WO2010012840A1

WO2010012840A1 - Use of nucleoside analogs as anti-koi herpes virus molecules

Info

Publication number: WO2010012840A1
Application number: PCT/EP2009/060039
Authority: WO
Inventors: Berenice Costes; Johan Neyts; Alain Vanderplasschen
Original assignee: Katholieke Universiteit Leuven, K.U.Leuven R&D; Universite De Liege
Priority date: 2008-08-01
Filing date: 2009-08-03
Publication date: 2010-02-04
Also published as: GB0814065D0

Abstract

The present invention relates to the use of substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1'-ylmethyl nucleobase derivatives as active agents for the treatment or prevention of Koi Herpes virus infections in fish, especially in carps.

Description

USE OF NUCLEOSIDE ANALOGS AS ANTI-KOI HERPES VIRUS

MOLECULES

FIELD OF THE INVENTION

The present invention relates to substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives for use in the treatment or prevention of Koi Herpes virus (KHV) infections in fish, especially in carps.

BACKGROUND OF THE INVENTION

Common carp (Cyprinus carpio carpio) is a fish species that is widely cultivated for human food; 1.5 million metric tons is harvested annually, principally in China and many other Asian and European countries (www.fao.org). Unlike common carp, the koi subspecies (Cyprinus carpio koi) is a beautiful and colorful fish, and it has become part of a worldwide hobby consisting of keeping the fish in backyard ponds and large display aquaria for personal pleasure and competitive showing.

Koi herpes virus (KHV) is an emerging virus that infects common carp and its varieties such as mirror, leather, koi and ghost carp. The disease is of global significance because of the extensive international trade in highly prized and expensive ornamental koi.

Koi carp is an important ornamental fish of very high added value in the fish farming industry in Asia. The practice of selectively breeding common carp for striking body colors, leading to a type called koi, has created a worldwide market for the ornamental fish hobby and competitive showing, where individual fish may exceed $100,000 in value. In recent years, the industry has been suffering from a severe blow through the infection of Koi Herpesvirus (KHV). The outbreak of KHV is found in some Asian countries, such as Taiwan, Hong Kong and Indonesia, as well as in Israel and Europe. The KHV disease process is highly contagious and is characterized by causing massive morbidity and mortality rate ranging between 80% and 90%. According to an Indonesian research analysis, the process of KHV infection in East Java has affected over 5000 fish farmers and caused a loss of US$ 0.5 million from March to October in 2002.

The KHV infection spread is furthermore of great importance because of the high economic importance of common carp as a food fish in countries such as China. Common carp also represent a significant resource in the UK as a major target species for freshwater anglers.

Following the initial identification of KHV in the late 1990's the virus has spread across the world. There is now a major concern over the potential spread of KHV into wild stocks of common carp which sustain carp fishing as an industry estimated to be worth over £1.5 billion pounds a year.

With the growth of angling and the demand for larger numbers of expensive, specimen carp, the number of stocking events (which generally occur during the colder months of the year to minimize transport stress) increases the risk of introducing unhealthy fish or fish harboring covert KHV infections. This creates a problem for the carp fisheries that may not be easily resolved. Screening tools based on detection of viral genome by PCR or on ELISA to detect specific antibodies for KHV produced by common carp after exposure to the virus are being used to detect fish carrying the infection and so prevent their introduction into fisheries. Therapies for KHV-infections have not been developed up to date. It was found that temperature is the predetermining factor that controls whether KHV develops into a lethal infection and therefore, it has only been advised to control the temperature of the water.

Acyclovir has already been tested for the treatment of KHV-infections, but this anti-herpes simplex virus (HSV) drug widely used in humans is inactive against KHV infections as demonstrated and explained by llouze et al. in FEBS Letters (2006) 580(18) 4473-4478. Acyclovir is an antiviral drug for the treatment of herpes virus infections in man and is highly effective against various human herpesviruses, including herpes simplex virus type 1 and type 2. Many compounds that exhibit selective antiviral activity against herpes viruses that cause disease in man are known and in clinical use. These include, besides acyclovir (ACV) and its oral prodrug form valacyclovir (valACV) also ganciclovir (GCV) and its oral prodrug form valganciclovir (valGCV), brivudin (BVDV), foscarnet (PFA), cidofovir (HPMPC).

EP-A-502.690 describes a class of cyclopropane derivatives wherein a purine or pyrimidine residue is attached to the cyclopropyl ring via a methylene or bismethylene unit. Table 1 of this document shows that two derivatives of this class exhibit, in an in vitro assay, an inhibitory activity of HSV-1 which is higher than the activity of acyclovir. Intravenous, oral and transdermal administration are suggested.

EP-A-649.840 describes a class of bis(hydroxymethyl)cyclopropyl- methyl pyrimidine derivatives. Table 1 of this document shows that fifteen derivatives of this class exhibit a similar cytotoxicity but a lower anti-HSV-1 activity than acyclovir. Table 2 of this document shows that three derivatives of this class exhibit, in an in vitro assay, an inhibitory activity of varicella zoster virus which is higher than the activity of acyclovir. Intravenous, oral and subcutaneous administration are suggested.

EP-A-654.473 describes a class of cyclopropane derivatives wherein a purine residue is attached to the cyclopropyl ring via a methylene unit. Table 2 of this document shows that one derivative of this class exhibits, in an in vivo assay in HSV-1 infected mouse, a higher efficacy than a bis(hydroxymethyl)- cyclopropylmethylguanine reference compound. Intravenous, oral, transdermal and ophthalmic administration are suggested.

An alternative therapy or a therapy with high activity for KHV infections in fish, especially in carps, would be of high economic importance in order to reduce the number of fish that succumb because of the infection. Therefore, there is a clear need in the art for therapeutic or preventive (prophylactic) methods against KHV-infections in fish. One goal of the present invention is therefore to provide the use of a class of compounds as anti-Koi Herpes virus (KHV) active agents for the manufacture of a medicament for treating or preventing KHV-infections in fish, especially in carps.

SUMMARY OF THE INVENTION

It was surprisingly found that substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-yl-methyl nucleobase derivatives, such as one or more compounds described in EP-A-502.690, EP-A-649.840 or EP-A- 654.473, more in particular (VS, 2'R)-9-{[1\ 2'-bis(hydroxymethyl)cycloprop-1'-

yl]methyl}guanine (hereinafter referred as A-5021 ) are highly active against KHV-infections in cell culture and in fish, especially carps, while it is shown that other compounds known as effective anti-herpes agents in mammals and man, such as acyclovir, brivudin, foscarnet and ganciclovir exhibit very weak activity or are not active against KHV-infections in fish, especially in carps.

Without wishing to be bound by theory, there are at least three possible explanations to this unexpected behaviour:

- KHV belongs to a different sub-group of herpes viridae than human herpes simplex virus, and includes nucleotide sequences significantly different from the latter;

- the nucleotide sequence of fish such as carps significantly differs from that of mammals; and

- the immune system significantly differs in structure from the immune system of mammals.

The present invention thus provides the use of substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives, such as one or more compounds described in EP-A-502.690, EP- A-649.840 or EP-A-654.473, pro-drugs, pharmaceutically or veterinary acceptable salts, tautomers, isomers and solvates thereof as active agents, e.g. for the manufacture of a medicament, for the prevention or treatment of KHV-infections in fish, especially in carps. The present invention furthermore provides a method of treating or preventing KHV-infections in a fish, especially a carp, comprising administering to the fish in need of such treatment a therapeutically effective amount, e.g. a KHV inhibiting amount, of a substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivative, such as one or more compounds described in EP-A-502.690, EP- A-649.840 or EP-A-654.473, pro-drugs, pharmaceutically or veterinary acceptable salts, tautomers, isomers and solvates thereof. The invention also provides for the use of substituted or unsubstituted bis(hydroxymethyl)- cycloprop-1 '-ylmethyl nucleobase derivatives, such as one or more compounds described in EP-A-502.690, EP-A-649.840 or EP-A-654.473, as inhibitors of KHV, e.g. for the prevention or treatment of KHV-infections, especially in the form of a combination with one or more other agents being

biologically active against KHV in fish. The present invention also provides for the in vitro use of a substituted or unsubstituted bis(hydroxymethyl)-cycloprop- 1 '-ylmethyl nucleobase derivative, such as one or more compounds described in EP-A-502.690, EP-A-649.840 or EP-A-654.473, pro-drugs, pharmaceutically or veterinary acceptable salts, tautomers, isomers and solvates thereof as anti-Koi Herpes virus compounds.

The substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '- ylmethyl nucleobase derivatives referred to in the different aspects of the present invention are bis(hydroxymethyl)-cycloprop-1 '-ylmethyl derivatives of purine or pyrimidine bases or analogs of these bases known in the art, especially from EP-A-502.690, EP-A-649.840 and EP-A-654.473, as well as pharmaceutically acceptable salts, tautomers or isomers thereof. In a particular embodiment the bis(hydroxymethyl)-cycloprop-1 '-ylmethyl derivatives of purine or pyrimidine bases are according to formula I, isomers, solvates, veterinary acceptable salts or prodrugs thereof,

wherein,

- B is an optionally substituted pyrimidin-1-yl, pyrimidin-3-yl, purin-3-yl, purin-7-yl or purin-9-yl residue, or an aza, deaza, deoxy or deamino analogue thereof, including salts thereof with alkali metals, ammonia or amines;

- each of R¹ and R² is independently hydrogen or a hydroxyl protecting group such as, but not limited to, benzyl, benzoyl, tetrahydropyranyl, acyl (e.g. acetyl or pivaloyl) and silyl; and

- each of m, n and k is independently selected from 1 and 2. In a preferred embodiment of the present invention, each of m, n and k is 1.

In a particular embodiment of the present invention, the B-residue may be an aza, deaza, deoxy or deamino analogue of uracil, thymine, cytosine, guanine, adenine, hypoxanthine or xanthine. In another particular embodiment of the present invention, the residue B is 6-alkylpurin-3-yl, 6-alkylpuhn-7-yl or 6-alkyl-9-yl or a derivative thereof. In yet another particular embodiment of the present invention, the B residue is substituted with a methyl, amino, bromo, fluoro, hydroxy and/or a thio group. In still another more particular embodiment of the present invention, the residue B is selected from Uracil-1- yl, Thymin-1 -yl, Cytosin-1 -yl, 6-Methylpuhn-9-yl, Guanin-9-yl, Hypoxanthin-9- yl, Adenin-9-yl, 2-Aminoadenin-9-yl, 8-Bromoadenin-9-yl, 2-Aminopurin-9-yl, 6-Hydrazinopurin-9-yl, 7-Deaza-8-azaadenin-9-yl, 7-Deaza-8-azahypoxanthin- 9-yl, 5-Methylcytosin-1 -yl, 5-Fluorouracil-1-yl, Guanin-7-yl, Adenin-3-yl, Hypoxanthin-9-yl, 2-Methyladenin-9-yl, 2-Methylthioadenin-9-yl, N⁶- Dimethyladenin-9-yl, 8-Hydroxyadenin-9-yl, 6-Hydroxylaminopurin-9-yl, 6- Thiopurin-9-yl, Purin-9-yl, or Xanthin-9-yl. In a more particular embodiment of the present invention, B is guanine (guanin-9-yl).

In another particular embodiment of the present invention, the bis(hydroxymethyl)-cycloprop-1 '-ylmethyl derivatives of purine or pyrimidine bases are according to any one of formulae M-A and M-B below, wherein R¹ and R² are as defined in formula (I), including isomers, solvates, veterinary acceptable salts or prodrugs thereof, and wherein more particularly the purin- 9-yl moiety can be further substituted with hydroxy, amino, Ci-β alkyl, Ci-β alkenyl or halogen, particularly at positions 2 or 6:

In yet another particular embodiment of the present invention, the bis(hydroxymethyl)-cycloprop-1 '-ylmethyl derivatives of purine or pyrimidine bases are according to any one of formulae Ml-A, Ml-B, Ml-C or Ml-D below, wherein R¹ and R² are as defined in formula (I), including isomers, solvates, veterinary acceptable salts or prodrugs thereof.

In a particular embodiment of the present invention, the bis(hydroxymethyl)-cycloprop-1 '-ylmethyl derivatives of purine or pyrimidine bases are according to formula IV below, including isomers, solvates, veterinary acceptable salts or prodrugs thereof,

wherein B, R¹ and R² are as defined in formula I (i.e. carbon atoms 1 ' and 2' of the cyclopropyl group are in the 1¹S, 2¹R conformation).

In another particular embodiment of the present invention, the bis(hydroxymethyl)-cycloprop-1 '-ylmethyl derivatives of purine or pyrimidine

bases are according to any one of formulae V-A and V-B wherein R¹ and R² are as defined in formula I, including isomers, solvates, veterinary acceptable salts or prodrugs thereof, wherein more particularly the purin-9-yl moiety can be substituted with hydroxy, amino, Ci_-6 alkyl, Ci_-6 alkenyl or halogen, particularly at positions 2 or 6:

(V-A) (V-B)

In a particular embodiment of any one of the formulae herein, B may be a purine-based moiety, more particularly is may be selected from the group consisting of purin-9-yl, guanin-9-yl, 2-aminopurin-9-yl, adenin-9-yl and 2- amino-adenin-9-yl.

In another particular embodiment of the present invention, the bis(hydroxymethyl)-cycloprop-1 '-ylmethyl derivatives of purine or pyrimidine bases are according to any one of formulae Vl-A, Vl-B, Vl-C or Vl-D below, wherein R¹ and R² are as defined in formula (I), including isomers, solvates, veterinary acceptable salts or prodrugs thereof.

In a particular embodiment of each of the compounds represented by any one of the above formulae of the invention, each of R¹ and R² is hydrogen. In another particular embodiment of the present invention, the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivative of the invention is A-5021 ((1'S, 2'R)-9-{[1 \ 2'- bis(hydroxymethyl) cycloprop-1 '-yl]methyl}guanine) or 9-[1 'α, 2'α- bis(hydroxymethyl)cyclopropan-1 'β-yl]methylguanine with the detailed structure represented as follows:

Other specific examples of compounds useful for the present invention include, but are not limited to: 9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methylguanine;

9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyladenine;

9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyl-2,6-diamino-purine;

9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-puhne;

9-[1 'α, 2'α-bis(acetoxymethyl)cyclopropan-1 'β-yl]methylguanine; 9-[1 'α, 2'α-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methylguanine;

9-[1 'α, 2'α-bis(acetoxymethyl)cyclopropan-1 'β-yl]methyladenine;

9-[1 'α, 2'α-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyladenine;

9-[1 'α, 2'α-bis(acetoxymethyl)cyclopropan-1 'β-yl]methyl-2,6-diamino-puhne; θ-ti 'α^'α-bisφenzoyloxymethyOcyclopropan-i 'β-yllmethyl^.e-diamino-puhne; 9-[1 'α, 2'α-bis(acetoxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-purine;

9-[1 'α, 2'α-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-purine;

9-[1 'α-(hydroxymethyl)-2'α-(hydroxyethyl)cyclopropan-1 'β-yl]methylguanine;

9-[1 'α, 2'β-bis(hydroxymethyl)cyclopropan-1 'β-yl]methylguanine;

9-[1 'α, 2'β-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyladenine; 9-[1 'α, 2'β-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-6-chloro- purine;

9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-6-chloro- purine;

9-[1 'α, 2'β-bis(hydroxymethyl)cyclopropan-1 'β-yl]methylhypoxanthine; 9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methylhypoxanthine;

9-[1 'α, 2'β-bis(hydroxymethyl)cyclopropan-1 'β-yl]methylthymine;

9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methylthymine; 9-[1 'α, 2'β-bis(hydroxymethyl)cyclopropan-1 'β-yl]nnethylcytosine; 9-[1 'α, 2'α-bis(hydroxymethyl)cyclopropan-1 'β-yl]methylcytosine; 9-[1 'α, 2'β-bis(hydroxynnethyl)cyclopropan-1 'β-yl]methyl-2,6-diamino-purine; 9-[1 'α, 2'β-bis(hydroxynnethyl)cyclopropan-1 'β-yl]methyl-2-amino-purine; 9-[1 'α, 2'β-bis(hydroxynnethyl)cyclopropan-1 'β-yl]methyluracil; 9-[1 'α, 2'a-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyluracil; 9-[1 'α, 2'β-bis(hydroxymethyl)cyclopropan-1 'β-yl]nnethyl-5-fluorouracil; 9-[1 'α, 2'a-bis(hydroxymethyl)cyclopropan-1 'β-yl]methyl-5-fluorouracil; 9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]nnethylguanine; 9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]nnethyladenine;

9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]methyl-2-amino-6-chloro- purine;

9-[1 'α, 2'α-bis(acetoxymethyl)cyclopropan-1 'β-yl]nnethyl-2-annino-6-chloro- purine;

9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]nnethylhypoxanthine;

9-[1 'α, 2'a-bis(acetoxymethyl)cyclopropan-1 'β-yl]methylhypoxanthine;

9-[1 'α, 2'β-bis(acetoxymethyl)cyclopropan-1 'β-yl]nnethylthymine;

9-[1 'α, 2'a-bis(acetoxymethyl)cyclopropan-1 'β-yl]methylthymine; 9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]methylcytosine;

9-[1 'α, 2'a-bis(acetoxymethyl)cyclopropan-1 'β-yl]nnethylcytosine;

9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]methyl-2,6-diamino-purine;

9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]methyl-2-amino-purine;

9-[1 'α, 2'β-bis(acetoxymethyl)cyclopropan-1 'β-yl]methyluracil; 9-[1 'α, 2'α-bis(acetoxymethyl)cyclopropan-1 'β-yl]methyluracil;

9-[1 'α, 2'β-bis(acetoxynnethyl)cyclopropan-1 'β-yl]methyl-5-fluorouracil;

9-[1 'α, 2'a-bis(acetoxymethyl)cyclopropan-1 'β-yl]nnethyl-5-fluorouracil;

9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methylguanine;

9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]nnethyladenine; 9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-6-chloro- purine;

9-[1 'α, 2'α-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-6-chloro- purine; 9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]nnethylhypoxanthine; 9-[1 'α, 2'α-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methylhypoxanthine; 9-[1 'α, 2'β-bis(benzoyloxynnethyl)cyclopropan-1 'β-yl]methylthynnirιe; 9-[1 'α, 2'a-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]nnethylthynnine; 9-[1 'α, 2'β-bis(benzoyloxynnethyl)cyclopropan-1 'β-yl]nnethylcytosine; 9-[1 'α, 2'a-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]nnethylcytosine; 9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyl-2,6-diannino- purine;

9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyl-2-amino-purine; 9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyluracil; 9-[1 'α, 2'a-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyluracil;

9-[1 'α, 2'β-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]methyl-5-fluorouracil;

9-[1 'α, 2'a-bis(benzoyloxymethyl)cyclopropan-1 'β-yl]nnethyl-5-fluorouracil; 1 -[1 ' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1 ' beta-yl]methyl-5- bromo-2,4(1 H,3H)-pyrimidinedione;

1 -[1' alpha ,2' beta -bis^ydroxymethyOcyclopropan-i' beta -yl] methyl -5- bromo-2,4(1 H,3H)-pyrinnidinedione;

1 -[1 ' alpha ,2' alpha -bis^ydroxymethyOcyclopropan-i ' beta -yl]methyl-5- chloro-2,4(1 H,3H)-pyrinnidinedione;

1 -[1 ' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1 ' beta -yl]methyl-5-chloro-

2,4(1 H,3H)-pyrinnidinedione;

1 -[1 ' alpha ,2' alpha -bis(hydroxynnethyl)cyclopropan-1 ' beta -yl]methyl-5- fluoro-2,4(1 H,3H)-pyrimidinedione; 1 -[1 ' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1 ' beta -yl]methyl-5-fluoro-

2,4(1 H,3H)-pyrinnidinedione;

1 -[1 ' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1 ' beta -yl]methyl-5-iodo-

2,4(1 H,3H)-pyrimidinedione;

1 -[1 ' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-iodo- 2,4(1 H,3H)-pyrimidinedione;

1 -[1 ' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1 ' beta -yl]methyl-5- bromo-2,4(1 H,3H)-pyrinnidinedione;

1 -[1 ' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1 ' beta -yl]methyl-5- bromo-2,4(1H,3H)-pyrimidinedione;

1-[V alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- chloro-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-chloro- 2,4(1 H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- fluoro-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-fluoro- 2,4(1 H,3H)-pyrinnidinedione; 1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-iodo- 2,4(1 H,3H)-pyrinnidinedione; 1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-iodo-

2,4(1 H,3H)-pyrinnidinedione; 1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-ethyl-

2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis^ydroxymethyOcyclopropan-i¹ beta -yl]methyl-5-ethyl-

2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5- propyl-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-propyl-

2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-ethyl-

2,4(1 H,3H)-pyrimidinedione; 1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-ethyl-

2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- propyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-propyl- 2,4(1 H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5- trifluoromethyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5- trifluoromethyl-2,4(1H,3H)-pyrimidinedione;

1-[V alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- trifluoromethyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- trifluoromethyl-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- bromoethyl)-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- bromoethyl-2,4(1H,3H)-pyrinnidinedione; 1-[1' alpha ,2' alpha -bis(hydroxynnethyl)cyclopropan-1' beta -yl]methyl-5-(2- chloroethyl-2,4(1H,3H)-pyriπnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- chloroethyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- fluoroethyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- fluoroethyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(hydroxynnethyl)cyclopropan-1' beta -yl]methyl-5-(2- iodoethyl-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- iodoethyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- bromoethyl)-2,4(1H,3H)-pyrinnidinedione; 1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- bromoethyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- chloroehtyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- chloroethyl-2,4(1 H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- fluoroethyl)-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- fluoroethyl-2,4(1H,3H)-pyrimidinedione;

1-[V alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- iodoethyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(2- iodoethyl-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bisChydroxymethylJcyclopropan-i' beta -yl]methyl-5- ethenyl-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bisChydroxymethylJcyclopropan-i' beta -yl]methyl-5- ethenyl-2,4(1H,3H)-pyrimidinedione; 1-[1' alpha ,2' alpha -bis(hydroxynnethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-

1 -propenyl]-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-1- propenyl]-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-

ethenyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- ethenyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)- 1 -propenyl-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-1- propenyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-

2-bromoethenyl]-2,4(1H,3H)-pyrinnidinedioπe; 1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- bromoethenyl]-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-

2-chloroethenyl]-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- chloroethenyl]-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-

2-fluoroethenyl]-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- fluoroethenyl]-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-

2-iodoethenyl]-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- iodoethenyl]-2,4(1 H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(acethoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-

2-bromoethenyl]-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- bromoethenyl]-2,4(1H,3H)-pyrinnidinedione; 1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-

2-chloroethenyl]-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- chloroethenyl]-2,4(1H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)- 2-fluoroethenyl]-2,4(1 H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- fluoroethenyl]-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)- 2-iodoethenyl]-2,4(1 H,3H)-pyrimidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-[(E)-2- iodoethenyl]-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bisChydroxymethyOcyclopropan-i' beta -yl]methyl-5- ethynyl-2,4(1H,3H)-pyrimidinedione; 1-[1' alpha ,2' beta -bisChydroxymethyOcyclopropan-i' beta -yl]methyl-5- ethynyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' alpha -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(1- propynyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(hydroxymethyl)cyclopropan-1' beta -yl]methyl-5-(1- propynyl)-2,4(1 H,3H)-pyrimidinedione;

1-[1' alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- ethynyl-2,4(1H,3H)-pyrinnidinedione;

1-[1' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5- ethynyl-2,4(1 H,3H)-pyrimidinedione;

1 -[V alpha ,2' alpha -bis(acetoxymethyl)cyclopropan-1' beta -yl]methyl-5-(1 - propynyl)-2,4(1 H,3H)-pyrimidinedione;

1 -[1 ' alpha ,2' beta -bis(acetoxymethyl)cyclopropan-1 ' beta -yl]methyl-5-(1 - propynyl)-2,4(1 H,3H)-pyrinnidinedione;

9-[1 ' alpha ,2' alpha -Bis(O-L-alanyloxymethyl)cyclopropan-1' beta - yl]methylguanine;

9-[1 ' alpha , 2' alpha -Bis(O-L-2-aminobutyloyloxymethyl)cyclopropan-1 ' beta - yl]methylguanine; 9-[1' alpha , 2' alpha -Bis (O-L-valyloxymethyl )cyclopropan-1' beta - yl]methylguanine;

9-[1 ' alpha , 2' alpha -Bis^-L-Ieucyloxymethyljcyclopropan-I¹ beta - yl]methylguanine;

9-[1 ' alpha , 2' alpha -Bis^-L-isoleucyloxymethylJcyclopropan-i ¹ beta - yl]methylguanine;

9-[1 ' alpha , 2' alpha -Bis (O-L-alanyloxynnethyl)cyclopropan-i ' beta -yl]methyl-

2-aminopurine;

9-[1 ' alpha , 2' alpha -'Bis(O-L-2-aminobutyloyloxymethyl)cyclopropan-1 ' beta - yl]methyl-2-aminopurine;

9-[1 ' alpha ,2' alpha -Bis(O-L-valyloxymethyl)cyclopropan-1 ' beta -yl]methyl-2- aminopurine;

9-[1 ' alpha ,2' alpha -Bis^-L-Ieucyloxymethyljcyclopropan-I ¹ beta -yl]methyl-

2-aminopurine; 9-[1 ' alpha ,2' alpha -Bis^-L-isoleucylmethylJcyclopropan-i ¹ beta -yl]methyl-2- aminopurine;

9-[1 ' alpha ,2' alpha -Bis(O-L-alanyloxymethyl)cyclopropan-1 ' beta -yl]methyl-

2-amino-6-chloropurine;

9-[1 ' alpha ,2' alpha -Bis(O-L-2-aminobutyloyloxymethyl)cyclopropan-1 ' beta - yl]methyl-2-amino-6-chloropurine;

9-[1 ' alpha ,2' alpha -Bisp-L-valyloxymethyOcyclopropan-i ¹ beta -yl]methyl-2- amino-6-chloropurine;

9-[1 ' alpha ,2' alpha -Bis^-L-Ieucyloxymethyljcyclopropan-I ¹ beta -yl]methyl- 2-amino-6-chloropurine; and

9-[V alpha ,2' alpha -Bis^-L-isoleucylmethy^cyclopropan-i' beta -yl]methyl-2- amino-6-chloropurine.

The fish that can be treated by the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives of the present invention are preferably carps, more particularly may be selected from the common carp Cyprinus carpio carpio and its varieties such as mirror, leather, koi {Cyprinus carpio koi) and ghost carp.

Thus, a first aspect of the invention relates to the use of substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives as described in the different embodiments herein above, a prodrug or solvate thereof for the manufacture of a medicament or veterinary or pharmaceutical composition for the prevention, improvement and/or treatment of a KHV-infection in fish, more in particular carps, yet more in particular Koi. This aspect of the invention relates to the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives as described in the different embodiments herein above, for the treatment, improvement or

prevention of KHV-infections in fish. Another aspect of the present invention relates to a method of prevention, improvement or treatment of a KHV-infection in a fish, especially a carp, comprising administering to the fish in need of such treatment a therapeutically effective amount, i.e. a KHV inhibiting amount of a substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives as described in any one of the different embodiments herein above, a pro-drug or solvate thereof as an active ingredient, preferably in admixture with at least a veterinary acceptable carrier.

The present invention also relates to the use of substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives in a combination preparation with one or more other anti-KHV compounds. Therefore, the present invention also relates to veterinary compositions comprising a substituted or unsubstituted bis(hydroxymethyl)- cycloprop-1 '-ylmethyl nucleobase derivative as referred in any one of the above embodiments of the description herein, and another anti-KHV compound.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the experiment of example

3. All carps were inoculated by balneation (40 pfu/ml) with KHV FL LUC strain expressing luciferase at day 0 then treated with the drug A-5021 (50 mg/kg) or mock treated at indicated times (V). Carps were then analysed by in vivo imaging system (IVIS) at indicated times post-infection (®). Figure 2 shows cumulative survival rates of carps infected with the

KHV FL LUC strain. On day 0, two groups, each consisting of 6 koi carps, were inoculated by balneation with KHV FL LUC strain (40 pfu/ml). The first group (KHVLUC) was used as a postive control of infection. The second group (KHVLUC-A-5021 ) was infected and then treated with the drug A-5021 (50 mg/kg) at different time post-infection as described in figure 1. Percentages of surviving carps are expressed according to days post-infection.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term " pyhmidine and purine bases " includes, but is not limited to, adenine, thymine, cytosine, uracyl, guanine and 2,6- diaminopurine and analogues thereof. A purine or pyrimidine base as used herein includes a purine or pyrimidine base found in naturally occurring nucleosides as mentioned above. An analogue thereof is a base which mimics such naturally occurring bases in such a way that their structures (the kinds of atoms and their arrangement) are similar to the naturally occurring bases but may either possess additional or lack certain of the functional properties of the naturally occurring bases. Such analogues include those derived by replacement of a CH moiety by a nitrogen atom (e.g. 5-azapyhmidines such as 5-azacytosine) or vice versa (e.g., 7-deazapurines, such as 7- deazaadenine or 7-deazaguanine) or both (e.g., 7-deaza, 8-azapuhnes). By derivatives of such bases or analogues are meant those bases wherein ring substituents are either incorporated, removed, or modified by conventional substituents known in the art, e.g. halogen, hydroxyl, amino, (d-CβJalkyl and others. Such purine or pyrimidine bases, and analogues thereof, are well known to those skilled in the art, e.g. as shown at pages 20-38 of WO 03/093290 . In yet another particular embodiment of the present invention, B may be selected from the group comprising pyrimidine bases represented by the structural formula (B):

and purine bases represented by the structural formula (D):

wherein:

- R⁷ and R⁹ are independently selected from the group consisting of H, -OH, -SH, -NH₂, and -NH-CH₃; - R⁸ and R¹⁰ are independently selected from the group consisting of H, methyl, ethyl, isopropyl, hydroxyl, amino, ethylamino, trifluoromethyl, cyano and halogen; and

- X and Y are independently selected from CH and N.

Just as a few non-limiting examples of pyrimidine analogues, can be named substituted uracils with the formula (B) wherein X is CH, R⁷ is hydroxyl, and R⁸ is methyl, ethyl, isopropyl, amino, ethylamino, trifluoromethyl, cyano, fluoro, chloro, bromo and iodo.

It was previously shown that certain substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives have an anti- human herpes virus effect. The in vitro anti-human herpes virus activity of these compounds is similar to the anti-herpes virus activity of commercial molecules such as acyclovir, ganciclovir, penciclovir, cidofovir and brivudin. It was already known and we further show that selective anti-herpes virus molecules, such as acyclovir, HPMPC (cidofovir), BVDU (brivudin) and ganciclovir (GCV) have no or a very limited effect on KHV replication. The 50% effective concentration for inhibition of KHV replication of these molecules is 100 to 100.000 fold higher (thus less effective) than their IC50 values for inhibition of the replication of other herpes viruses (e.g. human herpes simplex virus). However, in the present invention, we show that surprisingly these substituted or unsubstituted bis(hydroxymethyl)-cycloprop- 1 '-ylmethyl nucleobase derivatives are much more efficient in inhibiting KHV replication than antiviral drugs such as acyclovir, ganciclovir, cidofovir and brivudin, i.e. other known anti-human herpes virus compounds. In fact the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives of the present invention are presently the most potent anti-KHV compounds among known antiviral compounds. More particularly, A- 5021 has been found to be highly active against KHV.

Looking at the in vitro use of the anti-Koi Herpes virus compounds, such compounds are very useful for the decrease or inhibition of KHV- infections in cells in vitro, for example in cell culture.

The compounds of the invention with a high potency against KHV infections have a chemical structure in accordance with any one of the formulae described herein, such as formulae I, II, III, IV, V or Vl, including isomers, solvates, pharmaceutically acceptable salts or prodrugs thereof. The present invention provides for the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives as described herein for use as a veterinary medicine for the prevention or treatment of KHV-infections in fish, more in particular in carps. The invention thus provides for the use of substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '- ylmethyl nucleobase derivatives as described herein for the manufacture of a medicament for the prevention or treatment of KHV-infections in fish, more in particular in carps.

The present invention furthermore provides for a KHV-inhibiting veterinary composition comprising a therapeutically effective amount of a substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivative as described herein optionally in combination with one or more veterinary acceptable carriers. The present invention provides furthermore such a veterinary composition for the prevention or treatment of a KHV-infection in fish, especially in carps.

It will also be appreciated that the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives and pharmaceutically or veterinary acceptable compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutically acceptable compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '- ylmethyl nucleobase derivatives may be administered concurrently with another agent used to treat the same disorder), or they may achieve different

effects (e.g., control of any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated".

As an example, the substituted or unsubstituted bis(hydroxymethyl)- cycloprop-1 '-ylmethyl nucleobase derivatives may be combined with antiviral compounds acting on other viral or non-viral targets.

The amount of additional therapeutic agent present in the compositions of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

The substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives are employed for the treatment or prophylaxis of KHV- infections in fish. When using substituted or unsubstituted bis(hydroxymethyl)- cycloprop-1 '-ylmethyl nucleobase derivatives:

- the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '- ylmethyl nucleobase derivative may be administered to the fish to be treated by any means well known in the art, i.e. orally, intraperitonealy, subcutaneously, intramuscularly, intradermally, intravenously, intra- arterially, parenterally or by catheterization; the preferred route of administration for the infected fish is the intramuscular, intraperitoneal or subcutaneous route whereby a sterile solution of the compound of the invention are injected; and/or - the therapeutically effective amount of the preparation of the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivative in fish is a KHV-inhibiting amount. This can be achieved by administration of the required dosage to obtain predetermined plasma levels. Suitable dosage ranges are from about 15 to about 300 mg/kg, or from about 40 to about 250 mg/kg, or from about 10 to about 50 mg/kg. The said effective amount may be divided

into several sub-units per day or may be administered daily or at more than one day intervals, e.g. over a treatment period of 1 day to about 5 days or about 30 days.

The substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '- ylmethyl nucleobase derivatives, prodrugs or solvates of the present invention as described herein also comprise their pharmaceutically or veterinary acceptable salts, tautomers or isomers of the compounds of formula I as described hereunder.

The term "pharmaceutically acceptable salts" or "veterinary acceptable salts" as used herein means the therapeutically active non-toxic acid addition salt forms which the compounds of formula (I) are able to form and which may conveniently be obtained by treating the base form of such compounds with an appropriate acid. Examples of such appropriate acids include, for instance, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, 2-hydroxypropanoic, 2-oxopropanoic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic (i.e. 2-hydroxybenzoic), p-aminosalicylic and the like. This term also includes the solvates which the compounds of formula (I) as well as their salts are able to form, such as for example hydrates, alcoholates and the like. Also included within the scope of this invention are the salts of the parental compounds with one or more amino acids, especially the naturally-occurring amino acids found as protein components. The amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine.

Further examples of veterinary acceptable salts of the compounds of the invention include salts derived from an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth (for example, magnesium), ammonium and NX⁴⁺ (wherein X is CrC₄ alkyl). Physiologically acceptable salts of an hydrogen atom or an amino group include salts of organic

carboxylic acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic and p- toluenesulfonic acids; and inorganic acids, such as hydrochloric, sulfuric, phosphoric and sulfamic acids. Physiologically acceptable salts of a compound containing a hydroxy group include the anion of said compound in combination with a suitable cation such as Na⁺ and NX⁴⁺ (wherein X typically is independently selected from H or a Ci-C₄ alkyl group). However, salts of acids or bases which are not physiologically acceptable may also find use, for example, in the preparation or purification of a physiologically acceptable compound. All salts, whether or not derived form a physiologically acceptable acid or base, are within the scope of the present invention.

The term " isomers " as used herein means all possible isomeric forms, including tautomeric forms, which the compounds of formula (I) may possess. Unless otherwise stated, the chemical designation of compounds denotes the mixture of all possible stereochemical^ isomeric forms, said mixtures containing all diastereomers and enantiomers (since the compounds of formula (I) may have at least one chiral center) of the basic molecular structure. More particularly, stereogenic centers may have either the R- or S- configuration, and substituents may have either cis- or trans-configuration.

Pure isomeric forms of the said compounds are defined as isomers substantially free of other enantiomeric or diastereomehc forms of the same basic molecular structure. In particular, the term "stereoisomerically pure" or "chirally pure" relates to compounds having a stereoisomeric excess of at least about 80% (i.e. at least 90% of one isomer and at most 10% of the other possible isomers), preferably at least 90%, more preferably at least 94% and most preferably at least 97%. The terms "enantiomerically pure" and "diastereomehcally pure" should be understood in a similar way, having regard to the enantiomeric excess, respectively the diastereomeric excess, of the mixture in question.

Consequently, if a mixture of enantiomers is obtained during any of the following preparation methods, it can be separated by liquid chromatography using a suitable chiral stationary phase. Suitable chiral stationary phases are, for example, polysaccharides, in particular cellulose or amylose derivatives. Commercially available polysaccharide based chiral stationary phases are

ChiralCel™ CA, OA, OB, OC, OD, OF, OG, OJ and OK, and Chiralpak™ AD, AS, OP(+) and OT(+). Appropriate eluents or mobile phases for use in combination with said polysaccharide chiral stationary phases are hexane and the like, modified with an alcohol such as ethanol, isopropanol and the like. The terms cis and trans are used herein in accordance with Chemical

Abstracts nomenclature and refer to the position of the substituents on a ring moiety. The absolute stereochemical configuration of the compounds of formula (I) may easily be determined by those skilled in the art while using well-known methods such as, for example, X-ray diffraction.

Those of skill in the art will also recognize that the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivatives may exist in many different protonation states, depending on, among other things, the pH of their environment. While the structural formulae provided herein depict the compounds in only one of several possible protonation states, it will be understood that these structures are illustrative only, and that the invention is not limited to any particular protonation state, any and all protonated forms of the compounds are intended to fall within the scope of the invention.

The compounds of the invention may be formulated with conventional carriers and excipients, which will be selected in accordance with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. Formulations optionally contain excipients such as those set forth in the "Handbook of Pharmaceutical Excipients" (1986) and include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.

Subsequently, the term "pharmaceutically acceptable carrier" or "veterinary acceptable carrier" as used herein means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, suspensions, ointments, creams, tablets, pellets or powders. Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents may also be prepared by inicronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 gm, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients. Suitable surface-active agents, also known as emulgent or emulsifier, to be used in the pharmaceutical compositions of the present invention are non-ionic, cationic and/or anionic materials having good emulsifying, dispersing and/or wetting properties. Suitable anionic surfactants include both water-soluble soaps and water-soluble synthetic surface-active agents. Suitable soaps are alkaline or alkaline-earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures obtainable form coconut oil or tallow oil. Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulphonates and sulphates; sulphonated benzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates or sulphates are usually in the form of alkaline or alkaline-earth metal salts, unsubstituted ammonium salts or ammonium salts substituted with an alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium or calcium salt of lignosulphonic acid or dodecylsulphonic acid or a mixture of fatty alcohol sulphates obtained from natural fatty acids, alkaline or alkaline- earth metal salts of sulphuric or sulphonic acid esters (such as sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are the sodium, calcium or alcanolamine salts of dodecylbenzene sulphonic acid or dibutyl- naphtalenesulphonic acid or a naphtalene-sulphonic acid/forrnaldehyde condensation product. Also suitable are the corresponding phosphates, e.g. salts of phosphoric acid ester and an adduct of p-nonylphenol with ethylene and/or propylene oxide, or phospholipids. Suitable phospholipids for this purpose are the natural (originating from animal or plant cells) or synthetic phospholipids of the cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycehne, lysolecithin, cardiolipin, dioctanylphosphatidyl-choline, dipalmitoylphoshatidyl - choline and their mixtures.

Suitable non-ionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides containing at least 12 carbon atoms in the molecule, alkylarenesulphonates and dialkylsulphosuccinat.es, such as polyglycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives preferably containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Further suitable non-ionic surfactants are water-soluble adducts of polyethylene oxide with poylypropylene glycol, ethylenediaminopolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ether groups. Such compounds usually contain from 1 to 5 ethyleneglycol units per propyleneglycol unit.

Representative examples of non-ionic surfactants are nonylphenol - polyethoxyethanol, castor oil polyglycolic ethers, polypropylene/polyethylene oxide adducts, thbutylphenoxypolyethoxyethanol, polyethyleneglycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable non-ionic surfactants.

Suitable cationic surfactants include quaternary ammonium salts, particularly halides, having 4 hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy; for instance quaternary ammonium salts containing as N-substituent at least one C8C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl and the like) and, as further substituents, unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy- lower alkyl radicals.

A more detailed description of surface-active agents suitable for this purpose may be found for instance in "McCutcheon's Detergents and Emulsifiers Annual" (MC Publishing Crop., Ridgewood, New Jersey, 1981 ), "Tensid-Taschenbucw¹, 2 d ed. (Hanser Verlag, Vienna, 1981 ) and "Encyclopaedia of Surfactants, (Chemical Publishing Co., New York, 1981 ).

While it is possible for the active ingredients to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations for veterinary use of the present invention comprise at least one active ingredient, as above described, together with one or more veterinary acceptable carriers therefore and optionally other therapeutic ingredients. The carrier(s) optimally are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations include those suitable for oral or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of veterinary pharmacy.

Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and

intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

The formulations are optionally applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1 % and 20% in increments of 0.1 % w/w such as 0.6% w/w, 0.7% w/w, etc), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Optionally, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus the cream should optionally be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the veterinary art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Compounds of the invention can be used to provide controlled release pharmaceutical formulations containing as active ingredient one or more compounds of the invention ("controlled release formulations") in which the release of the active ingredient can be controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given invention compound. Controlled release formulations adapted for oral administration in which discrete units comprising one or more compounds of the invention can be prepared according to conventional methods. Additional ingredients may be included in order to control the duration of action of the active ingredient in the composition. Control release compositions may thus be achieved by selecting appropriate polymer carriers such as for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like. The rate of drug release and duration of action may also be controlled by incorporating the active ingredient into particles, e.g. microcapsules, of a polymeric substance such as hydrogels, polylactic acid, hydroxy- methylcellulose, polyniethyl methacrylate and the other above-described polymers. Such methods include colloid drug delivery systems like liposomes, microspheres, microemulsions, nanoparticles, nanocapsules and so on. Depending on the route of administration, the veterinary composition may require protective coatings. Pharmaceutical forms suitable for injection use in fish include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation thereof. Typical carriers for this purpose therefore include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol and the like and mixtures thereof in suitable proportions. According to a particular embodiment, the substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylmethyl nucleobase derivative of the present invention may be administered to fish, e.g. carps, as an injectable sterile solution comprising a suitable liquid medium (e.g. DMEM), whereby the said derivative may be present at a concentration ranging from about 10 mg/ml to about 80 mg/ml.

When several active ingredients are used in combination, they do not necessarily bring out their joint therapeutic effect directly at the same time in the fish to be treated, the corresponding composition may also be in the form of a medical kit or package containing the two ingredients in separate but

adjacent repositories or compartments. In the latter context, each active ingredient may therefore be formulated in a way suitable for an administration route different from that of the other ingredient, e.g. one of them may be in the form of an oral or parenteral formulation whereas the other is in the form of an ampoule for intravenous injection.

Another embodiment of this invention relates to various precursor or "pro-drug" forms of the compounds of the present invention. It may be desirable to formulate the compounds of the present invention in the form of a chemical species which itself is not significantly biologically-active, but which when delivered to the fish will undergo a chemical reaction catalysed by the normal function of the body of the fish, inter alia, enzymes present in the stomach or in blood serum, said chemical reaction having the effect of releasing a compound as defined herein. The term "pro-drug" thus relates to these species which are converted in vivo into the active pharmaceutical ingredient.

The pro-drugs of the present invention can have any form suitable to the formulator, for example, esters are non-limiting common pro-drug forms. In the present case, however, the pro-drug may necessarily exist in a form wherein a covalent bond is cleaved by the action of an enzyme present at the target locus. For example, a C-C covalent bond may be selectively cleaved by one or more enzymes at said target locus and, therefore, a pro-drug in a form other than an easily hydrolysable precursor, inter alia an ester, an amide, and the like, may be used. The counterpart of the active pharmaceutical ingredient in the pro-drug can have different structures such as an amino acid or peptide structure, alkyl chains, sugar moieties and others as known in the art.

For the purposes of the present invention the term "therapeutically suitable pro-drug" is defined herein as "a compound modified in such a way as to be transformed in vivo to the therapeutically active form, whether by way of a single or by multiple biological transformations, when in contact with the tissues of the fish to which the pro-drug has been administered, and without undue toxicity, irritation, or allergic response, and achieving the intended therapeutic outcome ".

KHV inhibitory activity can be readily detected by using one or more of the assays described herein, as well as assays generally known to those of ordinary skill in the art. In general, carp cells are infected with KHV and are incubated with or without the compounds to be tested for their activity.

The following examples are provided for the purpose of illustrating the present invention and by no means are meant and in no way should be interpreted to limit the scope of the present invention.

EXAMPLE 1 - INHIBITION OF KOI HERPES VIRUS INFECTIONS IN CELL

CULTURE

Materials and Methods The compounds were dissolved in DMSO (Merck) at a concentration of

20 mg/ml, then filtrated through a 0.45 μm filter to obtain a sterile solution. Common carp brain (CCB) cells were grown in minimum essential medium (MEM, invitrogen) containing 4.5 g/l glucose (D-glucose monohydrate, Merck) and 10% fetal calf serum (FCS, BioWhittaker). Cells were incubated at 25°C in a humid atmosphere containing 5% CO₂. Confluent cell monolayers grown in 24 wells cluster dishes were inoculated with 200 plaque forming units (PFU) of KHV in 0.2 ml of serum free Dulbecco's modified eagle medium (DMEM, Invitrogen). After an incubation period of 2 hours (h), cells were overlaid with DMEM (4.5 g/l glucose, 10% FCS) containing increasing doses of the compounds (3; 6.5; 12.5; 25; 50; and 100 μg/ml). Infected cells treated with DMSO without compounds (ND = no drugs) were included as negative controls. Five days post infection (pi), viral plaques or virus induced cytopathic effects were observed by staining cells with a crystal violet (Merck) solution.

Results

The results are summarized in Table 1 showing IC50 values for the compounds tested. The compound of the present invention, herein referred as example A-5021 , shows excellent activity against KHV replication, i.e. at a concentration as low as 3 μg/ml viral replication is inhibited by about 99%. None of other reference anti-herpes virus compounds was, in contrast to expectations, able to efficiently inhibit KHV replication in vitro.

Table 1

EXAMPLE 2 - IN VIVO KOI CARPS TOXICITY EXPERIMENT WITH A-5021

Naϊve adult koi carps were kept in 60-liter tanks with filtrated water maintained at 23°C. Four groups (5 koi per group) were treated for 10 consecutive days, by intramuscular injection of A-5021 (20 mg/kg and 50 mg/kg) dissolved in 0.1 ml of propyleneglycol (33,3%)/ethanol(8,3%) (PE buffer). Two other groups (5 koi per group), used as negative controls, were injected with 0.1 ml of DMEM or PE buffer. After 10 days, all fish were examined and weighed. Three fishes of each group were euthanized. Liver and kidney explants were treated for anatomopathology analysis.

This experiment did not reveal any toxic effect of A-5021 (see table 2). None of the fishes injected with A-5021 died nor exhibited clinical signs. The mean weight of fishes infected with drugs or controls were statistically similar.

Table 2

Mean initial Mean final Survival Microscopic

Fish Clinical signs weight (g) weight (g) (%) lesions

DMEM 20.9 20.2 100 None None

PE 17.8 17.11 100 None None

A-5021 (20) 20.9 19.4 100 None None

A-5021 (50) 23.6 23.2 100 None None EXAMPLE 3: EXPERIMENT WITH KHV FL LUC RECOMBINANT EXPRESSING THE FIREFLY LUCIFERASE IN VIVO KOI CARPS Materials and Methods

Recombinant virus. A KHV FL LUC recombinant expressing the firefly luciferase was produced as follows. Firstly, a KHV FL BAC plasmid carrying a galactokinase gene {galK) into the intergenic region between KHV ORF136 and ORF137 (GenBank accession no. DQ177346) was produced using a KHV FL BAC plasmid (Costes et al., 2008) and a galK positive selection of bacteria as previously described (Warming et al., 2005). Secondly, the ga/K gene was replaced by the LUC cassette encoding the firefly luciferase gene under control of the human cytomegalovirus immediate-early promoter (kindly provided by Dr Bremont, France) leading to the KHV FL BAC LUC plasmid. Thirdly, to reconstitute infectious particles expressing the LUC protein but deleted for the BAC cassette, the KHV FL BAC LUC plasmid was co- transfected into CCB cells with the pGEMT-TK vector previously produced (Costes et al., 2008) leading to the KHV FL LUC strain used in this study. Antiviral compound. The antiviral compound used in this study is the guanosine analog (19S,29R)-9{[19,29-bis(hydroxymethyl) cycloprop-19- yl]methyl}guanine (A-5021 ). The compound was dissolved in propylene glycol (33.3%) / ethanol (8.3%) (PE buffer) at a concentration of 10 mg/ml. Subsequently, compound was filtrated through a 0.45 μm filter to obtain a sterile solution.

Infection and treatment of fish. Specific-pathogen-free koi carps, with an average weight of 28 g, were kept in 60-liter tanks with filtrated water maintained at 23°C. Two groups (6 koi per group) were infected by balneation with 40pfu/ml of KHV FL LUC strain. One of the group was then treated at different time post-infection (0, 2, 4, 6, 8, 10, 14 and 18 days pi) by intramuscular injection of A-5021 (50 mg/kg). The second group was used as positive control of infection and was injected with buffer. Fishes were examined daily for clinical signs of KHV disease, and dead fishes were removed. Fishes were analysis by in vivo imaging system (IVIS) at different time post-infection (1 , 2, 3, 4, 6, 8, 10, 14, 18, 30 days pi). The animal study was accredited by the local ethics committee of the University of Liege (Belgium).

Bioluminescence imaging.

Imaging of firefly luciferase in fish was performed on a Xenogen in vivo imaging system (IVIS) which consists of a cooled charge-coupled device camera (Xenogen). Briefly, 150 mg/kg of D-luciferin (Xenogen) was administrated to fish by intraperitoneal injection. Fish were then anesthetized with benzocaϊne, and imaging began 10 min after administration of D-luciferin. Images were acquired using 1 min exposure, a binning factor of 4 and a f/stop of 1. Images were acquired for the left and the right side of each fish. Relative intensities of transmitted light from in vivo bioluminescence were represented as a pseudocolor image ranging from violet (least intense) to red (most intense). Corresponding gray-scale photographs and color luciferase images were superimposed with Livinglmage analysis software (Xenogen).

Figure 2 and table 3 show the results of this experiment as the cumulative survival rates of carps infected with the KHV FL LUC strain.

Table 3

EXAMPLE 4

This example aims at evaluating the safety of a high dosage formulation containing (1 'S,2'f?)-9-[[1 ',2'-bis(hydroxymethyl)cycloprop-1 '-yl]methyl]guanine (A-5021 ) following administration to healthy koi carp.

An initial pre-trial period (D-35) allowed acclimation of trial animals and adaptation to their new diet and surroundings and allowed stabilisation of water parameters in each tank. The 8-day pre-trial period (D-8) started after having demonstrated that water parameters had been stabilised for five consecutive days.

Detailed individual clinical examinations and observations were started as of D-8 and were conducted twice daily until DO which was the start of the experiment. These data provided pre-treatment base-line data. As of DO, detailed individual clinical examinations and observations were conducted three times daily until the end of the experiment (D21 ). The treatment schedule is as indicated in Table 4.

Table 4

Specifications of the animals

Species Fishes

Breed Koi carp (Cypήnus Carpio koi) (specific pathogen

Number 30

Sex male and female

Physiological state Healthy animals

Age 1 year

Bodyweight approximately 10 grams

Origin Hazorea Aquatics, Kibbutz Hazorea 36581 , lsrae

To be eligible for participation in the study, the animals had to come from a batch of specific pathogen free animals that had undergone the following four- step testing procedure:

• Clinical examination performed at the time of importation from Israel and during at least 4 months before entering the experiment;

• Anatomo-pathological examination performed on 1 % of the animals;

• Parasitic examination performed on 1 % of the animals; and

• Sensitivity check to koi herpes virus (KHV) infection.

Moreover, the selected animals from this batch were in good health on day D- 35 based on physical examination.

Exclusion criteria

The animals were ineligible for participation in the study if they:

• Did not adhere to the specification hereinbefore,

• Had been treated with similar compounds within 30 days prior to the onset of investigational treatment in this study, or

• Exhibited pre-study complicating disease conditions that may interfere with or prevent the evaluations and analyses in this study. Acclimation of test animal

Animals included were acclimated to the test facility for 35 days where feed, water and ambient conditions were identical to those expected during the study. Feed was purchased from Aquatic Science food. Water was maintained at a temperature of 21 -24°C. Ambient conditions were maintained at a temperature of 20-22⁰C. Artificial daylight was provided from 8 am to 5 pm. No routine prophylactic treatments were given prior to treatment or during the study, apart from the treatments foreseen in this study. No treatment similar to the test formulations was administered.

Housing and general maintenance

The animals were kept in a single facility.

Concurrent medication No concurrent medication was given during the experimental phase of the study.

Removal of subjects from the study

Three animals of group 1 (22, 74, 130) and three animals of group 2 (10, 34, 108) were selected at random for anatomopathological examination on D21. Of these six animals liver, kidney, muscle, heart, brain and gut were sampled for detailed anatomopathological analysis.

Test product The test product contains 250 mg/kg of A-5021 in a suitable formulation.

Dose and treatment frequency

Group 1 (safety/toxicity group) received 250 mg/kg of A-5021. The treatment was administered on D1 , D7, D13 and D18. On day D21 , the animals were euthanized.

Group 2 (control group) was left untreated. On day D21 , the animals were euthanized.

Pre-trial acclimation period The animals were photographed at the beginning of the period for identification and allocation to the different treatment groups. Each animal was examined by the Study Director or his delegate for pathological signs. During the pre-trial acclimation period, the animals were observed twice daily.

Allocation to treatment groups Random allocation was ensured via the "draw from the hat" technique. Allocation was such that the groups had a similar group arithmetic mean bodyweight and a similar range of bodyweights within the group. The animals were re-weighed on D-4 to determine the arithmetic mean bodyweight for treatment.

Parameters

Clinical examinations of all study animals were conducted twice daily from day D-8 until day D-1 and three times daily from day DO until the end of the study on day D21 for any signs of ill health. Local and general examination (Table 6) was realised at approximately the same hour each day.

Table 5 - Listing of parameters for clinical examination

Each animal was observed for signs of adverse drug events (ADE). The Study Director documented and described the reaction, categorised the severity and theorised on the relationship of the ADE to the drug article.

Post-mortem examination At the end of the study, all animals were euthanized but an anatomopathological examination was performed only on three animals of group 1 (22, 74, 130) and three animals of group 2 (10, 34, 108). Samples were transported to the testing laboratory by suitable means using appropriate shipping containers to maintain required storage conditions until arrival at the laboratory. The tissue samples were subjected to conventional histopathological evaluation. Body weights

On D-35, the mean bodyweight was 9.97 ± 1.14 g and 9.99 ± 1.10 g for groups 1 and 2 respectively. The mean bodyweight was 9.89 ± 1.00 g in group 1 and 9.72 ± 1.25 g in group 2 on D-4. Following an analysis of variance, it appeared that both groups did not differ significantly in bodyweight (p>0.05).

Clinical examinations

Local and general examination was realised at approximately the same hour each day. No abnormal signs were detected during the clinical examination in the two groups.

Adverse drug events (ADE)

No ADE was detected in groups 1 or 2.

Post-mortem examination

Six randomly selected animals (three of group 1 and three of group 2) were euthanized and submitted to a post-mortem examination on D21 (i.e. anatomopathological examination of the liver, kidney, muscle, heart, brain and gut). No significant differences were observed between both groups following histological examination of three animals per group.

All remaining animals were euthanized and removed from the study without performing a post-mortem examination.

As a conclusion, multiple administrations of 250 mg/kg of A-5021 to healthy koi carp is safe.

1. A substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '-ylnnethyl nucleobase derivative, an isomer, a solvate, or a veterinary acceptable salt thereof, for the prevention or treatment of Koi Herpes virus infections in fish.

2. A nucleobase derivative according to claim 1 , being represented by the formula :

(I) wherein,

- B is an optionally substituted pyrimidin-1-yl, pyrimidin-3-yl, purin-3-yl, puhn-7-yl or puhn-9-yl residue, or an aza, deaza, deoxy or deamino analogue thereof or a salt thereof with alkali metals, ammonia or amines; and

- each of R¹ and R² is independently hydrogen or a hydroxyl protecting group.

3. A nucleobase derivative according to claim 2, wherein B is a guanine.

4. A nucleobase derivative according to claim 2, wherein each of R¹ and R² is hydrogen.

5. A nucleobase derivative according to any of claims 1 to 4, wherein the carbon atoms 1 ' and 2' of the cyclopropyl group are in the 1 'S, 2'R conformation.

6. A nucleobase derivative according to claim 1 , being (1¹S, 2'R)-9-{[1 \ 2'-

Claims

43CLAIMS

(I) wherein,

- each of R¹ and R² is independently hydrogen or a hydroxyl protecting group.

3. A nucleobase derivative according to claim 2, wherein B is a guanine.

6. A nucleobase derivative according to claim 1 , being (1¹S, 2'R)-9-{[1 \ 2'- 44

bis(hydroxymethyl) cycloprop-1 '-yl]methyl}guanine.

7. A nucleobase derivative according to claim 1 , wherein the fish is a carp.

8. A nucleobase derivative according to claim 7, wherein the carp is a Cyprinus carpio koi.

9. A nucleobase derivative according to any one of claims 1 to 8, being administered via an intramuscular, intraperitoneal or subcutaneous route.

10. A nucleobase derivative according to any one of claims 1 to 9, in the form of a sterile injectable solution in one or more veterinary acceptable carriers.

11. A method for the prevention or treatment of KHV infections in fish, comprising the step of administering to said fish a therapeutically effective amount of a substituted or unsubstituted bis(hydroxymethyl)-cycloprop-1 '- ylmethyl nucleobase derivative, an isomer, a solvate or a veterinary acceptable salt thereof.

12. A method according to claim 11 , wherein said amount ranges from 15 to 300 mg per kg bodyweight fish per day.

13. A method according to claim 11 or claim 12, wherein said administration is continued for a period of time of from 1 to 30 days.