Synthesis and SAR studies on azetidine-containing dipeptides as HCMV inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.12.052

SAR studies on an azetidine-containing dipeptide prototype inhibitor of HCMV are described. Three series of structurally modified analogues, involving substitutions at the N- and C-terminus, and at the C-terminal side-chain were synthesized and evaluated

Full text of DOI:10.1016/j.bmc.2010.12.052

Bioorganic & Medicinal Chemistry 19 (2011) 1155–1161
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and SAR studies on azetidine-containing dipeptides
as HCMV inhibitors
Paula Pérez-Faginas a, M. Teresa Aranda a, M. Teresa García-López a, Robert Snoeck b, Graciela Andrei b,
Jan Balzarini b, Rosario González-Muñiz a,
⇑
a Instituto de Química Médica (CSIC), Juna de la Cierva, 3, 28006 Madrid, Spain
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
SAR studies on an azetidine-containing dipeptide prototype inhibitor of HCMV are described. Three series
of structurally modified analogues, involving substitutions at the N- and C-terminus, and at the C-terminal
side-chain were synthesized and evaluated for antiviral activity. Aliphatic or no substituents at the
C-carboxamide group, an aliphatic C-terminal side-chain, as well as a benzyloxycarbonyl moiety at the
N-terminus were absolute requirements for anti-HCMV activity. The conformational restriction induced
Received 18 October 2010
Accepted 23 December 2010
Available online 30 December 2010
by the 2-azetidine residue into the dipeptide derivatives, identified by 1H NMR as a
seems to have influence on the activity of these molecules.
c-type reverse turn,
Keywords:
Cytomegalovirus
Antiviral agents
Azetidine-containing peptides
Solid-phase synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
c
-Turn conformation
1. Introduction
ligands for the chemokine receptor US28, thus also acting at an early
stage of the replication cycle.8 Quite recently, some
c-sultone deriv-
Human cytomegalovirus (HCMV) is a prevalent b-herpesvirus
that can cause serious, life-threatening diseases in immunologi-
cally immature or immunocompromised individuals, like neonates,
AIDS patients, and transplant recipients.1 Moreover, infection with
HCMV is suggested to be associated with certain vascular diseases
(atherosclerosis, restenosis, etc.).2,3
The current antiviral agents approved for HCMV infection,
ganciclovir, acyclovir, their Val pro-drugs valganciclovir and vala-
cyclovir, cidofovir, foscarnet and the antisense RNA fomivirsen,
demonstrate in general suboptimal efficacy and safety profiles.4,5
Moreover, these drugs suffer from unfavourable pharmacokinetic
properties, and for some of them antiviral drug resistance have
emerged.6 Therefore, there is still a need for more effective and safe
therapeutic agents for HCMV disease.
atives with anti-HCMV activity were shown to keep full antiviral
sensitivity against a panel of mutant HCMV strains, selected for
resistance against approved drugs, and accordingly suggesting a
new mechanism of action, which is still unclear.9
The HCMV protease, essential for capsid assembly and viral
maturation, is being considered an attractive target for anti-HCMV
chemotherapy, and several structurally diverse inhibitors have
been described.10 Most of them include an activated carbonyl
group or are mechanism-based inhibitors that covalently interact
with the Ser residue within the enzyme active site.11,12 Among
the latter, we and others have discovered simple and small
b-lactam derivatives with anti-HCMV activity.13,14 In our case, a
simple deletion of the carbonyl group of the b-lactam ring resulted
in the corresponding azetidine derivatives, which behave as non-
covalent inhibitors of HCMV replication.14b Preliminary struc-
ture–activity relationship studies suggested the importance of a
certain hydrophobic environment by the carboxylate in position
2 of the azetidine ring. Moreover, within this series, some C-termi-
nal carboxamide derivatives showed restricted rotation around the
While most of the anti-HCMV approved drugs, except fomivir-
sen, are inhibitors of the viral DNA polymerase, compounds with
different mechanisms of action have also appeared. Among them,
the L-ribose benzimidazole derivative maribavir, targeting to the
viral UL97 protein kinase, is currently under clinical trials.7 A series
of thiourea small-molecules were identified as potent anti-HCMV
agents, inhibiting virion envelope fusion, and some piperidine-de-
rived compounds were described as the first class of small-molecule
N1–CO bond, due to the stabilization of a c-turn-like folded confor-
mation.14b To investigate further the structural and conformational
issues within these azetidine derivatives and to determine their
interest as antiviral agents, we initiated a program directed to
the modification of compound 1, selected as a prototype (Fig. 1).
To this end, diverse substituents have been built-in the C-terminal
carboxamide moiety (A), amino acids with side-chains of different
⇑
Corresponding author. Fax: +34 915644853.
E-mail addresses: rosario.gonzalezmuniz@iqm.csic.es, iqmg313@iqm.csic.es
(R. González-Muñiz).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.052

1156
P. Pérez-Faginas et al. / Bioorg. Med. Chem. 19 (2011) 1155–1161
B
O
Me
O
Me
O
Me
NH2
NH2
NH2
(S)
N
H
O
(S)
(R)
(R)
H
N
N
H
N
H
(R)
(S)
+
N
O
A
Fmoc
N
O
N
O
Z
Z
O
Fmoc-Rink amida
a), b)
7a
7a
(S,R)
(R,R)
(S,S)1
C
d)
O
Z
R3
R3
H
H
N
Figure 1. Prototype and points of modification (A–C).
a), c)
N
Fmoc
N
N
H
H
N
O
O
Ph
character were selected as C-terminal residues (B), and the
N-terminal benzyloxycarbonyl group was replaced by different
urethane, acyl and urea moieties containing aromatic groups (C).
This paper deals with the solution/solid-phase synthesis,
conformational analysis by NMR, and biological assays of the com-
pounds resulting from the three indicated series A–C of azetidine-
containing dipeptides.
5a-f
6a-f
CO2H
Xaa = D-Ala,Phe, Val,
Leu, Lys (Boc),
Glu(tBu)
N
Z
d)
2
R3
O
R3
O
NH2
NH2
(S)
(S)
N
H
N
(S)
(R)
+
H
N
O
N
O
2. Results and discussion
2.1. Synthesis
Z
Z
7b-f
7b-f
(R,S)
(S,S)
: R3 = CH2Ph
c: R3 = CH(CH3)2
b
To increase the hydrophobic character of compound 1, different
alkyl groups were incorporated at the C-terminal carboxamide to
generate derivatives 4 (Series A). The preparation of these com-
pounds was achieved by coupling the 2-carboxy azetidine 2 with
d: R3 = CH2CH(CH3)2
: R3 = (CH2)4NH2
e
f
: R3 = (CH2)2CO2H
the corresponding L-alanyl carboxamides 3 under standard solu-
Scheme 2. Synthesis of C-terminal side-chain-modified compounds (B). Reagents:
(a) 20% pip/DMF; (b) Fmoc-Xaa-OH/DIC/HOBt/DMF; (c) 2/DIC/HOBt/DMF; (d) TFA/
H2O/TIPS (95:4:1).
tion methodologies (Scheme 1). All final compounds 4a–e were ob-
tained in good yield as mixtures of two diastereoisomers that could
be chromatographically resolved. Since the starting azetidine was a
ꢀ2:1 S:R enantiomeric mixture,15 the major components of these
mixtures were assigned as the S,S-diastereoisomers.
dipeptides 6a–f were finally detached from the polymeric support
by treatment with a 95:4:1 TFA/H2O/TIPS cleavage cocktail. As for
the previous series, compounds 7 were obtained in excellent yields
as mixtures of two diastereoisomers that were separated by
column chromatography, and configurationally assigned as
previously indicated for analogues 4.
Taking into account the inactivity of the N-methoxycarbonyl-
substituted analogue of 1,14b which suggested a role for the aro-
matic phenyl ring of the Z-group, different Ar-containing urethane,
acyl and urea groups were envisaged as substituents at the N1
position of the azetidine moiety (series C). The good results in
the synthesis of compounds 7 prompted us to apply a similar
solid-phase approach for the preparation of the third series of
To understand the role of the C-terminal amino acid side-chain,
D-Ala-, Phe-, Val-, Leu-, Lys- and Glu-bearing derivatives 7a–e were
designed (series B). Solid-phase synthetic methodologies, employ-
ing a Rink-amide polystyrene resin as insoluble matrix, were used
for the preparation of these compounds (Scheme 2). After removal
of the Fmoc-protecting group from the commercial resin, the
corresponding Fmoc-Xaa-OH amino acids were coupled, using
DIC/HOBt, to afford intermediates 5a–f. A similar procedure was
followed for the subsequent incorporation of the 1-benzyloxycar-
bonyl azetidine 2. The resulting resin-bound azetidine-containing
derivatives (Scheme 3). In this case, the Fmoc-L-Ala resin 5g was
R1
O
Me
deprotected and coupled to the Fmoc-azetidine derivative 8 to pro-
vide key intermediate 9. Unfortunately, this coupling step was dif-
ficult and, despite it was repeated, the Kaiser test indicated an
incomplete incorporation. The direct cleavage of 9 afforded after
purification the Fmoc-azetidine dipeptide 10a. Removal of the
Fmoc-protecing group from 9 followed by reaction with different
chloroformates or acyl- or sulfonyl-chlorides in the presence of
propylene oxide gave to derivatives 10b–g, after cleavage. Alterna-
tively, the treatment with isocyanates, followed by exposure to the
cleavage cocktail, allowed the preparation of urea analogues
10h–i. In all these cases, only the major S,S-diastereoisomer of
the mixture was isolated in enough purity for biological assays.
The deficient coupling of azetidine derivative 8 could be responsi-
ble for the formation of the complex reaction crudes, making the
purification step more difficult.
N
(S)
R2
CO2H
Z
N
H
(S)
N
N
O
Z
2
a)
4a-e
(S,S)
+
+
R1
N
R1
N
Me
O
Z
Me
R2
R2
(S)
H2N
N
H
(R)
O
N
O
3a-e
4a-e
(R,S)
: R1 = tBu, R2 = H
a
: R1 = Bzl, R2 = H
b
c: R1 = iBu, R2 = H
d: R1 = Chx R2 = H
: R1 = Me, R2 = Me
e
All prepared compounds were characterised by standard ana-
lytical and spectroscopic methods. The 1H NMR spectra always
showed cis/trans rotamer around the CON1 bond, fluctuating
Scheme 1. Synthesis of C-terminal amide-modified compounds (A). Reagents: (a)
BOP/TEA/THF.

P. Pérez-Faginas et al. / Bioorg. Med. Chem. 19 (2011) 1155–1161
1157
Table 1
H
N
Me
H
a, b
Temperature coefficients of amide protons in the azetidine-containing dipeptide
derivatives 4, 7, 10
Fmoc
N
Fmoc
N
H
O
Fmoc-Rink amida
5g
O
Z
Me R1
O
Z
R3
O
Me
Ph
N
NH2
NH2
N
H
R2
N
H
N
H
(S)
(S)
(S)
a, c
O
CO2H
Fmoc
(S)
N
N
O
N
O
N
O
8
R4
T (ppb/K)
CONHa,b
4
7
10
Me
O
Compd
R1, R2
Dd/D
H
N
NH2
(S)
a
-NHa
N
H
(S)
N
H
Fmoc
d
O
N
N
O
(S,S)-4a
(R,S)-4a
(S,S)-4b
(R,S)-4b
(S,S)-4c
(S,S)-4d
(R,S)-4d
(S,S)-4e
(R,S)-4e
H, C(CH)3
H, C(CH)3
H, CH2Ph
H, CH2Ph
H, CH2CH(CH3)2
H, Chx
H, Chx
CH3, CH3
CH3, CH3
R3
CH3
CH3
CH2Ph
ꢁ2.5
ꢁ2.0
ꢁ2.5
ꢁ1.6
ꢁ3.5
ꢁ3.0
ꢁ2.5
ꢁ2.5
ꢁ2.5
ꢁ3.5
ꢁ4.0
ꢁ4.5
ꢁ2.9
ꢁ4.0
ꢁ4.0
ꢁ5.5
—
O
O
9
10a
a, e, d
a, e or f, d
—
Compd
(S,R)-7a
(R,R)-7a
(S,S)-7b
(R,S)-7b
(S,S)-7c
(R,S)-7c
(S,S)-7d
(R,S)-7d
Me
O
O
ꢁ3.3
ꢁ2.4
ꢁ3.6
ꢁ2.2
ꢁ2.5
ꢁ1.8
ꢁ3.5
ꢁ2.2
ꢁ5.1
NH2
NH2
(S)
N
H
(S)
(S)
N
ꢁ4.1, ꢁ5.4
ꢁ4.5
(S)
O
S O
H
O
N
O
N
O
CH2Ph
ꢁ5.3
CH(CH3)2
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
R4
Fmoc
CO2CH2C6H4Cl
CO2CH2C6H4NO2
CO2Ph
CO2C10H7
SO2C10H7
ꢁ8.1
X
R4
ꢁ4.8, ꢁ5.2
ꢁ5.6
10b: X = O; R4 = (o-Cl)Bzl
ꢁ4.6
10c: X = O; R4 = (o-NO2)Bzl
10f
4
Compd
10d
10e
: X = O; R = Ph
4
(S,S)-10a
(S,S)-10b
(S,S)-10c
(S,S)-10d
(S,S)-10e
(S,S)-10f
(S,S)-10g
(S,S)-10h
(S,S)-10i
ꢁ2.8
ꢁ3.0
ꢁ3.0
ꢁ2.1
ꢁ3.1
ꢁ4.0
ꢁ2.7
ꢁ4.0
ꢁ3.8
ꢁ4.7
: X = O; R = 1-Nph
10g: X = CH2; R4 = Bzl
ꢁ4.3, ꢁ5.5
ꢁ4.7, ꢁ5.1
ꢁ4.0
10h: X = NH; R4 = Bzl
: X = NH; R4 = (1S-Me)Bzl
10i
ꢁ4.5
ꢁ4.6, ꢁ5.7
ꢁ4.9, ꢁ5.6
ꢁ4.8
Scheme 3. Synthesis of N-terminal-modified compounds (C). Reagents: (a) 20%
pip/DMF; (b) Fmoc-Ala-OH/DIC/HOBt/DMF; (c) 8/DIC/HOBt/DMF; (d) TFA/H2O/TIPS
(95:4:1); (e) R4XCOCl or R4SO2Cl/propylene oxide/DMF; (f) R4NCO/DMF.
CO2(CH2)2Ph
CONHCH2Ph
CONHCH(Me)Ph
ꢁ4.7
a
Determined by least-squares linear regression analysis from measurements
over the range 30–60 °C (seven points), in DMSO-d6.
between 10% and 20% of cis conformer in CDCl3 and increasing up
to 40% in DMSO-d6.
b
CONH2 for series B and C.
2.2. Conformational analysis by 1H NMR
3 ppb/K (in absolute value) are indicative of solvent-protected
NH, probably implicated in a hydrogen bond, while values above
4 ppb/K indicated accessibility to solvent or non-hydrogen-
bonded states, and those between 3 and 4 ppb/K are in the range
of uncertainty.18 As shown in the table, most compounds showed
In our previous work on anti-HCMV N-benzyloxycarbonylazeti-
dines, we found that some active 2-carbamoyl derivatives had low
conformational flexibility, due to the formation of a reverse c-turn,
stabilized through an intramolecular hydrogen bond between the Z
carbonyl group and the NH proton of the 2-carboxamide.14 Recent
studies on 2-alkyl-2-carboxy azetidine-containing model peptides
confirmed that these non-proteinogenic amino acids are valuable
D DT values for the a-NH amide proton <3 ppb/K, while for a
d/
small number of them the values were not conclusive. These data
point towards the stabilization of a -turn structure through the
formation of an intramolecular hydrogen bond implicating the
a-NH amide proton. In contrast, the temperature coefficients for
c
c
-turn inducers.16 To ascertain whether this particular folded con-
formation could play or not a role in the antiviral activity of the
new prepared compounds, their chemical shifts and temperature
coefficients for amide protons were evaluated.
the C-terminal amide protons are large enough, indicating total
accessibility to the solvent. The only exception was compound
In CDCl3, the chemical shift values for the
a
-NH amide protons
(R,S)-4b, for which the
gested either the coexistence of two conformational states, b- and
-turn or a single double-turned conformation.
Dd/DT values for both amide protons sug-
in the major trans-rotamer were always >7 ppm, and the variation
when changing to DMSO-d6 was very small (0.01–0.3 ppm in most
cases), suggesting the participation of these protons in intramolec-
ular stable hydrogen bonds that protect them from solvent influ-
ence.17 On the contrary, the C-terminal CONH2 or CONHR amide
protons showed chemical shifts in the range of 5.27–6.82 ppm in
CDCl3, while the change of solvent resulted in an important fluctu-
ation of d values, indicative of solvent exposure (normally >1 ppm,
see Table 1S, Supplementary data).
The variation of amide proton chemical shifts with the tem-
perature was measured in DMSO-d6 for all compounds 4, 7 and
10, and the calculated temperature coefficients are recorded in
Table 1. It is established that, in small peptides, values below
c
2.3. Antiviral activity
The azetidine-derived dipeptides of the three series, A–C, were
evaluated for their ability to inhibit the replication of HCMV in
vitro.19 The results were compared to those of the reference com-
pound 1 and the commercial drug ganciclovir (Tables 2–4).
As shown in Table 2, derivatives (S,S)-4a, (R,S)-4a and (S,S)-4c,
with tert-butyl and iso-butyl groups at the C-terminal amide, retain
the anti-HCMV activity with EC50 values comparable to model
dipeptide 1. Benzyl- and cyclohexyl-substituted analogues were

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P. Pérez-Faginas et al. / Bioorg. Med. Chem. 19 (2011) 1155–1161
Table 2
Table 4
Activity of 1-Z-Azf-Ala-NR1R2 derivatives against human cytomegalovirus (HCMV)
Activity of 1-R4-Azf-Ala-NH2 derivatives against human cytomegalovirus (HCMV)
O
Me R1
O
Me
N
NH2
R2
N
H
(S)
(S)
N
H
O
(S)
N
O
N
O
R4
O
Compd
R4
EC50
(lM)
Cytotoxicity (
lM)
HCMVa
MCCb
CC50
c
Compd
R1, R2
EC50
(lM)
Cytotoxicity (lM)
c
O
HCMVa
MCCb
CC50
O
(S,S)-4a
(R,S)-4a
(S,S)-4b
(R,S)-4b
(S,S)-4c
(R,S)-4c
(S,S)-4d
(R,S)-4d
(S,S)-4e
(R,S)-4e
(S,S)-1
11
H, C(CH)3
H, C(CH)3
H, CH2Ph
45
49
>20
>4
63
>100
>100
>20
>100
>100
32
>100
>100
7.0
>100
>100
100
P20
>100
>100
>100
100
>100
>100
>50
>100
>100
P1575
>100
>100
42
(S,S)-10a
>4
20
14
H, CH2Ph
42
H, CH2CH(CH3)2
H, CH2CH(CH3)2
H, Chx
H, Chx
CH3, CH3
>100
>100
>100
46
>100
>100
50
>100
>100
580
Cl
O
O
(S,S)-10b
(S,S)-10c
>20
>20
>100
100
>100
>100
O
O
CH3, CH3
H, H
Z-Phe-Ala-NH2
NO2
12
GCV
Z-Phe-Ala-NHtBu
O
O
(S,S)-10d
(S,S)-10e
>20
45
>100
>100
>100
>100
a
Effective concentration required to reduce virus plaque formation by 50% (Davis
strain). Virus input was 100 plaque forming units (PFU).
Minimum cytotoxic concentration that causes a microscopically detectable
alteration of cell morphology.
O
b
c
O
O
Cytotoxic concentration required to reduce cell growth by 50%.
O
S
Table 3
(S,S)-10f
>100
>100
>100
Activity of 1-Z-Azf-Xaa-NH2 derivatives against human cytomegalovirus (HCMV)
O
O
O
R3
(S,S)-10g
(S,S)-10h
>100
>100
100
>100
>100
NH2
N
H
O
N
O
>100
N
H
O
R3
EC50
(lM)
Cytotoxicity (
lM)
Me
Compd
O
O
c
HCMVa
MCCb
CC50
(S,S)-10i
>100
>100
>100
N
H
(S,R)-7a
(R,R)-7a
(S,S)-7b
(R,S)-7b
(S,S)-7c
(R,S)-7c
(S,S)-7d
(R,S)-7d
7e
CH3
CH3
CH2Ph
CH2Ph
CH(CH3)2
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
(CH2)4NH2
(CH2)2CO2H
CH3
63
>100
>100
100
36
>100
33
37
100
85
50
66
>100
>100
50
>100
>20
>20
76
>100
>20
45
>100
>100
32
(S,S)-1
32
>50
50
63
100
O
>100
>100
100
>100
>100
>100
>50
GCV
5.6
1575
a, b and c as defined in Table 2.
7f
(S,S)-1
GCV
As indicated in the previous section, the described azetidine
dipeptides adopt a -turn-like conformation, centred at the azeti-
c
7.0
P1575
580
dine residue. To explore to which extent this structural character-
istic could be important for HCMV antiviral activity, dipeptides
Z-Phe-Ala-NH2 (11) and Z-Phe-Ala-NHtBu (12), non-constrained
analogues of active compounds 1 and 4a, respectively, were syn-
thesized and evaluated. All the amide protons of peptides 11 and
12 showed temperature coefficients higher than 4.5 ppb/K, in
absolute value, indicating random coil conformations, and there-
fore higher flexibility than azetidine-containing analogues. Both
dipeptides were unable to inhibit the replication of HCMV in cell
culture (Table 2), suggesting a key function of the azetidine ring
in the observed antiviral activity. A direct role, in which the meth-
ylene groups of the four-member ring interact with the target, or
an indirect function to spatially pre-organise the molecule to
facilitate the interaction through other functionalities, could be
envisaged.
a, b and c as defined in Table 2.
inactive, indicating a preference for aliphatic non-cyclic substitu-
ents at the C-terminal position. The lack of antiviral activity of
the N,N-dimethyl derivatives 4e could suggest the need for a pro-
ton at the amide group and its possible implication in the direct
interaction with the biological target. This could explain the inac-
tivity of the ester-substituted analogues found in our previous
work.14b Although only a few compounds showed significant activ-
ity, a potential preference for the S,S-configuration could be de-
duced from the comparison of the inhibitory potency between
the diastereoisomeric pairs of compounds 4a and 4c (and data ob-
tained from the B and C series of compounds).

P. Pérez-Faginas et al. / Bioorg. Med. Chem. 19 (2011) 1155–1161
1159
In series B (Table 3), only compounds (S,R)-7a, (S,S)-7c and
(R,S)-7d, derived from -Ala, -Val and -Leu, respectively, maintain
solvent was removed and the residue dissolved in water and
washed with EtOAc. The aqueous layer was lyophilised and the
resulting residue was dissolved in a mixture of Na2CO3 (10%)/diox-
ane (20 mL/12 mL). The reaction was cooled at 0 °C and a solution
of Cl-Fmoc (1.97 g, 7.64 mmol) in dioxane (12 mL) was added.
After 1 h, the temperature was allowed to raise at rt and the reac-
tion was stirred for 3 h. Then, water was added and the mixture
was washed with Et2O. The organic layer was acidified with 1 M
HCl up to pH 2 and extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4 and the solvent removed un-
der vacuum. The residue was purified by flash chromatography
employing a mixture of EtOAc/CH2Cl2 (1:7) to afford 1.89 g (60%)
D
measurable anti-HCMV activity, while the Phe, Lys, and Glu ana-
logues were inactive. Again, a preference for aliphatic groups at
the C-terminal residue is observed. However, neither an increase
of the aliphatic side-chain volume (Val or Leu vs Ala) nor a change
in the configuration of the Ala residue succeeded in improving the
activity of prototype 1. Active compounds in the B series are—as a
rule—also somewhat cytotoxic, specially the
taining analogues.
D-Ala- and Phe-con-
As shown in Table 4, most prepared derivatives in compound ser-
ies C were totally inactive at 100 M, except for compound (S,S)-10e,
l
bearing a 1-naphtylcarbamate group. This group can be considered
as a conformationally restricted analogue of the benzyloxycarbonyl
substituent present in prototype 1. These results suggest that the Z
group plays a key role in the activity of these azetidine-derived com-
pounds. In fact, small changes, such as the incorporation of different
types of substituents at the aromatic phenyl ring (10b, 10c), the
shortening of the benzyl group to phenyl (10d) and the replacement
of the urethane oxygen atom by CH2 or NH (10g, 10h, 10i), led to
inactive analogues. Compound 10a, being inactive against HCMV,
was the most cytotoxic/cytostatic compound among the three series
of azetidine derivatives (Table 3).
of the title azetidine 8 as a solid. Mp: 88–90 °C. HPLC:
Rt = 5.78 min (A/B = 35:65). 1H NMR (CDCl3) rotamers ratio: 3.2:1.
Major rotamer d: 7.85–7.18 (m, 13H, Ar), 4.56–4.48 (m, 2H, CH2-
Fmoc), 4.34 (m, 1H, CH-Fmoc), 3.75 (m, 1H, 4-H), 3.56 (d, 1H,
J = 14.2, 2-CH2), 3.18 (m, 1H, 4-H), 3.09 (d, 1H, J = 14.2, 2-CH2),
2.63 (m, 1H, 3-H), 2.32 (m, 1H, 3-H). Anal. Calcd for C26H23NO4:
C, 75.53; H, 5.61; N, 3.39. Found: C, 75.78; H, 5.73; N, 3.42.
4.2. Synthesis of azetidine-derived dipeptides
4.2.1. Series A
To assess HCMV selectivity, all compounds were also evaluated
for antiviral activity against a wide panel of DNA and RNA viruses.
Some active derivatives against HCMV also inhibited the replica-
tion of varicella-zoster virus (VZV, Table 2S within Supplementary
data), while they were inactive against a wide variety of other DNA
General procedure for the coupling reaction: A solution of the cor-
responding H-Ala-NR1R2 trifluoroacetate (0.36 mmol) in anhy-
drous THF, was treated with azetidine 2 (58 mg, 0.18 mmol), BOP
(0.16 g, 0.36 mmol) and Et3N (0.1 mL, 0.72 mmol). The reaction
was stirred overnight at rt. The solvent was evaporated and the
resulting residue was dissolved in EtOAc and washed with citric
acid (10%), NaHCO3 (10%) and brine. The organic layer was dried
over Na2SO4 and evaporated affording a residue, which was puri-
fied by flash chromatography (EtOAc/Et2O/hexane, 1:1:1).
or RNA viruses at subtoxic concentrations (20–100
lM). Dual anti-
VZV and anti-HCMV activity was also described for non-nucleoside
4-benzyloxy-c-sultones and bicyclic furanopyrimidine-derived
nucleosides.9b,20
4.2.1.1. (2S,10S)-2-Benzyl-1-benzyloxycarbonyl-2-[(10-tert-
butylcarba-moyl)ethyl]carbamoylazetidine, (S,S)-4a
3. Conclusion
Syrup. Yield: 58%.
[a]D = +7.3 (c 0.49, CHCl3). HPLC:
Structure–activity studies on a new family of azetidine-contain-
ing dipeptide inhibitors of HCMV are described. Compounds
modified at the C-terminal residue (carboxamide and amino acid
side-chain) and at the N-terminus were easily synthesized by appli-
cation of both solid-phase and solution methodologies. The results
of the biological assays indicated quite strict structural require-
ments for activity. Thus, only derivatives with aliphatic groups at
the C-terminal residue, either at the carboxamide moiety or at the
side-chain showed significant inhibitory activity against HCMV rep-
lication in cell cultures. Additionally, at the C-carboxamide group,
the presence of a hydrogen atom is an absolute requirement. Substi-
tutions at N-terminus recommended the initial benzyloxycarbonyl
(Z) group as the best choice at this position. In addition, the confor-
Rt = 19.79 min (A/B = 50:50). 1H NMR (DMSO-d6), rotamers ratio:
5.2:1. Major rotamer d: 8.20 (br d, 1H, J = 7.7, 10-NH), 7.79 (br d,
1H, J = 7.3, NHtBu), 7.45–7.13 (m, 10H, Ph), 5.24 (d, 1H, J = 12.4,
CH2-Z), 5.04 (d, 1H, J = 12.4, CH2-Z), 4.29 (m, 1H, 10-H), 3.53 (m,
1H, 4-H), 3.31 (d, 1H, J = 13.9, 2-CH2), 3.03 (d, 1H, J = 13.9, 2-CH2),
2.88 (dd, 1H, J = 8.1 and 16.5, 4-H), 2.32 (m, 1H, 3-H), 2.10 (m,
t
1H, 3-H), 1.27 (s, 9H, Bu),1.20 (d, 3H, J = 6.9, 10-CH3). MS: 452.2
[M+1]+, 474.2 [M+Na]+. Anal. Calcd for C26H33N3O4: C, 69.16; H,
7.37; N, 9.31. Found: C, 68.87; H, 7.61; N, 9.29.
4.2.1.2. (2R,10S)-2-Benzyl-1-benzyloxycarbonyl-2-[(10-tert-
butylcarba-moyl)ethyl]carbamoylazetidine, (R,S)-4a
Syrup. Yield: 25%.
[
a
]
D = ꢁ47.5 (c 0.61, CHCl3). HPLC:
mational restriction induced by the reverse turn (c-type) seems
Rt = 17.60 min (A/B = 50:50). 1H NMR (DMSO-d6), rotamers ratio:
4.6:1. Major rotamer d: 8.14 (d, 1H, J = 7.7, 10-NH), 7.81 (br d, 1H,
J = 7.6, NHtBu), 7.46–7.13 (m, 10H, Ph), 5.23 (d, 1H, J = 12.4, CH2-
Z), 5.06 (d, 1H, J = 12.4, CH2-Z), 4.29 (m, 1H, 10-H), 3.50 (m, 1H, 4-
H), 3.36 (d, 1H, J = 13.9, 2-CH2), 2.98 (d, 1H, J = 13.9, 2-CH2), 2.79
(m, 1H, 4-H), 2.30 (m, 1H, 3-H), 2.11 (m, 1H, 3-H), 1.25 (s, 9H,
tBu), 1.23 (d, 3H, J = 7.3, 10-CH3). MS: 452.2 [M+1]+, 474.2
[M+Na]+. Anal. Calcd for C26H33N3O4: C, 69.16; H, 7.37; N, 9.31.
Found: C, 68.94; H, 7.42; N, 9.28.
important for activity, since more flexible dipeptide analogues were
totally inactive. Further SAR studies are needed to improve the
activity of the present series of inhibitors.
4. Experimental
For details on general methods and complete 1H, 13C and MS
characterisation of all intermediates and final compounds, see
the Supplementary data. Azetidine 2 was synthesized according
to the previously reported method.14b
4.2.1.3. (2S,10S)-2-Benzyl-1-benzyloxycarbonyl-2-[(10-
isobutylcarba-moyl)ethyl]carbamoylazetidine (S,S)-4c
4.1. (2R,S)-2-Benzyl-1-(9-fluorenylmethoxy)carbonyl-2-
carboxyazetidine (8)
Foam. Yield: 60%.
[a]D = +14.7 (c 0.5, CHCl3). HPLC:
Rt = 22.29 min (A/B = 47:53). 1H NM (DMSO-d6), rotamers ratio:
2.8:1. Major rotamer d: 8.29 (d, 1H, J = 7.3, 10-NH), 7.85 (m, 1H, NHi-
Bu), 7.44–7.10 (m, 10H, Ph), 5.25 (d, 1H, J = 12.4, CH2-Z), 5.04 (d,
1H, J = 12.4, CH2-Z), 4.34 (m, 1H, 10-H), 3.54 (m, 1H, 4-H), 3.31 (d,
A solution of (2R,S)-2-benzyl-2-methoxycarbonylazetidine15a
(1.57 g, 7.64 mmol) in MeOH (20 mL) was treated with 2 N NaOH
(19.1 mL, 9.55 mmol). The reaction was stirred at rt for 24 h. The

1160
P. Pérez-Faginas et al. / Bioorg. Med. Chem. 19 (2011) 1155–1161
1H, J = 13.9, 2-CH2), 3.01 (d, 1H, J = 13.9, 2-CH2), 2.95 (m, 1H, 4-H),
2.84 (m, 2H, CH2-iBu), 2.32 (m, 1H, 3-H), 2.01 (m, 1H, 3-H), 1.72
(m, 1H, CH-iBu), 1.26 (d, 3H, J = 7.1, 10-CH3), 0.85 (d, 6H, J = 6.6,
CH3-iBu). MS: 452.3 [M+1]+, 474.3 [M+Na]+. Anal. Calcd for
rotamers ratio: 2.2:1. Major rotamer d: 7.94 (d, 1H, J = 8.5, 10-
NH), 7.48–7.15 (m, 11H, Ph, CONH2), 6.99 (br s, 1H, CONH2), 5.24
(d, 1H, J = 12.4, CH2-Z), 5.07 (d, 1H, J = 12.4, CH2-Z), 4.35 (m, 1H,
10-H), 3.48 (m, 1H, 4-H), 3.28 (d, 1H, J = 13.9, 2-CH2), 3.02 (d, 1H,
J = 13.9, 2-CH2), 2.78 (m, 1H, 4-H), 2.35 (m, 1H, 3-H), 2.14 (m, 2H,
3-H, 20-H), 1.58 (m, 2H, 20-H and 30-H), 0.87 (d, 3H, J = 6.6, 30-
CH3), 0.83 (d, 3H, J = 6.7, 40-H). MS: 438.3 [M+1]+, 460.2 [M+Na]+.
Anal. Calcd for C25H31N3O4: C, 68.63; H, 7.14; N, 9.60. Found: C,
68.71; H, 7.23; N, 9.52.
C26H33N3O4: C, 69.16; H, 7.37; N, 9.31. Found: C, 68.91; H, 7.29;
N, 9.15.
4.2.2. Series B and C
General procedure for the solid-phase coupling reactions: A solu-
tion of the corresponding Fmoc-Xaa-OH (0.3 mmol) and HOBt
(0.3 mmol) in anhydrous DMF (1 mL), was treated with DIC
(0.3 mmol) and the resulting mixture was added over the Fmoc-
deprotected resin (0.1 mmol). After 18 h of slow stirring at rt, the
excess of reagents was eliminated by successive washes with
DMF/DCM/DMF/DCM (4 ꢂ 0.5 min) to afford intermediates 5a–g.
The efficiency of the coupling reactions was monitored by the Kai-
ser0s test. A similar procedure was followed for the subsequent
incorporation of azetidines 2 and 8.
Synthesis of N-substituted derivatives of series C: After removal of
Fmoc group from resin 9, propylene oxide (15 equiv, except for the
reaction with isocyanates) was added. The reaction was cooled at
0 °C and 10 equiv of the corresponding isocyanate, chloroformate,
acyl- or sulfonyl-chloride were added. After 18 h of slow stirring
at room temperature, the excess of reagents was eliminated by
successive washes with DMF/DCM/DMF/DCM (4 ꢂ 0.5 min) to af-
ford compounds 10b–i. The efficiency of the coupling reactions
was monitored by the chloranyl test.
4.2.2.4. (2S,10S)-2-Benzyl-2-[(10-carbamoyl)ethyl]carbamoyl-1-
(1-naphthyloxy)carbonylazetidine, (S,S)-10e
Syrup. Acetone/CH2Cl2, 1:6. Yield: 52%. [a]D = +18.1 (c 0.8,
CHCl3). HPLC: Rt = 13.31 min (A/B = 40:60). 1H NMR (DMSO-d6),
rotamers ratio: 2.2:1. Major rotamer d: 8.16 (d, 1H, J = 7.7, 10-
NH), 7.72–7.35 (m, 13H, Ar, CONH2), 7.31 (br s, 1H, CONH2), 4.32
(m, 1H, 10-H), 3.64 (m, 1H, 4-H), 3.27 (d, 1H, J = 13.8, 2-CH2), 3.18
(d, 1H, J = 13.8, 2-CH2), 2.98 (m, 1H, 4-H), 2.33 (m, 1H, 3-H), 2.28
(m, 1H, 3-H), 1.28 (d, 3H, J = 7.6, 10-CH3). MS: 432.2 [M+1]+. Anal.
Calcd for C25H25N3O4: C, 69.59; H, 5.84; N, 9.74. Found: C, 69.66;
H, 5.88; N, 9.68.
4.2.2.5. (2S,10S)-2-Benzyl-2-[(10-carbamoyl)ethyl]carbamoyl-1-
(2-naphtyl)-sulfonylazetidine, (S,S)-10f
Solid. Acetone/CH2Cl2, 1:6. Yield: 42%. Mp: 154–156 °C.
[
a
]
D = ꢁ41.2 (c 0.3, CHCl3). HPLC: Rt = 17.07 min (A/B = 40:60). 1H
NMR (DMSO-d6): 8.51 (br s, 1H, 10-NH), 8.21–7.64 (m, 7H, Naph),
7.41 (br s, 1H, CONH2), 7.28–7.18 (m, 5H, Ph), 7.21 (br s, 1H,
CONH2), 4.33 (m, 1H, 10-H), 3.73 (m, 1H, 4-H), 3.62 (m, 1H, 4-H),
3.48 (d, 1H, J = 13.8, 2-CH2), 3.24 (d, 1H, J = 13.8, 2-CH2), 2.48 (m,
1H, 3-H), 2.27 (m, 1H, 3-H), 1.36 (d, 3H, J = 7.3, 10-CH3). MS:
452.2 [M+1]+. Anal. Calcd for: C24H25N3O4S: C, 63.84; H, 5.58; N,
9.31. Found: C, 63.88; H, 5.61; N, 9.28.
Cleavage reactions: The resin-bound compounds were cleaved
by treatment with the TFA/H2O/TIPS (95:4:1) cocktail for 3 h at
rt. The filtrate was kept and the resin was washed with DCM
(3 ꢂ 3 mL). The filtrates, containing final products, were combined
and evaporated under vacuum. The resulting residue was dissolved
in water and lyophilised before chromatography, using the solvent
system indicated in each case.
4.2.2.6. (2S,10S)-2-Benzyl-2-[(10-carbamoyl)ethyl]carbamoyl-1-
phenetyl-carbonylazetidine, (S,S)-10g
4.2.2.1. (2S,10R)-2-Benzyl-1-benzyloxycarbonyl-2-[(10-
carbamoyl)ethyl]-carbamoylazetidine, (S,R)-7a
Foam. 15–33% acetone in CH2Cl2. Yield: 28%. [a]D = +14.2 (c 0.2,
Foam. 5–17% Acetone in CH2Cl2. Yield: 70%. [
a
]
D = +20.0 (c 1.3,
CHCl3). HPLC: Rt = 10.03 min (A/B = 40:60). 1H NMR (DMSO-d6),
rotamers ratio: 4.5:1. Major rotamer d: 8.67 (d, 1H, J = 7.6, 10-
NH), 7.2 (br s, 1H, CONH2), 7.28–7.16 (m, 10H, Ph), 7.05 (br s, 1H,
CONH2), 4.26 (m, 1H, 10-H), 3.63 (m, 1H, 4-H), 3.33 (d, 1H,
J = 13.6, 2-CH2), 2.98 (d, 1H, J = 13.6, 2-CH2), 2.98 (m, 1H, 4-H),
2.82 (m, 2H, CH2CH2Ph), 2.34 (m, 2H, CH2CH2Ph), 2.24 (m, 1H, 3-
H), 2.07 (m, 1H, 3-H), 1.36 (d, 3H, J = 7.4, 10-CH3). MS: 394.2
[M+1]+. Anal. Calcd for C23H27N3O3: C, 70.21; H, 6.92; N, 10.68.
Found: C, 70.09; H, 7.07; N, 10.57.
CHCl3). HPLC: Rt = 8.72 min (A/B = 35:65). 1H NMR (DMSO-d6),
rotamers ratio: 2.2:1. Major rotamer d: 8.06 (d, 1H, J = 7.2, 10-
NH), 7.41–7.13 (m, 11H, Ph, CONH2), 7.05 (br s, 1H, CONH2), 5.23
(d, 1H, J = 12.5, CH2-Z), 5.03 (d, 1H, J = 12.5, CH2-Z), 4.28 (m, 1H,
10-H), 3.49 (m, 1H, 4-H), 3.37 (d, 1H, J = 13.8, 2-CH2), 2.99 (d, 1H,
J = 13.8, 02-CH2), 2.79 (m, 1H, 4-H), 2.30 (m, 1H, 3-H), 2.11 (m,
1H, 3-H), 1.16 (d, 3H, J = 6.7, 10-CH3). MS: 396.1 [M+1]+. Anal. Calcd
for C22H25N3O4: C, 66.82; H, 6.37; N, 10.63. Found: C, 66.73; H,
6.43; N, 10.55.
4.2.2.7. (2S,10S,100S)-2-Benzyl-2-[(100-carbamoyl)ethyl]carbamoyl-
1-[(10-phenyl)ethyl]carbamoylazetidine, (S,S)-10i
4.2.2.2. (2R,10S)-2-Benzyl-1-benzyloxycarbonyl-2-(10-carbamoyl-
20-methyl)propyl]carbamoylazetidine, (R,S)-7c
Solid. Acetone/CH2Cl2, 1:4. Yield: 35%. Mp: 137–139 °C.
Syrup. 5–17% Acetone in CH2Cl2. Yield: 24%. [
a
]
D = ꢁ9.3 (c 1.2,
[a]
D = +4.0 (c 0.9, CHCl3). HPLC: Rt = 6.67 min (A/B = 40.60). 1H
CHCl3). HPLC: Rt = 19.84 min (A/B = 35:65). 1H NMR (DMSO-d6),
rotamers ratio: 2.5:1. Major rotamer d: 7.88 (d, 1H, J = 8.7, 10-
NH), 7.48 (br s, 1H, CONH2), 7.38–7.10 (m, 10H, Ph), 7.07 (br s,
1H, CONH2), 5.25 (d, 1H, J = 12.3, CH2-Z), 5.06 (d, 1H, J = 12.3,
CH2-Z), 4.21 (m, 1H, 10-H), 3.45 (m, 1H, 4-H), 3.36 (d, 1H, J = 13.9,
2-CH2), 2.98 (d, 1H, J = 13.9, 2-CH2), 2.78 (m, 1H, 4-H), 2.37 (m,
1H, 3-H), 2.16 (m, 2H, 20-H, 3-H), 0.87 (d, 3H, J = 6.6, 20-CH3), 0.76
(d, 3H, J = 6.8, 30-H). MS: 424.2 [M+1]+, 446.3 [M+Na]+. Anal. Calcd
for C24H29N3O4: C, 68.06; H, 6.90; N, 9.92. Found: C, 68.17; H, 7.01;
N, 9.85.
NMR (DMSO-d6), rotamers ratio: 4.7:1. Major rotamer d: 8.12 (br
s, 1H, 100-NH), 7.38–7.14 (m, 12H, 10-NH, Ph, CONH2), 7.06 (br s,
1H, CONH2), 4.97 (m, 1H, 10-H), 4.34 (m, 1H, 100-H), 3.49 (m, 1H,
4-H), 3.35 (d, 1H, J = 13.6, 2-CH2), 3.27 (d, 1H, J = 13.6, 2-CH2),
2.82 (m, 2H, 4-H and 3-H), 2.36 (m, 1H, 3-H), 1.36 (d, 3H, J = 7.4,
10-CH3), 1.24 (d, 3H, J = 7.1, 100-CH3). MS: 409.2 [M+1]+. Anal. Calcd
for C23H28N4O3: C, 67.63; H, 6.91; N, 13.72. Found: C, 67.69; H,
7.02; N, 13.67.
4.3. Antiviral assays
4.2.2.3. (2R,10S)-2-Benzyl-1-benzyloxycarbonyl-2-[(10-
carbamoyl-30-methyl)butyl]carbamoylazetidine, (R,S)-7d
The antiviral assays, other than the anti-HIV, -HCMV and -VZV
assays, were based on inhibition of virus-induced cytopathicity in
HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vac-
cinia virus, vesicular stomatitis virus, cytomegalovirus (HCMV)
Syrup. 5–25% acetone in CH2Cl2. Yield: 27%. [
a]
D = ꢁ6.3 (c 0.9,
CHCl3). HPLC: Rt = 13.54 min (A/B = 40:60). 1H NMR (DMSO-d6),

P. Pérez-Faginas et al. / Bioorg. Med. Chem. 19 (2011) 1155–1161
1161
and varicella-zoster virus (VZV), Vero (parainfluenza-3, reovirus-1,
Sindbis and Coxsackie B4), HeLa (vesicular stomatitis virus, Cox-
sackie virus B4, and respiratory syncytial virus), CrFK (feline coro-
navirus (FIPV) and feline herpes virus) or MDCK [influenza A
(H1N1; H3N2) and influenza B] cell cultures. Confluent cell cul-
tures (or nearly confluent for MDCK cells) in microtiter 96-well
plates were inoculated with 100 CCID50 of virus (1 CCID50 being
the virus dose to infect 50% of the cell cultures). After a 1-h virus
adsorption period, residual virus was removed, and the cell cul-
tures were incubated in the presence of varying concentrations
(fivefold compound dilutions) of the test compounds. Viral cyto-
pathicity was recorded as soon as it reached completion in the con-
trol virus-infected cell cultures that were not treated with the test
compounds. The minimal cytotoxic concentration (MCC) of the
compounds was defined as the compound concentration that
caused a microscopically visible alteration of cell morphology.
The methodology of the anti-HIV assays was as follows: human
CEM (ꢀ3 ꢂ 105 cells/cm3) cells were infected with 100 CCID50 of
pre-doctoral I3P fellowship and a postdoctoral I3P contract, respec-
tively. Thanks to Mrs. Lies Van den Heurck, Mrs. Anita Camps, Mr.
Steven Carmans, Mrs. Leentje Persoons, Mrs. Leen Ingels and Mrs.
Frieda De Meyer for excellent technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.052.
References and notes
1. Trincado, D. E.; Scott, G. M.; White, P. A.; Hunt, C.; Rasmussen, L.; Rawlinson, W.
D. J. Med. Virol. 2000, 61, 481.
2. Melnick, J. L.; Hu, C.; Burek, J.; Adam, E.; DeBakey, M. E. J. Med. Virol. 1994, 42,
170.
3. Zhou, Y. F.; Leon, M. B.; Waclawiw, M. A.; Popma, J. J.; Yu, Z. X.; Finkel, T.;
Epstein, S. E. N. Eng. J. Med. 1996, 335, 624.
4. Griffiths, P. D. J. Antimicrob. Chemother. 2002, 49, 243.
5. Biron, K. K. Antiviral Res. 2006, 71, 154.
6. Baldanti, F. J. Antimicrob. Chemother. 2003, 52, 324.
HIV(IIIB) or HIV-2 (ROD)/mL and seeded in 200 lL wells of a micro-
7. Biron, K. K.; Harvey, R. J.; Chamberlain, S. C.; Good, S. S.; Smith, A. A., III; Davis,
M. G.; Talarico, C. L.; Miller, W. H.; Ferris, R.; Dornsife, R. E.; Stanat, S. C.; Drach,
J. C.; Townsend, L. B.; Koszalka, G. W. Antimicrob. Agents Chemother. 2002, 46,
2365.
titer plate containing appropriate dilutions of the test compounds.
After 4 days of incubation at 37 °C, HIV-induced CEM giant cell for-
mation was examined microscopically.
8. Jones, T. R.; Lee, S. W.; Johann, S. V.; Razinkov, V.; Visalli, R. J.; Feld, B.; Bloom, J.
D.; O’Connell, J. J. Virol. 2004, 78, 1289.
9. (a) De Castro, S.; Peromingo, M.-T.; Naesens, L.; Andrei, G.; Snoeck, R.; Balzarini,
J.; Velázquez, S.; Camarasa, M.-J. J. Med. Chem. 2008, 51, 5823; (b) De Castro, S.;
García-Aparicio, C.; Andrei, G.; Snoeck, R.; Balzarini, J.; Camarasa, M.-J.;
Velázquez, S. J. Med. Chem. 2009, 52, 1582.
4.3.1. Anti-HCMV assays
Confluent human embryonic lung (HEL) fibroblasts were grown
in 96-well microtiter plates and infected with the human cytomeg-
alovirus (HCMV) strain Davis at 100 PFU per well. After a 2-h incu-
bation period, residual virus was removed and the infected cells
were further incubated with medium containing different concen-
trations of the test compounds (in duplicate). After incubation for
7 days at 37 °C, virus-induced cytopathogenicity was monitored
microscopically after ethanol fixation and staining with Giemsa.
Antiviral activity was expressed as the EC50 or compound concen-
tration required to reduce virus-induced cytopathogenicity by 50%.
EC50’s were calculated from graphic plots of the percentage of cyto-
pathogenicity as a function of concentration of the compounds.
10. (a) Welch, A. R.; Woods, A. S.; McNally, L. M.; Cotter, R. J.; Gibson, W. A. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 10792; (b) Shieh, H.-S.; Kurumbail, R. G.; Stevens,
A. M.; Stegeman, R. A.; Sturman, E. J.; Pak, J. Y.; Wittwer, A. J.; Palmier, M. O.;
Wiegand, R. C.; Holwerda, B. C.; Stallings, W. C. Nature 1996, 383, 279; (c)
Waxman, L.; Darke, P. L. Antiviral Chem. Chemother. 2000, 11, 1.
11. LaPlante, S. R.; Bonneau, P.; Aubry, N.; Cameron, D. R.; Déziel, R.; Grand-Maitre,
C.; Plouffe, C.; Tong, L.; Kawai, S. J. Am. Chem. Soc. 1999, 121, 2974.
12. (a) Pinto, I. L.; Jarvest, R. L.; Clarke, B.; Dabrowski, C. E.; Fenwick, A.; Gorczyca,
M. M.; Jennings, L. J.; Lavery, P.; Sternberg, E. J.; Tew, D. G.; West, A. Bioorg. Med.
Chem. Lett. 1999, 9, 449; (b) Borthwick, A. D.; Crame, A. J.; Erlt, P. F.; Exall, A. M.;
Haley, T. M.; Hart, G. J.; Mason, A. M.; Pennell, A. M. K.; Sing, O. M. P.;
Weingarten, G. G.; Woolven, J. M. J. Med. Chem. 2002, 45, 1.
13. (a) Yoakim, C.; Ogilvie, W.; Cameron, D. R.; Chabot, C.; Guse, I.; Haché, B.; Naud,
J.; O’Meara, J. A.; Plante, R.; Déziel, R. J. Med. Chem. 1998, 41, 2882; Bonneau, P.;
Hasani, F.; Plouffe, C.; Malenfant, E.; Laplante, S. R.; Guse, I.; Ogilvie, W. W.;
Plante, R.; Davidson, W. C.; Hopkins, J. L.; Morelock, M. M.; Cordingley, M. G.;
Déziel, R. J. Am. Chem. Soc. 1999, 121, 2965.
14. (a) Gerona-Navarro, G.; Pérez de Vega, M. J.; García-López, M. T.; Andrei, G.;
Snoeck, R.; De Clercq, E.; Balzarini, J.; González-Muñiz, R. Bioorg. Med. Chem.
Lett. 2004, 14, 2253; (b) Gerona-Navarro, G.; Pérez de Vega, M. J.; García-López,
M. T.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J.; González-Muñiz, R. J.
Med. Chem. 2005, 48, 2612.
15. (a) Gerona-Navarro, G.; Bonache, M. A.; Alías, M.; Pérez de Vega, M. J.; García-
López, M. T.; López, P.; Cativiela, C.; González-Muñiz, R. Tetrahedron Lett. 2004,
45, 2193; (b) Gerona-Navarro, G.; Bonache, M. A.; Herranz, R.; García-López, M.
T.; González-Muñiz, R. J. Org. Chem. 2001, 66, 3538.
4.3.2. Cytostatic toxicity assays
Cytostatic activity measurements were based on the inhibition
of HEL cell growth. HEL cells were seeded at 5 ꢂ 103 cells/well into
96-well microtiter plates and allowed to proliferate for 24 h. Then,
medium containing different concentrations of the test compounds
was added. After 3 days of incubation at 37 °C, the cell number was
determined with a Coulter Counter. The cytostatic concentration
was calculated as the CC50 or compound concentration required
to reduce growth by 50% relative to the number of cells in the un-
treated controls. CC50 values were estimated from graphic plots of
the number of cells (percentage of control) as a function of the con-
centration of the test compounds. Cytotoxicity was expressed as
minimum cytotoxic concentration (MCC) or compound concentra-
tion that causes a microscopically detectable alteration of cell
morphology.
16. (a) Baeza, J. L.; Gerona-Navarro, G.; Pérez de Vega, M. J.; García-López, M. T.;
González-Muñiz, R.; Martín-Martínez, M. Tetrahedron Lett. 2007, 48, 3689; (b)
Baeza, J. L.; Gerona-Navarro, G.; Pérez de Vega, M. J.; García-López, M. T.;
González-Muñiz, R.; Martín-Martínez, M. J. Org. Chem. 2008, 73, 1704; (c)
Baeza, J. L.; Gerona-Navarro, G.; Thomson, K.; Pérez de Vega, M. J.; Infantes, L.;
García-López, M. T.; González-Muñiz, R.; Martín-Martínez, M. J. Org. Chem.
2009, 74, 8203.
17. Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.; Scolastico, C. Eur. J. Org. Chem.
1999, 389.
18. Stevens, E. S.; Sugawara, N.; Bonora, G. M.; Toniolo, C. J. Am. Chem. Soc. 1980,
102, 7048.
Acknowledgements
19. Snoeck, R.; Andrei, G.; Bodaghi, B.; Lagneaux, L.; Daelemans, D.; De Clercq, E.;
Neytes, J.; Schols, D.; Naesens, L.; Michelson, S.; Bron, D.; Otto, M. J.; Bousseau,
A.; Nemecek, C.; Roy, C. Antiviral Res. 2002, 55, 413.
20. McGuigan, C.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J.
Bioorg. Med. Chem. Lett. 2003, 13, 4511.
This work was supported by the Spanish Ministry of Science
and Innovation (SAF 2006-01205 and SAF 2009-09323) and the
K.U.L. (GOA no. 10/14). P.P.-F. and M.T.A. thank the CSIC for a