Synthesis and anti-HCMV activity of 1-acyl-β-lactams and 1-acylazetidines derived from phenylalanine

Article abstract of DOI:10.1016/j.bmcl.2004.02.010

Different Phe-derived 1-acyl-β-lactams, analogous to a series of 2-azetidinones acting as HCMV serine protease inhibitors, were synthesized. Some of these compounds were modest inhibitors of the HCMV replication. Interestingly, removal of the carbonyl gro

Full text of DOI:10.1016/j.bmcl.2004.02.010

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2253–2256
Synthesis and anti-HCMV activity of 1-acyl-b-lactams and
1-acylazetidines derived from phenylalanine
a
Guillermo Gerona-Navarro, M Jesus Perez de Vega, M Teresa Garcıa-Lopez,
a
a
a
a
ꢀ
ꢀ
ꢀ
ꢀ
Graciela Andrei,b Robert Snoeck,b Jan Balzarini,b Erik De Clercqb
a,
*
ꢀ
~
and Rosario Gonzalez-Muniz
aInstituto de Quꢀımica Mꢀedica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 2 September 2003; revised 30 December 2003; accepted 3 February 2004
Abstract—Different Phe-derived 1-acyl-b-lactams, analogous to a series of 2-azetidinones acting as HCMV serine protease inhib-
itors, were synthesized. Some of these compounds were modest inhibitors of the HCMV replication. Interestingly, removal of the
carbonyl group of the b-lactam ring, most likely acting as the serine trap, resulted in an azetidine derivative with anti-HCMV
activity comparable to that of the reference compound ganciclovir.
Ó 2004 Elsevier Ltd. All rights reserved.
Human cytomegalovirus (HCMV) is a ubiquitous
member of the herpes virus family. Although most
infections are asymptomatic, severe manifestations of
HCMV can be seen in individuals whose immune system
has been weakened by a disease, such as late-stage
cancers and AIDS, or by immunosuppressive therapy
following organ transplantation.1–3 At present there are
four drugs that have been approved for the treatment of
HCMV infections, namely the viral DNA polymerase
inhibitors ganciclovir (DHPG), cidofovir [(S)-HPMPC]
and foscarnet, and the antisense oligonucleotide fomi-
virsen.1–3 However, because of toxicity and bioavail-
ability problems, together with the emergence of
resistance to these compounds, the need for effective,
and safe anti-HCMV drugs based on other mechanisms
continues.
thought to stabilize the tetrahedral intermediate during
catalysis.5;7 Since the active site of this enzyme has been
identified, the search for protease inhibitors has been a
high priority. Most of these inhibitors contain classical
serine protease inhibitor motifs based on an activated
carbonyl group such as peptidyl ketoamides,8;9 as well as
mechanism-based inhibitors such as oxazazinones10;11
and pyrrolidine-5,5-trans-lactams.12–14 Among these
latter inhibitors, a series of monocyclic b-lactams 1
(compound 1a being the prototype) has resulted in
highly potent derivatives in the isolated enzyme assay,
but their efficacy in cell culture was quite limited, as for
all described inhibitors of this enzyme.15–19
Due to its critical role in capsid assembly and viral
maturation, HCMV serine protease has become an
attractive target for the development of anti-HMCV
drugs.2–4 The three dimensional structure of HCMV
protease shows that it has a unique catalytic triad
composed of Ser132, His63, and His157.5;6 The structure
also suggests that two conserved arginine residues at
positions 165 and 166 form the oxyanion hole that is
CO2R1
X
CO2 Bu
O
t
N
N
H
N
N
O
O
R2
O
O
O
R
2: X = NH, O
3k
1a: R= H
1b: R = (R )-Me
1c: R = (S)-Me
R
R
1 = tBu, H
2 = Ph, CH(CH3)Ph,
tBu, CH2Ph
This paper deals with the synthesis and evaluation of a
series of new azetidinones 2, derived from phenylala-
nine, which were designed on the basis of the structure
of the reported b-lactam inhibitors16 and of the residues
Keywords: Anti-HCMV activity; b-lactams; Azetidines.
* Corresponding author. Tel.: +34-91-5622900; fax: +34-91-5644853;
e-mail: iqmg313@iqm.csic.es
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.010

2254
G. Gerona-Navarro et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2253–2256
1.49 ppm
CO2Me
implicated in the active site of the HCMV protease.5 In
comparison with model compounds 1, these new b-lac-
tams have an additional 4-carboxylate group that could
be suitable for extra interactions with the guanidine
group of the Arg165 or Arg166 residue of the viral
protease. In addition, we have also synthesized and
evaluated the azetidine derivative 3k, analogous to the
resulting most active b-lactam within this series.
S
HN
O
S
a
5g
2g
N
O
NH
O
1.36 ppm
S
HN
O
R
a
CO2Me
The reaction of the Phe-derived 2-azetidinone 420 with
phenyl isocyanate under different conditions was first
investigated, since acylation of related b-lactams with
isocyanates generally proceeded in low yield (Scheme
1).16 Among TEA/DMAP, DBU, and Cs2CO3, the latter
base was found to be the most effective for the synthesis
of compound 2a (82%). Reaction of compound 4 with
(S)-1-phenylethyl isocyanate under these conditions
afforded a mixture of the 1-acyl-b-lactams 2b and 2d in
an approximately 1.5:1 ratio (74% total yield),21 which
were separated by column chromatography. In a similar
way, compounds 2c (50%) and 2e (34%) were prepared
by treatment of 4 with (R)-1-phenylethyl isocyanate,
followed by chromatographic resolution.
5i
2i
N
O
NH
O
Scheme 2. Reagents and conditions: (a) H-L-Ala-OMe/BOP/TEA/
THF.
Compounds 2 were evaluated against HCMV in human
embryonic lung (HEL) cells,26 and the results compared
to those obtained for the reference compounds DHPG
(ganciclovir) and HMPC (cidofovir) (Table 1). For
comparative purposes, the reported anti-HCMV activity
of the model b-lactam 1a was also included in the table.
As shown in Table 1, trisubstituted b-lactams 2a–e were
found to exhibit some antiviral activity, slightly higher
than that reported for the prototype 2-azetidinone 1a.
No appreciable influence of the absolute configuration,
either at C4 or at the 1-phenylethyl substituent, on the
inhibition of viral replication was observed. However,
the lack of activity of the free carboxylic acid derivatives
2f–i suggests a role for the tert-butyl ester moiety. In a
similar way, the presence of an aromatic group at the
1-acyl substituent seems important for the antiviral
activity, as deduced from the lack of activity of the tert-
Compounds 2b–e were hydrolyzed to the corresponding
free carboxylic acids 2f–i, respectively, by treatment with
(1:4) TFA/CH2Cl2. Compounds 2j and 2k were prepared
by treatment of the 2-azetidinone derivative 4 with tert-
butyldicarbonate and benzyl chloroformate, respec-
tively, as previously described.22
The assignment of the absolute configuration at C4
position in compounds 2g and 2i, was based on the
chemical shifts of the b-H protons of the aliphatic resi-
due of dipeptide derivatives 5g and 5i (Scheme 2),23;24
and on their respective retention times in HPLC.25 The
knowledge of the configuration in compounds 2g and 2i
allowed the subsequent configurational assignment of
their enantiomers 2h and 2f, respectively, and hence of
their respective precursors 2b–e.
Table 1. Activity of compounds 2 and 3 against human cytomegalo-
virus in HEL cell culturesa
Compounds Configu-
rations
AD-169
strain EC50
(lM)b
Davies strain CC50
EC50 (lM)b
(lM)c
2a
2b
4R,S
50
35
50
35
118
184
4S,10S
4S,10R
4R,10S
4R,10R
4S,10S
4S,10R
4R,10S
4R,10R
4R,S
2c
2d
32
32
33
33
70
164
t
t
CO2 Bu
t
CO2 Bu
a
e
CO2 Bu
NH
2e
100
>50
>200
>200
>50
>200
11
107
>50
>200
>200
>50
>200
13
>200
187
H
N
N
O
2f
2g
N
O
O
O
>200
>200
>200
>200
36
O
4
O
d
2h
2i
2k
2a
b or c
2j
2k
3k
1ad
DHPG
4R,S
2R,S
0.74
53
0.9
0.62
––
0.8
34
4S
4R
CO2tBu
CO2R
H
CO2R
H
4S
––
>750
>150
>150
+
N
N
N
N
N
O
O
O
*
O
*
(S)-HPMPC ––
0.16
0.5
1'
1'
O
O
O
a The anti-HCMV assay was based on scoring the virus-induced
cytopathicity in HEL cell cultures at 7 days post infection as
described in Ref. 26.
2b, 2c: R = tBu
2f, 2g: R = H
2d, 2e: R = tBu
2h, 2i: R = H
2j
TFA
TFA
Scheme 1. Reagents and conditions: (a) PhNCO/Cs2CO3/THF; (b)
(S)-PhCH(CH3)NCO/Cs2CO3/THF; (c) (R)-PhCH(CH3)NCO/
Cs2CO3/THF; (d) (tBuO)2CO/TEA/DMAP/CH2Cl2; (e) BzlOCOCl/
DBU/CH2Cl2.
b Effective concentration required to reduce virus plaque formation by
50%. Virus input was 100 plaque forming units (PFU).
c Cytotoxic concentration required to reduce cell growth by 50%.
d From Ref. 16.

G. Gerona-Navarro et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2253–2256
2255
butoxycarbonyl derivative 2j and the presence of activity
Acknowledgements
of the benzyloxycarbonyl analogue 2k. The latter com-
pound showed the highest anti-HCMV activity within
the b-lactam series, but it had a very narrow therapeutic
window, with a cell toxicity value close to the IC50 data
for viral inhibition.
This work was supported by CICYT (SAF 2000-0147
and 2003-07207-C02) and Geconcerteerde Onderzoek-
sacties–Vlaanderen (GOA-00/12). G.G.-N. holds a pre-
doctoral fellowship from the Spanish Ministry of
Science and Technology.
Considering the structural analogy between b-lactams 2
and model compounds 1, it is expected that compounds
2 could inhibit HCMV replication in an irreversible
manner, through acylation of the virus serine protease.
It is well known that irreversible enzyme inhibitors
could bind to many nucleophiles en route to the
intended target, most likely resulting in toxic side
effects.27 Taking into account that we have recently
reported that compound 2k is highly reactive toward O-
and N-nucleophiles,22 this reactivity could be masking,
at least to some extent, its real antiviral effect. With this
in mind, we thought that the elimination of the elec-
trophilic carbonyl lactam group, while maintaining
enough hydrogen bonds, ionic, and/or van der Waals
interactions with the enzyme, could led to more active
and safe HCMV inhibitors. A similar approach has
successfully guided the design of noncovalent inhibitors
of some mammalian serine proteases.27;28 Following this
idea, we deleted the carbonyl group in the supposedly
covalent inhibitor 2k to obtain the corresponding
azetidine derivative 3k (Scheme 3).29;30
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6
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obtained as diastereoisomeric mixtures. b-Lactam deriva-
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ꢀ
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showed that the methyl or methylene protons of the
29. Compound
6 was synthesized by reduction of the
carbonyl group of the corresponding 1-p-methoxybenzyl-
2-azetidinone with diphenylsilane followed by removal
of the p-methoxybenzyl group by hydrogenolysis:
Gerona-Navarro, G.; Bonache, M. A.; Herranz, R.;
aliphatic residue are more shielded in the
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D–L and L–D
ꢀ
L
–L
~
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1249.
ꢀ
ꢀ
~
24. The above rule was unambiguously confirmed for one of
ꢀ
30. All new compounds showed analytical and spectroscopic
data according to the proposed structures.
our b-lactam derivatives: Gerona-Navarro, G.; Garcıa-