ANTI-HERPESVIRUS THERAPIES: BASIC STRATEGIES AND APPLICATIONS

Erik DE CLERCQ Rega Institute for Medical Research, K.U.Leuven B-3000 Leuven, Belgium Human herpesviruses (HHV)

HHV-1: type 1 (HSV-1) HHV-2: Herpes simplex virus type 2 (HSV-2) HHV-3: Varicella-zoster virus (VZV) HHV-4: Epstein-Barr virus (EBV) HHV-5: Human (HCMV) HHV-6: Human herpes simplex virus type 6 (HSV-6) HHV-7: Human herpes simplex virus type 7 (HSV-7) HHV-8: Kaposi’s sarcoma herpesvirus (KSHV) O

N HN

N H2N N

HO O

Acyclovir, (ACV), Acycloguanosine 9-[(2-Hydroxyethoxy)methyl] Zovirax® O O

N N HN HN

N N H2N N H2N N

H2N CH CO O HO O O CH H3CCH3 Acyclovir Acyclovir valyl ester

O O N N HN HN N N H2N N H2N N

H2N CH CO O H2N CH2 CO O O O CH3

Acyclovir glycyl ester Acyclovir alanyl ester O

N HN

N H2N N O H H2N C C O O CH H3CCH3

Valaciclovir Valine ester of acyclovir Valtrex®, Zelitrex® Antiviral activity spectrum of valaciclovir (acyclovir)

Herpesviridae ! Herpes simplex virus type 1 (HSV-1) ! Herpes simplex virus type 2 (HSV-2) ! Varicella-zoster virus (VZV) ! Epstein-Barr virus (EBV) " Human cytomegalovirus (HCMV) " Human herpesvirus type 6 (HHV-6) " Human herpesvirus type 7 (HHV-7) " Human herpesvirus type 8 (HHV-8) " Thymidine kinase-deficient HSV (TK- HSV) " Thymidine kinase-deficient VZV (TK- VZV) Major clinical indications of valaciclovir

Systemic (oral) treatment of HSV and VZV : • primary and recurrent genital herpes, mucocutaneous HSV-1 and HSV-2 infections in immuno-compromised and –competent patients • varicella-zoster in immuno-compromised and –competent patients O

N N N HN

N N H2N N H2N N O

H3C O HO

H3CO OH

O

Famciclovir Famvir® N N N N N N

N N N H2N N H2N N H2N N O O

H3C O Esterase H3C O Esterase HO

H3C O OH OH

O

Xanthine Xanthine Xanthine oxidase oxidase oxidase

O O O

N N N HN HN HN

N N N H2N N H2N N H2N N O O Esterase Esterase H3C O H3C O HO

H3C O OH OH

O Intravenous penciclovir versus acyclovir in the treatment of HSV infections in immunocompromised patients

Kaplan-Meier plot of time to healing of lesions for penciclovir at 5 mg/kg q12 h (long-dashed line), penciclovir at 5 mg/kg q8h (short-dashed line), and acyclovir at 5 mg/kg q8h (solid line) Lazarus et al., Antimicrob. Agents Chemother. 43: 1192-1197 (1999) O

N HN

N H2N N

HO O

OH (GCV) 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) Cymevene®, Cytovene® O

N HN

N H2 N N

O H H2N C C O O CH H3CCH3 OH

Valganciclovir Valcyte® Nucleoside kinase O O + -O P C 3 Na O- O-

Foscarnet Phosphonoformate (trisodium salt) PFA Foscavir® O

N NH

N N NH2

HO HO HO O O

OH OH Acyclovir Ganciclovir H2G HO HO HO O

OH OH OH Penciclovir Lobucavir Anhydrohexitol guanine

HO HO HO

HO OH Synguanol A-5021 D/L-Cyclohexenyl guanine O

N HN

N H2N N

HO

OH

Lobucavir O

HN N N H2N N

HO

OH

(-) 2HM-HBG H2G (-)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine NH2 O

N N N NH

HO N HO N N N NH2

Synadenol Syngenol

Methylene cyclopropane nucleoside analogues O

N NH

N N NH2 HO

OH

D/L-Cyclohexenyl guanine O

N NH

N N NH2 HO

HO

A-5021 O

N N N NH

N N N NH2 N NH2

HO HO

OH OH

AV-038 A-5021 Comparative potency of different antiherpetic compounds against different herpesviruses

Compound 50% Effective concentration EC50 (µM) HSV-1 HSV-2 VZV HCMV Acyclovir +++ +++ ++ + Penciclovir +++ ++ ++ (+) Ganciclovir +++++ +++++ ++ ++ Lobucavir ++++ ++++ +++ ++ H2G ++++ ++++ ++++ (+) A-5021 ++++ +++ +++ + Synguanol + - + ++ Anhydrohexitol G ++(+) +++(+) ++ ++ D-Cyclohexenyl G ++++ +++ ++ ++ L-Cyclohexenyl G +++(+) +++ ++ ++

EC50 (µM)= 10-100 (+), 1-10 (++), 0.1-1 (+++), 0.01-0.1 (++++), 0.001-0.01 (+++++) H2N N N

N N

HO O

OH

S2242 2-Amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine O OH CH3 OH C O O OCH3

CH3

Mycophenolic acid O OH CH3 O C N O O O OCH3

CH3

Mycophenylate mofetil (Cellcept®) 2-Morpholinoethyl ester of mycophenolic acid MPA-IMPDH interactions

Sintchak et al., Cell 85: 921-930 (1996) Phosphoribosylamine (PRA)

+ Mycophenolic acid H2O + NAD IMP

IMP dehydrogenase

+ NADH + H XMP Deoxyguanosine Guanosine analogues

GMP dGMP dGMP analogues

GDP dGDP dGDP analogues

GTP dGTP dGTP analogues

RNA DNA synthesis synthesis Inhibitory effects of mycophenolate mofetil (MMF) and lobucavir (LBV) on cytopathicity of HCMV in human embryonic lung (HEL) cells

Virus Control 2.5 µg/ml MMF

1 µg/ml LBV 1 µg/ml LBV + 2.5 µg/ml MMF Inhibitory effects of topical mycophenolate mofetil (MMF) 5% and topical acyclovir (ACV) 0.1% on HSV-1-induced lesions in hairless mice Virus Control MMF

ACV MMF + ACV O I HN

O N HO O

OH

Idoxuridine 5-iodo-2’-deoxyuridine (IDU) Stoxil® O

CF3 HN

O N HO O

OH

Trifluridine, Trifluorothymidine (TFT) 5-Trifluoromethyl-2’-deoxyuridine Viroptic®, TFT-Ophthiol® O Br HN

O N

HO O

HO

Brivudin, Bromovinyldeoxyuridine (E)-5-(2-bromovinyl)-2’-deoxyuridine (BVDU) Zostex®, Zonavir® Antiviral activity spectrum of BVDU

De Clercq, Recent Advances in Nucleosides: Chemistry and Chemotherapy, pp 433-454 (2002) O O O Br Br Br HN HN HN

O N O N O N

HO HO HO O O HO

HO HO OH HO

BVaraU BVriboU C-BVDU

De Clercq, Recent Advances in Nucleosides: Chemistry and Chemotherapy, pp 433-454 (2002) O NH2 O Br Br Br HN N HN

O N O N O N

HO HO S O HO OH O HO HO

S-BVDU BVDC L-BVDU

De Clercq, Recent Advances in Nucleosides: Chemistry and Chemotherapy, pp 433-454 (2002) N

O N N Br O HN Br N Br HN O N O N O N HO S HO O HO O

HO O HO

S-BVMU Triazolyl BVDC L-BVODDU derivative

De Clercq, Recent Advances in Nucleosides: Chemistry and Chemotherapy, pp 433-454 (2002) O O O Br Br Br HN HN HN

O N O N O N

HO O HO O H3C HO

HO OH OH

BMS-181165 4’-methyl BVDU AV-100

De Clercq, Recent Advances in Nucleosides: Chemistry and Chemotherapy, pp 433-454 (2002) Mechanism of action of BVDU HSV-1 dThd kinase DNA polymerase

BVDU BVDUMP BVDUDP BVDUTP DNA

BVDU BVDUMP

HSV-2 dThd kinase O O H Br H Br C C C C HN H HN H

O N O N H

HO HO O O O P + Pi BVU + Thymidine phosphorylase HO HO BVDU BVU O O F F HN HN Dihydrothymine O N O N H H dehydrogenase

5-Fluorouracil 5-Fluorodihydrouracil Major clinical indications of BVDU (Brivudin)

Topical: - herpetic keratitis (BVDU eyedrops) - herpes labialis (BVDU cream)

Systemic: - mucocutaneous HSV-1 and VZV infections (oral) - immunocompromised patients - immunocompetent patients Oral brivudin versus intravenous acyclovir in the treatment of severe herpes zoster in cancer patients

BRIVUDIN: BVDU: (E)-5-(2-bromovinyl)-2’-deoxyuridine orally 125 mg x 4 per day for 5 days

ACYCLOVIR: ACV: 9-(2-hydroxyethoxymethyl)guanine intravenously 10 mg/kg x 3 per day for 5 days

Multicentered, double-blind, randomized BVDU group (24 patients) also received placebo i.v. ACV group (23 patients) also received placebo p.o.

Wutzler et al., J. Med. Virol. 46: 252-257 (1995) Double-blind study BVDU versus ACV

100 ---- ACV (n = 23) — BVDU (n = 24) 80

60

40

% Patients with new vesicles new with Patients % 20

0 1 2 3 4 5 6 7

Number of days following start of therapy Current antiviral therapy for zoster

Valaciclovir oral 1000 mg 3 x daily 7 days

Acyclovir oral 800 mg 5 x daily 7 days

Acyclovir intravenous 5-10 mg/kg 3 x daily 7-10 days

Famciclovir oral 250 mg 3 x daily 7 days

Brivudin oral 125 mg 1 x daily 7 days

Gross et al., J. Clin. Virol., in press (2003) Brivudin compared to acyclovir Incidence of postherpetic pain

A randomized, double-blind post-study survey on the effect of brivudin in the prevention of postherpetic pain in comparison with acyclovir

Survey patients (n = 608)

Brivudin Acyclovir (n = 309) (n = 299) Patients with postherpetic pain n(%) 101 (32.7) 130 (43.5)

Odds ratio (95% Cl) 1.61 (1.15 – 2.25) p value for difference 0.006

Prof. Dr. S.W. Wassilew Brivudin compared to famciclovir

Brivudin compared to famciclovir in the prevention of postherpetic neuralgia: a prospective randomized, double-blind multicenter trial

Prevalence of postherpetic pain ITT (n = 1954)

Brivudin Famciclovir (n = 980) (n = 974) Patients with postherpetic pain n(%) 109 (11.1) 90 (9.2)

Odds ratio (95% Cl) 1.23 (0.92 – 1.65) p value for non-inferiority 0.0102

Prof. Dr. S.W. Wassilew Brivudin compared to famciclovir

Brivudin compared to famciclovir in the prevention of postherpetic neuralgia: a prospective randomized, double-blind multicenter trial

Duration of postherpetic pain ITT (n = 199)

Brivudin Famciclovir (n = 109) (n = 90) Duration of postherpetic pain Mean ± SD 69.3 ± 62.8 72.9 ± 63.7 Median 47.0 54.0 Risk ratio (95% Cl) 1.05 (0.76 – 1.45) p value for non-inferiority 0.049

Prof. Dr. S.W. Wassilew Brivudin compared to famciclovir Duration of ZAP (Zoster-Associated Pain), n = 1712

1 Brivud in 0,9 Fam ciclovir 0,8

0,7

0,6

0,5 Probability 0,4

0,3 0,2

0,1

0 0 30 60 90 120 150 180 210 240 270 Duration of ZA P [days, after start of tre a tment]

Prof. Dr. S.W. Wassilew Nucleoside Analogues Non-nucleoside Analogues 4-Hydroxyquinoline-3-carboxamides (4-HQC)

OH O

HO N H N Cl

PNU-181465 inhibit replication of HSV-1, VZV and HCMV, through inhibitory effect on HSV-1, VZV and HCMV DNA polymerase

Oien et al., Antimicrob. Agents Chemother. 46: 724-730 (2002) O H N N

N O N H3C NH2 S

BILS 179 BS inhibits replication of HSV (and other herpesviruses ?) through inhibitory effect on helicase/primase (UL5, UL8 and UL52 gene products)

Crutz et al., Nature Medicine 8: 386-391 (2002) CH3 O NON SNH2 N S O

CH3

BAY 57-1293

N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)- phenyl]acetamide inhibits replication of HSV (and other herpesviruses ?) through inhibitory effect on helicase /primase (UL5, UL8 and UL52 gene products)

Kleymann et al., Nature Medicine 8: 392-398 (2002) BAY 57-1293 in the rat lethal challenge model

_#_ Placebo _≤_ Valaciclovir 45 mg/kg _X_ Valaciclovir 135 mg/kg _π_ BAY 57-1293 0.5 mg/kg _′′′_ BAY 57-1293 2 mg/kg

Lewis rats were inoculated with HSV-1walki intranasally and were treated via oral gavage t.i.d. from day 0 to day 4 post . Five animals for each group were used.

Betz et al., Antimicrob. Agents Chemother. 46: 1766-1772 (2002) BAY 57-1293 in the murine lethal challenge model

12

Mice were infected intranasally with HSV-2 MS and were treated via oral gavage once-daily from day 0 to day 4 post infection. Ten animals for each group were used.

Betz et al., Antimicrob. Agents Chemother. 46: 1766-1772 (2002) 1,4-Dihydroxynaphthalene 1,4-Naphthoquinone

OH O

OH O

inhibit HSV-1 and HCMV proteases, could be considered as scaffold for development of non-peptidic anti-herpesvirus agents

Matsumoto et al., Biol. Pharm. Bull. 24: 236-241 (2001) CMV423

O NH2

Cl N

N

2-Chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1- carboxamide inhibits HCMV replication, acts at an early stage (coinciding with IE antigen synthesis) of the HCMV replication cycle

Snoeck et al., Antiviral Res. 55: 413-424 (2002) Pyrrolopyrimidines

S S NH NH NH NH NH 2 2 2 2 2 CN

N N N NH2 N N N N N N

H3CO O

H3CO H3C 828 951 1028 inhibit HCMV replication, target a viral protein that is expressed early in the HCMV replication cycle

Jacobson et al., Antimicrob. Agents Chemother. 43: 1888-1894 (1999) Naphthyridine and dihydroisoquinoline derivatives

N H N

O N H

7,8-Dihydroisoquinoline-6-carboxyl-[2-(1-indol-3-yl) ethyl]amide inhibits HCMV (and HSV) replication, affects early (post-adsorption) event(s) of HCMV replication cycle

Bedard et al., Antimicrob. Agents Chemother. 44, 929-937 (2000) BAY 38-4766

H3CCH3 N

CH3 H N OH

O CH3 S O O N H

3-Hydroxy-2,2-dimethyl-N-[4({[5-dimethylamino)-1-naphthyl] sulfonyl}amino)-phenyl]propanamide inhibits HCMV replication, targets HCMV DNA maturation via UL89 and UL56 gene products, inhibits cleavage of high-molecular-weight DNA concatemers and packaging of monomeric genomes into procapsids

Buerger et al., J. Virol. 75: 9077-9086 (2001) BDCRB

Cl N Br Cl N

HO O

HO OH 1-(β-D-Ribofuranosyl)-2-bromo-5,6-dichlorobenzimidazole inhibits HCMV replication, targets HCMV DNA maturation via UL89 and UL59 gene products which are responsible for cleavage of high-molecular-weight DNA concatemers and packaging of monomeric genomes into procapsids

Biron et al., Antimicrob. Agents Chemother. 46: 2365-2372 (2002) 1263W94

Cl N CH3 N H CH3 Cl N

OH O

HO OH

1-(β-L-Ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole

inhibits HCMV replication, through interaction with the UL97 gene product (which is required for egress of viral nucleocapsids from the nucleus)

Biron et al., Antimicrob. Agents Chemother. 46: 2365-2372 (2002) [Krosky et al., J. Virol. 77: 905-914 (2003)] Indolocarbazoles

H O N

N N

CH3

CN Gö 6976 inhibit HCMV replication, target UL97 gene product (which is required for egress of viral nucleocapsids from the nucleus) Zimmermann et al., Antiviral Res. 48: 49-60 (2000) [Krosky et al., J. Virol. 77: 905-914 (2003)] CONCLUSION

Marketed antiviral drugs for treatment of HSV, VZV and CMV infections:

HSV VZV CMV

Acyclovir Ganciclovir Valaciclovir Famciclovir Brivudin