Recent strategies in the development of new human cytomegalovirus inhibitors

Recent Strategies in the Developmentof NewHuman Cytomegalovirus

Inhibitors

Ana Martinez, Ana Castro, Carmen Gil, Concepcion Perez

Instituto de QuõÂmica MeÂdica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

!

Abstract: Human cytomegalovirus (HCMV) is one of the most common opportunistic infections

in immunucompromised individuals, such as AIDS patients and organ transplant recipients, and is

the most frequent congenital viral infection in humans. Despite a reduction of the incidence of

AIDS-related opportunistic infections in patients under highly active antiretroviral treatment,

attention should be paid to the HCMV risk factor in these individuals. Furthermore, HCMV may

have an important role in atherosclerosis. Existing antiviral treatments for the HCMV infection

suffer from poor bioavailability, toxicity, and limited effectiveness, mainly due to the development

of drug resistance. Fortunately there are novel and potentially very effective new compounds

undergoing pre-clinical and clinical evaluation. This review provides an overview in the last ®ve

years of new HCMV inhibitors (chemical structures, SAR and new mechanisms of action) with

the aim to provide new clues for the development of the future drugs against this opportunistic

virus. ß 2001 John Wiley & Sons, Inc. Med Res Rev, 21, No. 3, 227±244, 2001

Key words: HCMV inhibitors; HCMV protease; opportunistic infection; immunocompromised

patients

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

Human cytomegalovirus (HCMV) is a highly species-speci®c DNA virus member of the beta-

herpesvirinae, a subfamily of the herpesviridae.1 Like other herpesviruses, primary infection with

HCMV is followed by persistence of the virus in a latent form. The sites of latency are still largely

unde®ned,2 but they probably include bone marrow progenitor cells and peripheral blood

monocytes. From these sites, the virus can reactivate,3 resulting in renewed shedding of the virus, or,

in immunocompromised persons, development of disease. HCMV-infected cells may become

enlarged (cytomegalia) showing intranuclear inclusions, and co-survival of cells and virus is often

established. Except for a mononucleosis-like illness in some persons, infection with HCMV rarely

227

Correspondence to: Dr. AnaMartinez, Instituto deQuimicaMedica (CSIC), Juande la Cierva 3, 28006Madrid,Spain;E-mail: [email protected] sponsor:Fondode Investigaciones Sanitarias; Contractgrant number:FIS 98/253; Contractgrant sponsor: Comuni-daddeMadrid; Contractgrant number: 82/36.1/1999.

Medical Research Reviews,Vol. 21,No. 3, 227^244, 2001ß 2001 John Wiley & Sons, Inc.

causes disease in inmunocompetent individuals.4 However, reactivation of virus is of signi®cant

concern in the immunocompromised individual,5,6 particularly when the de®ciency affects cell-

mediated immune responses such as in HIV-infected patients. Immunosupressed individuals,

including solid organ and allogeneic bone marrow recipients,7 are also at great risk of developing

HCMV disease. This pathogen is the most common congenital viral infection in humans due to the

high prevalence of the virus in the general population, with up to 90% of the urban population

infected.8 Although it is well known that highly active antiretroviral therapy has decreased

substantially the incidence of HCMV retinitis in AIDS patients,9 this change does not indicate that

HCMV in AIDS patients has abated and is no longer a concern.10,11 On the contrary, HCMV disease

in solid organ recipients, newborns, and even in AIDS patients still accounts for considerable

morbidity, and drug resistance to the anti-HCMV compounds offers a major concern.12±14 Further-

more, HCMV may have an important role in the development of metabolic diseases, such as

atherosclerosis.15,16

Antiviral agents currently licensed for the treatment of HCMV infections include ganciclovir,17

foscarnet,18 cidofovir,19 and fomivirsen.20 All of these drugs, either directly or indirectly inhibit

viral polymerase and are able to reduce viral proliferation in patients who develop the clinical

symptoms associated with HCMV disease. However, such therapy cannot clear the virus comple-

tely. Additionally, these agents exhibit toxicity21 and require intravenous administration to obtain

therapeutic drug levels, both of which obviate their use for long-term treatment. Incomplete

suppression of viral replication during chronic antiviral treatment predisposes to the development of

viral drug resistance and consequent drug failure. Indeed, the emergence of CMV strains with

reduced susceptibility to ganciclovir, foscarnet, cidofovir and/or fomivirsen during long-term

therapy has been documented.22±24

There is renewed interest in the search for new HCMV inhibitors because of the growing

population of inmunocompromised individuals. This is mainly due to two facts: ®rstly, improve-

ments on transplantation techniques have increased the number of people living with a transplanted

organ; and secondly, the advent of new retroviral therapies has converted AIDS into a chronic

immunocompromised disease. Additionally, advances in gene therapy25,26 have stimulated the

search for new anti-HCMV inhibitors. Fortunately there are novel and potentially very effective new

compounds27,28 undergoing pre-clinical and clinical evaluation. These developments point the way

toward new therapies and also to clearer understanding of the biology of HCMV replication,

infection, and disease. This review provides an overview in the last 5 years of new HCMV inhibitors

(chemical structures and new mechanisms of action) with the aim to provide new clues for the

development of the future drugs against this opportunistic viral infection.

A. New Prodrugs of Existing Clinical Agents

Much effort has been made to overcome the poor pharmacokinetics of the approved drugs for the

treatment of HCMV infections. The bioavailability of orally administered ganciclovir in humans

ranged from 2.6 to 7.3%,29 although giving the drug with food increases absorption. Thus, the

search for a prodrug that is orally well-absorbed and then readily converted to ganciclovir (1) is of

high priority. It has been recently reported that the mono-3-(2 0-acetoxy-4 0,6 0-dimethylphenyl)-3,

3-dimethylpropanoic ester of ganciclovir (2), showed a 4-fold increase in oral bioavailability over

the parent drug in rats.30 It has been reported that 6-¯uoropurine acyclonucleosides are converted to

ganciclovir in the presence of calf intestinal mucosal adenosine deaminase.31 Recently, the

monoester (3) has shown a 15-fold higher oral gancicolovir bioavailability in rats.32

The serious irreversible nephrotoxic effects associated with cidofovir (4) treatment has been

improved in the recently described tetrahydrofuran diethyl phosphonate analogue (5). It has a

potency comparable to that of cidofovir against HCMV and a greater safety index in vivo.33 Prodrug

approaches have also been investigated to circumvent problems associated with the bioavailability

228 * MARTINEZETAL.

and permeability of phosphonoformate (PFA, foscarnet (6)). Synthetic methods have been developed

in order to incorporate the PFA moiety within biomimetics of nucleotides, carbohydrates,

and phospholipids.34 Cyclic PFA esters (7±9) are much more reactive towards hydrolysis than

their acyclic analogs. This approach holds promise when combined with a strategy for activating

the prodrug to subsequent de-esteri®cation at phosphorous. Some other pronucleotide design

approaches have been recently reviewed35 offering important tools for the in vivo delivery of

antiviral nucleotides.

B. Nucleoside and Nucleotide Derivatives as HCMV Inhibitors

Nucleoside and nucleotide analogs are the focus of current interest as antiviral and antitumor agents.

A great number of HCMV inhibitors are concentrated in this group of compounds. In general, the

new nucleotide/nucleoside analogs described are derived from the natural purine or pyrimidine

heterocycle nucleus, although derivatives of synthetic halo-benzimidazoles seem to be very

promising inhibitors.

1. Purine and Pyrimidine Nucleoside Analogs

Modi®cations of some ribofuranosyl derivatives of purine and pyrimidine related heterocycles such

as imidazoisothiazole36 (10), pyrrolopyridazinone37 (11) or pyranoic acid derivatives38 (12), have

Figure 1. Newprodrugs ofclinical agents.

NEWHUMANCYTOMEGALOVIRUS INHIBITORS * 229

been performed in order to increase anti-HCMV activity. However, the antiviral potency of these

compounds was tempered by their toxicity to uninfected cells (Fig. 2).

Anti-HCMV activity has been found for a series of carbohydrate-modi®ed nucleoside analogs

such as those comprising methylenecyclopropane function39 (13±14) or cyclohexene rings40 (15±16)

(Fig. 3). In both cases, the two enantiomeric nucleosides exhibited similar activity against several

herpesviruses. As the viral thymidine kinase (TK) plays an important role in their biological activity

(through phosphorylation of 5 0-hydroxyl mimic), these data con®rm previous observations that TK

could accommodate a con®gurational changes in the nucleoside upon binding, resulting in phos-

phoration of both pairs of enantiomers.

Natural endogenous nucleosides must be phosphorylated to the corresponding 5 0-triphosphated

to be incorporated into the DNA strand being synthesized in the cell. The ®rst phosphorylation step

leading to the formation of nucleoside 5 0-monophosphate, is commonly catalyzed by a nucleoside

kinase, such as TK, encoded by the host cell or the virus infecting the host cell. Inactive nucleoside

analogs can be activated by conversion to lipophilic pronucleotides, thus bypassing the ®rst

phosphorylation step in their activation mechanism. These pronucleotides are usually phosphate-

esther prodrugs of nucleoside 5 0-monophosphates. The phenyl phosphoranilate 17 increased over

50-fold its activity against HCMV compared to the parent spironucleoside 18, whereas the cyto-

toxicity remained low (Fig. 3).41 These results con®rm the hypothesis that phosphorylation is

indispensable for the activity of this new kind of nucleoside.

The area of oligonucleotide chemical synthesis has recently opened up a source of antiviral

agents.42 The working hypothesis developed during these studies was that the ability to form an

ordered, non-hydrogen-bonded array in solution was an important criterion for activity.43 The poly-

and oligonucleotides containing triazolo[2,3-a]purines (19±20) (Fig. 3) have been shown to be more

potent inhibitors of HCMV than ganciclovir.44 Their mechanism of action is currently under study.

More interesting are the inhibitors that exert their activity against HCMV by a novel mechanism

of action. This is the case of the novel pyrimidine and purine derivatives of L-ascorbic acid (Fig. 3).

Compound 21 exhibited a potent (yet speci®c) antiviral effect against TK� and TKÿ varicella zoster

virus and HCMV.45 The mechanism of action of these new anti-HCMV inhibitors, although different

from DNA-polymerase inhibition, actually remains unknown.

Analogs of the pyrrolo[2,3-d]pyrimidine nucleosides tyocamycin (22), sangivamycin (23), and

thiosangivamycin (24) have been shown to be potent inhibitors of HCMV (Fig. 3). The parent

nucleosides possess signi®cant activity against HCMV but are highly toxic to uninfected cells.46 In

an effort to decrease the cytotoxic effects and obtain more selective antiviral agents, a diverse class

of 7-substituted 4-aminopyrrolo[2,3-d]pyrimidines were prepared,47 including non-nucleoside

analogs as acyclic48 (25) or benzylic derivatives49 (26). Structure-activity data established that the

N-7 substituent in this family of compounds is important to modulate the toxicity against the

uninfected cells, while the 5-thioamide moiety is crucial in conferring antiviral activity against

HCMV. Phosphorylation of a side chain hydroxyl group is not required for antiviral activity, thus

indicating a different mechanism of action than current nucleoside clinical drugs. Biological studies

Figure 2. Cytotoxic HCMVribonucleoside inhibitors.

230 * MARTINEZETAL.

established that the pyrrolopyrimidines are active earlier in the replication cycle than ganciclovir,

which targets viral DNA polymerase, so the target of this new kind of HCMV inhibitor may be an

immediate-early protein.50

2. Halo-benzimidazoles

Riboside analogs derived from 2-substituted 5,6-dichlorobenzimidazole have proven to be potent

and selective inhibitors of the replication of HCMV at noncytotoxic concentrations. 2,5,6-trichloro-

1-(b-D/-ribofuranosyl)benzimidazole (TCRB) (27) and 2-bromo-5,6-dichloro-1-(b-D-ribofuranosyl)

benzimidazole (BDCRB) (28) were the ®rst lead compounds discovered (Fig. 4).51 Although these

compounds have a 5 0-OH in the riboside moiety that could be triphosphorylated to provide

inhibitors of DNA polymerase, biological evaluations of TCRB and BDCRB have established that

their antiviral action involves neither phosphorylation of the compound nor inhibition of DNA, RNA,

Figure 3. Carbocyclic, oligonucleotide, andnucleosideanalogswith HCMV inhibitoryactivity.


or protein synthesis. Rather they act by a unique mechanism which involves the inhibition of viral

DNA processing and virus assembly.52 Further studies revealed that certain benzimidazoles

ribonucleosides are less toxic to hemopoietic progenitors than ganciclovir53 which could

circumvent one of the toxic effects of this clinical drug in prolonged maintenance therapy.

Chemical modi®cations in the 5 0-position of these benzimidazole derivatives (29, 30) indicate

that the HCMV receptor for the TCRB tolerates signi®cant modi®cation in bulk and electro-

negativity in this region without adversely affecting antiviral activity,54 including the lack of the

5 0-hydroxymethyl group exempli®ed by the potent erythrofuranosyl compound (31).55 In addition

to these D-carbohydrate derivatives, several acyclic56 (32), L-carbohydrate57,58 (32, 34) and benzyl59

(35) derivatives have been synthesized and evaluated (Fig. 4). From these data, the carbohydrate

moiety (ribo-, lyxo-, arabino-, D- or L-) is necessary for anti-HCMV activity as the acyclic or

benzylic derivatives are completely devoid of anti-HCMV activity. The addition of ribose to a

polyhalogenated benzimidazole reduces toxicity and retains or increases activity against HCMV.60

However, pharmacokinetics studies of TCRB in rat and monkeys revealed that the nucleosides

are metabolized to a certain extent in vivo by glycosidic bond cleavage. The instability of 27 and 28is detrimental to their use as antiviral agents because the half-lives of the parent compounds are less

than 1 h. Several structural modi®cations have been explored to improve pharmacokinetic

properties. Some approaches, like the synthesis of C-nucleoside61 (36) or ¯uorosugar analogs62 (37)

failed, as they do not provide more potent compounds. However, carbocyclic benzimidazoles (38)

resulted in very stable, potent, and interesting inhibitors.63

Concerning the base-like heterocycle moiety, several changes have been made including

3-deaza analogs (39).64 The order for activity against HCMV among the 5,6-dihaloribonucleosides

was I�Br�Cl>>F>H compared to I>Br>Cl>F, H for cytotoxicity, establishing that the size and

electronegativity of substituents in the 5- and 6-positions are critical for activity against HCMV and

for toxicity to uninfected cells.65 Structure-activity relationships have revealed the importance of

substitution at the 2-position.66 From among these studies, 5,6-dichloro-2-isopropylamino-1-

(b-L-ribofuranosyl)benzimidazole (1263W94) (40) was chosen for clinical trials due to its potent

and selective activity in vitro and its superior pharmacokinetic properties.67 This L-ribofuranosyl

derivative inhibits HCMV DNA synthesis by a mechanism that does not require phosphorylation

and does not involve DNA polymerase.68 Compound 1263W94 is in clinical development by Glaxo

Wellcome for the treatment of HCMV infections.69

Genotypic characterization of HCMV resistant to TCRB and BDCRB has identi®ed two viral

genes, UL56 and UL89, which mutate to give drug-resistant virus.70 Thus, the proteins encoded by

these two genes are likely the target(s) TCRB and related analogs. However, little is known

regarding their overall three-dimensional structure nor of benzimidazole binding pocket(s) on

the proteins. To probe the spatial limitation of the target enzyme, a serie of tricyclic nucleosides71

(41±44) have been synthetized leading to active but very cytotoxic compounds. A three-dimen-

sional QSAR study will be desirable in the future to speci®cally delineate structure-activity

relationships.

C. Nonnucleoside HCMV Inhibitors

The clear medical need for HCMV drugs with a more desirable pro®le and/or new mechanism of

action has initiated different screening programs to discover new leads. Broad screening of the

Pharmacia and Upjohn chemical library led to identi®cation of the acyloxyamidine 45 as a

competitive inhibitor of the HCMV DNA polymerase.72 Preliminary SAR investigation charac-

terized this class by two aryl groups connected by an acyloxyamidine linker. Initial studies

in which the two aryl groups were varied revealed little tolerance for modi®cation of the left hand

2,4-dichlorophenyl group, but replacing the right hand benzothiazole ring with a disubstituted

isoxazole ring as in 46 (Fig. 5) led to a 30-fold enhancement of activity.

232 * MARTINEZETAL.

Targeting alternative viral protein functions led to the discovery of the simple, unsubstituted

indolocarbazole Arcyria¯avin A 47 (Fig. 5) as a potent, selective inhibitor of HCMV replication in

vitro.73 A series of novel symmetrical analogs have been prepared to investigate SAR, affording a

number of very active compounds, such as the 2,10-di¯uoro derivative 48, which is approximately

100-times more potent than ganciclovir in vitro. The study of their mechanism of action is under

study. The anti-HCMVactivity does not appear to be related to the inhibition of protein kinase C, the

pharmacophore of compounds 47 and 48 are related to bis-indolylphtalimides, such as

staurosporine, which are known to be potent protein kinase C (PKC) inhibitors. The anti-HCMV

mode of action of indolocarbazoles could be inhibition of UL97,74 an alternative viral kinase, or

inhibition of an induced or elevated host kinase.

The identi®cation of 1,6-naphthyridine 49 (Fig. 5) as a potent HCMV inhibitor is the result of

the screening program of BioChem Pharm. SAR investigation on this class of compounds has

de®ned the structural requirements for potency and selectivity.75 On the benzylamine side of the

molecule, a number of modi®cations can be tolerated, however, the 2 0-alkoxy benzylamines seem to

be the most promising in terms of potency and selectivity. Substitution on the C-8 of the

napththyridine ring enhances potency whereas introduction of a methyl at C-4 did not result in any

improvement. The isoquinoline 50, a deaza derivative of the lead, showed also interesting anti-

HCMV activity.76 However, during preliminary pharmacological evaluation of 49 and 50, it was

found that these compounds were not stable in mouse or monkey S9 liver preparations, indicating

that they are likely to be subjected to ®rst pass of metabolism. Additionally, in vivo studies in mice

Figure 4. Benzimidazolenucleosidesactiveagainst HCMV.


con®rmed that no parent compounds were detected 20 min after 49 or 50 were given orally. In an

effort to circumvent these metabolism issues, the tetrahydroisoquinoline 51 provided a meta-

bolically stable and orally bioavailable alternative to the 1,6 naphthyridines and isoquinolines.77

The favourable pharmacokinetic pro®le of this class of compounds will allow their future evaluation

as potential oral-anti HCMV agents with a new mechanism of action implicated on events at the

early and late stage of virus replication.78

Some imidazo[1,2-a]pyridine and pyrimidine derivatives (52, 53) proved quite promising anti-

HCMV and anti-VZV agents.79 SAR studies de®ned the nature of the thioether side chain which

must contain an aromatic cyclic ring.80 The fact that they show selective activity against these two

viruses suggests that they are preferentially metabolized by the virus infected cells and/or show

particular af®nity for virus-speci®c enzymes.

Recently the benzothiadiazine dioxide (BTD) modi®ed acyclonucleosides (54), has been

reported to exhibit marked activity against HCMV and varicella-zoster virus (VZV).81 The structure

of this compound is quite unique, not only with respect of the nature of the heterocyclic base, but

also because of the lack of the 5 0-OH mimetic group present in GCV and other current anti-CMV

drugs, which points to a different mechanism of action.82 Initial structure±activity data showed the

necessity of a double substitution in the heterocycle, while monosubstituted compounds were

completely devoid of antiviral activity.83 Simultaneously, lipophilicity in the acyclic side chain is

essential to preserve the anti-HCMV activity. These two factors were considered when preparing

second-generation analogs of BTD. The chlorophenylmethyl BTD derivatives (55, 56) proved to be

potent non-nucleoside HCMV inhibitors.84 In order to obtain further insights into the structural

requirements for the biological activity of BTD as new anti-HCMV drugs, a CoMFA model was

developed which identi®ed the most signi®cant steric and electrostatic interactions involved in anti-

HCMV activity. The model suggested that the steric component is a predominant factor in the

antiviral activity of these analogs with electrostatic factors playing a smaller yet signi®cant role.85

Figure 5. NonnucleosideHCMV inhibitors.

234 * MARTINEZETAL.

The BTD compounds do not inhibit the HCMV protease. Time-of-addition experiments performed,

revealed that the anti-HCMVaction may be targeted at an early stage of the viral replicative cycle.86

Further experiments are in progress to elucidate the mechanism of inhibition and molecular target of

action of these novel compounds.

D. HCMV Protease Inhibitors

The cytomegalovirus protease (or assemblin) is essential for proper assembly of viral capsids and, as

a consequence, viral replication.87 A great deal of attention is being paid to these enzymes as

therapeutic targets for the development of novel antiherpetic agents,88,89 specially for the highly

prevalent HCMV as the existing antiviral treatments suffer from toxicity and limited effectiveness.

All herpesvirus proteases are synthesized as a fusion with capsid assembly protein processing

two speci®c proteolytic cleavages sites.90 One of these takes place at the maturation site (M-site)

near the C-terminus of the assembly protein while the others occurs at the realease site (R-site)

separating the amino-terminal protease from the assembly protein domain. More recently, yet a third

HCMV protease autoprocessing site has been identi®ed.91 This cleavage site, called the internal-site

(I-site) is limited to simian- and human-cytomegalovirus proteases and is located within the protease

domain.

The quest for new inhibitors of HCMV protease92 has been paralleled by much activity

focusing on the structural biology of the target,93,94 which has elucidated the highly novel structure

of this enzime.95 X-ray crystallographic studies96 have revealed HCMV protease to possess a

protein fold previously unseen among serine proteases as well as a unique catalytic triad (Ser, His,

His).97 The crystal structure revealed that it is an homodimeric enzyme with two well-separated

active sites on opposite faces of the dimer.98 This biological and structural knowledge99 has been

used to rationally design both mechanism- and nonmechanism-based inhibitors of HCMV

protease.100

Development of peptidomimetic inhibitors of HCMV protease based on amino acid sequences

of the major cleavages sites of the protease has been one of the strategies employed for the design of

new inhibitors resulting in derivatives 57, 58 (Fig. 6), which showed sub-micromolar enzymatic

inhibitory activity.101 Further investigation on these compounds concluded that the HCMV protease

is an induced-®t enzyme, undergoing a conformational change upon complexation of electrophilic

ketone peptidic inhibitors such as 57.102, 103 Nevertheless cell culture assay of some members of this

series showed EC50 values in the 100 mM range probably due to the peptidic nature of the inhibitors

and poor cell penetrability. This poor activity has prompted up the design of new mechanism-based

derivatives incorporating a b-lactam ring.104 The peptidic series of monobactam inhibitors of

HCMV protease, illustrated with derivative 59, exhibited good in vitro potency (IC50 values against

HCMV protease N0 were between 0.01 and 1 mM) and were selective when tested for inhibitory

activity using a number of serine and cysteine proteases.105 Further NMR and mass spectrometry

studies demonstrated that monocyclic b-lactam derivatives inhibit HCMV protease by acting as

alternatedcomprised substrate inhibitors through the formation of an acyl enzyme intermediate.106

Moreover, some of the compounds in a smaller series bearing an aromatic ring linked by a

methlylene group at C-4 compounds 60, 61, exhibited activity in a viral replication assay which

suggested that these compounds were cell penetrable and could potentially be useful antiviral

agents.107,108

Directed mechanism-based screening, targeted to compounds that could afford a stable acyl-

enzyme adduct of the active site serine, identi®ed a novel class of serine protease inhibitors, the

spirocyclopropyl oxazolones represented by 62±64 as submicromolar inhibitors of HCMV

protease,109 and the known serine protease inhibitor class of 2-substituted benzoxazinones.110,111

A series of 6-substituted 2-aminobenzoxazinone analogs (65±67) has been prepared that show

potent inhibitory activity toward the target enzyme (IC50� 0.46±4 mM) and demonstrate antiviral


Figure 6. Inhibitors of HCMV protease.

236 * MARTINEZETAL.

activity in cell culture (IC50� 23 mM). Modi®cations of the substituent at the 6-position modulates

both enzyme selectivity and plasma stability without adversely affecting potency toward the viral

protease. Further chemical structure optimization led to the thiophene oxazinones (68, 69), depicted

in Figure 6, which are potent and selective mechanism based inhibitors of the HCMV protease with

enhanced stability relative to benzoxazinones.112 A parallel synthesis approach was utilized to

prepare a range of N-acyl analogs113 (70, 71) and further studies revealed that these potent and

selective inhibitors of HCMV act by not only acylating the catalytic serine 132 (opening of the

oxazine ring of the inhibitor) but also by alkylating cysteine 161 of the protease (Michael addition to

an enedione side chain).114 Incorporation of moieties with H-bonding potential in an attempt to

increase potency vs. the enzyme, led to the thienoxazinone derived nitroso and hydroxylamine

compounds (72, 73) which are some of the most potent, non-peptide inhibitors of HCMV protease

prepared to date.115 The inhibition of the enzyme is through 1:1 adducts at Ser 132, although the

reason for the increased potency of these last compounds has not been de®nitively established.

A high-throughput screen to identify inhibitors of HCMV protease activity was devel-

oped116,117 to enable a variety of samples types, including natural products, to be screened. During

the search of potential new anti-viral drugs a series of tripepsides, cytonic acids A (74) and B (75),

was isolated from solid-state fermentation of the fungus Cytonamea sp,118 while the naphtha-

ceneaquinone glycosides, quanolirones I (76) and II (77), were isolated from an ethyl acetate extract

of Streptomyces sp WC76535.119 Assemblin protease contains several surface accessible cysteine

sulphhydryl groups. Moreover, this protease must be in a reduced state to maintain its catalytic

activity. Thus, many reported inhibitors which contain polyphenolic groups (e.g., 74, 75) or quinone

moieties (compounds 76, 77) may inhibit assemblin via an oxidative mechanism. Additionally, the

previous HTS identi®ed benzothiopyran-S,S-dioxides (78, 79) and 1,4-naphthoquinones (80, 81) as

potent inhibitors of HCMV protease. Studies of benzothiopyrane compounds supports a reversible

mode of inhibition that does not involve oxidative inactivation of the enzyme,120 while the naphtho-

quinone derivatives 80 and 81 are irreversible protease inhibitors which covalently modify Cys

202.121 Certain examples retain activity in viral plaque assays, but have limited chemical stability

toward biologically relevant nucleophiles.

E. Vaccines

The most important rationale for development of a HCMV vaccine is prevention of congenital

HCMV disease by preventing primary maternal infection. This is complicated by the fact that

maternal infection usually passes unnoticed.

The presence of maternal antibodies before conception clearly is effective in reducing the rate

of congenital infection and preventing severe disease. It has been estimated that vaccination of

HCMV seronegative mothers would prevent 80±85% of congenital HCMV disease. Additionally,

effective vaccination strategies may also be useful in speci®c high-risk adult populations, e.g.,

seronegative transplant recipients.

In initial efforts to identify an effective vaccine, the Towne strain of HCMV has been used,

which is a laboratory-adapted, attenuated virus variant, originally isolated from a newborn with

congenital disease. Initial vaccines conferred limited ef®cacy,122 so alternative strategies with

improved protective action were investigated. Thus included the use of subunit vaccines, directed

toward speci®c antigens,123,124 or DNA vaccination. This last methodology provides an elegant

vaccination approach, in which immunogenic proteins are synthesized by host cells after intra-

cellular delivery of genes encoding these proteins. The potential advantages of DNA vaccination

include the lack of an infectious agent or live vector, the ease of production, manipulation and

storage of DNA vaccines, and potentially long-lasting responses to DNA vaccination. DNA

vaccination against cytomegalovirus125 is currently undergoing evaluation in animal models with

good results.126,127


F. Conclusions

Existing antiviral treatments for the high prevalent HCMV suffer from toxicity and limited

effectiveness. Additionally, long-term therapies needed for managing HCMV infections in

immunosupressed patients provided the background for the emergence of resistance to currently

approved antivirals agents.

From the above reported studies, it is evident that several new HCMV inhibitors with less

adverse effects or/and new mechanism of action than current clinical drugs have been discovered.

Nonnucleosidic compounds show a great potential for the future treatment of HCMV infections

because of their potent and selective HCMV inhibition, and different mechanisms of action than that

of current clinically used drugs. Several of these promising candidates are in different clinical phase

studies which will greatly enable the advent of novel HCMV therapy.

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Ana Martinez received her Ph.D from Complutense University of Madrid in 1987. In 1988, she began a CSIC

post-doctoral fellowship at Medicinal Chemistry Institute with Dr. P. Goya where she applied the molecular

modeling techniques to different ®elds of drug design. In 1990 she joined Medicinal Chemistry Institute as a

tenured scientist. Currently, her laboratory is interested in the design, synthesis, and evaluation of antiviral

drugs, specially those for opportunistic infections, and in the discovery of new therapeutic strategies for the

treatment of neurodegenerative disease.

Ana Castro graduated from Autonoma University of Madrid and received her Ph.D. in 1996, also from

Autonoma University, under the supervision of Dr. A. Martinez. Following 2 years as a postdoctoral fellow in

the laboratory of Professor W. Graham Richards at Oxford University, she joined the Medicinal Chemistry

Institute. Her current research interest includes rational development of bioactive molecules focused on two

different ®elds: antiviral agents and neurodegenerative disorders.

Carmen Gil received her B.S. degree in pharmacy at Complutense University of Madrid in 1995. She

completed her graduate education in medicinal chemistry under the supervision of Dr. A. Martinez at Medicinal

Chemistry Institute where She is currently a pre-doctoral fellow. She current research interests include the

synthesis of new anti-HCMV inhibitors.

Concepcin PeÂrez obtained her Ph.D from Complutense University of Madrid in 1995, investigating the

differentiation of U937 promonocytic cells by antitumor DNA topoisomerase II inhibitors. She joined the

Medicinal Chemistry Institute in 1998. Her current interests include antiviral agents and Alzheimer disease.

244 * MARTINEZETAL.

Recent strategies in the development of new human cytomegalovirus inhibitors

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