Recent advances in the chemistry of parainfluenza-1 (Sendai) virus inhibitors

Recent Advances in the Chemistryof Parain£uenza-1 (Sendai)

Virus Inhibitors

Raffaele Saladino,1 Umberto Ciambecchini,1 Lucia Nencioni,2

Anna Teresa Palamara 3

1Agrobiological and Agrochemical Department, University of Tuscia, via San Camillo de Lellis snc,

00100, Viterbo, Italy2Department of Experimental Medicine and Biochemical Sciences, Faculty of

Medicine, University of Rome ‘‘Tor Vergata,’’ Via Montpellier, 1, 00133, Rome, Italy3Institute of Microbiology, Faculty of Pharmacy, University of Rome

‘‘La Sapienza,’’ P.le Aldo Moro 5, 00185, Rome, Italy

DOI 10.1002/med.10036

!

Abstract: Purine and pyrimidine derivatives, antioxidants, fusion inhibitors, statins, prostaglandins,

antibiotic nucleosides, inhibitors of Ca2þ homeostasis, carbohydrate derivatives, antisense poly-

nucleotides and chimeras, are described as inhibitors of parainfluenza-1 (Sendai) viral infections.

� 2003 Wiley Periodicals, Inc. Med Res Rev, 23 No. 4, 427–455, 2003

Key words: antivirals; synthesis; parainfluenza-1 (Sendai) virus; therapy

1 . I N T R O D U C T I O N

The family paramyxoviridae, which includes the parainfluenza, respiratory syncytial, measles

and mumps viruses, cause disease in humans and animals. Two subfamilies have recently been

distinguished: the paramyxovirinae, which comprises the genera respirovirus, rubulavirus, and

morbillivirus, and the pneumovirinae, which includes the genera pneumovirus and metapneumo-

virus.1 The parainfluenza viruses (PIV) belong to the genus respirovirus and include four human

types, hPIV1, hPIV2, hPIV3, and hPIV4, which were discovered between 1956 and 1960. The first

isolated PIVwas Sendai virus (SV), which is now classified as themurine counterpart of human PIV1,

to which it is antigenetically related. Many of the basic biochemical and molecular biological

properties of the paramyxoviruses have been identified through the study of SV. Respiratory syncytial

Contract grant sponsor: Italian Ministry of Health; Contract grant sponsor: Italian National Research Council; Contract

Grant number: CNRC007A72 _ 003.

Correspondence to: Prof. Raffaele Saladino, Dipartimento A.B.A.C., Universita' dellaTuscia, Via S. Camillo de Lellis, s.n.c., 00100,

Viterbo, Italy.E-mail: [email protected]

Medicinal Research Reviews, Vol. 23, No. 4, 427^455, 2003

� 2003 Wiley Periodicals, Inc.

virus (RSV), a member of the pneumovirus genus, shares many structural, pathogenic, epide-

miological and clinical features with the PIV, and for this reason, data on RSV are in many cases

relevant to our understanding of PIVas well.

The PIVare enveloped RNAviruses with linear, nonsegmented, negative-sense, single-stranded

genomes containing six to ten tandemly linked genes. The genome is enclosed within a helical

nucleocapsid containing nucleocapsid (NP), phospho- (P) and large polymerase complex (L)

proteins, which initiate intracellular virus replication. Protruding through the pleomorphic lipid-

containing envelope, the glycoproteins, hemagglutinin-neuraminidase (HN), and fusion protein (F),

mediate the virus’s entry into and exit from host cells. Another structural protein, the viral matrix

protein (M), is extremely important in virion architecture. It is found between the envelope and the

core and is released when the virus enters a host cell.

Parainfluenza virus replication occurs entirely within the cytoplasm of infected cells (Fig. 1).

It begins with transcription of the genomic RNA into 5 0-capped and 3 0-polyadenylated mRNAs.

Transcription occurs in a sequential manner, terminating and reinitiating at each gene junction.

The genome contains, in 3 0 to 5 0 order, the NP, P, M, F, HN, and L genes (named for the six

structural proteins they encode). After primary transcription and translation, when sufficient

amounts of unassembled NP are present, viral RNA synthesis becomes coupled to the concomitant

NP-encapsidation of the nascent (þ ) RNA chain. Under these conditions, viral RNA polymerase

ignores all the junctions, to produce an exact complementary antigenome chain. By a process known

as RNA editing, which involves the use of an overlapping open reading frame (ORF), the PIV P gene

also gives rise to multiple nonstructural protein species: the V protein and four carboxy-coterminal

proteins (C 0, C, Y1, and Y2) referred to collectively as the C proteins.

All of these proteins play functional roles in PIV replication and pathogenesis. The NP is a

58-kDa protein, and each of itsmonomers is associatedwith six nucleotides. It forms the nucleocapsid

in complex with RNA and the P protein, which represents the smaller subunit of RNA polymerase.

The P protein is a 68-kDa tetramer phosphorylated on one or two serine residues in the N-proximal

module. The third nucleocapsid-associated protein, the L protein, contains approximately 2200

amino acids and represents the major RNA polymerase subunit. Present in a small number of copies

per virion (about 20–40 in each virus particle), the L protein hasmultiple functions in RNA synthesis,

including catalytic activity, capping and methylation. The M protein, with a molecular weight of

about 40 kDa, is abundantly expressed within the virion. It interacts with both the viral envelope

and the nucleocapsid and is thought to play a role in virus maturation. The attachment protein, HN,

is an integral membrane glycoprotein with molecular weight of 72 kDa. It binds the virion to sialic

acid-containing receptors on the host cell surface (hemagglutinating activity), thus initiating the

process of infection. It also causes cleavage (neuraminidase activity) of sialic acid residues from

glycoproteins and glycolipids, which prevents self-aggregation of virus particles during their release

from infected cells. The second membrane glycoprotein, the F protein, is synthesized as an inactive

precursor (F0) that is post-translationally cleaved by a host cell protease to form the biologically active

molecule, which has a molecular weight of approximately 63 kDa. Following attachment, the F

protein mediates the fusion of the virion and host cell surface membranes. This fusion occurs at a

neutral pH and allows delivery of the nucleocapsid into the cell cytoplasm, where gene expression

begins. Later in infection, F protein is expressed on the surface of infected cells,mediating their fusion

with adjacent uninfected cells and allowing the virus to spread.2

As mentioned above, the P gene also encodes the nonstructural V and C proteins. The former

consists of a P module attached to a V carboxyl-terminal region containing seven highly conserved

cysteine residues that form zinc finger motifs and bind Zn2þ .3 The V protein is not necessary for viral

replication in tissue culture cells: its main purpose is the maintenance of high viral loads. This

function, which is involved in pathogenesis in vivo, has been mapped to carboxyl-terminal portion of

the V protein4 and is associated with its zinc-binding capacity.3 Its presence is associated with severe

pneumonia in mice.5 The C proteins, C 0, C, Y1, and Y2, are carboxyl-coterminal proteins consisting,

428 * SALADINO ET AL.

Figure

1.Shematic

representationofthelifecycleofa

paramyxovirus.

PARAINFLUENZA-1 (SENDAI) VIRUS * 429

in SV, of 215, 204, 181, and 175 amino acids, respectively. They are extremely versatile: they

counteract the antiviral action of interferons, inhibit viral RNA synthesis and are involved in virus

assembly. The C species is expressed in infected cells at a molar ratio several-fold higher than the

other three.1

A. Clinical Aspects and Pathogenesis

PIV and RSV have been known primarily as respiratory pathogens in young children, but now

they are also recognized as important pathogens in adults. Both replicate in the nasopharyngeal

epithelium after an incubation period of 2–8 days. They spread from cell to cell in the upper

respiratory tract, arriving in the lower airways 1–3 days later. The typical inflammation of RSV

bronchiolitis is characterized by necrosis of the bronchiolar epithelium and sloughing of the ciliated

epithelial cells of the small airways. Subsequently, lymphocytes migrate into affected tissues,

producing peribronchiolar infiltrates. The submucosal and adventitial tissues become edematous,

and secretions frommucous-producing cells increase. Plugs ofmucous, cellular debris, fibrin strands,

and DNA-like materials can occlude the smaller bronchioles and obstruct the flow in small airways.6

The clinical picture at this point has all the hallmarks of bronchiolitis: hyperinflation, atelectasis, and

wheezing.7

Among children, RSV infections account for 50–90% of hospitalizations for bronchiolitis,

5–40% of those for pneumonia and 10–30% of those for tracheobronchitis. PIV produce a similar

spectrum of respiratory illnesses but fewer hospitalizations. These infections generally involve the

upper respiratory tract, and 30–50% are complicated by otitis media. In about 15% of all cases,

however, PIV infections progress into the lower respiratory tract. Croup is the most typical clinical

manifestation of PIV infection (especially type 1) and is the principal cause of hospitalization in PIV-

infected children from 2–6 years of age. In older children and adults, PIVand RSV infections are not

as well recognized because other respiratory-tract infections can cause similar clinical manifesta-

tions.7 Some authors feel that there may be a pathogenic link between asthma and viral infections,

especially those caused by RSV. The inflammatory response elicited by asthmatic attacks is in fact

quite similar to that observed in viral infections. Furthermore, roughly half of the infants hospitalized

with RSV bronchiolitis later experience episodes of wheezing.

The number of patients subjected to intense immunosuppression for bone-marrow or solid-

organ transplantation is growing, and this increase has revealed another aspect of PIVand RSV, their

ability to cause opportunistic infections.8 Immunocompromised patients in transplantation units can

be exposed to these viruses through contact with staff members and/or visitors with mild upper

respiratory tract infections. Depending on the patient’s general condition and immune status, the type

of virus, and the time of exposurewith respect to the transplantation procedure, the results can be quite

severe, particularly with RSV.8

Many viruses, including the PIV, have evolved strategies to impede host defenses mediated by

interferon (IFN).9 Alpha/beta IFNs (IFN-a and IFN-b), the principal antiviral cytokines, act directlyon target cells to block viral replication.10Most of IFN’s antiviral effects require IFN-inducedmRNA

and protein synthesis. The complete signal transduction pathway from the IFN receptors to the

nucleus has been identified.11 IFN-a and IFN-b bind to heterodimeric IFN-a/b receptors consisting

of IFN-a receptor I and IFN-b receptor II. This interaction activates two cytoplasmic protein

tyrosine kinases, which consecutively phosphorylate tyrosine residues of the receptors. These

phosphotyrosines then bind to src homology 2 (SH2) domains of signal transducer and activator of

transcription (STAT) 1 and STAT2. The phosphorylation of these transducers leads to the formation

of heterodimers or homodimers through mutual SH2-domain-phosphotyrosine interactions. The

STAT1-STAT2 heterodimer associates with a third protein, interferon regulatory factor-9 (IRF9),

that allows DNA recognition. The result is an active transcriptional complex known as ISGF3 that

enhances transcription of target genes.


PIV neutralization of IFN-mediated cellular defenses seems to be related to the nonstructural

C and/or V proteins. The ability of SV to counteract the antiviral action of exogenous or endo-

genously produced IFNs is mediated by its C proteins,12 and it has recently been demonstrated that

the smallest of these, Y2, is as active as the C and Y1 proteins in this context.13 Recent data indicate

that the ISGF3 complex is the direct target of the anti-IFN strategies employed by certain negative-

stranded RNA viruses. The paramyxovirus, Simian virus 5, for example, provokes proteolytic

degradation of the STAT1 component of the complex, while hPIV2 preferentially degrades STAT2.14

Both of these effects aremediated by theviruses’Vproteins,15 andwhile the underlyingmechanism is

not entirely understood, it appears to involve subjugation of the host cell’s proteasome degradation

systems.15

B. Transmission

Most evidences indicate that PIV transmission depends upon direct contact with secretions,

fomites or large-particle aerosols. The major portals of entry for RSV infection are the eyes and nose;

the oral route is less permissive. Clinical observations suggest that PIV are transmitted similarly.

Parainfluenza virus type 1 has been recovered from air samples collected near infected patients.6

The high rates of primary infection and the frequency of reinfection suggest that these viruses spread

readily, that reinfected persons may be infectious and that a small inoculum is necessary to produce

an infection. Both viruses can survive for prolonged periods on skin, cloth, and other objects.

Fomites thus play an important role in the nosocomial diffusion of PIVand RSV, and hand washing

is essential in controlling infection.7

2 . C U R R E N T T H E R A P Y , N O V E L A N T I V I R A L A G E N T S A N D S T R A T E G I E S

The only antiviral agent currently licensed for treatment of RSV infections is aerosolized 1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Ribavirin, 1) (Fig. 2),16–21 a synthetic guanosine

analogue with broad-spectrum antiviral activity. Ribavirin prophylaxis is usually recommended for

children and adults undergoing bone-marrow transplantation.22

Ribavirin therapy for RSV infections is limited to treatment of infants. Improved oxygenation

and clinical scores and decreases in the levels of secretory mediators of inflammation associated

with severe bronchospasm have been observed in treated patients.23 However, its mode of action is

not well understood and it is also quite expensive. For these reasons, its use in RSV disease is the

object of continuing debate.24 Ribavirin exerts antiviral effects against PIV in vitro, and it has been

tested for treatment and prophylaxis of lower respiratory tract infections in immunocompromised

patients. Recently, Ramasamy and co-workers25 have described an efficient synthesis of triazole

L-ribofuranosyl nucleosides, comprising the lead compound (1) (Scheme 1). 1,2,3,5-tetra-O-acetyl-L-

ribose (3), easily prepared from L-ribose (2), was treated with methyl 1,2,4-triazole-3-carboxylate

to provide nucleosides (4) and (5). Treatment of (4) with ammonia at room temperature give (1) in90% yield.

Figure 2. Ribavirin (1).


New antiviral agents being evaluated for RSVand PIV treatment include purine and pyrimidine

derivatives, antioxidants, fusion inhibitors, statins, prostaglandins, antibiotic nucleosides, inhibitors

of Ca2þ homeostasis, carbohydrate derivatives. Studies are also underway to evaluate antisense

polynucleotides and chimeras. Thus far, however, no antivirals for RSV have reached the clinical-trial

stage of testing.26

A. Pyrimidine and Purine Derivatives

Pyrimidines active against SV were prepared by oxidation of uracil derivatives with dimethy-

ldioxirane (DMD).27,28 The reaction proceeds through selective C-5,6 double bond oxidation to give

5,6-dihydro-5,6-oxiranyl uracils.29 As an example, when 1,3-dimethyl uracil (6), 1,3,5-trimethylur-

acil (13), and 1,3,6-trimethyluracil (14) were treated with DMD (1.0 M, acetone solution), the

corresponding 5,6-epoxides (7, 15, and 18) were obtained in good yields (Scheme 2) beside to cis- and

trans-diols (8, 9, 16, and 17) as side products.The reaction performed in the presence of nucleophiles (alcohols or amines) give 6-substituted-

5,6-dihydro-5-hydroxy uracils (10, 11, 22, 23, and 24) by ‘‘one-pot’’ ring-opening functionalizationof the epoxide moiety (Scheme 3).29

6-Substituted-5,6-dihydro uracils (6–24) were tested as antiviral agents. Among them trans-1,3-

dimethyl-5-hydroxy-6-ethylamino uracil (24) (Fig. 3) showed activity against SV, measured by

decreased haemagglutinin units (HAU).30

The dose-response effect of (24) is shown in Figure 4 (panel A). Concentrations of (24) lowerthan 1.0 mg/mL had no effect on HAU production. At higher concentrations, the inhibitions were

dose-dependent and reached maximum values (87.5% inhibition) at 100 mg/mL. The maximal

antiviral dose was not toxic for the cell.

Thiopurine and thiopyrimidine derivatives are also useful starting materials to synthesize new

SV inhibitors. The thioketo moiety may be easily functionalized by formation of the corresponding

sulphinic or persulphinic acid intermediates. As for example, treatment of 2-thiouracils,31a

pyrimidine-2-thione,31b 4-thiopyrimidine, and 6-thiopurine nucleosides31c with ozone afforded

Scheme 1.


desulfurized products or products of nucleophilic substitution depending on the experimental

conditions. The oxidation of the thioketo moiety with DMD is faster than other possible reactions.32

Thus, starting from thiopyrimidine or thiopurine nucleosides, alkoxy or alkylamino derivatives were

obtained in the presence of alcohols or amines as nucleophiles (Scheme 4).

The efficacy of this procedure is illustrated by the synthesis of 1-(b-D-arabinofuranosyl)pyrimidine derivatives with antiviral and antitumor properties (Scheme 5).

These compounds have been assayed for antiviral activity against parainfluenza 1 (Sendai)

virus,33 according with the HAU procedure.30 The 1-(b-D-arabinofuranosyl) derivative (27c) (Fig. 5)was found to inhibit virus replication at all the doses of the experiments. Unfortunately, toxic effects

have been found at dose of 100 mg mL�1 on uninfected cells.

In contrast to the extensive studies about 5-substitued pyrimidines, less attention has been

devoted to the 6-substituted isomers, probably because of the difficulty in their synthesis and

their supposed lack of biological activity. In the last few years, 6-substitued pyrimidines, for example

1-[(2-hydroxyethyl)methyl]-6-(phenylthio)thymine (HEPT)34a,b and 3,4-dihydro-2-alkoxy-6-ben-

zyl-4-oxopyrimidines (DABOs),35 showed potent and selective activity against human immunode-

ficiency virus type-1 (HIV-1). For this reason new synthetic procedures to obtain 6-substitued

pyrimidines are of great interest. Racemic and chiral 1,3-dimethyl-6-oxiranylpyrimidin-2,4-dione

(28a–b)36 showed remarkable anti-ASFV (African Swine Fever Virus) activity (Fig. 6).37,38

On the basis of these data, uracil and pyrimidinone derivatives (30a–g, 31a–c, 33a–e, 34a–j, 36,39a–c) bearing the oxiranyl moiety in the C-6 position of the pyrimidine ring were prepared by

selective metalation of 6-methyl-4(3H)-pyrimidinones39 and evaluated for their activity against

SV.40–43

Scheme 2.

Scheme 3.


As an example, the reaction of (29b) with chloroacetone, 2-chloroacetophenone, 1-chloro-

pinacolone, 3-chloro-2-butanone, 2-chlorocyclopentanone, and 2-chlorocyclohexanone, gave

6-methyloxiranyl uracil derivatives (30a–g) in good yields (Scheme 6). In a similar way, the

reaction of (29a) with 4-chlorobutyraldheyde, and 5-chloro-2-pentanone afforded tetrahydrofur-

anylmethyl uracil derivatives (31a–c) (Scheme 6).

The metalation-alkylation sequence performed on 2-alkyloxy-3,6-dimethyl-4(3H)-pyrimidi-

nones (32a–d)44 or (35) gave compounds (33a–e), (34a–j) (Scheme 7), and (36) (Scheme 8).

Under similar experimental conditions 6-oxiranyluracil derivatives have been prepared by

reaction of the lithium derivative of 1,3-dimethyl-6-chloromethyluracil (37) with several carbonyl

compounds as electrophiles (Scheme 9).

Figure 3. Structure ofcompound (24).

Figure 4. Effect of compound (24) on Sendai virus replication. Panel A: Dose-response curve at 48 hr after virus challenge.

PanelB: Time dependentactivityat concentrationof10 mg/mLand100 mg/mL.C, control.


Most of the synthesized compounds showed inhibitory activity against SV. An ED50 lower

than micromolar was found in compounds (30a–c), (30f), (33e), and (39a–b), while most of the

remaining compounds were about 3 order of magnitude less potent (Table I). Moreover, (30b)presented a micromolar ED50 (1.1 mM) with a selectivity index of about 200.

On the basis of the results reported in Table I, the following structure-activity relationships

may be formulated: (i) The N,N-dimethyluracil scaffold, unusual in antiviral agents, along with

the 6-substitution on the uracil ring, seems to be an important property for active compounds. (ii) The

substitution pattern and the stereochemistry of the oxirane ring plays a role in modulating both

the activity and the toxic effect of the products. (iii) The substitution of the oxiranyl moiety with

a tetrahydrofuranyl ring resulted in less active agents, with the exception of (31b) which shows

an interesting selectivity index (about 200) as a consequence of a decreased toxic effect. (iv)

Unsubstituted derivatives (31a, 34h–j) show very low values of inhibitory activity and relevant

toxicity.

Computational studies,42 using the Catalyst software,43 have been performed to obtain a pharma-

cophore model describing the three dimensional structural properties for profitable interactions with

the binding site on SV. This computational approach has been applied to a set of 22 SV inhibitors

Scheme 4. Top:Method A:Dimethyldioxirane (DMD) 1.0 equiv.mol�1,CH2Cl2, 25�C.Method B:Dimethyldioxirane (DMD),1.0 equiv.

mol�1, dry alcohol-CH2Cl2(1:1 v/v), 25

�C. Method C: Dimethyldioxirane (DMD) 1.0 equiv. mol�1, amine, CH2Cl2, 25

�C. Bottom:Method B: R¼OR1; R1,:CH3,CH2CH3,CH2(CH2)2CH3.Method C:R¼NHR2; R2:CH3,CH2CH2CH3,C6H4Me-p.

Scheme 5.


(Table I), chosen according to the Catalyst guideline. The resulting pharmacophore hypotheses

use chemical functions and their spatial location to explain the differences in inhibitory activity

within the training set. All but 1 of the 10 generated hypothesis have in common the presence of

three hydrogen bond acceptor groups (HBA) and one hydrophobic region (HY). Hypothesis 1,

characterized by the highest scoring and statistical parameters, has been chosen to represent the

‘‘pharmacophore model.’’ As a representative example, Figure 7 shows the best-fitted conformer of

(30b) into the pharmacophore model. The phenyl moiety fits within the region of the hydrophobic

group (HY), the carbonyl groups at the 2- and 4-position occupy two hydrogen bond regions (HBA2

and HBA3), and the oxirane oxygen is located in the third hydrogen bond acceptor region (HBA1) of

the hypothesis.

Deazapyrimidine nucleosides also show biological activity against SV. These derivatives

have extensive biochemical and medicinal applications.45,46 In particular, the effectiveness of 3-

deazauridine (deaza UR) (40) and 3-deazacytidine (deaza CT) (41) (Fig. 8) on replication of variousRNA viruses was measured by the extent of HAU production.47

The HAU experiments show that 3-deazacytidine (41) was more active than deaza UR (40).47

As illustrated in Figure 9, c.a. 100% inhibition in cytopathogenic effect (CPE) on chicken embryo

cells was obtained in the presence of deaza CT (41).A new synthesis48 of 3-deazapyrimidine derivatives (Scheme 10) has been proposed as a

synthetic alternative to previously reported methods49,50 which require dangerous materials such as

Figure 5. Structure ofcompound (27c).

Scheme 6.


1-methoxy-1-butene-3-yne. In this procedure 2-chloropyridine (42) was oxidized to the correspond-ing 2-chloropyridine-N-oxide (43). After selective nitration and reduction of the nitro moiety, the

4-amino-2-chloropyridine (45) was treated with sodium hydroxide to give 4-amino-2-pyridone (46)in good yield.

3-Deazacytidine (41) could be obtained starting from (46) using Vorbruggen’s procedure51 as

described in Scheme 11. This procedure has also been used to prepare 3-deaza-2 0-deoxycytidinephosphoramidite.48

Triazine derivatives and their nucleosides,52,53 such as 6-azauridine, show a wide spectrum of

antiviral activity against DNA and RNAviruses.54 Several compounds with this structure have been

synthesized and evaluated against parainfluenza type-1 SV. Salicylhydrazones of asymmetrical

Scheme 7.

Scheme 8.

Scheme 9.


Table I. Measured and Calculated Activity Against Parainfluenza 1 (Sendai) Virus and

Cytotoxicity of the Compounds 30a–g, 31a–c, 33a–e, 34a–j, 36, 39a–c

aInhibitoryconcentrationrequiredtoreducevirusyieldby50%.

bMinimuntoxicconcentrationrequired tocauseamicroscopicallydetectablealterationofnormalcellmorphology.

cConcentrationrequired toinhibitcellproliferationby50%.


triazines (47–49) (Fig. 10) and the corresponding copper complexes show anti-SVactivity associated

to a superoxide radical scavenger activity.55

The inhibition of viral multiplication is probably due to the capture of oxygen radicals generated

after the contact of the virus with the host cell. The (47-Cu) complex was the most active compound

(Fig. 11).

Pseudonucleotidic derivatives of as-triazines (50–53) were also tested against SV (Fig. 12).56

B. Antioxidants

The pathogenesis of viral diseases depends not only on the characteristics of the infective agent, but

also on the metabolic state and defensive capacities of the host cell. Shifts in the intracellular redox

state towards pro-oxidant conditions have been observed during several viral infections in both invitro

and in vivo studies.57–60 Although, the role of the oxidative environment in viral replication is not

completely understood, there is reason to believe that the pro-oxidant state observed after infection is

important for completion of the life cycle of the virus.61–63

We have demonstrated that glutathione (GSH, 54) (Fig. 13), the major intracellular antioxidant,

can inhibit, in vitro and in vivo, the replication of SV, as well as that of other viruses with different

mechanisms of replication (herpes simplex-1 virus, human immunodeficiency virus type 1 (HIV-1))

(Fig. 14).30,64–67

Figure 6. Structures ofcompounds (28a^b).

Figure 7. Compound (30b) mapped into thepharmacophoremodel for Sendai virus inhibitors.


The antiviral effect of exogenous GSH on different viruses is shown in Figure 15.

This effect seems to be related to inhibition of the post-transcriptional stages of viral replication,

in particular the correct folding and maturation of specific viral envelope proteins. The normal

assembly of these proteins involves oligomerization based on disulfide bonds,68 which are typically

affected by reducing agents.

C. NA Inhibitors

PIV share a number of strategical characteristics with the influenza virus, such as attachment to

host-cell surfaces via a sialic acid-containing receptor, fusion with host-cell lipid bilayers, and

NA-dependent viral release from infected cells. In addition, influenza virus NA and paramyxovirus

HNglycoproteins share structural similarities.69 The recently developed antiviral drug, Zanamivir (4-

guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid, 55) (Fig. 16) is a sialic acid transition-state analog that produces potent and selective inhibition of influenza virus NA.

Its clinical efficacy has been demonstrated.70 In light of the functional and structural similarities

of influenza virus and PIV, it is reasonable to expect that Zanamivir (55)might also be effective against

the NA of PIV HN. Greengard et al.71 showed the drug was indeed capable of inhibiting PIV

neuraminidase activity, hemadsorption and fusion of infected and uninfected cells. More recently,

however, this group also reported a paramyxovirus variant that appears to be relatively resistant

to Zanamivir.72 Nonetheless, inhibition of NA activity seems to be a promising strategy, and the

development of more effective molecules of this type could have an important impact on the

Figure 8. 3-Deazapyrimidinenucleosides (40,41).

Figure 9. Trendof CPE inhibitionby sequential aditionof 3-deazacytidine (41) on Sendai virus infectedchickenembryo cells.


prophylaxis and treatment of PIV infections. An efficient synthesis of Zanamivir (55) has been

proposed73 starting from N-acetyl neuraminic acid. Other potent inhibitors of influenza

Neuraminidase are Tamiflu (Oseltamivir),74 the cyclopentane variant BCX-1812,75 and compounds

based on a pyrrolidine motif, such as A-315675.76

D. (HMG-CoA) Reductase Inhibitors

Lovastatin (56, Fig. 17) is used in the treatment of hypercholesterolemia. It inhibits hydro-

xymethylglutaryl coenzyme A (HMG-CoA) reductase, which plays an important role in cholesterol

biosynthesis.77

Since a branch of this pathway leads to the formation of isoprenyl groups, lovastatin has also

been used to study isoprenylation and membrane localization of proteins such as RhoA,78 a small

GTP-binding member of the Ras superfamily that is ubiquitously expressed in mammalian cells.

Interaction between the RSV F protein and RhoA has been demonstrated.79 RhoA activation, which

involves geranylgeranyltransferase-mediated isoprenylation at the protein’s carboxy terminus,80

leads to the production of several cytokines, such as interleukin (IL)-1-beta, IL-6, and IL-8, and

structural alterations of the cytoskeleton mediated by the organization of actin stress fibers and the

formation of focal adhesion plaques. RhoA activation has been demonstrated in RSV-infected cells.81

Gower et al.81 have suggested that RhoA-mediated signaling might be involved in various aspects of

RSV pathogenesis, including cell-to-cell fusion, induction of cytokine secretion, and airway

hyperreactivity. Indeed, they demonstrated lovastatin inhibits both in vitro and in vivo replication of

RSV and suggested that drugs like lovastatin that prevent isoprenylation or other pharmacological

approaches for preventing RhoA membrane localization might represent a novel approach to the

prevention of RSV-related disease in high risk groups.81 Lovastatin (56) is generally produced by a

fermentation process.82,83 However, total or semi-synthetic chemical routes have been consid-

ered.84,85 As an example, the preparation of lovastatin (56) and analogue compounds has been

reported86 via a regioselective enzymatic esterification of the commercially available diol (57) withthe (S)-(þ )-2-methylbutyric acid (58) (Scheme 12).

The reaction was performed using nylon-immobilized lipase from Candida Rugosa.

Scheme 10.

Scheme 11.


E. Prostaglandins

Prostaglandins (PGs), a class of cyclic fatty acids, synthesized from polyunsatured precursors of the

phospholypids of the cell membrane, are involved in many biological and phatological phenomena

including regulation of cell cycle,87–89 immunization,90 inflammation,91 interferon action,92 and viral

replication. In the last 20 years more and more attention has been devoted on the antiproliferative and

antiviral property of PGs and related compounds. In particular, certain cyclopentenone prostaglandins

(PGAs and PGJs) have demonstarted potent antitumor effects in vitro and in vivo93–96 and antiviral

activity against awide range ofDNAandRNAviruses.97–100 In the case of SV, PGAs, both PGA1 (59)and PGA2 (60) were found to inhibit specifically the viral replication and to prevent persistent

infections, while PGs of the B, E, and F series were inactive (Fig. 18).101

The antiviral action of PGA1 (59) has been related to the selective block of viral protein synthesisoccurring at the translational level. Such a block has been associated to the synthesis of a 70-kDa heat

shock protein (HSP70) induced by PGA1(60) treatment in both uninfected and infected cells.102

PGJ2 (61), a dehydration product of PGD2, was also found to be a potent inhibitor of SV

replication at doses not toxic for uninfected cells.103

F. Inhibitors of Ca2þ -ATPase and Ca2þ -Perturbants

Treatment of cells with agents which interfere with Ca2þ -homeostasis has been shown to influence

the process of maturation and secretion of membrane proteins, being Ca2þ ions required in several

steps of the proteic pathway from endoplasmic reticulum (ER) to the plasma membrane.104

Thapsigargin (TG, 62) is a sequiterpene-g-lactone isolated from Thapsia garganica L.105,106that

Figure 10. Asimmetrical triazine derivatives (47^49).

Figure 11. The kinetics of the HA activity with virus cultivated in the absence of the complex compound 47-Cu (a) and with virus

incubated for 30minwith the complex conpound 47-Cu (b).


inhibits the ubiquitous sarcoplasmic and ER Ca2þ -dependent ATPases (SERCA’s) (Fig. 19).

Evaluation of its effects on SV infection in mouse fibroblast BALB3T3 cells demonstrated an

inhibition of virus replication due to a block in the transport of envelope proteins HN and F0 to host

cell membrane.107

Other inhibitors of Ca2þ pump such as Di-tert-butyl-hydroquinone (BHQ, 63) and cyclo-

piazonic acid (CPA, 64) and Ca2þ -ionophores such as Ionomycin (65) and Calcymicin (66) (Fig. 20)were found to inhibit SV by influencing the process of maturation of the glycoprotein HN.

G. Long Chain Fatty Acids

Although lipid bilayers are generally not considered to be involved inmembrane protein functionality,

the inactivation of viral infectivity has been reported by alteration of the lipid bilayer by various

means.108 In particular, treatment of SV with phospholipase A2 or B give reduction of its hemolytic

property.109 Of the different ways inwhich these phospholipases could inactivate the virus, the effects

of free fatty acids produced by the enzymes action have been evaluated in successive studies110 and

has been found that these compounds, especially cis-unsatured acids, are potent inhibitors of SV

induced hemolysis. Their action is ca. two orders of magnitude more potent than that of others

described membrane dissolving amphiphiles.111

H. Antiviral Glycosides

Several studies on the biological activity of carbohydrate derivatives as immunostimulant, antivirus or

interferon-inducing agents have been reported.112 Among them, many alkyl, aryl, or alkylaryl D-

glycopyranoside derivatives have been prepared and tested against enveloped viruses.113

The structure-activity relationships revealed that p-alkylphenyl-6-halogeno-6-deoxy-b-D-gluco-sides (67) (Fig. 21) were themost effective agents and the removal of aglyconmoiety resulted in a loss

of virucidal activity.114

When this series of compound was tested on SV, the p-(sec-butyl)-6-chloro-6-deoxy-b-D-glucopyranoside (68) results the most effective agent inhibiting the cell fusion capability and

infectivity without affecting hemagglutinating or neuramidase activities.114,115

Figure 13. Glutathione (GSH)54.

Figure 12. Structuresofcompounds (50^53).


Figure 14. Different viruses inducean intracellular pro-oxidant state viadecrease of GSH (54).

Figure 15. ExogenousGSH (54) inhibits replicationof several viruses.


I. Antibiotic Nucleosides

Tunicamycins (TM, 69),116 a family of nucleosides isolated from Streptomyces lysosuperficus,

exhibit antibiotic and antiviral activity, inhibiting the biosynthesis of specific polysaccharides,

glycolipids, and glycoproteins. In particular, these antibiotics are involved in the enzyme inhibition of

processing of UDP glucose and UDP galactose derivatives. Structurally, the most characteristic

backbone is a sugar of 11 carbon atoms (undecose) formed by a furanose and a pyranose rings

separated by an ‘‘ethano’’ spacer. The uracil residue is attached to the ribose ring and an

additional carbohydrate residue, a D-2-deoxy-2-acetamidogalactose unit, is linked to the pyranose

moiety of the undecose (Fig. 22).

Several research groups have reported the effects of the TM on the growth of different viruses

such as Rous sarcoma virus,117a influenza virus,117b measles virus,117c vesicular stomatitis virus,117d

Sindbis virus.117e,118

SV replication is strongly inhibited byTM(69).118 Themechanismof the antiviral effect has been

related to an inhibition of HN and F0 envelope protein glycosilation.

J. Antisense Oligonucleotides

The first reported antiviral effects of antisense oligonucleotides were published in 1978 by Zamecnik

and Stephenson,116 who showed that Rous sarcoma virus replication could be inhibited by

oligonucleotides complementary to reiterated terminal sequences of the viral 35S RNA. Since then,

antisense oligonucleotides have been used against a variety of viruses, including negative-stranded

Figure 16. Zanamivir (55).

Figure 17. Lovastatin (56).

Scheme 12.


RNA viruses (such as influenza virus, RSV, and rabies virus).119–121 Chimeric oligonucleotides

consisting of 2 0,5 0 oligoadenylate (2–5A) covalently linked to antisense (2–5A-antisense) have beendesigned to bind to the ubiquitous intracellular enzyme, endoribonuclease RNase L.122 Activation

of RNase L requires the presence of 2 0,5 0-oligoadenylates (2–5A), which are produced by cells afterexposure to IFN, and it results in the nonselective degradation of single-stranded RNA.123 The 2–5A-

antisense approach harnesses this enzyme to produce selective cleavage of individual RNA targets:

the antisense compound associates with complementary RNA sequences in the cell and activates

RNase to produce cleavage of the proximal RNA.

High levels of RNase have been demonstrated in human alveolar macrophages infected with

RSV,119 suggesting that this virus might be susceptible to the effects of 2–5A-antisense. The chimera

has been shown to produce potent inhibition of RSV replication in cultured human tracheal cells,119

and, more recently, dose-dependent reductions in nasal replication of the virus have been observed

following intranasal administration of a 2–5A-anti-RSV compound to African green monkeys.123

K. Chimeras

The cowpea mosaic virus (CPMV) has recently been developed as a biomolecular platform for the

display of heterologous peptide sequences. Khor et al.124 utilized a CPMV-chimera to create an

antiviral against measles virus (MV). This peptide sequence displayed on the CPMV platform

Figure 18. Antiviral prostaglandins (59^61).

Figure 19. Inhibitors of Ca2þ -ATPase (62^64).


corresponds to a portion of the MV binding site on the human MV receptor, CD46. The displayed

sequence retains its virus-binding activity and is capable of inhibiting viral entry in vitro and in vivo.

CPMV has also been used with promising results to produce vaccines against several viral

epitopes. In experimental animals, strongly protective antibody responses have been elicited

with chimeric particles expressing epitopes from HIV-1, human rhinovirus, canine parvovirus, foot-

and-mouth disease virus, mink enterititis virus, and the bacterium Staphylococcus aureus.125–128

CPMV-based antivirals, such as the one produced by Khor et al.,124 could also be co-administered

with vaccines to provide protection until full vaccine-induced immunity develops.

3 . I M M U N I Z A T I O N

The immune response to RSV and PIV is complex and not fully understood. Naturally acquired

immunity is neither complete nor durable, as reflected by the high rate of recurrent infections. These

factors have hindered attempts to produce effective vaccines, which must provide greater protection

than that afforded by natural infection and be effective during the early weeks of life.

The first RSVvaccine trials in the 1960s129were a dismal failure although important lessonswere

learned. The formalin-inactivated whole RSV vaccine used in these studies caused enhanced disease

in naive hosts following natural infection, and morbidity and mortality rates among vaccinated

children were greater than those of unvaccinated controls. Subsequent analysis revealed deficiencies

in the humoral response to this vaccine, compared to that elicited by natural infection, suggesting that

important surface glycoprotein epitopes had been selectively modified by formalin inactivation.

In addition, enhanced cell-mediated responses were observed in some of the vaccinated subjects.

These findings highlighted the importance of balance between the two components of immunity in

protection against RSV infection.

Later efforts focused on the development of attenuated vaccines. The first RSV vaccines

containing temperature-sensitive or cold-passaged mutants were effective in adults. In children,

Figure 20. Structuresof Ca2þ -ionophores (65^66).

Figure 21. Antiviral glycosides (67^68).


however, they proved to be excessively virulent, too attenuated or unstable with reversion to wild-

type virus. More recent strategies have centered around the use of purified surface glycoproteins,

genomicmaterial, and synthetic peptides.With reverse genetics, a recombinant RSVhas been created

that expresses therapeutic levels of Interferon-g, and it seems to protect mice against reinfection

without inhibiting the immune response to vaccine.130

Attenuated PIV vaccines have been developed from both human and bovine strains.7 Bovine

PIV3, which is antigenically related to human PIV3, replicates poorly in humans and protects against

human PIV3 challenge.131 Reverse genetics has been use to produce an attenuated chimeric PIV1

containing internal proteins of PIV3 and surface glycoproteins F and HN of PIV1.132

4 . C O N C L U S I O N S A N D P E R S P E C T I V E S

Several new antiviral agents and therapeutic strategies against Parainfluenza virus infection are

actually under investigation. The ‘‘classical’’ approach is mainly focused on the synthesis of new

compounds characterized by high activity and selectivity in the inhibition of specific viral targets.

Among the pyrimidine derivatives, the synthesis of new 6-substituted uracils and 4-(3H)-

pyrimidinones, with an high degree of superimposition on the hydrophobic and hydrogen bond

acceptor regions present on the pharmacophore model, could be a relevant result.

Moreover, a ‘‘non-classical’’ approach based on the design of derivatives able to interfere with

cellular structures and/or metabolic pathways involved in supporting virus life-cycle (e.g., redox

state, cellular enzymes, transcription factors) appears to be a promising way to block virus replication

and to avoid the emergence of resistant virus strains.

A C K N O W L E D G M E N T S

Thisworkwas partially supported by ItalianMinistry ofHealth and ItalianNational ResearchCouncil

(CNRC007A72 _ 003) grants. INFM is acknowledged.

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Raffaele Saladino graduated in Chemistry at the University of Rome ‘‘La Sapienza,’’ Italy in 1989. He received

his Ph.D in 1993 at the University of Rome ‘‘La Sapienza’’ and carried out a period of postdoctoral research

with Prof. S. Hanessian (Universite de Montreal, Montreal, Quebec, Canada). He was appointed as research

assistant in 1992 at the University of Tuscia, Viterbo (Italy). In 2000, he was appointed as associate professor of

Organic Chemistry at the same university. His current research interests include the chemistry of natural

substances and the synthesis of new antiviral and antitumor agents.

Umberto Ciambecchini studied Medicinal Chemistry at the University of Rome ‘‘La Sapienza’’ and received

his degree in 1999. His thesis work was on 6-substituted pyrimidine derivatives. After a period, post lauream,

at University of Rome, focused on synthesis of new phosphoramidite nucleosides, he moved in 2000 to

University of Tuscia, Viterbo where he is currently engaged in synthesis of purine derivatives as antiviral

agents.

Lucia Nencioni graduated in Biology at the University of Florence in 1993. She received her Ph.D in 2002

at the University of Rome ‘‘Tor Vergata’’ under the direction of Prof. E. Garaci. Currently, she is carrying out

her post-doctoral research in the Department of Experimental Medicine and Biochemical Sciences of the

University of Rome ‘‘Tor Vergata.’’ Her research interests include the identification of molecular mechanisms

involved in the control of viral replication and apoptosis, and the study of antiviral agents.


Anna Teresa Palamara graduated in Medicine at the Catholic University of Rome, Italy in 1982. She started

her research activity in the Department of Experimental Medicine and Biochemical Sciences of the University

of Rome ‘‘Tor Vergata,’’ where she was appointed as research assistant in 1985. In 1998, she was appointed as

associate professor of Microbiology at the University of Naples ‘‘Federico II’’ and then full professor of

Microbiology at the University of Rome ‘‘La Sapienza’’ in 2001. Her current research interests include the

identification of the virus/host cell interaction involved in the control of viral replication and the study of new

antiviral therapeutic strategies.


Recent advances in the chemistry of parainfluenza-1 (Sendai) virus inhibitors

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