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Figure 21 Comparison of volume map for HRV-14 generated from x-ray data

(left) and small-molecule energy-minimized structures (right).

methyltetrazole analogue with a three-carbon linker (Fig. 23) appeared to

provide good chemical stability and improved biological activity in

comparison to WIN54954 [41]. However, when this compound was

administerd to dogs, hepatotoxicity was observed which was attributed

to metabolic instability. Further modifications resulted in the synthesis of

the 5-methyl 1,2,4-oxadiazole analogue, which was selected as a possible

Figure 22

Homologous series of compounds.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 23 Structure of the 4-methyltetrazole analogues.

development candidate based on potency and spectrum of activity [42]. To

address metabolic stability, however, a monkey liver microsomal assay

was established by means of which the half-life, the extent of metabolism,

and the nature of the metabolic products could be determined [43]. Initially, WIN54954 was incubated at 37jC with a liver microsomal mixture

for 30 min and the incubate was extracted with hexane. The extracts were

analyzed by high performance liquid chromatography (HPLC), which

revealed 18 metabolic products. When the oxadiazole analogue was subjected to the same conditions, two major peaks, metabolites A and B, were

observed by HPLC (Fig. 24), in addition to six minor ones. The rate of

metabolism was similar to that of WIN54954, however, with a half-life of

27 vs 20 min.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 24 HPLC spectrum resulting from the incubation of the oxadiazole with

monkey liver microsomes.

The question at this point was whether modifications could be made

to the oxadiazole molecule to enhance metabolic stability and achieve

comparable activity. This approach required knowledge of the site of

metabolism and the nature of the metabolic products. This information

was obtained from ion mass spectrometry. The identity of these products

was determined by comparing the fragmentation pattern of metabolites A

and B with the parent compound and the corresponding daughter ions

(Fig. 25).

Analysis of the metabolic products indicated that hydroxylation

occurred to a greater extent (30%) on the methyl group attached to the

isoxazole ring than to the methyl group on the oxadiazole ring (10%). The

methyl group in this postion was replaced with a trifluormethyl group to

prevent hydroxylation. The result of the incubation of this compound

indicated that although this position was protected, three metabolic

products were produced; in addition, the half-life was not substantially

different from the parent compound.

A similar replacement on the oxadiazole ring (Fig. 26) not only prevented metabolism at this position but also protected the entire mole-

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 25 Biotransformation of WIN 61893 and WIN 64172.

Figure 26 Metabolism of the trifluoromethylisoxazole analogue.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Figure 27

Structure of second properties pleconaril.

cule, resulting in two minor metabolites, and substantially increased the

half-life. In addition, pleconaril has exhibited a broad spectrum of antipicornavirus activity and has shown good bioavailability (Fig. 27) and is

undergoing clinical trials for upper respiratory rhinovirus infections.


x-Ray crystallography has added a new dimension to antirhinovirus drug

design. It has enabled us to examine the molecular interactions within

the compound binding site and to better understand the mechanism of

binding. We have been able to devise a model based on x-ray crystallography that qualitatively describes properties of molecules that are beneficial

for antirhinovirus activity. Also, by comparison to a volume map based

on x-ray conformations, we have developed a comparable model based on

small-molecule energy-minimized structures exclusive of x-ray data.

Finally, we have been able to apply our results to the synthesis of compounds active against both HRV-1A and HRV-14. Aside from the design

aspects, we have dealt with the more practical considerations such as

metabolic stability and bioavailability, which have led to a clinical candidate. Now certain unanswered mechanistic questions can be addressed.

How does the drug enter the binding site? Is there a recognition site, which

may explain some anomalous results that remain a mystery? Hopefully,

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

future work in this regard will eventually lead to an understanding of the

binding process and its relationship to biological activity.


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Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.


Profiles of Prototype Antiviral Agents

Interfering with the Initial Stages

of HIV Infection

Erik De Clercq

Rega Institute for Medical Research, Katholieke Universiteit Leuven,

Leuven, Belgium



The initial stages of the human immunodeficiency virus (HIV) infection

could be defined as the steps of the viral growth cycle that precede the

integration of the proviral DNA into the host cell genome. These stages

occur during the acute phase of the HIV infection, that is, when the virus

has invaded new cells. Once the proviral DNA has been integrated into

the host genome, the host cell and all its progeny cells can be considered to

be persistently or chronically infected. Expression of the integrated viral

genome will follow the classical flow of gene expression: that is, transcription, translation, and post-translational modifications under the concerted regulatory action of both cellular and viral factors.

This chapter reviews prototypes of single chemical entities that

interfere with the initial stages of the acute HIV infection, at steps that

are predominantly, if not solely, determined by specific viral proteins.

Thus, the compounds interacting with these steps in the HIV replicative

cycle may be expected to display a reasonably high specificity in their

mode of action. The targets that could be envisaged for such chemotherapeutic attack are the following: (1) virus adsorption, involving the

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

viral envelope glycoprotein gp120, (2) virus – cell fusion, involving both

viral glycoproteins gp120 and gp41, (3) viral uncoating, involving the viral capsid proteins, (4) the substrate (dNTP) binding site of the viral reverse

transcriptase, and (5) an allosteric non – substrate binding site of the HIV-1

reverse transcriptase. HIV inhibitors interacting with these targets have

been the subject of some earlier reviews [1 –4]. The chemokine receptors

CXCR4 and CCR5 used as coreceptors by X4 and R5 HIV-1 strains are

not discussed here; for reviews on inhibition of HIV infection by these receptor antagonists, see Refs. 5 and 6.

The compounds are highlighted from the following viewpoints: antiHIV potency and selectivity, mechanism of action, antiviral activity spectrum, clinical or therapeutic potential, and risk of resistance development.



Various polyanionic substances (viz., polysulfates, polysulfonates, polycarboxylates, and polyoxometalates) have been reported to block HIV

replication; for a review on the polysulfates, see Ref. 7. These substances

inhibit HIV-induced cytopathicity at a concentration of 0.1 to 1 Ag/mL,

while not being toxic to the host cells at concentrations up to 2 or 5 mg/mL,

thus achieving selectivity indexes of approximately 10,000 [7]. The target of

interaction for the polysulfates would be the V3 loop of the viral gp120

glycoprotein [8 –10]. This loop contains a highly basic region with which

the polyanionic substances could interact electrostatically. Thus, polyanions such as dextran sulfate may be assumed to block virus adsorption by

shielding the viral envelope glycoproteins [8]. Alternatively or additionally, polyanionic substances may also interact with the cellular CD4 receptor [11], thus preventing the viral envelope gp120 from anchoring to

the outer cell membrane.

Depending on their molecular weight, the nature of their anionic

groups, and the density/distribution of their negative charges, the polyanionic substances exhibit an activity spectrum that extends to several

enveloped viruses other than HIV: among the retroviruses, SIV (simian

immunodeficiency virus); among the herpesviruses, HSV (herpes simplex

virus) and CMV (cytomegalovirus); among the orthomyxoviruses, influenza A; among the paramyxoviruses, RSV (respiratory syncytial virus);

and toga-, flavi-, arena-, bunya-, and rhabdoviruses. Among the different

HIV strains, rather striking differences have been noted with regard to

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

susceptibility to polyanionic substances (e.g., dextran sulfate) [12], and this

differential susceptibility may be related to differences in the composition

of the viral glycoprotein portions with which the compounds interact.

Because of their broad activity spectrum, encompassing various

enveloped viruses, polyanionic substances may be of practical utility in

the prophylaxis and/or therapy of a number of important virus (e.g., HIV,

HSV, CMV, RSV, influenza A) infections. Yet, there is little, if any,

evidence for the in vivo efficacy of these compounds following either

parenteral or topical administration. Polyanions, and dextran sulfate in

particular, are poorly absorbed upon oral administration [13], and, in

addition, sulfated polysaccharides are notorious for their anticoagulant

activity. However, these problems can be overcome by the appropriate

chemical modifications (Fig. 1). Thus, h-cyclodextrin sulfate becomes

orally bioavailable following substitution of benzyl groups at either C-2

or C-6 of the sugar residues mCDS71 [14] and mCDS11 [15], respectively,

and heparin loses anticoagulant activity when acylated at the C-3 position

of the sugar rings [16]. These favorable features (oral bioavailability, loss of

anticoagulant activity) were obtained without impairing the anti-HIV

activity of the products (mCDS71, mCDS11, or O-acylated heparin).

The polyanionic substances may be expected to yield their greatest

promise when put in contact with the virus under the conditions that

Figure 1 Modified sulfated polysaccharides: (A) O-acylated heparin (m = 2, 4,

6, . . .) and (B) mCDS71 (a modified h-cyclodextrin sulfate).

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

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