
d
3.69 (d, J¼8.2 Hz). Hydrogenolysis of the hydroxylamines 15a,b
was not conducted under the standard conditions, catalytic hy-
drogenation with Pd/C at room temperature, since the method
did not allow for the clean cleavage of the NeO bond and mix-
tures of unidentified compounds were formed. The mild re-
duction with Al(Hg) in THF/H2O 10:1 as solvents at 0 ꢀC for 8 h
afforded the aminolic structures 16a,b in 92% and 90% yields,
Scheme 4.
The structures of cycloadducts 14a,b were assigned based on
their analytical and spectroscopic data. The 1H NMR spectrum

M. Savion et al. / Tetrahedron 68 (2012) 1845e1852
1847
HO
Br
HO
NH2
O
NH2
N
N
Br
COPh
COPh
O
NC
OH
CN
HO
N
N
16a
16b
O
14a
14b
O
MeO-CH
CH-CO-NCO
C, MS, 12h
NaOH
THF/H2O 1:1
O
O
Br
O
N
N
NH
NH
Br
O
HO
NH
NH
HO
MeO
O
MeO
CN
HN
O
HN
O
15a
15b
O
NC
OH
17b
HO
Al(Hg)
THF/H2O 10:1
17a
N
p.TsOH
C, 2h
HO
N
HO
O
O
NH2
NH2
H
N
H
N
OH
16a
16b
OH
O
O
HO
HO
Scheme 5.
N
N
NC
OH
CN
HO
18a
18b
respectively. NeO bonds in bicyclic HDA adducts are readily
cleaved on exposure to excess Al(Hg) in aqueous THF at 0 ꢀC for
several hours.13 However, the structure assignments revealed
that also the NeO bond belonging to the isoxazoline ring was
cleaved and a new hydroxy group and a nitrile were found in
Scheme 6.
The 3-methoxy-2-propenoyl isocyanate was easily obtained
starting from the commercially available methyl 3-methoxy-2-
propenoate through basic hydrolysis to the acid,15 conversion to
the chloride with thionyl chloride,16 and coupling with silver
cyanate in benzene.15 The addition reactions of the aminols 16a,b
to isocyanate were conducted according to the procedure re-
ported in the literature15 by performing the reactions at ꢁ20 ꢀC
in DMF solutions for 12 h in the presence of MS. After chro-
matographic purification, the urea adducts 17a,b were obtained
in fair yields (40% and 44%, respectively). Their structures rely
upon the analytical and spectroscopic data. Neat distinctive
bands corresponding to the OH (3200, 3290 cmꢁ1) and the two
NH (3462, 3490 cmꢁ1) groups were evident in the IR spectra as
well as the bands corresponding to the nitrile groups (2250,
2247 cmꢁ1). The NMR spectra showed the signals of the methoxy
propenoyl chains as well as those of the carbocyclic moiety in the
usual ranges.
Cyclization of the ureas 17a,b took place smoothly upon heating
in dioxane solutions for 2 h in the presence of catalytic amounts of
p-toluensulfonic acid (p-TsOH). The uracil nucleosides 18a,b were
isolated from these solutions after pH adjustment to 7 and ex-
traction with dichloromethane. The yields of the cyclization steps
were satisfactory (94% and 96%, respectively) and the structures of
the nucleosides 18a,b rely upon their analytical and spectroscopic
data. The IR spectra of nucleosides 18a,b showed OH and NH bands
in the usual range, along with the nitrile bands at 2246 cmꢁ1. The
1H NMR spectra of the uracil nucleoside 18a,b showed the char-
acteristic coupled vinyl protons of the uracil unit as doublets at
a typical b-hydroxynitrile arrangement.
The IR spectrum of the aminol 16a showed an intense and broad
band centered at 3330 cmꢁ1 corresponding to the hydroxy groups
and covering the signals of the NH2 group. A sharp and intense
band at 2245 cmꢁ1 was clearly attributable to the CN group. In the
1H NMR spectrum the methylene protons gave splitted signals as
multiplets at
d 1.30 and 2.34 because of the coupling with the ad-
jacent protons of the cyclopentane ring and anisotropic effects in-
duced by the nitrile group.
The proton adjacent to the nitrile group gave a double-doublet
(J¼8.0, 5.0 Hz) at
found at
3.57 as a double-doublet (J¼15.0, 8.0 Hz). The two
CHeOH protons were found as multiplets at 3.82 and 3.95.
d 2.96 while the one linked to the NH2 was
d
d
Similarly, the IR spectrum of the aminol 16b showed an intense
and broad band centered at 3340 cmꢁ1, corresponding to the
hydroxy groups and covering the signals of the NH2 group. The
CN group gave a sharp and intense band at 2245 cmꢁ1 in the IR
spectrum. In the 1H NMR spectrum the methylene protons gave
splitted signals because of anisotropic effects induced by the
nitrile group and appear as multiplets at
of the coupling with the adjacent protons of the cyclopentane
ring. The protons adjacent to the nitrile group and to the NH2
d 1.21 and 2.29 because
gave a multiplet at
d 3.04 while the two CHeOH protons were
found as double-doublets at
(J¼15.0, 7.0 Hz).
d
3.88 (J¼9.0, 5.0 Hz) and 3.95
The stereoisomeric aminols 16a,b were then converted into the
uracil nucleosides14 through the linear construction of these het-
erocycles.15 The synthetic route to uracil nucleosides involves the
steps illustrated in Scheme 6 and started with the preparation of
the appropriate isocyanate.
d
5.73 and 7.71 (J¼8.0 Hz) and at
d
5.63 and 7.69 (J¼8.0 Hz), re-
spectively, for 18a and 18b. The other signals are in the expected
range.

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M. Savion et al. / Tetrahedron 68 (2012) 1845e1852
3. Modeling and docking studies
antiviral agent known as Brivudin (BVDU),20 molecular docking and
molecular dynamics (MD) studies have been conducted on Thy-
midine Kinase (TK). From the X-ray structure of TK co-crystallized
with BVDU21 a model of the enzyme was developed by us and
optimized through MD simulations carried out for 5 ns with the
SANDER module of the AMBER 10 package22 using explicit water as
solvent.
The binding mode of BVDU, that remained unchanged after
MD simulations, is depicted in Fig. 3A. The deoxyribose moiety
binds to Tyr101 and Glu225 via hydrogen bonds of its OH in
position 30, and to Glu83 via the OH in 50. Moreover, the uracil
moiety interacts with the side chain amide group of Gln125 by
means of a couple of hydrogen bonds involving the carbonyl and
the NH groups. The bromovinyl chain occupies an available hy-
drophobic pocket in the neighborhood of residues Trp88, Tyr132,
Arg163, and Ala167.
The structures of uracil nucleosides 18a,b belong to a class of
nor-nucleosides,17 lacking the hydroxymethylene side-arm syn to
the nucleobase. They keep the conformational mobility of the
cyclopentane spacer because of the isoxazoline ring opening.5
When fused to an isoxazoline ring or similar rings,5e the cyclo-
pentane moiety usually adopts an envelope conformation with the
flap directed toward the isoxazoline ring thus giving a boat-like
appearance to the structure. A comparison with this well-known
conformational array and the one corresponding to the novel nu-
cleosides 18a,b has been made with the aid of the optimized
structures at the B3LYP/6-31G(d) level,18 which are represented in
Fig. 2.
Then, using this optimized model of TK deprived of BVDU,
docking calculations were performed with the aim of determining
the binding mode of compounds 18a and 18b. The best docking
solutions were selected on the basis of their attained goldscore
values. As shown in Fig. 3B the binding mode of 18a and 18b
matches well with the one of BVDU. In detail, the results obtained
for compound 18a put in evidence that it creates a H-bond network
with the same four TK residues found for BVDU. The pairwise in-
teractions with Gln125 and those with Glu225, Tyr101 and Glu83
are retained.
On the other hand, compound 18b maintains the hydrogen
bonds with Gln125 and Glu83, but loses the positive interactions
with Glu225 and Tyr101, because of steric repulsion between the
nitrile at position 3’ and the side chain of Glu225.
Fig. 2. Conformations of nucleosides 18a,b and relative energies in water. Curved ar-
rows specify the dihedral angles in degrees while the dashed lines indicate hydrogen
ꢁ
bonds and the distances are given in A.
Finally, in order to verify the stability of the poses and to better
simulate the induced-fit effect of the ligands on the enzyme, opti-
mization and MD simulations were performed on the complexes of
18a and 18b within TK. Compared to the docking pose, new binding
modes were observed, as highlighted by the superimposition of 18a
and 18b with BVDU (Fig. 4A).
In the case of compound 18a the stabilizing pairwise interaction
of the nitrogen basis with Gln125 is maintained, some interactions
are lost and some new interactions are observed. Adaptation of 18a
into the TK binding site seems to be driven by the proximity of the
CN group to the side chain of Tyr101, that causes a change in the
hydrogen bond network found for the cyclopentane moiety. Actu-
ally, the OH at C3’ remains in contact with the side chain of Tyr101,
while the OH at C4’ establishes an interaction with Glu225. More-
over, the lone pair of the latter hydroxyl group accepts a hydrogen
bond from the charged side chain of Arg222, a residue in turn
blocked by salt bridges with Glu225 and Glu83.
The structures of nucleosides 18a,b are conditioned by the
strong intramolecular hydrogen bonds between the OH and the
uracil carbonyl,19 helping the cyclopentane ring to assume a typical
envelope conformation with the flap bearing the uracil ring shifted
downward with respect to the average plane of the defined by the
other four carbon atoms of the spacer.
The dihedral angles between the hydrogen atoms opposite to
the uracil ring are about ꢁ80 ꢀC. The dihedral angles between the
methyne linked to the uracil and the adjacent CHeCN or CHeOH in
18a and 18b, respectively, are 155ꢀ. The overall effect of these arrays
in the 1H NMR spectra is the presence of couplings between the
cyclopentane hydrogen atoms and the coupling constant (J) values
are in the expected range for a cyclopentane ring in an envelope
conformation, as previously described.
In order to understand the possible interactions of the regioi-
someric nucleosides 18a and 18b, that are structurally similar to an
Fig. 3. (A) Binding mode of BVDU after 5 ns of MD simulations, starting from the co-crystallized structure of TK. Hydrogen bonds are shown as yellow dashed lines. (B) Overlay of
BVDU (green) with the best docking solutions found for the regioisomeric compounds 18a (cyan) and 18b (magenta).

M. Savion et al. / Tetrahedron 68 (2012) 1845e1852
1849
Fig. 4. (A) Overlay of BVDU (green) with 18a (cyan) and 18b (magenta) after 5 ns of MD simulations, starting from the best docking solutions. The shown residues of the active site
belong to the complex with BVDU. (B) Predicted binding mode of compound 18a after MD simulation. (C) Predicted binding mode of compound 18b after MD simulation. Hydrogen
bonds are shown as yellow dashed lines.
Conversely, in the case of compound 18b, only one of the two
hydrogen bonds of the nucleobase with Gln125 is maintained.
Moreover, the OH groups at C2’ and C4’ of the cyclopentane ring
interacts with Glu225 and Glu83, respectively (Fig. 4C). As a result
of the dynamics simulations, the reallocation allows the removal of
the repulsive interaction of the CN group with the Glu225 side
chain present in the docking pose.
to the cleavage of two different types of NeO bonds. Scheme 7 gives
a representation of a possible mechanism to explain this double
cleavage and the obtaining of the
b-hydroxynitrile aminols for
regisomer 15a; similarly happened for 15b.
Furthermore, it is worthy pointing out that both compounds 18a
and 18b, compared with BVDU, lack the bromovinyl chain with the
consequent loss of the related hydrophobic interactions that might
exert an anchoring affect in the pocket of the enzyme shaped by
Trp88, Tyr132, Arg163, and Ala167.
Δ
4. Discussion and conclusions
Scheme 7.
The regioisomeric cycloadducts 14a,b of the bromonitrile oxide
13 to the N-benzoyl-2,3-oxazanorborn-5-ene 1 were easily ob-
tained and easily elaborated into a novel class of uracil nor-
nucleosides by applying know procedures5 adapted to the sub-
strates at hand. The alkaline hydrolysis of the cycloadducts 14a,b
took place in the presence of NaOH in THF/H2O 1:1 allowing for the
easy detachment of the benzoyl group. Conversely, the NeO bond
cleavage had to be tuned up and the mild reduction with Al(Hg) in
THF/H2O 10:1 as solvents at 0 ꢀC was necessary to prepare the re-
quired aminolic structures 16a,b in high yields. The peculiarity of
this step is represented by the concurrent double ring opening due
The Al(Hg) promoted cleavage13 of the hydroxylamine moiety
occurred as expected affording the bicyclic aminolic structure 19
that does not survive even under these mild reaction conditions.
The NeO bond belonging to the isoxazoline ring was also cleaved
and the formal loss of HBr from the intermediate 20 afforded the
final aminol 16a. A comparable mechanism is reported in literature
for the trimethylsilylnitrile oxide cycloadducts of type 21, which are
thermally decomposed under hydrolytic conditions to the
b-
hydroxynitriles 22,23 as depicted in the inset of Scheme 7.

1850
M. Savion et al. / Tetrahedron 68 (2012) 1845e1852
Alternatively, the isoxazoline rings are transformed into
hydroxynitriles by heating in the presence of bases.24
b
-
3200e3400; nC]N 1400; 1H NMR (
d C
, DMSO): 12.6 (s, 1H, ]NeOH); 13
NMR ( , DMSO): 94.5.
d
With the aminols 16a,b in hand the synthesis of the uracil de-
rivatives was secured by applying the known protocol.5 The con-
densation of the isocyanate with the aminols was the more delicate
step since isolation of pure derivatives 17a,b required a chromato-
graphic separation from the reaction mixtures, with somewhat
decrease of product yields. However, the final acid catalyzed cy-
clization worked nicely affording the uracil derivatives 18a,b in
high yields.
5.4. Cycloaddition of bromonitrile oxide 13 to the N-benzoyl-
2,3-oxazanorborn-5-ene 1
To a stirred solution of 5.28 g (26.24 mmol) of N-benzoyl-2,3-
oxazanorborn-5-ene 1 in 200 mL of AcOEt, 3.93 g (47.2 mmol) of
NaHCO3 is added. To this suspension 7.98 g (39.4 mmol) of bro-
moxime 12 dissolved in 50 mL of AcOEt is added dropwise. The
reaction proceeds under stirring for 48 h at room temperature.
Upon filtration and evaporation of the solvent, an oily residue is
obtained and submitted to chromatographic separation, allowing
for the isolation of the regioisomeric cycloadducts 14a,b.
Structurally similar to
a known antiviral agent, Brivudin
(BVDU)20, the possible interactions of the regioisomeric nucleo-
sides 18a and 18b were evaluated through molecular docking and
MD studies, conducted on TK. The binding mode of 18a and 18b
matches well with the one of BVDU. The results obtained for
compound 18a put in evidence that it creates a H-bond network
similar to that found for BVDU. The pairwise interactions with
Gln125 and Glu225 are retained. However, OH at C4’ establishes
a new interaction with Arg222. Compound 18b maintains only one
hydrogen bond with Glu125 and those with Glu83 and Glu225.
These promising observations suggest the importance of testing
these novel nucleoside compounds against Herpes viruses in order
to confirm the theoretical evaluations. Samples of compounds 18a
and 18b will be soon submitted for the biological tests on Herpes
viruses to the National Institute of Health (NIH) within the Antiviral
Evaluation program.
5.4.1. Compound 14a. Yield 3.82 g (45%), mp 125e130 ꢀC, colorless
crystals from ethanol. Rf (cyclohexane/AcOEt 70:30) 0.76. IR: nC]O
1657 cmꢁ1. 1H NMR (
d, CDCl3): 2.01e2.04 (m, 2H, CH2), 3.99 (d, 1H,
J¼8.4 Hz, H4 isox), 4.88 (s, 1H, CHeN), 5.01 (dt, 1H, J¼8.4, 1.7 Hz, H5
isox), 5.17 (s, 1H, HCeO), 7.41e7.47 (m, 2H, arom), 7.50e7.54 (m, 1H,
arom), 7.77e7.79 (m, 2H, arom). 13C NMR (
d, CDCl3): 32.6, 58.0, 60.3,
80.4, 83.1, 128.1, 129.1, 132.1, 132.2, 136.9, 171.9. Elemental analysis:
calcd for C13H11BrN2O3 (MW¼323.14) C, 48.32; H, 3.43; N, 8.67.
Found: C, 48.2; H, 3.1; N, 8.7.
5.4.2. Compound 14b. Yield 3.73 g (44%), mp 75e80 ꢀC, colorless
crystals from ethanol. Rf (cyclohexane/AcOEt 70:30) 0.81. IR: nC]O
5. Experimental section
5.1. General
1663 cmꢁ1. 1H NMR (
d, CDCl3): 2.09e2.11 (m, 2H, CH2), 3.90 (d, 1H,
J¼8.0 Hz, H4 isox), 4.96 (s, 1H, CHeN), 5.15 (dt, 1H, J¼8.0, 1.4 Hz, H5
isox), 5.14 (s, 1H, CHeO), 7.43e7.47 (m, 2H, arom), 7.54e7.56 (m, 1H,
arom), 7.73e7.76 (m, 2H, arom). 13C NMR (
d, CDCl3): 32.5, 61.4, 61.9,
All melting points are uncorrected. Elemental analyses were
done on a C. Erba 1106 elemental analyzer available in our De-
partment. IR spectra (Nujol mulls) were recorded on an FT-IR Per-
kineElmer RX-1. 1H and 13C NMR spectra were recorded on a Bruker
AVANCE 300 in the specified deuterated solvents. Chemical shifts
79.2, 82.8, 128.2, 128.8, 132.1,132.4, 136.0, 170.6. Elemental analysis:
calcd for C13H11BrN2O3 (MW¼323.14) C, 48.32; H, 3.43; N, 8.67.
Found C, 48.3; H, 3.5; N, 8.7.
are expressed in parts per million (d) from internal tetramethylsi-
5.5. Alkaline hydrolysis of cycloadducts 14a,b
lane. Column chromatography and TLC: silica gel 60
(0.063e0.200 mm) (Merck); eluant cyclohexane/ethyl acetate from
9:1 to 5:5. MPLC: Biotage FMP apparatus equipped with KP-SIL
columns, eluant cyclohexane/ethyl acetate from 9:1 to 7:3. The
identification of samples from different experiments was secured
by mixed mps and superimposable IR spectra.
To a stirred solution of the cycloadducts 14a,b (1.13 g, 3.5 mmol)
in 40 mL of THF/H2O 1:1,1.1 equiv (0.15 g, 3.8 mmol) of solid ground
NaOH was added portionwise at room temperature. After keeping
the reaction mixture overnight at room temperature, the solvent
was evaporated and the residues were taken up with DCM and
washed twice with brine and finally the organic phases were dried
over anhydrous Na2SO4. The evaporation of the DCM left yellowish
oils corresponding to the hydroxylamines 15a,b in nearly quanti-
tative yields.
5.2. Materials
Glyoxylic acid monohydrate, solvents, and all the reagents re-
quired for the preparation of the starting materials are from Sig-
maeAldrich and were used without purification.
5.5.1. Compound 15a. Yield 0.74 g (97%), yellowish oil. Rf (cyclo-
hexane/AcOEt 70:30) 0.85. IR: nNH 3229 cmꢁ1 nC]N 1572 cmꢁ1 1H
, .
5.3. Preparation of the bromoxime 12
NMR (d, CDCl3): 1.87 (d, 1H, CH2), 2.07 (d, 1H, CH2), 3.69 (d, 1H,
J¼8.4 Hz, H4 isox), 4.02 (d, 1H, J¼1.0 Hz, CHeN), 4.80 (dt, 1H, J¼8.4,
To a solution of 30.0 g (0.39 mol) of glyoxylic acid monohydrate
dissolved in 240 mL of water, 28.2 g (0.41 mol) of hydroxylamine
hydrochloride was added portionwise under stirring and the so-
lution left at room temperature for 24 h. After this period of time,
70.6 g (0.85 mol) of NaHCO3 was added carefully and portionwise
followed by 300 mL of DCM. The solution was then cooled around
5 ꢀC and a bromine solution (28.9 mL of Br2 in 150 mL DCM) was
added dropwise and the solution left under stirring for 3 h. The
organic phase was then separated and the water phase extracted
with DCM (twice with DCM). The collected organic layers were
dried over anhydrous Na2SO4. Evaporation to dryness of the solvent
afforded the crude bromoxime 12, used without any further puri-
fication: mp 65e70 ꢀC (Ref. lit. Mp 65e66 ꢀC);9 IR (cmꢁ1): nOH
2.0 Hz, H5 isox), 4.64 (t, 1H, J¼2.0 Hz, HCeO), 5.60 (b, 1H, NH). 13
C
NMR (d, CDCl3): 34.7, 58.6, 60.9, 77.0, 83.6, 137.6. Elemental analy-
sis: calcd for C6H7BrN2O2 (MW¼219.04) C, 32.90; H, 3.22; N, 12.79.
Found C, 32.8; H, 3.1; N, 12.8.
5.5.2. Compound 15b. Yield 0.73 g (95%), yellowish oil. Rf (cyclo-
hexane/AcOEt 70:30) 0.87. IR: nNH 3231 cmꢁ1 nC¼N 1571 cmꢁ1 1H
, .
NMR (d, CDCl3): 1.85e1.88 (m, 1H, CH2), 2.16 (m, 1H, CH2), 3.69 (d,
1H, J¼8.2 Hz, H4 isox), 4.07 (d,1H, J¼1 Hz, CH-N), 4.94 (dt,1H, J¼8.2,
2.0 Hz, H5 isox), 4.72 (t, 1H, J¼2.0 Hz, HCeO), 5.67 (br s, 1H, NH). 13
C
NMR (d, CDCl3): 34.6, 61.3, 62.4, 75.7, 84.1, 136.8. Elemental analy-
sis: calcd for C6H7BrN2O2 (MW¼219.04) C, 32.90; H, 3.22; N, 12.79.
Found C, 32.7; H, 3.4; N, 12.8.

M. Savion et al. / Tetrahedron 68 (2012) 1845e1852
1851
5.6. Reductive cleavage of hydroxylamines 15a,b
solutions were heated at 80 ꢀC for a couple of hours. After cooling,
the pH was adjusted at 7 with NaHCO3 and the water phase
extracted with DCM. Evaporation of the dried organic phase
afforded the uracil derivatives 18a,b.
To a stirred solution of the hydroxylamines 15a,b (2.6 g,
11.86 mmol) in 200 mL of THF/H2O 10:1 under nitrogen and at 0 ꢀC,
3.0 g of Al(Hg) is added portionwise and the reaction is conducted
until complete consume of the starting materials (monitoring by
TLC). After completion, the solution is diluted with THF and filtered
over Celite. The organic phase was then evaporated to dryness to
leave oily residues corresponding to the aminols 16a,b.
5.8.1. Compound 18a. Yield 0.42 g (96%), yellow oil. Rf (AcOEt) 0.90.
IR: nNH/OH 3374 cmꢁ1 nCN 2246 cmꢁ1 nC]O 1682 cmꢁ1 1H NMR (
, , . d,
DMSO): 1.51e1.55 (m, 1H, CH2), 2.50e2.53 (m, 1H, CH2), 3.44e3.48
(m, 1H, CHeCN), 3.94 (d, 1H, J¼5.0 Hz, CHeOH), 4.06 (d, 1H,
J¼5.0 Hz, CHeOH), 5.30e5.32 (m,1H, CHeN), 5.73 (d,1H, J¼8.0 Hz, ]
5.6.1. Compound 16a. Yield 1.55 g (92%), pale yellow oil. Rf (cyclo-
CH), 7.71 (d, 1H J¼8.0 Hz, NeCH]), 11.37 (s, 1H, NH). 13C NMR (
d,
hexane/AcOEt 50:50) 0.95. IR: nOH,NH2 3330 cmꢁ1
;
nCN 2245 cmꢁ1
.
DMSO): 36.5, 43.1, 62.4, 71.1, 72.2, 101.7, 119.1, 143.4, 151.0, 163.0.
Elemental analysis: calcd for C10H11N3O4 (MW¼237.21) C, 50.63; H,
4.67; N, 17.71. Found C, 50.5; H, 4.8; N, 17.5.
1H NMR (
d, DMSO): 1.26e1.32 (m, 1H, CH2), 2.32e2.36 (m, 1H, CH2),
2.96 (dd, 1H, J¼8.0, 5.0 Hz, CHeCN), 3.57 (dd, 1H, J¼15.0, 8.0 Hz,
CHeNH2), 3.80e3.83 (m,1H, HCeO), 3.93e3.97 (m,1H, HCeO), 5.00
(br, 3H, NH2 and OH). 13C NMR (
d, DMSO): 40.0, 41.7, 53.7, 75.6, 77.2,
5.8.2. Compound 18b. Yield 0.42 g (96%), mp 150e2 ꢀC, colorless
120.0. Elemental analysis: calcd for C6H10N2O2 (MW¼142.16) C,
crystals from ethanol. Rf (AcOEt) 0.93. IR: nNH 3182 cmꢁ1
;
nOH 3412
50.69; H, 7.09; N, 19.71. Found C, 50.7; H, 7.1; N, 19.7.
and 3050 cmꢁ1
,
nCN 2246 cmꢁ1
,
nC]O 1716 and 1673 cmꢁ1. 1H NMR (
d,
DMSO): 1.72e1.74 (m, 1H, CH2), 2.30e2.32 (m, 1H, CH2), 3.20 (t, 1H,
J¼7.0 Hz, CHeCN), 4.26e4.29 (m, 1H, CHeOH), 4.36e4.39 (m, 1H,
CHeOH), 4.47e4.49 (m, 1H, CHeN), 5.98 (br, 1H, OH), 5.63 (d, 1H,
J¼8.0 Hz, ]CH); 7.69 (d, 1H, J¼8.0 Hz, NeCH]), 11.30 (s, 1H, NH). 13C
5.6.2. Compound 16b. Yield 1.55 g (92%), pale yellow oil. Rf (cyclo-
hexane/AcOEt 50:50) 0.96. IR: nOH,NH2 3340 cmꢁ1 nCN 2245 cmꢁ1
; .
1H NMR (
d, DMSO): 1.20e1.22 (m, 1H, CH2); 2.27e2.30 (m, 1H, CH2);
3.02e3.06 (m, 2H, CH-CN and CH-NH2); 3.88 (dd, J¼9.0, 5.0 Hz, 1H,
NMR (d, DMSO): 36.5, 43.1, 62.4, 71.1, 72.2, 101.7, 119.1, 143.4, 151.0,
HCeO); 4.27 (dd, 1H, J¼15.0, 7.0 Hz, HCeO); 3.3e5.6 (br, 3H, NH2
163.2. Elemental analysis: calcd for C10H11N3O4 (MW¼237.21) C,
and OH). 13C NMR (
d, DMSO): 40.0, 42.9, 57.0, 72.5, 76.1, 120.1. El-
50.63; H, 4.67; N, 17.71. Found C, 50.6; H, 4.7; N, 17.6.
emental analysis: calcd for C6H10N2O2 (MW¼142.16) C, 50.69; H,
7.09; N, 19.71. Found C, 50.8; H, 7.3; N, 19.9.
5.9. Docking and molecular dynamics
5.7. Synthesis of the isocyanate adducts 17a,b
The structure of Thymidine Kinase (TK) from Herpes Simplex
Virus type-1 have been reported in the Protein Data Bank co-
crystallized with BVDU (accession code 1KI8) and with the in-
hibitor Ganciclovir (accession code 1KI2). For our calculation we
decided to use the first one due to the similarity between our li-
gands and the co-crystallized one.
To solutions of the aminols 16a,b (4.42 g, 31.1 mmol) in 35 mL of
anhydrous DMF at ꢁ20 ꢀC, 38.8 mL of a 0.94 M solution of the
isocyanate in anhydrous benzene was added dropwise with stirring
ꢁ
under nitrogen atmosphere and in the presence of MS 4 A. After
keeping for one night at room temperature, the solutions were
filtered and the solvent removed under reduced pressure. The
residues were submitted to chromatographic separation to isolate
the adducts 17a,b.
Part of the missing residues in the crystal structure of BVDU/TK
were modeled taking into account the orientation found in the
structure of the TK co-crystallized with the inhibitor Ganciclovir.
Then, the residues not solved in both crystal structures were
completed through the Biopolymer option implemented in Sybyl
8.0 (Tripos Inc). The final model of the enzyme was geometrically
refined in solvent following standard procedures previously re-
ported: minimization, equilibration, and molecular dynamics sim-
ulation,25 using AMBER 10 package.22
5.7.1. Compound 17a. Yield 3.35 g (40%), yellow oil. Rf (AcOEt) 0.79.
IR: nNH 3462 cmꢁ1 nOH 3200 cmꢁ1 nCN 2250 cmꢁ1 nC]C 1668 cmꢁ1
; , , .
1H NMR (
d, DMSO): 1.33e1.36 (m, 1H, CH2), 2.02e2.06 (m, 1H, CH2),
3.16e3.18 (m, 1H, CHeCN), 3.69 (s, 3H, OeCH3), 3.85 (s, 1H, CH-OH),
3.98 (s,1H, CHeOH), 4.52 (m,1H, CHeNH), 5.20 (d,1H, J¼4.0 Hz, OH),
5.54 (d, 1H, J¼9.0 Hz, ]CH), 5.72 (d, 1H, J¼4.0 Hz, OH), 7.58 (d, 1H,
J¼9.0 Hz, OeCH]), 8.78 (d, 1H, J¼9.0 Hz, NH); 10.16 (s, 1H, NH). 13C
Ligands were built using GaussView and their conformational
space was studied using the Gaussian09 program package18 at
B3LYP/6-31G(d) level. Vibrational frequencies were computed at
the same level as above to verify that the optimized structures were
minima. The energy of the located conformations was recalculated
in water through single-point calculations using a continuum sol-
vent model (PCM). Then, docking calculations were performed on
the preferred conformations of compounds 18a and 18b by GOLD
4.1 software26 into the binding cleft depicted by the presence of
BVDU.
NMR (d, DMSO): 38.7, 40.8, 51.3, 58.0, 75.6, 76.9, 97.8, 119.8, 153.2,
162.9,167.4. Elemental analysis: calcd for C11H15N3O5 (MW¼269.25)
C, 49.07; H, 5.62; N, 15.61. Found C, 49.1; H, 5.8; N, 15.4.
5.7.2. Compound 17b. Yield 3.52 g (42%), mp 190e2 ꢀC, colorless
crystals from ethanol. Rf (AcOEt) 0.81. IR: nNH/OH 3290 cmꢁ1
,
nCN
2247 cmꢁ1 nC]C 1682 cmꢁ1 1H NMR (
, . d, DMSO): 1.38e1.42 (m, 1H,
ꢁ
CH2), 2.36e2.39 (m, 1H, CH2), 2.98 (t, 1H, J¼6.0 Hz, CHeCN), 3.68 (s,
3H, OeCH3), 3.86e3.90 (m, 1H, CHeOH), 4.12 (s, 1H, CHeOH), 4.31
(dd, 1H, J¼13.0, 6.0 Hz, CHeNH), 5.53 (d, 1H, J¼13.0 Hz, ]CH) 5.61
(br s, 1H, J¼4.0 Hz, OH), 5.85 (br s, 1H, OH), 7.58 (d, 1H, J¼13.0 Hz,
The cavity was detected with an active site radius of 15.0 A from
the sulfur atom of Met128. The goldscore fitness function and the
distribution of torsional angles were chosen to evaluate the quality
of the docking results. Van der Waals and hydrogen bonding radii
OeCH]); 8.66 (d, 1H, J¼7.0 Hz, NH), 10.11 (s, 1H NH). 13C NMR (
d
,
were set at 4.0 and 3.0 A, respectively; genetic algorithm parame-
ꢁ
DMSO): 38.6, 43.5, 55.8, 58.0, 72.6, 74.8, 97.9, 119.7, 153.2, 162.8,
167.4. Elemental analysis: calcd for C11H15N3O5 (MW¼269.25) C,
49.07; H, 5.62; N, 15.61. Found C, 49.0; H, 5.5; N, 15.7.
ters were kept at the default value. The resulting complexes were
submitted to molecular dynamics simulations with the sander
module of the AMBER 10 package. FF0327 and GAFF28 force field
were applied for the protein and the ligands, respectively. The
complexes were immersed in a box containing about 8600 water
molecules and the TIP3P29 model was employed to explicitly rep-
resent the solvent. Naþ counterions were added to maintain neu-
trality on the systems. At first, the energy of the water molecules
5.8. Construction of the uracil nucleosides 18a,b
Adducts 17a,b (0.5 g (1.86 mmol)) were dissolved in dioxane
(6 mL) in the presence of a catalytic amount of p-TsOH. The

1852
M. Savion et al. / Tetrahedron 68 (2012) 1845e1852
(o) Li, F.; Warshakoon, N. C.; Miller, M. J. J. Org. Chem. 2004, 69, 8836e8841; (p)
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ꢁꢁ1
a convergence criterion on the gradient of 10ꢁ4 kcal molꢁ1
A .
Prior to starting the MD simulations, the system was equilibrated
for 40 ps at 300 K in isocore conditions (NVT). Subsequently, 5 ns of
MD simulations in isothermaleisobaric (NPT) ensemble were car-
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systems were performed in periodic boundary conditions. Van der
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Acknowledgements
Financial support by the University of Pavia, MIUR (PRIN 2008,
CUP F11J10000010001) and EUTICALS S.p.a.dV.le Milano, 86/
88d26900 Lodi, Italy is gratefully acknowledged. The authors
wish to thank the Fondazione Banca del Monte di Lombardia for
a research grant. Authors warmly thank Prof. L. Toma and Prof. P.
Caramella for helpful discussion. Thanks are due to Dr. Massimo
Boiocchi (CGS) for X-ray data collection.
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Supplementary data
Crystal structure analysis of cycloadduct 14a, CCDC 844826.
Cartesian Coordinates of optimized compounds 18a,b. Supplemen-
tary data related to this article can be found online at doi:10.1016/
j.tet.2011.12.086. These data include MOL files and InChIKeys of the
most important compounds described in this article.
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