CA1257256A - Antiviral agents - Google Patents

Abstract

NOVEL ANTIVIRAL AGENTS

Abstract Phosphonate analogues of mono-, di-, and tri-phosphates of antiviral nucleoside analogues are dis-closed. These materials are represented structurally as wherein Z1 and Z2 are the same or different and selected from the group made up of hydrogen, the one to six car-bon alkyls, phenyl and benzyl, X is H, OH, or together with Y = O, Y is H or together with X = O, n is an integer, O, 2 or 4, R1 and R2 together complete a .beta.-pentofuranose sugar or R1 is H and R2 is H or -CH2OH, R3 is H or OH and B is a purine or pyrimidine base.
These materials have antiviral activity, expecially against herpes virus. Antiviral pharmaceutical prepara-tions and their use are disclosed as well.

Description

~ 2 ~2~

NOVEL_ANTIVIRAL AGENTS

Field of the Invention This invention concerns nucleotide ana-logues and their synthesis and use. More particu-5 larly, it concerns phosphonic acid analogues ofnatural and synthetic nucleoside phosphates and their preparation and use as antiviral agents.

Background of the Invention There is a recognized need for antiviral 10 agents. Herpes virus hominis alone infects between 50 and 150 million Americans at this time. A numher of the antiviral agents that are currently viewed as most e~fective against herpes are nucleoside analogues.
These materials include iododeoxyuridine, 2-hydroxy-15 ethoxymethylguanine, 2'-fluoro-5-iodo-1-arabino-furanosyl cytosine and 5-E-bromovinyldeoxyuridine. It is believed that these materials act through their conversion by viral thymidine kinase (but not by host TK) to the nucleotide which is then converted to the 20 triphosphate and incorporated into viral DNA. The incorporation of these analogues into the viral DNA
prevents its replication and thus is lethal to the virus. Two shortcomings of this antiviral mechanism have been recognized, however. First, thymidine 25 kinase negative herpes mutants (TK-) have been identified which are inherently inactive toward phosphorylating these analogues and thus permitting their incorporation in viral DNA. In addition, TK+
mutants that are resistant to 2-hydroxyethoxymethyl-30 guanine have been reported in mice by H. Field, et al,in J Inect Dis, 143 281 (1981). TK~ mutants resistant to iododeoxyuridine have been reported in humans by A. ~irano, et al, in Acta ViroI 23 226 (1979). It may be that these newly-discovered resis-tant viral strains do not undergo the monophosphoryla-tion or triphosphate formation needed to permit incor-5 poration in the ~NA.
References to these antiviral agents of the art and their use include Am J Med, 73 No lA, July 20, 1982 "Proceedings of a Symposium on Acyclovir";
Biochem Biophys Acta, 32 295-6 (1959); Antimicrob 10 Agents Chemother, 578-584 (1965); Science, 145 585-6 (1964); Science, 255 468-80 (1975); J Med Chem, 19 495-8 (1976) Proc ~atl Acad Sci, 76 4415-18 (1979)-and J Med Chem 22 21-24 (1979)~
The present invention provides antiviral 15 materials which can be lethally incorporated into DNA
without the dependence upon enzyme-moderated phosphor-ylation. The materials of the invention are phospho-nate analogues of the mono-, di- and triphosphates of the deoxynucleotide analogs. An article by Robert 20 Engel appearing at Chem Reviews, 11, ~3 pp 349-367 (1977) discusses phosphonate analoges of nucleotides and the like. Other representative references in this area, some of which are cited in the Chem Reviews article, are German O.L.S 2,350,608 (1974) of Syntex 25 (Jones and Moffatt inventors); German O.L.S. 2,009,834 1970 also of Syntex with Jones and Moffatt as inven-tors; British Patent 1,243,214 of Syntex, and US
Patent 3,560,478 of Myers.

Statement of the Invention A group of new materials have now been found. These materials in a broad sense are phospho-nate analogues of mono-, di-, and triphosphates of antiviral nucleoside analogues. These analogues i7~5~

differ from nonlethal natural nucleosides by varia-tions in their sugar ribose moiety and/or by varia-tions in their nucleosid~e base moieties. Such mater-ials are represented structurally as Z2 ~1l 1 1 ,~P-l (CH2!n 1 ,O
Y CH ~ CH

2 Rl wherein Zl and Z2 are the same or different and selec-ted from the group made up of hydrogen, the one to six carbon alkyls, phenyl and benzyl, X is H, OH, or to~ether with Y = O, Y is H or together with X = O, n is an integer, 0, 2 or 4, Rl and R2 together comple-te a ~-pentofuranose sugar or Rl is H and R2 is H or -CH2OH, R3 is H or OH and B is a purine or pyrimidine base.
In other aspects this invention relates to ~the preparation of these materials, their formulation into antiviral pharmaceutical compositions and the use of these formulations to treat viral infections, in particular herpes infections.

Detailed Description of the Invention 20The compounds The compounds of this invention are phos-phonates which have the structure set forth above in Statement of the Invention.

7;~

-CH B
I ~0 CH ~CH
R2 Rl The unit defines an antiviral nucleoside.
As previously noted, in this structure Rl and R2 can together complete a ~-pentofuranose sugar.
In this configuration they preferably complete a sub-stituted or unsubstituted ~-ribofuranose or ~-arabino-furanose such as ribose, 2-deoxyribose, 2,3-dideoxy-ribose, 3-deoxyribose, 2-fluoro-2-deoxyribose or arabinose or the like.
In this structure, Zl and Z2 preferably are each selected ~rom hydrogens and one to four carbon alkyls. More preferably Zl and Z2 are each hydro-gens. Further in this structure, the integer n is significant in defining whether the compound is in size equivalent to a nucleoside monophosphate, a diphosphate or a triphosphate.
Preferred bases include guanine, adenine, 5-iodouracil, 5-trifluorothymine, 5-iodocytosine, E-5-2-bromovinyluracil, 5-propyluracil, and 5-ethyluracil.
Preferred nucleoside analogues (e.g.

- CH B
I ,o~l CH CH
R2 Rl include 5-iodo-2'-deoxyuridine, 9-B-D-arabinofurano-syladenine, 5-trifluorothymidine, E-5-(2-bromovinyl)-2'-deoxyuridine, 1-(2'-deoxy-2'-fluoro-B-D-arabino-~ ~7~5~

furanosyl)-5-iodocytosine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 9-(2-hydroxy-ethoxymeth-yl)guanine, 9-(ethoxymethyl)guanine, 9-(2-hydroxy-ethoxymethyl)adenine and 9-(ethoxymethyl)adenine.
These nucleoside analogues yield phospho-nates (and di- and triphosphate-phosphonate analogues) having the ~ollowing general structures Z 20 ~ ~
.P-C-CH B
Z11 1 ,-o~J
Y CH CH
R2 Rl ~ P-C-(CH2)2-CH B
Z10 I I ~ I
Y CH ~CH , and R2 Rl 10~p - C - ( CH 2 ) 4 - CH B
Y CH fH
R2 Rl ~ Y' Zl~ Z2~ B~ Rl, R2 and R3 are as des-cribed above. In preferred embodiments, X is hydrogen or hydroxyl and Y is hydrogen. Thus, representative compounds can have the structures shown in Table I.

Table I
Representative Structures - Structure Structure Number zlO~P CH2-(CH2)n-TH2 1 \N~NH2 Z2 ~1l ~ N ~ \ N /~ NH2 2 5~ P-7H -(cH2)n-TH2 z O / -11 - (CH2)n-CH` N / NH2 3 5~

O ~Br HN~CH=C~H

2 ~P-CH2- ( CH2 ) n-CH2 4 Z1 ~0~

OH

O ~Br H~J~/CH=C~
I ~

~P-IH ~~CH2)n~'~ 5 OH

O /Br HN Jl~/CH=C~H

2 ~ I -C--( CH2 ) -C~J 6 OH

~ ~i'7256 ~L~ CH2CH2-CH3 P-CH2-(CH2)n~CH2 ZlQ ,0 OH

HN ~

~ P-CEI2-(CH2)n-CH2 8 Z10 l,o,~

OH

o~
2 ~II-CH2-(CH2)n-CH2 N 9 Z1/ l~

OH

5~

g N ~

_p-CH2-(C~2)n~C~ 10 OH

Z 1 / 2 ( CH 2 ) n -C~2 1 1 ~ ' OH

2 p _ CH 2 - ( CH 2 ) n - I H 2 ~ 1 2 Z 2 1I N~N ~
~p-CH ~(CH2)n-CH2 13 Z 1 OH ¦ O

O Br ll CH=C
HN~ ~ ~H

Z20~1i O N
Z 1--P CH 2 ~ ( CH2 ) n~C 1 20 ¦ 14 \~ .
OH

O /Br HN~CH=C~ H
I I
Z2O 11 O~N/
Z 1 ~P - ,CHH ~ ( CH 2 ) n ~ 1 5 OH

25~

O Br ~ CH=C~
HN ~ \EI

z2o~1l O~N~
Z1--P-~-(CH2)n CIH2o 1 16 OH

HN/~

z2` 1l O~N~
Z10-- CH2-(CH2)n-cH2 ¦ 17 OH

HN/~CF3 0 ,1 11 Z2~ 1¦ --N/

Z10~P~CH2~(CH2)n C1~20 ¦ 18 OH

~ ~72~6 N~2 H~ I

Z 1--P - CH 2 - ( CH 2 ) n - Cl~ 2 1 19 N ~
Z2` 11 N ~N~ NH2 P-CH2- ( CH2 ) n CH2 1 2 0 ~~ NH
Z O 1l <~ J`NH
2 ~P-ÇH -(CH2)n-CH2 1 21 2~6 Z2 1l < ~ ~HNH2 ~ p-C ~(CH2)n-CH2 ~ 22 Z 1 ll I O~ I
O CH~ CH

Table II
Representative Compounds Monophosphate Analogues Compound Number Structure Number Zl _ n la 1 H H O
lb 1 CH3 CH3 O
lc 1 H CH3 O
ld 1 H C2H5 O

2a 2 H H O
2b 2 CH3 CH3 O
2c 2 H CH3 O
2d 2 H C2H5 O

3a 3 H H O
3b 3 CH3 CH3 3c 3 H CH3 O
3d 3 H C2H5 22a 22 H H O
22b 22 CH3 CH3 22c 22 H CH3 o 22d 22 H C2~5 O

1 ~ -Diphosphate Analogs Compound Number Structure Number Zl Z2 n le 1 H H 2 lf . 1 CH3 CH3 2 5 lg 1 H CH3 2 lh 1 H C2H5 2 2e 2 H H 2 2f 2 CH3 CH3 2 2g 2 H CH3 2 102h 2 H C2H5 2 22a 22 H H 2 22b 22 CH3 CH3 2 22c 22 H CH3 2 22d 22 H C2H5 2 15 Triphosphate Analogues Compound Number Structure Number Zl Z2 n These are merely representative compounds as it will be apparent to those skilled in the art that other combinations of substituents and bases could be employed as well.

. ~

25~i ~ -15-Preparation The compounds of this invention can be prepared by the following general procedures: The non B-pentofuranose materials such as materials having structures l, 2, 12, 13, 20, 21, and 22 in Table I can be made with the represen1::ative reaation sequence I.

I.
' tRo)3P + BrCH2(CH2)nCH2Br ~ (RO)2 CH2(CH2)nCH2Br 1. NaOAc/DMFi 2. H+/EtOH

(Ro)2l~cH2(cH2)ncH2ocH2cl CH20 (RO)2PCH2(CH2)nCH2oH
~ HCl ¦tris(tri~ethyl Lsilyl) guanine R Gu. Gu.
(RO)2~cH2(cH2)ncH2OcH2 BrSiMe3 or (HO)2~cH2(cH2)ncH2OcH2 1. NaOCH2C6H5 2. Pd-H

Scheme I

In this sequence, a trialkyl (C2, C4 or C6) phosphite reacts with a dibromoalkane in an arbuzov reaction to give the bromoalkyl phosphonate (See J Am Chem Soc. 87 (2), 253 (l965)).

Displacement of the bromide using sodium acetate in DMF
followed by hydrolysis of the acetate ester gives diethyl 3-hydroxypropylphosphonate (i.e.) in sequence I, R = C2H5, n = 1).
This material is chloromethylated to the chloromethylester and then reacted with a suitably protected base such as tris-trimethylsilylguanine by the method of Kelley, et al, '`~;' s~i J Med Chem, 24 1528 (1981). This yields the phosphono product, e.g. 9-(3-phosphonopropyloxymethyl)guanine, directly. Phosphonate esters are smoothly cleaved by bromotrimethylsilane to the phosphonic ester by the method of McKenna, et al, Tet Letts, 155 (1977).
The syntheses of a representative deoxy-riboside is illustrated in Scheme II.
Oxidation of 2',3'-0-isopropylidine-5-propyluridine (1) by the Moffatt procedure, Pfitzner and Moffatt J Am Chem Soc, 85 3027 (1963) will yield the 5'-aldehyde. The reaction of (2) by the Wittig reagent prepared as shown in Scheme (2) gives the chain-extended, unsaturated "nucleotide" (4). Hydro-genation and deacetonation of the nucleotide (4) will give the partially unblocked nucleotide (5).
The conversion of the riboside (5) to the deoxyriboside (7) is accomplished by a strategy des-cribed recently by Lessor and Leonard, J Org Chem, ~6 4300 (1981), which was based on the selective partial deacylation of fully acylated nucleosides outlined by Ishido, et al, J Chem Soc, Perk I, 563 (1980). Thus benzoylation of (5) to the 2',3'-di-O-benzoate fol-lowed by treatment with hydroxylaminium acetate in dry pyridine will give the 3'-benzoate (6). Thiobenzoyla-tion of (6) followed by treatment with tributyl tinand debenzoylation using sodium benzyloxide, converts the 2'-hydroxyl to H, deacylates the 3'-O-benzoate, and substitutes the phenyl phosphate ester with benzyl phosphate, Jones and Moffatt J Am Chem Soc, 90 5337 (1968). Hydrogenolysis removes the benzyl ester and gives the desired phosphonic acid (7).

2~F~

Alternatively, compounds in the 2-~eoxy-ribose series are prepare!d from the 2'-deoxynucleoside by chemistry outlined in Scheme III. Thus, selective tritylation followed by mesylation of 5-propyl-2'-deoxyuridine (1) gives (8), which is converted to the2,3'-cyclonucleoside (9) using sodium benzoate in DMF
(Yung and Fox, J Am Chem Soc, 83 3060 (1961)). Oxida-tion to the aldehyde (10) followed by a l~ittig conden-sation gives the unsaturated phosphonate (11). Hydro-genation and ring-opening (Yung and Fox) gives the phosphonate ester (7), which can be deblocked, (U.S.
Patent 3,524,846 and J Am Chem Soc, 90 5337 ~1968)) to tha free phosphonic acid. Hydroboration of (11) followedby treat-ment with sodium benzoate in dimethyl formamide (DMF) gives a mixture of 5'-hydroxy and 6'-hydroxy isomers.
Oxygen functionality a to the p~osp~onate can be introduced by the chemistry outlined in Scheme IV. The appropriate propargyl phosphonic acid (13) can be prepared from tetrahydropyranyl-propargyl alco-hol (12) by the procedures outlined by Chattha andAquiar, J Org Chem, 36 2719-20 (1971)o Se'ective hydrogenation to the vinyl phosphonate followed by the sequence outlined in Scheme IV results in phosphonate analogues with one or two hydroxyls in the phosphonate chain.

2~

(RO)3P BrtCH2)n+1Br (RO)2P(CH2) +1Br (RO)2P(CH2) +1P-~3 R=C"H, I Br O l (RO) ~P(CH~-CH~P~3

3,C~ C~I17 /S ~,C~7 HOCU~ O~ CV'~¦ (R)P(CH2)nCH~CH O~

~IP 1 ~IP~ 2 ~
,C3H7 ¦ J~ C3H7 Ol (RO)2P(CH2)nCH2CH2 (RO)2P( CH 2)nCH2C,~O V \
~ f~l OBz OH H H

(R'O)lP(CH2)nCH2CH2 0 OH
7 n-O, 2, 4 R~'CGH5~ H
Scheme II

s~

3~C3H7 HOCH~ IrOC~ TrOl~

~ 2 . ~ D~ ~C3H7 (RO) 2P (CH2)nC~Ck~ O-C~ 1O

lI \ 1 hydrobora~ion O
1. IH] O \ 2. NaOBz/DM~
2. NaOBz/DMF ~JN~ O~J~

O ~ 11 OH
R H R=H
Scheme III

5~

HC-C(CH2)nCH2OR' l. BuLi ~ (RO)2PC--C(CH2)nCH2OP`~
l2 2. (RO)2PCl l3 11H] Lindlar catalyst O
(RO)2PCH-CH(CH2)nCH2Br ~ (RO)2PCHnCH(Q2)nCH20R' R'~tetrahydropvranyl R'-H
. 03p R'-Ms ~ r2. OH- ~ hydroboration O O
(RO)2PCH=CH(CH2)nHC8P(0)3 (RO)2PCHCH2(CH2)nCI120Ms ¦ 1~ H
2. LiBr O
Wittig reaction 11 (RO)2PCHCH2(CH2)nCH2Br O OH

(RO)2PCH~CH(CH2)nHC-CH O Base ~ 11 OH (RO)2PCHCH2(CH2)nCH=P(Cl)3 OH
Hydroboration . ~ittig reaction ~ . 2. Hydrogenate O H OH 1l ~
(RO)2PCIHCH2(CH2)nCH - C V Base (RO)2PCIHCHz(CHz)nCH2C ~ O Base ~ ~/1 R~alkyl Rsalkyl R-H R-H
Sch~me IV

~ ~ 5t~ 5 ~

Carbonyl analogs can be prepared by the chemistry outl.ined in Scheme V. This method involves Arbuzov reaction of triethylphosphite with ~-acetoxy-propionyl chloride (14) by the method of Yamashita, et al, Bull Chem Soc Japan, 53~6) 1625 (1980).

(EtO~3P t ClCCH2CH20Ac ol 1l 14 15 ¦ l. NaOEt -- 12. p3P-Br2 (EeO) 2P-CCH2CH=03 ~ - 2 OH- (EtO) zP-CCH2CH2Br OC~

NJ~C3H, ' ~3~

~E~O) ~ CCN~CIi~ $ 2, Na O /D~ ' (RO~ ) CH ,C~

lB
-- 19 R H, n 1 Scheme V

This gives the a-carbonyl p~osphonate ester (15).
Conversion of 15 to the bromoethyl derivative 16 followed by reaction with triphenylphosphine will give the Wi-ttig reagent ~17). Condensation of 17 with the appropriate aldehyde (e.g. 10) gives the olefin 18, which, after hydrogenation and deblocking, results in the desired product 19, a nucleoside diphosphonate analog. The nucleoside triphosphate analog 19 (wherein n = 3) can be prepared starting from 5-acetoxyvalerylchloride.
The nucleoside monophosphonate analog (20) can be prepared by the chemistry outlined in Scheme ~rI .
o ~C3H7r~J~C3H7 CI12 1 OC~ CU2 Hydroboration ¦ O
~ 0/ l~C3H7 ClCC~ " 1CrO3-pyridine HOCH2C~

(EtO)3P ¦ o N~C3H7 /~N~
l 1 NaOB~ DHI ~ (HO) P-CCH2 0 B
0 (EtO)2P-CC~ ~0\ 2 OH

Sche~e VI

5~

Salts Physiologically acceptable salts of com-pounds of this invention are prepared by methods known in the art. The salts include ammonium salts and salts of physiologically acceptable metals, partic-ularly Li+, K+, Na+, Ca+~ and Mg+~, and are novel compounds and comprise a further aspect of the inven-tion. Metal salts can be prepared by reacting a metal hydroxide with a compound of the invention. Examples of metal salts which can be prepared in this way are salts containing Li~, ~a+, and K+. A less soluble metal salt can be precipitated from a solution of a more soluble salt by addition of a suitable metal com-pound. Acid salts can be prepared by reacting a com-pound of the invention with an acid such as HCl, HBr,H2SO4, or an organic sulp~onic acid.

Pharmaceutical Preparations _ The compounds of this invention (including the physiologically acceptable salts thereof) have antiviral ac~ivity. They present activity against Herpes Simplex viruses and related viruses for example Herpes Simplex virus I, Herpes Simplex virus II, Epstein-Barr virus, varicella ~oster virus, and cyto-megalo virus. Thus the compounds can be formulated into p~armaceutical preparations. Such preparations are composed of one or more of the compounds in asso-ciation with a pharmaceutically acceptable carrier.
The book Remington's Pharmaceutical Sciences, 15th Ed by E.W. Martin (Mark Publ. Co., 197S) discloses typ-ical carriers and methods of preparation, The compounds may be administered topic-ally, orally, parenterally (e.g. intraveno~sly, by 25~

intramuscular injection, or by intraperitoneal injec-tion or the like depending upon the nature of the viral infection being treated.
For internal infections the compositions are administered orally or parenterally at dose levels of about 0.1 to 300 mg/kg, preferably 1.0 to 30 mg/kg of mammal body weight and can be used in man in a unit dosage form administered one to four times daily in the amount of 1 to 250 mg per unit dose. For oral administration, fine powders or granules may contain diluting, dispersing and/or surface active agents, and - may be presented in water or in a syrup, in ~apsules or sachets in the dry state or in a nonaqueous solu-tion or suspension, wherein suspending agents may be included; in tablets, wherein binders and lubrlcants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, pre-serving, suspending, thickening or emulsifying agents may be included. Tablets and granules are preferred oral administration forms and these may be coated.
For parenteral administration or for admin-istration as drops, as for eye infections, the com-pounds may be presented in aqueous solution in a con-centration of from about 0.1 to lO~, more preferably about 0.1 to 7%. The solution may contain antioxi-dants, buffers, etc.
~ lternatively, for lnfections of the eye, or other external tissues, e.g. mouth and skin the compositions are preferably applied to the infected part of the body of the patient topically as an oint-ment, cream, aerosol or powder, preferably as an oint-ment or cream. The compounds may be presented in an ointment, for instance with a water soluble ointment base, or in a cream, for instance with an oil in water 5~i cream base in a concentration of ~rom ahout 0.01 t~
10~, preferably 0.1 to 7%, most preferably about 0.5%
w/v. Additionally, viral infections of the eye, such as Herpetic keratitus may be treated by use of a sus-tained release drug delivery system as is described inthe art.
The exact regimen for administration of the compounds and compositions disclosed herein will nec-essarily be dependent upon the needs of the individual subject being treated, the type of treatment and, of course, the judgement of the attending practitioner.
The invention will be further described by the following nonlimiting examples.

Example I

O O O
Il 11 ~
15 (C2Hs)2P(cH2)3 >(C2H5O)2P(CH2)3occ~3 ~I .

(C2H50)2P(CH2)30H

Diethyl-3-hydroxypropylphosphonate Diethyl-3-bromophosphonate (12.0 g, 46 mmol, prepared by the method of Anatol ~berhard and 20 F.H. Westheimer, JACS 87 253-260 (1965)) was stirred with 12.0 g NaOAc 3H2O in 125 ml DMF heated in a steam bath. The reaction was evaporated to dryness in vacuo after 2 hours and partitioned between H2O and EtOAc, extracting the aqueous layer five times. The ethyl acetate extract was washed once with brine, dried with l~t~5~

~a2SO4 filtered, and evaporated to dryness in vacuo to yield 9.8 g light yellow oil (89~ H NMR (CDC13) ~ 1.3 (tr, 6 H), 1.5-2.0 (m, 4 H), 2.03 (s, 3 H), 4.1 (assym. quintet, 6 H)^ thin layer chromatography on SiGF developed with 2:1 EtOAc:CH2C12 gave Rf 0.30-The isolated diethyl-3-acetoxypropylphosphonate (9.8 g, 41 mmol) in 200 ml abs. EtOH was stirred with 30 ml Dowex 50 (H+) which had been rinsed three times each with H2O and EtOH. After 4-1/2 days at room temperature, another 10 ml of similarly prepared resin was added. Six hours later, the reaction was filtered and evaporated in vacuo. The quantitative yield of yellow oil was purified by dry column chromatography on 400 g silica packed in a 2.75 inch flat diameter nylon tube. The column was eluted with 1:9 MeOH:EtOAc and the appropriate fractions were cut and slurried with 1:1 MeOH:EtOAc. Filtration and evaporation ln vacuo afforded 5.33 g (66%) pale yellow oil. lH NMR
(CDC13 D20): ~ 1.30 (tr, 6 H), 1.60-2.08 (m, 4 H), 3.67 (tr, 2 H), 4.13 (quintet, 4 H); thin layer chro-rnatography on SiGF developed with 1:9 MeOH:~tOAc gave an R~ of 0.57.

9(3-phosphono-1-propyloxymethyl)guanine To 9.40 mmol silated guanine (James L.
Kelley, Mark P. Krochmal, and Howard J. Shaeffer, J Med Chem, 24 1528-1531 (1981)) in 9 ml dry toluene was added 7.60 mmol diethyl-3-chloromethoxypropylphos-phonate, prepared from diethyl-3-hydroxypropylp~os-phonate according to the procedure of Kelley, et al, followed by the addition of 2.2 ml triethylamine. The reaction was refluxed 24 hours and evaporated to dry-ness in vacuo~ The residue was digested with 70 ml EtOH and the voluminous tan solid was isolated by suc-25~i tion fil~ration. The solid was dissolved in water, made basic with conc. NH40H, and treated with excess aqueous lead diacetate. The lead salt was isolated ~y centrifugation and dissolved in 50% acetic acid fol-lowed by treatment with H2S for 20 minutes. The blacklead sulfide was removed by suction filtration through Celite. The filtrate was evaporated to dryness in vacuo, triturated in EtOH, and filtered. The residue was further triturated in DMF and filtered to yield 320 mg off white solid. This solid was dissolved in minimum water, acidified with 1 M HCl. Thereafter it was neutralized with 1 M NaOH and lyophilized. The solid residue was triturated in a mixture of D~F, H2O, and EtOH and filtered to yield 276 mg of 9(3-phospho-no-l-propyloxymethyl) quanine as a white solid (8 3~)- Anal- (CgHl2Nsosp-2~a-5H2o) C, H, N, W YmaX
(): pH 1, 255 (14, 700); pH 7, 251 (15, 600), pH 11, 257 (12, 800), 267 (12, 800), mass spectrum (T~S
derivative) m/e 591 (M+ of TMS4 derivative), lH NMR
(D2O): ~ 1.25-1.90 (m, 4 H), 3.42 (tr, 2 H), 5.50 (s, 2 H), 7.92 ~ (s, 1 H). Thin layer chromatography on SiGF developed with 7:3 CH3CN:0.1 N NH4Cl gave Rf 0.200 25~

Example II

N ~ O
(C2H50)2P(CH2)30H
H N ~ ~H2 lCl </ ~ N + (C2H5o)2p(cH2)3ocH2cl ->
N N NSi(CH3)3 Si(CH3)3 ~ ~ N

C2H5 ~\\ N--~N ~NH2 6~Chloro-9(3-diethylphosphono-1-propyloxy-_ methyl)guanine To 0.5 ~ (2.95 mmol) 2-amino-6-chloropur-ine, silated and treated with Hg(CN)2 according to the procedure of Robins and Ha~field (Morris J. Robins and 10 Peter W. Hatfield, Can J Chem, 60 547-553 (1982)) in 40 ml benzene was added a solution of 2.68 mmol ~;~5~25~j -29~

diethyl-3-chloromethoxypropylphosphonate prepared from (0.525 9) diethyl~3-hydroxypropylphosphonate according to the procedure of Kelley, et al, (James L. Kelley, Mark P. Krochmal and Howard J. Shaef.er, J Med Chem, 24 1528-1531 (1981)). The reaction was refluxed for 2 hours, cooled and 400 ml CHC13 was added. The organic phase was washed successively with 80 ml each of aque-ous saturated NaHCO3 and 1 M aqueous KI. The organic solution was dried over Na2SO4, filtered and evapora-ted to 790 mg of yellow gum. A portion of this crudematerial was used to conduct hydrolysis experiments.
The remaining material was chromatographed on a silica column. A solution of 574 mg of the crude reaction product was placed on 20 g silica packed in a column using 5:3 EtOAc:nPrOH. Elution with the same mixed solvent afforded sixteen fractions of 10-20 ml each.
Fractions 7-12 were combined to yield 258 mg of a colorless oil which spontaneously crystallized. Tri-turation in CH2C12 Et2O afforded two crops of white solid (207 mg), mp 109-110 (28~). A yield of 46% was obtained from a reaction performed on 45.2 mmol of the starting purine. Anal (C13H21ClNsO4P) ClHlN; W YmaX
( E) pH 1, 246 ( E 6600), 310 (7200) pH 7, 247 (6800), 308 (7400); pH 11 247 (6600), 308 (7100), mass spectrum: m/e 377 (M+); 1H NMR (CDC13): ~ 1.3 (tr, 6 H), 1.52-2.18 (m, 4 H), 3.58 (tr, 2 H), 4.09 (qu,

4 H), 5.48 (s. with broad base, 4 H), 7.89 (s, 1 H).
Thin layer chromatography on SiGF developed with 5:3 EtOAc:nPrOH gave Rf 0.40.

9(3-ethylphosphono-1-propyloxymethyl)guanine 6-Chloro-9(3-diethylphosphono-1-propyloxy-methyl)guanine (75 mg; 0.2 mmol) was combined with

5 ml 1 N aqueous NaOH and refluxed 1 hour. The cooled ~30-reaction was neutralized ~with Dowex 50X8 (pyridinium form) and filtered, rinsing liberally with water. The solution was partially evaporated to remove pyridine and was then lyophilized. The orange-colored residue (74 mg) was redissolved in H2O and centrifuged to remove insoluble material. The decanted solution (2 ml) was chromatographed on a 0.9 x 46 cm column of Whatman DE-52 Cellulose, HCO3 form, using a linear gradient of one liter each H2O and 0.2 M NH4H~03 after an lnitial H2O elution. Fractions (7 ml eac~) 43-47 yielded 25 mg (36~) of fluffy white solid ater three lyophilizations. Electron impact mass spectrum (T~S
derivative) showed m/l 547 (M+ of TMS derivative);
chemical ioni7ation mass spectrum (TMS derivative) showed m/l 543 (M+ + H of TMS3 derivative). lH NMR
(D2O) showed ~ 1.19 (tr, 3 H), 1.4-1.9 (m, 4H), 3.59 (tr, 2 H), 3.90 ~quintet, 2 H), 5.47 (s, 2 H), 8.2 (br-s, 1 H). Thin layer chromatograp~y behavior on 5iGF: Rf 0.40 when developed wi~h 7:3 CH3CH. 0.1 N
aqueous NH4Cl. The material had a formula of (CllH18N505P-~2O) Calc: C-37.82~ H-5.77%, N-20Ø
Found: C-38.27~, H-5.84%, N-19.65%. A W ~pectum was run on the material and 5howed W YmaX (~): pH 1~ 2~6 (10, 400), 278 shoulder; pH 7, 252 (11, 300) 271 shoulder; pH 11, 256-2~8 ~9, 600) 267 shoulder. The product was relyophilized.

* Trademark s~

Example III

N ~ O
<~ ,1 (C2H50)2P(CH2)70H

H

Cl r + (C2H50)2P(CH2)70CH2Cl -->
N N NSi(CH3)3 Si(CH3)3 P ( CH2 ) 5CH2 C2H50" 1 ~

6-Chloro-9(7-diethylphosphono-l-heptyl-methyl)guanine Diethyl 7-chloromethoxyheptylphosphonate was prepared from 1,7-dibromoheptane and triethyl-phosphite by the procedures used to prepare diethyl 3-chloromethoxypropylphosphonate (Example I). It was reacted with silated 2-amino-6-chloropurine, and mercuric cyanide as described for the preparation of 6-chloro-9(3-diethylphosphono-l-propyloxy-methyl)guanine (Example II) to give 32% of product as a colorless gum. UV Ymax pH 1: 246 nm (~ 6220), (~ 63~0); YmaX p~l 7, 247 nm (~ 5910), 310 nm ( F
6410), YmaX pH 11, 246 nm ( E 5950), 309 nm ( E 6380), mass spectrum 'H NMR (CDC13) ~ 1.9 (m, 18 H), 3.48 (t, 2 H), 4.10 (q, 4 H), 5.47 (s, 2 H), 5.88 (s, 2 H),

7.93 (s, 1 H). Thin layer chromatography on silica gel GF gave Rf 0.15 using ethylacetate:ethanol (100:1).

C 2H 50 t~ ~N J~ N ~ NH2 C2H50 ~2)5 ICH2 O

C2H50 \\ ~7 XJIN~ NH2 ~p-(CH2)5-CH2 9-(7-ethylphosp~ono-1-heptyloxymethyl)guanine:
6-chloro-9(7-diethylphosphono-1-heptyloxy-methyl)gua-nine was hydrolyzed by refluxing 1 N aqueous sodium hydroxide for 4 hours and isolated in 30% yield as described for the preparation of 9-(3-ethylphosphono-l-propyloxymethyl)guanine (Example II). ~t had R~ 0.5 on silica gel GF using acetonitrile (7:3) 0.1 N aque-ous ammonium chloride. Proton NMR (D2 ) 1.1-1.5 (m, 15 H), 3.5 (t, 2 H), 3.90 ~q, 2 H), 5.45 (s, 2 H) UV.

~L~5~ 5~

Biological Testing The compounds of Example I and II were evaluated _ vitro as antiviral agents against herpes virus.
The virus strain employed was Strain McCrae of type 1 herpes (thymidine kinase positive virus) prepared and titered in MA-104 cells and frozen at -90C until use.
Continuous passaged monkey kidney (MA-104) cells were used, with growth medium consisting of Minimum Essential Medium (MEM) supplemented with 0.1%
NaHC03 and 9% fetal calf serum. Test medium consisted of M~1 supplemented with 2% fetal bovine serum, 0.18%
NaHC03 and 50 ~ gentamicin.
The last compounds were added to test medium at a concentration of 2000 ~g/ml for use as a positive control.

Antiviral Test Method To a 96 well microtiter plate containing an established 24 hour monolayer of cells from which the medium has been decanted was added 0.1 ml of varying (one-half log10) concentrations of test compound, which incubated on the cell 15 minutes, after which 0.1 ml of virus in a concentration of 320 cell culture 50% infectious doses (CCID50)/0.1 ml was added. The plate was covered with plastic wrap and incubated at 37C, Included with the test were toxicity controls (each concentration of compound ~ test medium in place of virus), virus controls (virus + test medium in place of compound) and cell controls (test medium in place of compound and virus). The cells were examined microscopically after 72 hours for evidence of cyto-toxicity and for viral cytopathic effect (CPE).
Vidarabine was run on the same plate in parallel.

1~5~S~j Antiviral activity was determined by obser-vation of inhibition of viral CPE. This activity was expressed by minimum inhibitory concentration (MIC), defined as that dose range of compound causing 50% CPE
inhibition. A Virus Rating (VR) was also determined, which is a numerical expression of antiviral activity, weighted to take into account any cytotoxicity observed. Generally, a VR of 0.1 - 0.4 is usually indicative of slight antiviral effect, 0.5 - 0.9 indi-cates moderate antiviral effedt, and >1.0 impliesstrong antiviral activity.
The results of these are summarized in tables A and B. The test compound had a strong activity against the th~midine kinase positive type I
herpes virus. The activity was considered e~uivalent to that of vidarabine.

z~

TABLE A
Effect of compound c,f Example I and Vidarabine on Thymidine Kinase-Positive Type 1 Herpes Virus Infections in MA-104 Cells Compound of I Vidarabine cpEa cpEa Conc. Inhib. Conc. Inhib.
(~g/ml (%) (~/ml) (~) 103.2 28 3.2 28 1.0 31 1.0 56 O O
VRb 1.4 ~ 1.3 aCytopathic effect, % cell alteration or destruction.
bVirus rating, a numerical expression of antiviral activity (Sidwell et al, Appl Microbiol, 22:797, (1971), 0.1 - 0.4 = slight activity, 0.5 - 0.9 =
moderate activity, > 1.0 = strong activity.
CMinimum inhibitory concentration - that dosage range wherein a 50% CPE inhibition is seen.

5~i TABLE ~
Effect of compound oE Example II and Vidarabine on Thymidine Kinase Positive Type 1 Herpes Virus in MA-104 Cells (Two Tests) Compound of II Vidarabine (Control) Testl Test2 Testl Test2 cpEa cpEa cpEa cpEa Conc. Inhib. Inhib. Conc. Inhib. Inhib.
(~g/ml (%) ~ (~/ml) (%) (%) 103.2 96 52 3.2 2 0 1.2 69 48 1.0 0 0 O O
VRb >2.0 >1.4V~b 0.8 07 MIcc <1.0 <1.0MIcc 10 10 15MTDd 320 >1000 10 10 aCytopathic effect, ~ cell alteration or destruction.
bVirus rating, a numerical expression of antiviral activity (Sidwell et al, Appl Microbiol 22:797, (1971), 0.1 - 0.4 = slight activity, 0.5 ~ 0.9 =
moderate activity, > 1.0 = strong activity.
CMinimum inhibitory concentration - that dosage range wherein a 50% CPE inhibition is seen, ~g/ml.
dMaximum tolerated dose, ~g/ml.

The compound of Example II was further evaluated by the above-described ln vitro test method against cytomegalo virus. The compound was strongly active with a VR of 2.1-2.3.
The compound of Example II was tested ln vivo in guinea pigs as an agent against thymidine kinase positive herpes virus. The animals were innoculated with the virus. Eighteen hours later the material of Example II (0.4% solution in water or 1-2 solution), a 5% solution of acyclovir or a 1.4%
solution o poly(vinylalcohol) was administered and five days later blister diameters at the point of innoculation were measured. Satellite lesions were measured as well.
The results of these tests are given in Table C and show that the compound has superior activity against TK+ virus.

TABLE C

Effect of Compound of Example II
ln vivo against Herpes Virus Placebo, 1.4~ Acyclovir, Cx of II Cx of II
25 Virus poly(vinylalcohol) 5~ 1-2~* 0.4~
TK+ 1.7** 1.0 0.9 2.1 Satellite lesions 9 4 6 11 *saturated solution **average number of lesions .. . .
.

5~

Formulation_ The following formulations based on the compounds of the invention and their preparation are representa~ive.
A formulation suitable for injections intramuscularly or intraperitoneally is prepared by cGmbining the first four of the following materials Compound of the Invention 1 gram Poly(ethylene glycol)50 grams 10 Propylene glycol50 grams Tween 80 uspension agent 1.5 grams Injectable Saline 200 ml and then adding the last material. The material forms a clear solution which is filtered and sealed in sterile containers.
A simple intravenous injection formulation is formed b~ dissolving 1 gram of an active compound in 250 ml of injsctable saline which after filtering is packaged in sterile bottles.
A cream for topical administration is formulated by stirring 10 g of active compound of the invention with 20 g of mineral cil, 40 g of petroleum jelly, 0.3 g of mixed methyl/propyl paraben and 5 9 of nonionic surfactant at 50C. Then 150 ml of water are stirred into the mixture at 50C at high speed to orm a cream and the mixture is cooled and packaged in capped tubes.
An oral dosage form i5 prepared from 10 9 of compound of the invention, 100 g of lactose, and 1 g of ~tarch which are mixed with 0.1 g of magnesium stearate in methanol to granula~eO The methanol is removed by gentle heating with stirring. A portion of this material is retained as a granular powder for oral use while the remainder is hand formed into 250 mg tablets in a manual tabletiny machine.

* Trademark ~5~5~;

The foregoing examples and formulations have been presented to illustrate the present invention and are not to be construed as limitations on the inven-tion's scope which is instead defined by the following claims.

Claims (44)

1. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
   
   l. A process for preparing a compound having the structure wherein Z1 and Z2 are the same or different and selected from the group consisting of hydrogen, the one to six carbon alkyls, phenyl and benzyl, or represent the pharmaceutically acceptable salts, X is H, OH, or together with Y = O, Y is H
   or together with X = O, n is an integer, 0, 2 or 4, R1 and R2 together complete a beta-pentafuranose sugar or R1 is H and R2 is H or -CH2OH, R3 is H or OH and B is a substituted or unsubstituted purine or pyrimidine base, a substituent for said base being selected from the group consisting of C1-3 lower alkyl, halogen and C1-3 lower alkyl substituted with 1-3 halogen moieties with the proviso that X and Y
   are both H and n is O, R1 and R2 cannot complete a .beta.-pentafuranose sugar said process comprising, when R1 and R2 do not complete a beta-pentafuranose sugar, reacting a suitably protected purine or pyrimidine use with a halomethoxyalkylphosphonate of the formula wherein Z1' Z2' X, Y, R1, R2, R3 and B are as described above and Q is halo and thereafter removing the base-protecting groups; when R1 and R2 do complete a .beta.-pentafuranose sugar and n equals other than 0, reacting a suitable nucleoside 5'-aldehyde with a suitable Wittig reagent of a formula selected from (RO)2?(CH2)?CH=p?3 (RO)2?CH=CH(CH2)nHC=p(?)3 and (EtO)2?-?CH2CH=?3 to give the olefinically unsaturated nucleoside phosphonate and thereafter saturating the olefinic bond by adding hydrogen or water across it; and when R1 and R2 do complete a beta-pentafuranose sugar and n equals O and X and Y together constitute a carbonyl, reacting a suitable nucleoside 5'-alde-hyde with a methyl Wittig reagent to yield a methylene derivative, hydrogenating the methylene derivative's double bond to give a homonucleoside, converting the homonucleoside to the corresponding acid halide by oxidation and halogenation, and treating the acid halide with trialkyl phosphite and then deprotecting.
2. 2. The process of claim 1 wherein R1 and R2 do complete a beta-pentafuranose sugar and n equals other than 0, and wherein the process comprises reacting a suitable nucleoside 5'-aldehyde with a suitable Wittig reagent of a formula selected from (RO)2?(CH2)?CH=p?3 (RO)2?CH=CH(CH2)nHC=p(?)3 and (EtO)2?-?CH2CH=?3 to give the olefinically unsaturated nucleoside phosphonate and thereafter saturating the olefinic bond by adding hydrogen or water across it.
3. 3. The process of claim 1 wherein R1 and R2 do complete a beta-pentafuranose sugar and n equals 0 and X and Y together constitute a carbonyl, and wherein the process comprises reacting a suitable nucleoside 5'-aldehyde with a methyl Wittig reagent to yield a methylene derivative, hydrogenating the methylene derivative's double bond to give a homonucleoside, converting the homonucleoside to the corresponding acid halide by oxidation and halogenation, and treating the acid halide with trialkyl phosphite and then deprotecting.
4. 4. The process of claim 1 wherein R1 and R2 do not complete a beta-pentafuranose sugar and wherein the process comprises reacting a suitably protected purine or pyrimidine base with a halomethoxyalkylphosphonate of the formula Z1, Z2, X, Y, R1, R2, R3 and B are as described above and Q is halo and thereafter removing the base-protecting groups.
5. 5. A compound having the structure wherein Z1 and Z2 are the same or different and selected from the group consisting of hydrogen, the one to six carbon alkyls, phenyl and benzyl, or represent the pharmaceutically acceptable salts, X is H, OH, or together with Y = O, Y
   is H or together with X = O, n is an integer, 0, 2 or 4, R1 and R2 together complete a .beta.-pentofuranose sugar or R1 is H and R2 is H or -CH2OH, R3 is H or OH and B is a substituted or unsubstituted purine or pyrimidine base, a substituent for said base being selected from the group consisting of C1-3 lower alkyl, halogen and C1-3 lower alkyl substituted with 1-3 halogen moieties with the proviso that X and Y
   are both H and n is O, R1 and R2 cannot complete a .beta.-pentafuranose sugar.
6. 6. The compound of claim 5 wherein R1 and R2 together complete a .beta.-pentofuranose sugar.
7. 7. The compound of claim 6 wherein B is selected from guanine, adenine, 5-iodouracil, 5-trifluorothymine, 5-iodo-cytosine, E-5-2-bromovinyluracil, 5-propyluracil and 5-ethyluracil.
8. 8. The compound of claim 6 wherein n is 0.
9. 9. The compound of claim 6 wherein n is 2.
10. 10. The compound of claim 6 wherein n is 4.
11. 11. The compound of claim 6 wherein the .beta.-pentofuranose is a .beta.-ribofuranose.
12. 12. The compound of claim 6 wherein the .beta.-pentofuranose is a .beta.-arabinofuranose.
13. 13. The compound of claim 6 wherein Z1 and Z2 are each selected from the group made up from hydrogens and one to four carbon alkyls.
14. 14. The compound of claim 13 wherein n is 0.
15. 15. The compound of claim 13 wherein n is 2.
16. 16. The compound of claim 13 wherein n is 4.
17. 17. The compound of claim 6 wherein X and Y are each hydrogens.
18. 18. The compound of claim 6 wherein X and Y together are = 0.
19. 19. The compound of claim 6 wherein X is hydroxyl and Y is hydrogen.
20. 20. The compound of claim 5 wherein R1 is hydrogen and R2 is hydrogen.
21. 21. The compound of claim 20 wherein B is selected from guanine, adenine, 5-iodouracil, 5-trifluorothymine, 5-iodocytosine, E-5-2-bromovinyluracil, 5-propyluracil, 5-ethyluracil and 6-chloroguanine.
22. 22. The compound of claim 20 wherein Z1 and Z2 are each selected from the group made up of hydrogen and one to four carbon alkyls.
23. 23. The compound of claim 22 wherein X and Y are each hydrogens,
24. 24. The compound of claim 22 wherein X and Y together are = 0.
25. 25. The compound of claim 22 wherein X is hydroxyl and Y is hydrogen.
26. 26. The compound of claim 23 wherein n is 0.
27. 27. The compound of claim 23 wherein n is 2.
28. 28. The compound of claim 23 wherein n is 4.
29. 29. The compound of claim 5 wherein R1 is hydrogen and R2 is hydroxymethyl.
30. 30. The compound of claim 29 wherein B is selected from guanine, adenine, 5-iodouracil, 5-trifluorothymine, 5-iodo-cytosine, E-5-2-bromovinyluracil, 5-propyluracil, 5-ethyluracil and 6-chloroguanine.
31. 31. The compound of claim 29 wherein Z1 and Z2 are each selected from the group made up of hydrogen and one to four carbon alkyls.
32. 32. The compound of claim 31 wherein X and Y are each hydrogens.
33. 33. The compound of claim 31 wherein X and Y are = 0.
34. 34. The compound of claim 31 wherein X is hydroxyl and Y is hydrogen.
35. 35. The compound of claim 32 wherein n is 0.
36. 36. The compound of claim 32 wherein n is 2.
37. 37. The compound of claim 32 wherein n is 4,
38. 38. A metal salt of a compound of claim 5.
39. 39. A pharmaceutical preparation comprising a compound of claim 5 in a pharmaceutically acceptable carrier.
40. 40. The compound of claim 26 which is 9-(3-phosphono-1-propyloxymethyl) guanine or its pharmaceutically acceptable salts.
41. 41. The compound of claim 26 which is 6-chloro-9-(3-diethylphosphono-1-propyloxymethyl) guanine.
42. 42. The compound of claim 26 which is 9-(3-ethyl-phosphono-1-propyloxymethyl) guanine and its pharmaceutically acceptable salts.
43. 43. The compound of claim 26 which is 6-chloro-9-(7-diethylphosphono-1-heptyloxymethyl) guanine and its pharmaceutically acceptable salts.
44. 44. The compound of claim 26 which is 9-(7-ethyl-phosphono-1-heptyloxymethyl) guanine.