Chinese Pharmaceutical Association

Institute of Materia Medica, Chinese Academy of Medical Sciences

Acta Pharmaceutica Sinica B

Acta Pharmaceutica Sinica B 2012;2(6):535–548

2211-3835 & 2012 In

hosting by Elsevier B

http://dx.doi.org/10.1

nCorresponding au

E-mail address: e

Peer review under r

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REVIEW

Milestones in the discovery of antiviral agents: nucleosides

and nucleotides

Erik de Clercqn

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium

Received 20 June 2012; revised 18 August 2012; accepted 5 September 2012

KEY WORDS

Valacyclovir;

Brivudin;

FV-100;

Emtricitabine;

(S)-HPMPA;

(S)-HPMPC;

Cidofovir;

Adefovir;

Tenofovir;

Truvadas;

Phosphonoamidate

stitute of Materia M

.V. All rights rese

016/j.apsb.2012.10

thor. Tel.: þ32 16

rik.declercq@rega

esponsibility of Ins

Abstract In this review article, a number of milestones in the antiviral research field on

nucleosides and nucleotides are reviewed in which the author played a significant part, especially

in the initial stages of their development. Highlighted are the amino acyl esters of acyclovir,

particularly valacyclovir (VACV), brivudin (BVDU) and the valine ester of Cf1743 (FV-100), the

20,30-dideoxynucleosides (nucleoside reverse transcriptase inhibitors, NRTIs), the acyclic nucleoside

phosphonates (S)-HPMPA, (S)-HPMPC (cidofovir) and alkoxyalkyl esters thereof (HDP-, ODE-

CDV), adefovir and adefovir dipivoxil, tenofovir and tenofovir disoproxil fumarate (TDF),

combinations containing TDF and emtricitabine, i.e., Truvadas, Atriplas, Compleras/Evipleras

and the Quad pill, and the phosphonoamidate derivatives GS-7340, GS-9131, GS-9191 and

GS-9219.

& 2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical

Association. Production and hosting by Elsevier B.V. All rights reserved.

edica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and

rved.

.001

337367; fax: þ32 16 337340.

.kuleuven.be (Erik de Clercq).

titute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.

Figure 1 Prodrugs of acyclovir, ganciclovir and penciclovir.

Erik de Clercq536

1. Introduction

The era of antiviral drug therapy started with idoxuridine

(IDU) and trifluridine (TFT). IDU was first synthesized as a

potential anticancer drug by Prusoff in 19591; it was first

shown in 1961 to possess activity against herpes simplex virus

(HSV) and vaccinia virus by Herrmann2, before it was

launched in the clinic, for the topical treatment of herpetic

keratitis by Kaufman3. Two years later, Kaufman and

heidelbrger4 also unleashed trifluridine (TFT) for the topical

treatment of herpetic keratitis. As of today, IDU and TFT are

still used in the topical treatment of herpetic eye infections.

Still in the 1960s, arabinosyladenine (ara-A), originally

synthesized as a potential anticancer agent by Lee et al.5 was

first shown by Privat de Garilhe and de Rudder6 to be active

against HSV and vaccinia virus before it was further described

as an antiviral agent by Schabel7, before it became the first

antiviral drug to be used systemically, i.e., by Whitley et al.8 in

1976, in the therapy of varicella-zoster virus (VZV) infections.

Ara-A is no longer used in the clinic, essentially for a number

of reasons: it has low aqueous solubility, is rapidly deaminated

to the inactive arabinosylhypoxanthine (ara-Hx), but primar-

ily, because it was superseded from the 1980s by acyclovir.

Meanwhile, in the early 1970s ribavirin (virazole) had been

described by Sidwell et al.9 as a broad-spectrum antiviral

agent. For circa 30 years, ribavirin was looking for a disease

against which it could be useful, until it found its niche,

together with pegylated interferon, in the treatment of chronic

hepatitis C virus (HCV) infections. The combination of

pegylated interferon-a with ribavirin has since the last 10

years been the standard of care (SOC) for the treatment of

HCV infections, but is likely to be, first complemented and

then replaced by direct-acting antiviral agents (DAAs).

Of crucial importance in the treatment of herpesvirus (i.e.,

HSV and VZV) infections was the discovery of acyclovir, the

first truly specific antiviral agent, by Elion et al.10 and

Schaeffer et al11. Now, 35 years after it was originally

described, acyclovir can still be considered as the ‘‘gold

standard’’ for the treatment of HSV and VZV infections12.

How the antiviral research field, that started with the

nucleoside analogs IDU, TFT, ara-A, ribavirin and acyclovir,

further evolved (and thrived) will be the subject of the present

review. This review will focus specifically on nucleoside

analogs such as amino acyl acyclovir esters, bromovinyldeox-

yuridine and 20,30-dideoxynucleoside analogs, and nucleotide

analogs (i.e., acyclic nucleoside phosphonates (ANPs)).

Twenty-five years ago, the era of the ANPs started with the

birth of (S)-HPMPA. It has now grown to a large family of

marketed drugs, including cidofovir, adefovir and tenofovir,

and various prodrugs and drug combinations derived thereof.

2. Antiviral agents

2.1. Valacyclovir (VACV)

Acyclovir suffers from some drawbacks in that it is relatively

insoluble in aqueous medium and poorly absorbed after oral

administration. To circumvent the first problem, amino acid

(i.e., glycine, alanine) esters of acyclovir (Fig. 1) were synthe-

sized13. An advantage of such amino acyl esters is that for

topical use, i.e., for the treatment of herpetic keratitis, they can

be administered as eye drops14, whereas acyclovir has to be

applied as an eye ointment. A second advantage of such amino

acyl esters is that for systemic use they could be injected

intramuscularly or subcutaneously, in small volumes, whereas

the parent compound, acyclovir, has to be administered

intravenously in large volumes. However, from a practical

viewpoint, parenteral injection of the amino acid esters of

acyclovir has never been pursued.

Of the various amino acid esters of acyclovir that were

subsequently studied, the valine ester, valacyclovir (Zelitrexs,

Valtrexs) (Fig. 1) appeared to be the most suitable for

increasing the oral bioavailability of acyclovir15, and valacyclo-

vir has now replaced acyclovir in the oral treatment of HSV and

VZV infections. Likewise, to increase the oral bioavailability,

Discovery of antiviral agents 537

valganciclovir, the valine ester of ganciclovir (Fig. 1), and

famciclovir, the prodrug of penciclovir (Fig. 1), were introduced

for the oral treatment of, respectively, cytomegalovirus (CMV)

infections and HSV and VZV infections15.

VACV is the only antiviral drug approved for once-daily

suppressive therapy for genital herpes16. VACV has been FDA-

approved for the treatment of HSV and VZV infections in

immunocompromised patients. It is more effective than acyclovir

in the treatment of herpes zoster, particularly with regard to the

duration of post-herpetic neuralgia (mean duration of pain: 40

days as compared to 60 days)17. VACV at a dose of 1 g twice

daily from month 4 to 24 after hematopoietic stem cell

transplantation was found effective in suppressing herpes zos-

ter18. Oral VACV could also be recommended for early treat-

ment of HSV encephalitis in resource-limited settings19.

Single-day famciclovir (1000 mg administered twice daily)

proved as effective as a 3-day VACV regimen (500 mg adminis-

tered twice daily) for recurrent genital herpes20. One-year

suppressive therapy with oral VACV (500 mg daily) was shown

to be as effective and as well tolerated as acyclovir (400 mg twice

daily) in reducing the rate of recurrent ocular HSV disease21.

Administration of VACV (500 mg twice daily) beginning at

36 weeks’ gestation until delivery to women with a history of

recurrent genital HSV reduced the number of subsequent

clinical HSV recurrences22. Exposure to acyclovir or VACV

in the first trimester of pregnancy was not associated with an

increased risk of major birth defects23.

Preemptive valganciclovir and VACV prophylaxis would

be equally effective in the prevention of CMV disease

after renal transplantation24. Valganciclovir and VACV were

Figure 2 Synthetic scheme of FV-100.

similarly protective against CMV disease25. VACV reduced

the frequency of Epstein-Barr virus (EBV)-infected B cells

when administered for a long period (one year) and might

allow eradication of EBV from the body if reinfection does not

occur26. Whether VACV may be of clinical benefit in the

treatment of acute infectious mononucleosis remains subject

of large, placebo-controlled clinical trials27.

The conversion of VACV to acyclovir, and of valganciclovir

to ganciclovir, is catalyzed by valacyclovirase, an a-amino acid

ester hydrolase28. Curiously, VACV has been shown to inhibit

erythrocyte sickling in vitro29,30. Acyclovir is known to be

transported into human erythrocytes31, but what further

happens with acyclovir in the red blood cells is not known.

Ender et al.30 now suggest that VACV may be of potential

utility as an anti-sickling agent in sickle-cell anemia.

2.2. BVDU and FV-100

IDU (idoxuridine) and TFT (trifluridine), the first 5-

substituted 20-deoxyuridines (dUrds) to be recognized as

antiviral drugs (Fig. 2), have only proved useful for topical

administration, for the treatment of herpetic keratitis, as they

are too toxic for systemic administration32. The first (and so

far only) 5-substituted dUrd used in many countries (except

for the US and UK) for the treatment of herpes zoster, is

BVDU [(E)-5-(2-bromovinyl)-20-deoxyuridine, brivudin]. It

was first identified as a highly specific inhibitor of HSV type

133 and VZV. In fact, the efficacy of BVDU against herpes

zoster was first reported in patients34, before its potency and

Erik de Clercq538

selectivity as an anti-VZV agent in cell culture was demon-

strated by Shigeta et al35.

BVDU (Fig. 2) was first approved for clinical use (as

Helpins) in the former DDR (East Germany) in immunosup-

pressed patients, before it was licensed for clinical use in

immunocompetent persons for the treatment of herpes zoster.

It is now available in several countries, under different

Figure 3 Structures of ddN analogues.

trade names (Zostexs (Germany); Briviracs (Italy); Zerpexs

(Belgium)). While even more active than acyclovir against

both HSV-1 and VZV, BVDU is specifically used for the

treatment of VZV infections (herpes zoster)36. It owes its

selective anti-VZV activity to a specific phosphorylation by the

VZV-encoded thymidine kinase to the mono- and dipho-

sphate, which upon further phosphorylation by the (cellular)

Discovery of antiviral agents 539

nucleoside diphosphate (NDP) kinase to the triphosphate,

interferes with the viral DNA synthesis.

Recent review articles have pointed to FV-100 as an

emerging new candidate drug for the treatment of VZV

infections37–39. Migliore39 qualified FV-100 as the most potent

and selective anti-VZV agent reported to date.

Where did FV-100 originate from? The parent compound of

FV-100 (FV standing for ‘‘Fermavir’’) is Cf1743 (Cf standing for

‘‘Cardiff’’) (Fig. 2). Cf1743 belongs to the so-called BCNAs

(bicyclic furopyrimidine nucleoside analogs) originally described

by McGuigan et al40,41. Cf1743 is the most potent of a class of

compounds that are specifically active against VZV. The mechan-

ism of action of Cf1743 still needs to be elucidated but clearly

depends on a specific phosphorylation by the VZV-encoded

thymidine kinase42. Its potency against VZV exceeds that of all

other antiviral agents including BVDU43.

FV-100 is the 50-valine ester of Cf174344 (Fig. 2) and in this

sense related to the valine esters of acyclovir and ganciclovir

(Fig. 1). FV-100 has recently been the subject of 3 randomized,

double-blind, placebo-controlled clinical trials45. These studies

support further investigations with FV-100 (at a daily dosage

of 100 mg, 200 mg, 400 mg or 800 mg, for 7 days) for the

treatment of herpes zoster45. Herpes zoster patients can now

be monitored for VZV DNA in the saliva46. Such non-invasive

analysis would allow to follow the response of VZV infection

to anti-VZV agents such as VACV, BVDU and FV-100.

Figure 4 Structures of (S)-HPMPC and (S)-HPMPA.

2.3. 20,30-Dideoxynucleoside (ddN) analogs

The first 20,30-dideoxynucleoside (ddN) described, in 1985, for

its inhibitory effect on the infectivity and cytopathicity of HIV

(then called HTLV-III for human T-cell lymphotropic virus

type 3) was azidothymidine (AZT, retrovirs) (Fig. 3)47.

This compound was discovered serendipitously as an anti-

HIV agent, as it had been originally synthesized as a potential

anticancer agent by Horwitz et al48. In fact, in 1980 we had

described the anti-HSV activity of several ddN analogs,

including azidothymidine49. The mechanism of anti-HIV

action of azidothymidine was described by Furman et al50.

It resided in the inhibitory effect of its 50-triphosphate as an

HIV alternate substrate/competitive inhibitor on the reverse

transcriptase (RT).

Following azidothymidine, several other 20,30-dideoxynu-

cleoside analogs, including dideoxyinosine (ddI, didanosine,

Videxs, Videx ECs) and dideoxycytidine (ddC, zalcitabine,

Hivids) (Fig. 3) were found inhibitory to the infectivity and

cytopathicity of HIVal 51. Like AZT, ddI and ddC were found

to inhibit the HIV RT as chain terminators, ddI, following its

conversion to ddATP in competition with dATP, and ddC,

following its conversion to ddCTP in competition with dCTP,

both dATP and dCTP being regular substrates for the RT

reaction.

Following AZT, ddI and ddC, the fourth ddN analog to be

discovered was d4T (stavudine, Zerits), which was announced

successively by Baba et al52. Lin et al.53 and Hamamoto

et al54. The mode of action of d4T was resolved by Balzarini

et al55. It would become one of the most popular, world-wide

used anti-HIV drugs, later to be superseded only by tenofovir

(see infra).

Following stavudine (d4T), three more ddN analogs, lamivu-

dine (3TC), abacavir (ABC) and emtricitabine [(–)FTC] would

subsequently be licensed as anti-HIV drugs56,57. Lamivudine was

originally discovered by the late Belleau58 as a racemic mixture of

which the (–)enantiomer (3TC) was proven to be most active.

The anti-HIV activity of abacavir was originally described

by Daluge59, whereas the discovery of (–)FTC was credited to

Schinazi60. 3TC, ABC, and (–)FTC would later be commercia-

lized under the trade names of Epivirs, Ziagens and Emtrivas,

respectively.

Although many more ddNs than those reported above,

have been reported in the meantime, the number of approved

ddN analogs (now also referred to as nucleoside reverse

transcriptase inhibitors (NRTIs)) have been limited to the

seven ddN analogs shown in Fig. 3. In the past few years, no

more ddN analogs or NRTIs have been added to the current

anti-HIV drug armamentarium. Instead, further progress in

the field of anti-HIV therapy has been focused on the judicious

choice of combination of the appropriate NRTIs (such as

Emtrivas) with the appropriate NtRTIs (such as tenofovir

disoproxil fumarate) and NNRTIs (such as efavirenz and

rilpivirine) (see infra: Atriplas and Compleras/Evipleras).

2.4. (S)-HPMPA and (S)-HPMPC (Cidofovir)

The era of the acyclic nucleoside phosphonates (ANPs)61 started

in 1986 with the description of (S)-HPMPA [(S)-9-(3-hydroxy-2-

phosphonylmethoxypropyl)adenine] as a novel selective broad-

spectrum anti-DNA virus agent62. One year later followed the

description of (S)-HPMPC [(S)-1-(3-hydroxy-2-phosphonyl-

methoxypropyl)cytosine], the cytosine counterpart (cidofovir) of

(S)-HPMPA (Fig. 4)63. (S)-HPMPC showed an activity spectrum

similar to that of (S)-HPMPA; nine years later, in 1996, it would

be formally approved as Vistides for the treatment, by intrave-

nous injection, of CMV retinitis in AIDS patients. This indication

has virtually disappeared with the efficient treatment of AIDS.

Yet, cidofovir is still used off label in the treatment of poxvirus

infections (i.e., molluscum contagiosum) and human papilloma-

virus (HPV) infections. It has proven more efficacious than

smallpox vaccination upon lethal monkeypox virus infection64.

Cidofovir bears an hydroxyl group that is equivalent to the

30-hydroxyl group of dCMP and permits its incorporation into

DNA, creating a significant impediment to trans-lesion DNA

synthesis in a manner resembling DNA damage65. This leads

for vaccinia virus to an inhibition of genome encapsidation

and virus assembly66.

Cidofovir has proven highly effective in different models of

poxvirus infection, whether the compound was administered

either intraperitoneally, intranasally or topically67, including

Erik de Clercq540

camelpox in athymic nude mice68 as well as an aerosol

rabbitpox model69. Also, the sea lion parapoxvirus has proven

susceptible to cidofovir70. When co-administered with the

smallpox vaccine, cidofovir effectively reduced vaccination

side effects71.

In earlier reports, both topical and systemic cidofovir were

shown to resolve recalcitrant molluscum contagiosum virus

lesions72–75. The efficacy of intravenous cidofovir in the

treatment of giant molluscum contagiosum was most drama-

tically shown recently in a patient with HIV infection76.

Intralesional cidofovir has proven successful in the treat-

ment of acyclovir-resistant HSV infection77, whereas intrave-

nous cidofovir was used successfully in the treatment of dual

infection with polyomavirus BK and acyclovir-resistant HSV

in a bone marrow transplant recipient78. Cidofovir may be a

potentially effective therapy for the treatment of BK virus-

associated hemorrhagic cystitis, a severe complication after

allogeneic hematopoietic stem cell transplantation (HSCT)79.

BK virus-associated nephopathy in a kidney transplant reci-

pient was successfully treated with cidofovir, the first case in

Japan80.

Cidofovir markedly reduced the growth of HPV-positive

cervical cancer cell xenografts in nude mice81. This result was

confirmed in a clinical setting where cidofovir proved effective

in the topical treatment of cervical intraepithelial neoplasia

(CIN) stage 282.

The principal off-label use of cidofovir, however, is cur-

rently for intralesional injection for recurrent respiratory

papillomatosis in both children83 and adults84, and, even more

so, the local administration of cidofovir for HPV-associated

skin lesions in transplant recipients85. Successful treatment of

cutaneous warts has been reported with intravenous cidofovir

in an 11-year old girl86, and several case reports have pointed

to the successful use of topical cidofovir at 1% in the

treatment of HPV warts in children87–89.

2.5. CMX001 (HDP-CDV)

CMX001 (HDP-CDV) (Fig. 5) corresponds to hexadecylox-

ypropyl cidofovir, an ether lipid conjugate of cidofovir, which,

in principle, should be active against the same DNA viruses

against which cidofovir itself is active, but, in comparison with

CDV, CMX001, should have increased oral bioavailability

and increased cellular uptake, facilitated by the lipid moiety of

the molecule90. The increased cellular uptake accounts for the

increased antiviral activity of HDP-CDV91. Its oral activity

Figure 5 Structures of HDP-CDV and ODE-CDV.

has been demonstrated particularly for the treatment of

smallpox92 and in a lethal mousepox model93.

Preclinical studies have demonstrated the efficacy of

CMX001 in the treatment of vaccinia and cowpox virus

infections in mice, which suggested that the compound could

be used in the event of a bioterror attack by poxviruses94. The

advantage of CMX001 over its parent compound, cidofovir, is

that it could be administered orally (as a tablet or liquid)

(presumably) without nephrotoxicity, which limits the dosing

of the parent compound95. It has proven efficacious as a post

exposure antiviral in rabbits infected with rabbitpox virus, a

model for orthopoxvirus infection of humans96.

In pregnant guinea pigs, CMX001 improved he outcome

of congenital CMV infection97, suggesting the potential of

CMX001 for the treatment of congenital CMV infection in

humans. CMX001 may also be effective in the treatment of

acyclovir-resistant HSV infection, and combination of

CMX001 with acyclovir led to a synergistic inhibition of

HSV infections98.

CMX001 has been shown to prevent adenovirus-induced

mortality in a permissive immunosuppressed animal model

(cyclophosphamide-treated hamsters)99, and in a pediatric

hematopoietic stem cell transplantation recipient CMX001

led to the eradication of disseminated adenovirus infection100.

CMX001 also seems effective in the treatment of human

polyomavirus replication in primary human renal tubular

epithelial cells101, polyoma JC virus replication in human fetal

brain SVG cell cultures102 and polyomavirus JC replication in

human brain progenitor-derived astrocytes103.

In addition to CMX001, various other prodrugs of cidofo-

vir have been reported, i.e., Ala–Ser and Val–Ser prodrugs104.

Similarly, tyrosine-based prodrugs of cidofovir have been

synthesized which may be of potential use in the treatment

of the same DNA virus infections that are susceptible to

cidofovir105. The advantage of these prodrugs, if any, over

that of the parent compound, cidofovir, remains to be

demonstrated.

2.6. HDP-(S)-HPMPA

Alkoxypropyl, i.e., hexadecyloxypropyl (HDP) and octadecy-

loxyethyl (ODE) esters have been prepared from both

(S)-HPMPC and (S)-HPMPA. (S)-HPMPA was the prototype

of the acyclic nucleoside phosphonates62, which was never

commercialized for clinical use. The reason why (S)-HPMPC

(cidofovir), PMEA (adefovir), and (R)-PMPA (tenofovir) were

successfully introduced in clinical medicine, whereas the

Discovery of antiviral agents 541

prototype compound (S)-HPMPA was not, even not for any

of the DNA virus infections for which it was more effective

than (S)-HPMPC, has remained an enigma.

HDP-(S)-HPMPA and ODE-(S)-HPMPA (Fig. 6) were

recently reported to be active against HIV replication106.

This was not surprising given the broad-spectrum anti-

DNA virus activity of the parent compound, (S)-HPMPA62.

More surprising was the activity reported for ODE- and

HDP-(S)-HPMPA against hepatitis C virus (HCV), which,

being an RNA virus, was certainly not expected to be

sensitive to the inhibitory action of acyclic nucleoside

phosphonates107.

Of the alkoxyalkyl esters, the most active anti-HCV agent was

ODE-(S)-3-methoxy-2-(phosphonylmethoxy)propyl-guanine with

an EC50o0.01 nM and a selectivity index of 44.4 million106.

(S)-HPMPA has been found active against parasites, i.e., the

growth of Plasmodium falciparum and P.berghei108,109. The HDP-

cyclic(S)-HPMPA was found to have antischistosomal activity110,

and this has given a new impetus to a long forgotten therapeutic

potential of (S)-HPMPA and acyclic nucleoside phosphonates,

Figure 7 Structures of adefovir, adefovir dipivoxil, tenofovir and TD

Figure 6 Structures of HDP-(S)-HPMPA and ODE-(S)-HPMPA.

their unexplored therapeutical potential in the treatment of

malaria and other parasitic infections.

ODE and HDP esters of HPMPA are potent inhibitors of

HIV111, while its parent compound has not been recognized as an

anti-HIV agent. Alkoxyalkyl esters of (S)-HPMPAmay, like those

of cidofovir, exhibit wide spectrum antiviral activity against pox-,

adeno-, herpes-, cytomegalovirus infections, with HDP-(S)-

HPMPA being at least as promising as HDP-(S)-HPMPC as

orally active against various DNA virus infections112.

2.7. Adefovir and tenofovir

The oral bioavailability of cidofovir (CDV) is increased by linking

CDV to an alkoxyalkyl ester, as in hexadecyloxypropyl (HDP)-

CDV (see supra, section 2.5). The oral prodrugs designed for

adefovir (first described by de Clercq et al.62) and tenofovir (first

described by Balzarini et al.113) have been adefovir dipivoxil (AD,

Hepseras) and tenofovir disoproxil fumarate (TDF, Vireads)

(Fig. 7). The design of AD and TDF has made the parent

F.

Erik de Clercq542

compounds adefovir and tenofovir rapidly available, through

cleavage of the ester bonds, after oral administration.

Adefovir dipivoxil [bis(pivaloyloxymethyl)-9-(2-phosphonyl-

methoxyethyl)adenine] has been described by Starrett et al.114,

Cundy et al.115,116 and Shaw et al117.

The pioneering clinical studies of Hadziyannis et al.118 and

Marcellin et al.119 helped the approval of Hepseras, in 2002,

for the treatment of chronic hepatitis B. These results were

nicely confirmed by a cross-over study upon long-term

treatment with Hepseras in HBeAg-negative chronic HBV

patients120.

Tenofovir disoproxil [bis(isopropyloxycarbonyloxy-

methyl)-9-(R)-(2-phosphonylmethoxy-propyl)adenine] was

described by Robbins et al.121 and Naesens et al.122 Teno-

fovir disoproxil fumarate (TDF) was approved for clinical

use for the treatment of HIV infection (AIDS) in 2001, and

for the treatment of chronic HBV infection in 2008. As to

the latter, the key observation was that TDF at a daily dose

of 300 mg had superior antiviral activity with a similar

safety profile as compared to adefovir dipivoxil at a daily

dose of 10 mg123.

Twenty years after its original discovery, tenofovir has acquired

a crucial position in the fight against HIV: it is not only efficacious

against AIDS but also hepatitis B; it can be used in combination

with emtricitabine, efavirenz, rilpivirine, elvitegravir, atazanavir, or

darunavir, as a single once-daily oral pill; and it can be used

in combination with emtricitabine as a once-daily oral pill,

Truvadas, in the prophylaxis of sexual HIV transmission124.

The increased oral bioavailability and increased cellular

uptake noted for the hexadecyloxypropyl derivative of cido-

fovir (see supra, section 2.5) also holds for tenofovir. Hex-

adecyloxypropyl tenofovir (CMX157) shows markedly

increased oral bioavailability and cellular uptake as compared

with the parent compound125,126.

Figure 8 Drug combination of Quad.

For the treatment of HBV infections, there are officially six

compounds available: interferon-a, lamivudine (3TC), adefo-

vir dipivoxil, entecavir, telbivudine and TDF. For HIV

infections, however, 25 years after the discovery of HIV, there

are now more than 25 compounds approved as anti-HIV

drugs56,57. Tenofovir, in its oral prodrug form, TDF, is the

cornerstone for the treatment of HIV infections127,128, appar-

ently due to the magic of the phosphonate bond129.

2.8. Truvada, Atripla, Complera, Quad (Stribild TM)

When first approved in 2001, Vireads could hardly be

anticipated to grow to a double-drug combination (Truvadas)

in 2004, a triple-drug combination (Atriplas) in 2006, another

triple-drug combination (Compleras in the US, Evipleras in

the EU) in 2011, and even a quadruple-drug combination

Quad (Stribild TM) in 2012 (Fig. 8). The single once-daily oral

pill containing three active ingredients (Truvadas and efavir-

enz) was Atriplas130. Then followed Compleras/Evipleras,

like Atripla, containing three active ingredients: Truvadas

and rilpivirine (Edurants) (Fig. 8)131.

Scheduled for 2012 is the first Quad pill, containing in

addition to Truvadas, the integrase inhibitor (INI) elvitegra-

vir and the pharmacoenhancer cobicistat (Fig. 8). Also

expected to be approved in 2012 is Truvadas for prophylactic

use to prevent sexual transmission of HIV infection.

In addition to the ‘‘real’’ Quad (Quad no. 1 Stribild TM)

containing elvitegravir, cobicistat, emtricitabine and TDF, several

other Quads are forthcoming131: Quad no. 2, consisting of

Truvadas, cobicistat and atazanavir; Quad no. 3, consisting of

GS-7340, emtricitabine ((–)FTC), cobicistat and darunavir; and

Quad no. 4, consisting of GS-7340, emtricitabine ((–)FTC),

cobicistat and elvitegravir131.

Figure 9 Structures of GS-7340, (R)-PMPA, GS-9131 and GS-9148.

Discovery of antiviral agents 543

2.9. GS-7340, GS-9131, GS-9191 and GS-9219

GS-7340 (Fig. 9) (see supra, section 2.8) is an isopropylalaninyl

monoamidate phenyl monoester prodrug of tenofovir; it was first

described by Lee et al132. Phosphonoamidate prodrugs of

adefovir and tenofovir had originally been described by Ballatore

et al133. A novel one-pot synthesis of bis-amidate prodrugs of

acyclic nucleoside phosphonates was reported by Jansa et al134.

GS-9131 (Fig. 9) is the orally bioavailable phosphonoami-

date prodrug of GS-9148, a cyclic nucleoside phosphonate

analogue135 with a low nephrotoxic potential136. The 20-fluoro

group fulfills a specific role in this compound, as it may cause

steric hindrance to the side chain of the Q151L mutation137.

Cathepsin A is the major hydrolase catalyzing the intracellular

hydrolysis of GS-7340 to tenofovir and of GS-9131 to GS-

9148138. Cathepsin is also held responsible for the intracellular

hydrolysis of GS-9191 to cPrPMEDAP139. GS-9191 can be

considered as a ‘‘double’’ prodrug, being converted to PMEG

via cPrPMEDAP as intermediate, thus requiring hydrolysis

followed by deamination to yield PMEG, which is then

converted to PMEG diphosphate as the final active metabolite.

GS-9191 has been pursued for the topical treatment of

human papilloma virus (HPV) infections140. cPrPMEDAP

could also be directly applied topically or transdermally141.

Like GS-9191, GS-9219 (Fig. 10) is a double prodrug of

PMEG via cPrPMEDAP: it has potent antineoplastic activity

in dogs with spontaneous non-Hodgkin’s lymphoma142,143.

As for GS-9191, the final active metabolite of GS-9219 is

assumed to be PMEG diphosphate142.

3. Conclusions

The principal conclusions reached from this overview are as

follows:

3.1.

Valacyclovir (VACV) has succeeded acyclovir as the

gold standard for the oral treatment of both herpes

simplex virus (HSV) and varicella-zoster virus (VZV)

infections;

3.2.

BVDU (brivudin) and FV-100 (the valine ester of Cf1743)

have remained the most potent inhibitors of VZV

infections; Cf1743 does not suffer from the liability of

potentiating the toxicity of 5-fluorouracil, as noted for

bromovinyluracil, the degradation product of BVDU;

3.3.

Most of the 20,30-dideoxynucleoside (ddN) analogs that

have been licensed for clinical use, are still used for the

treatment of human immunodeficiency virus (HIV)

infections, i.e. (–)FTC (emtricitabine) in combination

with tenofovir disoproxil fumarate (TDF);

3.4.

The acyclic nucleoside phosphonates (S)-HPMPA and

(S)-HPMPC (cidofovir) possess broad-spectrum anti-

viral activity against DNA (pox, herpes, adeno, papil-

loma, polyoma) viruses;

3.5.

The alkoxyalkyl (HDP: hexadecyloxypropyl, ODE: octa-

decyloxyethyl) esters of (S)-HPMPA and (S)-HPMPC

show increased oral bioavailability and increased cellular

uptake as compared to the parent compounds;

3.6.

Adefovir dipivoxil, the prodrug of PMEA, is widely

accepted for the treatment of hepatitis B virus (HBV)

infections, whereas tenofovir disoproxil fumarate, the

prodrug of (R)-PMPA, is widely used for the treatment

of both HIV and HBV infections;

3.7.

Combinations containing TDF and (–)FTC (Truvadas),

if extended with efavirenz to Atriplas, or with rilpivirine

to Compleras (or Evipleras) are available as single pills

for once-daily use in the treatment of HIV infections.

was recently appeared is the Quad pill containing TDF,

(–)FTC, elvitegravir and cobicistat Quad (Stribild TM).

3.8.

In development are phosphonoamidate prodrugs,

i.e., GS-7340 and GS-9131, for the oral treatment of

HIV infections, GS-9191 for topical treatment of human

papilloma virus (HPV) infections, and GS-9219 for the

treatment of non-Hodgkin’s lymphoma (NHL) in dogs.

Figure. 10 Structures of prodrugs of PMEG.

Erik de Clercq544

Acknowledgments

The author thanks Mrs. Christiane Callebaut for her profi-

cient editorial assistance.

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