WO1996033201A1 - Acyclovir derivatives as antiviral agents - Google Patents

Abstract

Phosphotriester derivatives of acyclovir having potent antiviral inhibitory activity are provided. Compounds of the invention bear bioreversible protecting groups that can be cleaved intracellularly.

Description

ACYCLOVIR DERIVATIVES AS ANTIVIRAL AGENTS

CROSS REFERENCE TO PRIOR APPLICATIONS

This application is related to and claims priority of French patent application Serial Number 95-04797, filed April 21, 1995. This application is also related to United

States application Serial No. 465,450 filed June 5, 1995. Each of the foregoing is incorporated herein by reference.

BACKGROUND OF THE INVENTION

This application is directed to phosphotriester derivatives of the drug molecule known as acyclovir (ACV) , and their use as antiviral agents.

In a prior patent application, PCT/FR93/00498, which published as WO 93/24510, a very general approach that allows for intracellular delivery of ononucleotides using nucleosidic phosphotriesters bearing two bioreversible protecting groups was described.

It has now been found that this approach can be used for those nucleoside analogs which, in order to exert their biological activity, need to be phosphorylated to the corresponding triphosphates. Intracellular metabolism, such as phosphorylation, of nucleosides to active nucleotides reguires the successive action of three kinases, the first one being highly selective and strongly regulated. As a consequence, if a nucleosidic analog is not phosphorylated by the first kinase, it cannot exert its inhibitory activity.

Some viruses, such as herpes simplex virus (HSV) , encode for a viral kinase which is capable of phosphorylating some nucleoside analogs that are not phosphorylated by cellular kinases. In particular, some acyclonucleosides which are not substrates for the first cellular kinase mentioned above do not show any activity against viruses which do not provide their own kinase. This is the case for the known antiviral drug, acyclovir (ACV) , which has anti-herpetic activity due to its selective monophosphorylation by thy idine kinase of herpes simplex virus. However, acyclovir has no activity against many other viruses, such as human immunodeficiency virus (HIV) or hepatitis B virus (HBV) , which do not provide the necessary viral kinases. Moreover, ACV is poorly active against those herpetic viruses in which thymidine kinase activity is either decreased or absent, such as Epstein-Barr virus (EBV) or Cyto egalovirus (CMV) . Further, it has been well established that acyclovir-resistant herpes simplex viruses can appear both in vitro and in vivo, especially in highly immunodepressed patients. This resistance usually arises from a modification of the viral thymidine kinase, which results in a decrease or disappearance of the phosphorylation of ACV.

The intracellular delivery of acyclovir monophosphate (ACVMP) from various kinds of bioreversible phosphotriesters, as described in the patent application PCT/FR93/00498, is thus capable of enlarging the antiviral activity spectrum of this molecule.

Compared to ACV, phosphotriesters of ACV can be considered to be new therapeutic species since their spectrum of activity is much broader than that of ACV.

It is, therefore, an object of this invention to provide phosphotriester derivatives of acyclovir together with antiviral pharmaceutical formulations including such compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an exemplary synthetic scheme for certain preferred compounds of the invention.

Figure 2 is a graph showing the effect of acyclovir on vaginal lesion scores in mice infected with HSV-2. Intraperitoneal treatments were given twice daily for 5 days starting 24 hours after virus challenge. Figure 3 is a graph showing the effect of bis(SPTE)ACVMP on vaginal lesion scores in mice infected with HSV-2. Intraperitoneal treatments were given twice daily for 5 days starting 24 hours after virus challenge.

SUMMARY OF THE INVENTION

Phosphotriester compounds are provided corresponding to the general formula I:

(RO)₂-P(=0)-0-ACV I Wherein:

R is (CH₂)_n-S-X;

X is C(=Z)-Y or S-U;

Z is O or S;

Y and U, independently, are alkyl, aryl or a sugar moiety, optionally substituted with OH, SH or NH; n is 1 to 4, preferably 1 or 2; and

ACV is an acyclovir moiety having the formula:

In accordance with the present invention, Y and U are preferably C to C-, alkyl, phenyl, benzyl, glucose, mannose, rhamnose, or cyclofuranose. In one embodiment, when X represents S-U, U preferably represents the radical (CH_{J I}-X¹ where X¹ represents H, OH, SH or NH₂and n¹ is equal to 1 to 4, preferably 1 or 2.

It is preferred for some embodiments that compound (I) be such that R represents (CH₂)₂-S-S-(CH₂)₂-OH. In another preferred embodiment, X is C(=Z)-Y, and Y is CH₃ or (CH₃)₃C.

In other preferred embodiments, R represents (CH₂)_B-S- C(«0)-CH₃or (CH₂)_B-S-C(=0)-tBu, with n- l or 2.

Also preferred are compounds of formula la:

la

wherein R is alkyl, especially CH₃ or (CH₃)₃C.

In general, the compounds of the present invention are prepared by processes known to those skilled in the art. Such processes are described in patent application PCT/FR93/00498, published as WO 93/24510. In preparing compounds of the invention, functional groups such as R and, optionally, ACV, can be protected by suitable protecting groups, followed by deprotection of said functional groups to give compounds of formula I. For example, a compound of formula II:

O—P—0— - iACV

I

0^"

II in which ACV, which can be derivatized or protected, is reacted with a compound of formula III, X-S-(CH₂)_n-0H, wherein X represents the radical C(=Z)-Y or S-U, wherein Z is O or S and

Y and U are alkyl, aryl or saccharide moieties that may be optionally substituted, in particular with an OH, SH or NH₂ group, and suitably protected, in order to obtain the compound of formula I in a protected form that can then be deprotected.

In another embodiment, the ACV moiety of compound II

< is reacted with a phosphitylating reagent, then subjected to an oxidation reaction, and finally deprotected, in order to obtain a compound of formula I. An exemplary process for the preparation of the compounds of the present invention is illustrated in the following detailed description, in which other characteristics and advantages of the present invention are also discussed.

The compounds of the present invention can be included in antiviral pharmaceutical compositions that include a phosphotriester compound of the invention as an active substance together with a pharmaceutically acceptable diluent, excipient or carrier.

The present invention is further directed to antiviral compositions of increased therapeutic activity, for example, anti-HBV, anti-HIV, anti-CMV and anti-VZV compositions, together with other antiviral compositions directed against other viruses in which thymidine kinase activity is either decreased or absent. Other characteristics and advantages of the invention will be evident from a review of the detailed examples and descriptions that follow.

Compounds of formula la may be prepared in which the bioreversible protecting group, a substrate for cellular carboxyesterases, is an S-acylthioalkyl group. Acyclovir (preferably suitably protected at position 2) is coupled with an appropriate phosphitylating reagent. Deprotection yields compounds of formula la in which the R group can be, independently, alkyl, aryl, heterocyclyl, sugar, or another similar group. The following examples and procedures illustrate the present invention and are not intended to limit the same. I . SYNTHESIS

A. General Condition*

Thin layer chro atography was performed on Merck 6OF 254 silica plates (Art. 5554) . Column chromatography on silica gel were carried out with Merck 60 H silica (Art. 7719) or with RP2 Merck silanized silica (Art. 7719) .

Before analysis or lyophilization, solutions were filtered on Millex HV-4 filter (Millipore) .

UV spectra were recorded on a UVIKON 810 spectrophotometer.

Mass spectra were obtained using a JEOL JMS DX 300 apparatus by the FAB ionization method, in positive or negative mode, in a matrix of glycerol (G) , glycerol/thioglycerol (GT) or 3-nitrobenzyl alcohol (NBA) . Proton NMR spectra were recorded on a Varian EM 360 apparatus or on a Bruker AC 250 apparatus. The chemical shifts are expressed in ppm relative to the tetra ethylsilane (TMS) signal. The multiplicity and the appearance of the signals observed by NMR are indicated by one or more letter(s) : s(singlet), d(doublet), t(triplet), m(multiplet) , orb (broad). Phosphorus NMR spectra were recorded on a Bruker WP 200 SY apparatus with proton decoupling. The chemical shifts are expressed in ppm relative to the H₃P0₄ signal, which is taken as the external reference.

B. Synthetic Scheme

Figure I depicts an exemplary synthetic scheme for preparing certain compositions in accordance with the invention. Bis-S-acetylthioethyl, BIS(SATE), and bis-S- pivaloylthioethyl, BIS(SPTE), phosphotriester derivatives of acyclovir are shown. These compounds correspond to formula la wherein R is CH₃ and (CH₃)₃C.

EXAMPLE 1

Synthesis of 2,2-Dimethylthiopropanoic acid, _.

Hydrogen sulfide was bubbled, with stirring, for 1.5 hours into anhydrous pyridine (200 mL) maintained at -30 °C. Pivaloyl chloride (62 mL, 0.5 mol) was added dropwise, over 1 hour, to the resulting solution while maintaining the reaction mixture at -30°C. Sulfuric acid (5 N, 425 mL) was slowly added in order to obtain a solution Ph of 5. The two layers were separated and the organic layer was diluted with ether, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was then evaporated under reduced pressure to afford compound _. (47 g, 80%) .

Η NMR (CDC1₃) , δ: 3.69 (s, 1H, SH) ; 1.27 (s, 9H, (CH₃)₃).

Mass spectra (matrix = NBA), FAB negative: 117 (M-H) . Boiling point: 64°C (92 mm Hg) .

EXAMPLE 2

Synthesis of S-Acetylthioβthanol or S-2- hydroxyethylthioacetatβ, l,8-Diazabicyclo-(5,4,0)undec-7-ene (DBU) (33.7 mL, 0.23 ol) and iodoethanol (15.6 L, 0.20 mol) were consecutively added at 0°C to a stirred solution of commercial thioacetic acid (16.5 L, 0.23 mol) in toluene (80 mL) . The reaction mixture was allowed to react for two hours at room temperature, then diluted with methylene chloride and washed with water. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude residue 3_ (15.3 g, 64%) was used in the following condensation step without further purification.

*H NMR (DMSO-d , δ: 4.95 (t, 1H, CH₂Ofl, J - 5.5 Hz); 3.45 (m, 2H, CH-jCifcOH) ; 2.90 (t, 2H, SC&CH-j, J - 6.6 Hz); 2.31 (S, 3H, CH_j) .

EXAMPLE 3 Synthesis of s-pivaloylthioethanol or 8- (2,2- dimethylpropanoyl) thioethanol or 8-2-hydroxyβthylthio(2,2- dimethyDpropionate, ± l,8-Diazabicyclo-(5,4,0)undec-7-ene (DBU) (4.2 mL, 29 mmol) and iodoethanol (1.95 Ml, 25 mmol) were added consecutively, at 0^βC, to a stirred solution of 2,2- - 8 - di ethylthiopropanoic acid, 2. (3.43 g, 29 mmol) in toluene (12 Ml) . The reaction mixture was allowed to stand for two hours at room temperature, then diluted with methylene chloride and washed with water. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was dissolved in a small amount of methylene chloride and chromatographed on a silica gel column using a stepwise gradient of methanol (0-2%) in methylene chloride to give the title compound 4. (3.6 g, 89%). »H NMR (DMSO-dβ) , δ : 4.94 (t, 1H, CH₂0H, J - 5.5 Hz);

3.45 (m, 2H, CH₂Cfl₂θH) ; 2.88 (t, 2H, SC&CH-j, J - 6.6 Hz) ; 1.16 (s, 9H, (CH₃)₃)C).

EXAMPLE 4

Synthesis of 0,0'-Bis(8-acetylthioethyl)N,N-diisopropyl- phosphoramidite, £

A solution of S-acetylthioethanol, 3_ (4.81 g, 40 mmol) and triethylamine (6.1 mL, 44 mmol) in THF (100 mL) , cooled at -78^βC, was added over a period of 45 minutes to a solution of N,N-diisopropylphosphorodichloridite (4.04 g, 20 mmol) in THF (150 mL) . The reaction mixture was allowed to stand for two hours at room temperature, then filtered and evaporated under reduced pressure. The residue was dissolved in cyclohexane, filtered and evaporated under reduced pressure. The resulting pale yellow oil was dissolved in a small amount of cyclohexane containing 1% of triethylamine and chromatographed on a silica gel column that contained cyclohexane having 5% of triethylamine. The column was eluted with ethylacetate (0-2%) in cyclohexane containing 1% of triethylamine to yield the title compound, 5 (5.3 g, 72%). *H NMR (DMSO-d , δ : 3.59 (m, 6H, CH₂0H. or CH) ,* 3.04

(t, 4H, SC H₂CH₂, J - 6.4 HZ); 2.32 (s, 6H, CH₃C0) ; 1.10 (d, 12H, (CHj)₂CH, J = 6.8 Hz).

³¹P NMR (DMSO-d , δ : 147.9.

Mass spectra (matrix GT) , FAB positive: 370 (M+H)⁺; 103 (CH₃C0SCH₂CH₂)⁺. EXAMPLE 5 synthesis of θ,θ-Bis(β-(2,2-dimethylpropanoyl)thioethyl)N,N- diisopropylphosphora idite, £

A solution of S-(2 ,2-dimethylpropanoyl)thioethanol, 4. (5 g, 30.6 mmol) and triethylamine (4.7 mL, 33.7 mmol) in THF

(60 mL) , cooled at -78°C, was added, over a period of 15 minutes, to a solution of N,N-diisopropylphosphorodichloridite

(3.09 g, 15.3 mmol) in THF (130 mL) . The reaction mixture was allowed to stand for two hours at room temperature, then filtered and evaporated under reduced pressure. The residue was dissolved in cyclohexane, filtered and evaporated under reduced pressure. The resulting pale yellow oil was dissolved in a small amount of cyclohexane containing 1% of triethylamine, and chromatographed on a silica gel column containing cyclohexane having 5% of triethylamine. The column was eluted with ethylacetate (0-2%) in cyclohexane containing

1% of triethylamine to yield the title compound (3.2 g, 46%) . ^lH NMR (DMSO-d , δ : 3.59 (m, 6H, CH₂0H. or CH) ; 3.01 (t, 4H, SC IfcCHj, J =■= 6.3 Hz); 1.16 (s, 18H, (CH₃)₃C) ; 1.10 (d, 12H, (CH jCH, J = 6.8 Hz) .

³¹P NMR (DMSO-d , δ : 148.0.

Mass spectra (matrix GT) , FAB positive: 454 (M+H)⁺: 145 ((CH₃)₃CCOSCH₂CH₂)⁺; 85 ((CH₃)₃CCO)⁺; 57 ( (CH₃)₃C)⁺.

EXAMPLE 6 Synthesisof0-[l-(Methyl-(9-<N²-p-anisyldiphenylmethyDguanin- yl))l-hydroxyβthyl-2-yl]-0', O'^,-bis(8-acβtyl-2-thioβthyl)- phosphate, 2

Tetrazole (210 mg, 3.0 mmol) was added to a stirred solution of N²-(p-anisyldiphenylmethyl)-9-[ (-2-hydroxy- ethoxy)methyl] guanine (500 mg, 1 mmol) , prepared in accordance with Martin et al . _t J. Med . Chem . , 1986, 29 , 1384-1386, and compound 5 (445 mg, 1.2 mmol) in THF (3.0 mL) . After 35 minutes at room temperature, the reaction mixture was cooled to -40^βc and a solution of 3-chloroperbenzoic acid (407 mg, 1.3 mmol) in methylene chloride (5 mL) was added. The solution was then allowed to warm up to room temperature over one hour. Sodium hydrogen sulfite (10% solution, 3 mL) was added to reduce excess peracid. The organic layer was separated, diluted with methylene chloride (10 mL) , washed with a saturated solution of aqueous sodium bicarbonate (3 mL) , then with water (3x3 mL) , dried over Na₂S0₄ and evaporated to dryness. The residue was dissolved in a small amount of methylene chloride and chromatographed on a silica gel column (methanol (0-4%) in methylene chloride) . Compound 2 was obtained as a white foam (531 mg, 68%) . ^fH NMR (DMSO-d , δ : 10.63 (s, 1H, NH) ; 7.71 (s, 1H,

NH) ; 7.67 (s, 1H, H-8) ; 7.16-7.31 (m, 12H, aromatic); 6.87 (d, 2H, aromatic, J = 8.9 Hz); 4.86 (s, 2H, NCH₂0) ; 3.97 (m, 4H, OC CHjS) ; 3.71 (s, 5H, 0CH₃ or POC CH ) ; 3.10 (t, 4H, OCH-_jCH_jS, J = 6.3 Hz); 3.03 (m, 2H, POCH₂,CH₂0); 2.35 (s, 6H,CH₃COS). ^3lP NMR (DMSO-d , δ : -0.83.

Mass spectra (matrix GT) , FAB positive: 782 (M+M)⁺; 273 (trityl)⁺; 103 (CH₃C0SCH₂CH₂)⁺; FAB negative: 780 (M-H)'; 678 (M-CH₃C0SCH₂CH₂)^'.

EXAMPLE 7 Synthesisof0-[l-(Methyl-[9-<N²-p-anisyldiphenylmethyl)guanin- yl])l-hydroxyethyl-2-yl]-0', 0"-bis (S-pivaloyl-2-thioβthyl)- phosphate, a.

Tetrazole (126 mg, 1.8 mmol) was added to a stirred solution of N²-(p-anisyldiphenylmethyl)-9-[ (-2-hydroxy- ethoxy) ethyl] guanine (300 mg, 0.60 mmol), prepared in accordance with Martin et al . , J. Med. chem . , 1986, 29 , 1384- 1386, and compound £ (408 mg, 0.90 mmol) in THF (1.8 mL) . After 35 minutes at room temperature, the reaction mixture was cooled to -40°C and a solution of 3-chloroperbenzoic acid (342 mg, 0.99 mmol) in methylene chloride (4 mL) was added. The solution was then allowed to warm up to room temperature over one hour. Sodium hydrogen sulfite (10% solution, 3 mL) was added in order to reduce excess peracid. The organic layer was separated, diluted with methylene chloride (10 mL) , washed with a saturated solution of aqueous sodium bicarbonate (3 mL) , then with water (3x3 mL) , dried over Na₂S04 and evaporated to dryness. The residue was dissolved in a small amount of methylene chloride and chromatographed on a silica gel column (methanol (0-4%) in methylene chloride) to give compound 8_ as a white foam (372 mg, 71%) . *H NMR (DMSO-d , δ : 10.63 (s, 1H, NH) ; 7.71 (s, 1H,

NH) ; 7.68 (s, 1H, H-8); 7.17-7.30 (m, 12H, aromatic); 6.86 (d, 2H, aromatic, J - 8.9 Hz); 4.87 (s, 2H, NCH₂0) ; 3.97 (m, 4H, OCH_jCH_jS) ; 3.71 (Si, 5H, OCH₃ or POCH₂CH₂0) ; 3.09 (t, 4H, 0CH₂CH₂S, J = 6.4 Hz); 3.06 (m, 2H,

1.17 (s, 18H, (CH₃)₃C).

³¹P NMR (DMSO-dβ) , δ : -0.88.

Mass spectra (matrix GT) , FAB positive: 866 (M+M)⁺; 273 (trityl)⁺; 145 ( (CH₃)₃CCOSHC₂CH₂) ⁺; FAB negative: 864 (M-H)"; 720 (M-(CH₃)₃CCOSCH₂CH₂)-.

EXAMPLE 8

Synthesis of0-[l-(Methyl-[9-guanin-yl])-l-hydroxyethyl-2-yl]- O', 0"-bis(S-acetyl-2-thioethyl) phosphate, 2 [Bis(SATE)ACVMP]

A solution of compound 2 (450 mg, 0.57 mmol), in a mixture of acetic acid (32 mL) , methanol (4 mL) and water (4 mL) , was heated at 50°c for 18 hours, then evaporated to dryness. The residue was coevaporated three times with ethanol, twice with methylene chloride, then diluted in a small amount of methylene chloride and chromatographed on a silica gel column (methanol (0-10%) in methylene chloride) . The desired product, compound 9_, was obtained as a white foam (270 mg, 92%) .

UV: λ max (EtOH 95) 253 nm (e 14100). Η NMR (DMSO-dβ) , δ : 10.65 (s, 1H, NH) ; 7.81 (s, 1H, H-8); 6.52 (S, 2H, NH₂) ; 5.35 (s, 2H, NCH₂0) ; 4.05 (m, 2H, POCH_∑CHjO) ; 3.99 (m, 4H, OCH^C^S) ; 3.65 (m, 2H, POCH^C&O); 3.09 (t, 4H, OCH-_jC S, J = 6.3 Hz) ; 2.34 (s, 6H, CH₃C0S) . ³¹P NMR (DMSO-d , δ : -0.74.

Mass spectra (matrix GT) , FAB positive: 510 (M+M)⁺; 152 (BH₂)⁺; 103 (CH₃COSCH₂CH₂)⁺; FAB negative: 508 (M-H)^"; 406 (M- (CH₃COSCH₂CH₂)-; 150 (B)\ - 12 -

EXAMPLE 9

Synthesisof0-[l-(Methyl-[ -guanin-yl])-l-hydroxyethyl-2-yl]- O', 0"-bis(S-pivaloyl-2-thioethyl) phosphate, 10 [Bis(SPTE)ACVMP] A solution of compound 8. (360 mg, 0.42 mmol) , in a mixture of acetic acid (24 mL) , methanol (3 mL) and water (3 mL) , was heated at 50°C for 18 hours, then evaporated to dryness. The residue was coevaporated three times with ethanol, twice with methylene chloride, then diluted in a small amount of methylene chloride and chromatographed on a silica gel column (methanol (0-9%) in methylene chloride) . The desired product, compound _J_, was obtained as a white foam (210 mg, 85%) .

UV: λ max (EtOH 95) 253 nm (e 13400). ^lH NMR (DMSO-d , δ : 10.59 (s, 1H, NH) ; 7.79 (s, 1H,

H-8); 6.46 (s, 2H, NH₂) ; 5.36 (s, 2H, NCH₂0) ; 3.95-4.09 (m, 6H,

or OCHjCHjS) ; 3.67 (m, 2H, POCH₂CH₂θ) ; 3.08 (t, 4H, OCH₂,CH₂S, J - 6.3 HZ); 1.17 (s, 18H, (CH₃)₃C) . ^3,P NMR (DMSO-d , δ : -0.80. Mass spectra (matrix GT) , FAB positive: 594 (M+H)⁺;

152 (BH₂)⁺; 145 ((CH₃)₃CCOSCH₂CH₂)⁺; FAB negative: 592 (M-H)"; 448 (M-(CH₃)₃CCOSCH₂CH₂)-; 150 (B)\

II. BIOLOGICAL EVALUATION

The experimental procedure used for evaluation of activity against hepatitis B virus was as previously described by Korba and Milman in j ntiviral Res . , 1991, 217 , 217.

The experimental procedure used for evaluation of activity against human immunodeficiency virus (HIV) was as follows: HIV-1 replication (LAI isolate) in CEM cells was measured by assaying for reverse transcriptase (RT) in the culture supernatant after infection for five days. This activity reflects the presence of the virus released by the cells. After absorption of the virus, the test compounds were added at various concentrations in the culture medium. Antiviral activity was expressed as the lowest concentration of compound which reduced the viral production by at least 50% (ED_jo) . The toxic effect in non-infected CEM cells was assessed by a calorimetric reaction based on the capacity of living cells to reduce 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide into formazan after incubation for five days in the presence of various concentrations of the compounds. The results were expressed as the lowest concentration of compound which resulted in at least 50% inhibition of the formation of formazan (CD*,) . The experimental procedure used for evaluation of activities against herpes simplex virus type 1 (HSV-1) was as described by Genu-Dellac et al . in N cleosides and Nucleotides, 1991, 10 , 1345.

The experimental procedure used for the evaluation of compounds of the present invention against herpes simplex virus type 2 (HSV-2) involved the treatment of HSV-2 vaginitis in BALB/c mice, and is as follows:

BALB/c female mice, weighing approximately 20 g each at the start of the experiment, were intravaginally infected with HSV-2 (E194 strain) . This was accomplished in a 3-step process. First, the vagina of each mouse was swabbed for 5 seconds with a cotton-tipped applicator dipped in 0.1 N NaOH. Approximately 30-60 minutes later each vagina was dry swabbed for 5 seconds. Then an applicator dipped in virus medium (containing approximately 10⁷ plaque forming units of virus/mL) was used to swab each mouse for 20 seconds. Twenty-four hours after infection with virus, compounds were administered by i.p. injection. Treatments were given twice daily for 5 consecutive days. Lesion scores in infected mice were determined daily on days 3-10 of the infection. A score of 1+ to 4+ was given depending on the progression of lesion formation. Deaths were recorded daily for 21 days. Vaginal virus titers were made by titration of virus obtained from vaginal swabs (placed in 1 mL of cell culture medium) 3 days after virus inoculation. These titrations were conducted in MA-104 African green monkey kidney cells in 96-well plates. Calculation of virus titer was made by the 50% endpoint dilution method of Reed and Muench (Am. J. Hyg. , 1938, 27, 493). Statistical interpretation of survival (Fisher exact test) , mean day to death (Mann-Whitney U-test) and virus titer (Mann-Whitney U-test) were made by two-tailed analyses using the Instat™ computer program (GraphPad, Inc., San Diego, CA) .

The compounds of the present invention were also assayed for antiviral activity against murine gammaherpes virus type 68 (MHV-68) using a plaque reduction assay. MHV-68 is a mouse counterpart virus to EBV which infects mice, and may be used as a rodent model for studying anti-EBV compounds. The procedure for this assay was as follows:

Compounds were prepared as a 10 mM solution in DMSO, which was then diluted below 100 μM in cell culture medium. Acyclovir was initially prepared as a 4 mM solution in cell culture medium before diluting to micromolar concentrations. African green monkey kidney (Vero) cells were seeded into 12- well plates, then allowed to grow to confluency. The cells were infected with approximately 100 plaque forming units of MHV-68. After 1.5 hours, the medium was changed to include antiviral drug or drug-free medium (MEM, 2% FBS and 0.5% SeaPlaque agarose obtained from FMC Corp. , Rockland, MD) . At 7 days the plates were fixed for 15 minutes with 10% buffered formalin prior to removing the agar overlay. The cells were then stained with 0.2% crystal violet in 20% methanol. The plaques were counted at 17x magnification using a Plaque Viewer (Bellco Glass Co. , Vineland, NJ) . ECy-, values were determined by plotting inhibition versus drug concentration.

The compounds of the present invention were tested for their antiviral activity against human CMV using a plaque reduction assay. The procedure was as follows:

The antiviral activity of the compounds against human CMV was determined in MRC-5 cells (a continuous line of diploid, human (male) embryonic lung cells) . The compounds were diluted in MEM, 2% FBS, 0.1% NaHC0₃ and 50 μg/mL of gentamicin. The growth medium was decanted from established onolayers of MRC-5 cells in 24-well tissue culture plates. Human CMV (1 mL) , diluted in test medium, was placed in each well except those to be used for cell controls, which received 1 mL of sterile test medium. The virus was allowed to adsorb to the cells while the plates were centrifuged (2200 rpm, 30 minutes) at room temperature. The medium was aspirated from the plates and 0.8 mL of the compound (varying concentrations) was placed in each well. Test medium (0.8 mL) without any compound was added to each cell control and virus control well. The plates were placed in an incubator at 37°C in a moist atmosphere of 5% C0₂, 95% air until plaques could be distinguished in the virus control wells. The cells were observed microscopically for morphological changes due to compound cytotoxicity before the medium was aspirated from the wells. The cells were stained with crystal violet for 15 minutes, after which the staining solution was aspirated, the plates rinsed with water, inverted and dried at room temperature. Plaques were counted by the use of a dissecting microscope. ED_JQ and CD_M values were determined by regression analysis of the viral plaque data and the visual cell toxicity data, respectively. The antiviral activity of the compounds of the present invention against murine CMV (MCMV) was determined by a CPE (cytopathic effect) inhibition assay, which was performed as follows:

Growth medium was decanted from 96-well tissue culture plates containing monolayers of 3T3 cells (a continuous line of contact-inhibited mouse fibroblasts) . Varying concentrations of compound were added to the wells (4 wells/dilution, 0.1 mL/well of each compound). Compound diluent medium (MEM, without serum, 0.18% NaHC0₃, 50 μg of gentamicin/mL) was added to the cell and virus control wells

(0.1 mL/well). Virus, diluted in the test medium, was added to all the compound test wells of the plate (3 wells/dilution) and to virus control wells at 0.1 mL/well. Test medium without virus was added to all toxicity control wells ( 1 well/dilution) and to cell control wells at 0.1 mL/well. The plates were incubated at 37°C in a humidified incubator with 5% C0₂, 95% air until virus control wells had adequate CPE readings. Cells in test and virus control wells were then examined microscopically and virus CPE was graded on a scale of 0-4, with 0 being no CPE and 4 being 100% CPE. The cells in toxicity control wells were observed microscopically and graded for morphological changes due to cytotoxicity. ED_M and CD_JO values were determined by regression analysis of the viral CPE data and the visual toxicity data, respectively.

The compounds of the present invention were also assayed for their activity against varicella zoster virus (VZV) using a plaque reduction assay. The procedure was as described for the assay against human CMV, except that 0.5 mL of VZV was added to each well, and the virus was allowed to adsorb to the cells during a 1 hour stationary incubation period.

The compounds of the present invention were designed to be stable in biological fluids and various organs, thus allowing them to reach targeted cells. The significant stability of bis(SPTE)ACVMP in human serum was demonstrated by its half-life of 14 hours. The ex vivo procedure for half-life determination was as follows: Decomposition of the compound was studied at 37°C in human serum. During incubation, aliquots (80 μL) were removed and injected into a cleaning precolumn (Guard-Pak, Waters) which was equilibrated with triethylammonium acetate buffer (20 mM, pH 6.6). Protein and other undesired components were allowed to elute, while compounds to be analyzed were trapped. After 5 minutes, the precolumn was connected to an analytical column (Hypersil ODS, 100x4.6 mm, 3 μM particle size, Shandon) . The trapped analytes were transferred from the precolumn to the analytical column and eluted with 40 mM triethylammonium acetate:acetonitrile (1:1, v/v) . The structure of eluted analytes was confirmed by HPLC-UV-MS (a Waters HPLC module and a UV-diode array detector 996 were connected by two automated 7010 Rheodyne valves and controlled by a Millenium software program (Waters) . The mass spectrometer (SSQ 7000, Finnigan Mat) was directly coupled to the UV detector outlet) . RESULTS:

Anti-HBV activity, in transfected HepG2 (2.2.15) cells, of bis(S-acyl-2-thioethyl)phosphotriester derivatives of acyclovir, (compound 9_, Figure I) , as compared with those of the parent nucleoside and of 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC) are shown in Table 1.

TABLE 1

HBV virion

Composition

EC₅₀(μM) EC_w(μM) CCjo(μM) SI

ddC 1.3 ± 0.2 1.1 ± 1.2 219 ± 19 20

1 ddC 2.2 ± 0.3 8.3 ± 0.8 218 ± 19 30

Bis(SATE)ACVMP 0.7 ± 0.1 5.1 + 1 987 ± 99 194 2.

Bis(SPTE)ACVMP 0.2±0.04 7.1 ± 0.8 1593 ± 131 224 1G

Acyclovir 111 ± 15 >100 631 ± 40 ND

SI = CC^/EC,,,; ND = Not Determinable

In Table 1, EC^ and EC_W represent the molar concentrations which provide 90% inhibition and 50% inhibition, respectively, of HBV replication. CC^ represents the molar concentration which reduces the viability of non-infected cells by 50%. The data show that both acyclovir derivatives significantly inhibit HBV activity.

Synergistic anti-HBV activity was observed upon treatment with Bis(SATE)ACVMP (9_) and 3TC in combination. The results are shown in Table 2 (SI - CCso/EC^) . TABLE 2

I Composition EC_jo (μM) EC*, (μM) CC_JO (μM) SI

Bis(SATE)ACVMP + 0.03 0.19 913 4805 3TC

Acyclovir + 3TC 6.0 20 644 32

Analysis of the results shown in Table 2 by the

COMBOSTAT combination analysis program indicated that there was synergy between bis(SATE)ACVMP and 3TC at EC₅₀ and EC,,-,. In this combination treatment, bis(SATE)ACVMP was mixed with 3TC at a 10:1 molar ratio.

Anti-HIV activity, in CEM TK- cells, of bis(S-acyl-2- thioethyl) phosphotriesters derivatives of acyclovir, as compared with those of the parent nucleoside and of AZT are shown in Table 3.

TABLE 3

Composition EC₅o(μM) CCjo(μM)

AZT >100(20%) >100(5%)

Bis(SATE)ACVMP 77 >100(0%)

Bis(SPTE)ACVMP 3.6 >10(26%)

I Acyclovir >100(0%) >100(0%)

In Table 3, EC*, represents the molar concentration which produces a 50% inhibition of HIV replication. Significant inhibition of HIV replication was observed.

Anti-HSV-1 activity, in MRC-5 cells, of bis(S-acyl-2- thioethyl)phosphotriesters derivatives of acyclovir is compared to that of acyclovir in Table 4. TABLE 4

Composition HSV-1 HSV-1 TK-

Bis(SATE)ACVMP 1.25 100

Bis(SPTE)ACVMP 2.5 10

Acyclovir 0.63 >100

In Table 4, EC_W represents the molar concentration which produces a 50% inhibition of HSV-1 replication. Significant activity against HSV was observed.

Anti-HSV-2 activity of bis(SATE)ACVMP in BALB/c mice is demonstrated by the data shown in Tables 5 and 6, and Figures 2 and 3.

Table 5 shows the survival and vaginal virus titer data in mice infected with HSV-2. Compared to acyclovir, bis(SPTE)ACVMP elicited a superior response with 60% survival in the 100 and 50 mg/kg/day groups of bis(SPTE)ACVMP. Slightly less virus was recovered from lesions of bis(SPTE)ACVMP-treated mice compared to acyclovir-treated mice, however the differences were not significant.

Table 6 shows average daily lesion scores. Bis(SPTE)ACVMP, at 100 mg/kg/day produced a good response in lesion score reduction over the entire time frame. The data in Table 6 is also depicted graphically in Figures 2 and 3. It is clear from these figures that bis(SPTE)ACVMP is more effective in inhibiting lesion formation than acyclovir. The results discussed above indicate that bis(SPTE)ACVMP was effective against HSV-2 vaginal infection in mice. Acyclovir was effective at the outset, but its effect waned after the discontinuation of treatment. Bis(SPTE)ACVMP, at 100 and 50 mg/kg/day, reduced mortality, vaginal virus titers and vaginal lesion scores in HSV-2 vaginally infected mice when treatments were given twice daily for 5 days starting 1 day after virus inoculation. TABLE 5

Effects of Acyclovir and Bis(SPTE) CVMP on Survival and Vaginal virus Titers in Hβv-lnfected Mice

Compound Dose¹ Survivors/ Mean Day to Vaginal Virus (mg/Jcg/day) Total (%) Dβath^b Titers^e

Bis(SPTE)ACVMP 100 6/10 (60)* 12.5 ± 2.4** 2.5_ 0.7***

50 6/10 (60)* 11.7 ± 2.1* 2.8 ± 1.2***

25 4/10 (40) 10.7 ± 0.5** 2.3 + 0.8***

Acyclovir 100 3/8 (38) 16.2 ± 2.4^d*** 3.2 + 1.0⁴***

50 5/8 (63)** 12.0 ± 2.0* 2.3 ± 0.8***

25 1/10 (10) 11.0 ± 1.9** 3.0 + 0.7***

Placebo 0/10 (0) 7.8 + 1.6 5.3 ± 0.4

^• Intraperitoneal treatments were given twice daily for 5 days starting day 1 after virus infection. 10 ^b Of the mice that died (survivors lived through 21 days) . ^c Logio CCID50/lesion, determined 3 days after virus challenge. ^d Standard deviation.

* P<0.05 ** P<0.01 *** P<0.001

- 22 -

Antiviral activity against MHV-68 is exhibited by the data shown in Table 7. In the plaque reduction assay, the EC_W for bis(SPTE)ACVMP was 8 μM and the EC_M for acyclovir was 5 μM.

TABLE 7

Compound Cone. (μM) % Plaques*

Bis(SPTE)ACVMP 40 1

20 15

10 34

5 76

2.5 100

1.25 100

0 100

Acyclovir 40 0

20 6

10 22

5 54

2.5 82

1.25 100

0 100

Relative to the wells devoid of antiviral compound.

Antiviral activity of the compounds of the present invention against human CMV are shown in Table 8. Bis(SPTE)ACVMP was approximately 4-fold more effective in reducing plaques than was acyclovir.

Antiviral activity of the compounds of the present invention against murine CMV as determined by a CPE (cytopathic effect) inhibition assay is shown in Table 9. The data indicates that both bis(SATE)ACVMP and bis(SPTE) CVMP were active against murine CMV, with the latter compound being slightly more active. Antiviral activity of the compounds of the present invention against varicella zoster virus (VZV) is shown in Table 10. The data indicates that both bis(SATE)ACVMP and bis(SPTE)ACVMP were as active as acyclovir in the plaque reduction assay.

TABLE 8

Antiviral Activity (Plaque Reduction) of UA514, UA491 , UA486, or DHPG vs Human Cytomegalovirus, Strain AD-

169, In MRC-5 Cells

.UA514 UA491 UA486 DHPG

UA514. 491. & 486 Number Number Numbe ir Number DHPG

Conc'n. of % of % of % of % Concentration

⁽μM⁾ Plaques Reduction Plaques Reduction Plaques Reduction Plaques Reduction (ug/mll

100 0.0 100 28.5 31 12.0 71 0.0 100 100

32 3.0 93 42.5 0 50.5 0 0.0 100 32

10 32.5 0 49.0 0 43.0 0 8.5 80 10

3.2 42.5 0 40.0 0 62.0 0 30.5 27 3.2

1.0 32.5 0 32.0 0 36.5 0 37.0 0 1.0

0.32 35.5 0 33.0 0 37.0 0 37.0 0 0.32

0.1 40.5 0 40.0 0 28.5 31 44.0 0 0.1

ED50³: 18 μM >100 μM 71 μM 5.2 μg/ml

CD50^b: > 100 μM >100 μM > 100 μM >100 μg/ml

Tl^c: >5.6 N/D* >1.4 > 19 ^a The concentration at which the average number of plaques is reduced to 50% of that seen in the virus controls (Effective

Dose, 50% endpoint). ^b The concentration halfway between those at which 100% and 0% cytotoxicity are seen. ^c Therapeutic Index (CD50+ED50).

* Not Determinable UA514 is Bis(SPTE)ACVMP.

UA491 is Bis(SATE)ACVMP. UA486 is Acyclovir. DHPG is Ganciclovir.

TABLE 9

Antiviral Activity (CPE Inhibition) of UA514, UA491 , UA486, or Acyclovir vs. Murine

Cytomegalovirus, Strain Smith MSGV, In 3T3 Cells

UA514, 491, & 486 UA5 14 UA491 UA486

Conc'n. Visible Avg. Visible Avg. Visible Avg.

(μM) Cyotox, GEE CytQtox, CPE Cvtotox. CPE

100 0 0.5 0 0.5 0 0.0

32 0 0.3 0 0.5 0 0.0

10 0 0.5 0 2.5 0 0.5

3.2 0 4.0 0 4.0 0 1.8

1.0 0 4.0 0 4.0 0 4.0

0.32 0 4.0 0 4.0 0 4.0

0.1 0 4.0 0 4.0 0 4.0

4.0 (virus controls)

ED50^a: 6.1 μM 12 μM 2.8 μM

CD50&: >100 μM >100 μM >100 μM

Tic; > 16 >8.3 >36 ^a The concentration at which the average viral CPE is reduced to 50% of that seen in the virus controls

(Effective Dose, 50% endpoint). ^b The concentration halfway between those at which 100% and 0% cytotoxicity are seen. ^c Therapeutic Index (CD50+ED50).

CPE = Cytopathic Effect UA514 is Bis(SPTE)ACVMP.

UA491 is Bis(SATE)ACVMP. UA486 is Acyclovir.

Claims

What is claimed is:

1. A phosphotriester compound of the structure: (RO)₂-P(=0)-0-ACV wherein: R is a radical (CH₂)_β-S-X, X is C(=Z)-Y or S-U; Z is O or S;

Y and U are alkyl, aryl or a saccharide which is optionally substituted with OH, SH or NH₂; n is equal to 1 to 4, and ACV is an acyclovir moiety.

2. The compound of claim 1 wherein the acyclovir moiety has the structure:

3. The compound of claim 1 wherein: X is S-U and U represents the radical (CH₂)_nl-X¹;

X¹ is H, OH, SH or NH₂; and n¹ is 1 to 4.

4. The compound of claim l wherein:

X is C(=Z)-Y; and Y is CH₃ or (CH₃)₃-C.

5. The compound of claim 4 wherein:

R is (CH₂)_n-S-C(=0)-CH₃or (CH₂)_n-S-C(=0)-tBu; and n is 1 or 2.

6. The compound of claim 3 wherein R is (CH₂)₂-S-S-(CH₂)₂-OH.

7. A compound having the structure:

wherein R is CH₃ or (CH₃)₃C.

8. A pharmaceutical composition comprising the compound of claim 1 as an active ingredient and a pharmaceutically acceptable diluent, carrier or excipient.

9. The pharmaceutical composition of claim 8 exhibiting inhibitory activity against a virus whose thymidine kinase activity is either decreased or absent.

10. The pharmaceutical composition of claim 8 exhibiting inhibitory activity against human immunodeficiency virus, hepatitis B virus, herpes simplex virus type 1, herpes simplex virus type 2, or cytomegalovirus.

11. The pharmaceutical composition of claim 8 exhibiting inhibitory activity against varicella zoster virus.

12. An pharmaceutical composition comprising a compound of claim 7 as an active ingredient and a pharmaceutically acceptable excipient, diluent or carrier.

13. The pharmaceutical composition of claim 12 exhibiting inhibitory activity against a virus whose thymidine kinase activity is either decreased or absent.

14. The pharmaceutical composition of claim 12 exhibiting inhibitory activity against human immunodeficiency virus, hepatitis B virus, herpes simplex virus type 1, herpes simplex virus type 2, or cytomegalovirus.

15. The pharmaceutical composition of claim 12 exhibiting inhibitory activity against varicella zoster virus.

16. The compound of claim 1 wherein n is 1 or 2.