U.S. patent application number 09/731256 was filed with the patent office on 2001-08-09 for acyclovir diester derivatives.
This patent application is currently assigned to Drug Innovation & Design, Inc.. Invention is credited to Glazier, Arnold, Yanachkov, Ivan, Yanachkova, Milka.
Application Number | 20010012893 09/731256 |
Document ID | / |
Family ID | 27367691 |
Filed Date | 2001-08-09 |
United States Patent
Application |
20010012893 |
Kind Code |
A1 |
Glazier, Arnold ; et
al. |
August 9, 2001 |
Acyclovir diester derivatives
Abstract
Disclosed are novel prodrugs represented by the following
structural formula: 1 Z is oxygen or sulfur; Y is, together with a
hydroxy group, acyclovir or an analog of acyclovir; A is a
substituted benzyl group with one or more protected hydroxy or
protected amine groups in the ortho or para positions, relative to
the phosphate ester, which can be converted in vivo to a hydroxy or
amino group. Also disclosed is a method of treating a viral
infection in an individual or animal. The method comprises
administering to the individual or animal a therapeutically
effective amount of a prodrug represented by structural formula
shown above.
Inventors: |
Glazier, Arnold; (Newton,
MA) ; Yanachkova, Milka; (Newton, MA) ;
Yanachkov, Ivan; (Newton, MA) |
Correspondence
Address: |
N. Scott Pierce, Esq.
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Assignee: |
Drug Innovation & Design,
Inc.
|
Family ID: |
27367691 |
Appl. No.: |
09/731256 |
Filed: |
December 6, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09731256 |
Dec 6, 2000 |
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09459178 |
Dec 10, 1999 |
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6180790 |
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09459178 |
Dec 10, 1999 |
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09079647 |
May 15, 1998 |
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09079647 |
May 15, 1998 |
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09050220 |
Mar 27, 1998 |
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09050220 |
Mar 27, 1998 |
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08857150 |
May 15, 1997 |
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Current U.S.
Class: |
544/244 |
Current CPC
Class: |
C07F 9/65616
20130101 |
Class at
Publication: |
544/244 ;
514/81 |
International
Class: |
C07F 009/6524 |
Claims
What is claimed is:
1. A prodrug of acyclovir or an acyclovir analog represented by a
structural formula selected from the group consisting of: 14and
physiologically acceptable salts thereof; wherein Z is oxygen or
sulfur; Y, together with a hydroxy group, is acyclovir or an
acyclovir analog; and A is a substituted benzyl group with one or
more protected hydroxy or protected amine groups in the ortho or
para positions, relative to the phosphoester, which can be
converted in vivo to a hydroxy or amino group.
2. The prodrug of claim 1 wherein Y is represented by the following
structural formula: 15and physiologically acceptable salts thereof;
wherein: X is sulphur, --NH--, --N(lower alkyl)- or oxygen; R.sub.a
is hydrogen, halogen, hydroxy, (lower alkyl)--O--, azide, thio,
(lower alkyl)thio, amino, (lower alkyl)amino or di(lower
alkyl)amino; R.sub.b is hydrogen, halogen, (lower alkyl)thio,
(lower alkyl) --CO--NH--, amino or azide; R.sub.c is hydrogen,
lower alkyl, substituted lower alkyl, aryl and substituted aryl;
R.sub.d is hydrogen or lower alkyl; R.sub.e is hydrogen, lower
alkyl, substituted lower alkyl, aryl, substituted aryl, (lower
alkyl)O--, (substituted lower alkyl)O--, aryloxy and substituted
aryloxy; and A is a substituted benzyl group with one or more
protected hydroxy or protected amine groups in the ortho or para
positions, relative to the phosphoester, which can be converted in
vivo to a hydroxy or amino group.
3. The prodrug of claim 2 wherein Z and X are oxygen, R.sub.a is
--OH and R.sub.b is --NH.sub.2.
4. The prodrug of claim 3 wherein R.sub.c, R.sub.d and R.sub.e are
each --H.
5. The prodrug of claim 4 wherein the one or more groups in the
para or ortho positions of A relative to the phosphoester are
selected from the group consisting of R.sub.8--O--CO--O--,
R.sub.8NH--CC--O--, and R.sub.8CO--O--, wherein R.sub.8 is selected
from the group consisting of lower alkyl, substituted lower alkyl,
aryl and substituted aryl.
6. The prodrug of claim 5 wherein the benzyl group is
.alpha.-substituted with a moiety such that cleavage of the
phosphoester bond between A and a phosphate oxygen results in an
elimination reaction to form a carbon-carbon double bond between
the .alpha.-carbon and the moiety.
7. The prodrug of claim 5 wherein the one or more groups in the
para or ortho positions relative to the phosphoester are (lower
alkyl)CO--O--.
8. The prodrug of claim 4 wherein A is represented by the following
structural formula: 16wherein: R.sub.1, R.sub.3, or R.sub.6 are
independently selected from the group consisting of --H,
--O--CO--R.sub.8, --O--CO--OR.sub.8, --O--C(O)--NHR.sub.8,
--O--C(O)--N(R.sub.8).sub.2, --NH--CO--R.sub.8, --NH--CO--OR.sub.8,
--NH--CO--NHR.sub.8 and an inert group, with the proviso that at
least one of R.sub.1, R.sub.3 or R.sub.6, is not an inert group;
R.sub.2 and R.sub.7 are hydrogen, --O--CO--R.sub.8, or an inert
group and may be the same or different; R.sub.4 and R.sub.5 are
independently selected from the group consisting of hydrogen, a
lower alkyl group, a substituted lower alkyl group and a moiety
such that cleavage of the phosphoester bond between A and a
phosphate oxygen results in an elimination reaction to form a
carbon-carbon double bond between the benzylic position of A and
the moiety; and R.sub.8 is selected from the group consisting of a
lower alkyl group, a substituted lower alkyl group, an aryl group,
a substituted aryl group, and a group such that the resulting
ester, carbonate, amide, carbamate or urea moiety is degraded to
the free phenolic hydroxy group or free amine group in vivo.
9. The prodrug of claim 8 wherein at least one of R.sub.1, R.sub.3
or R.sub.6 is a (--O--CO--R.sub.8) group.
10. The prodrug of claim 9 wherein R.sub.4 is selected from the
group consisting --CH.sub.2COOR", --CH.sub.2COR",
--CH.sub.2CONH.sub.2, --CH.sub.2CONHR", --CH.sub.2NO.sub.2,
--CH.sub.2SO.sub.2R" and --CH.sub.2CN, wherein R" is selected from
the group consisting of lower alkyl, substituted lower alkyl, aryl
and substituted aryl.
11. The prodrug of claim 10 wherein R.sub.4 is a
--CH.sub.2--CO.sub.2--R".
12. The prodrug of claim 11 wherein R" is selected from the group
consisting of --H, methyl, ethyl, n-propyl, iso-isopropyl, n-butyl,
sec-butyl, t-butyl or --(CH.sub.2).sub.nCH.sub.3, wherein n is 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
13. The prodrug of claim 12 wherein R.sub.1 is a
--O--CO--R.sub.8.
14. The prodrug of claim 13 wherein R.sub.8 is selected from the
group consisting of methyl, ethyl n-propyl, iso-isopropyl, n-butyl,
sec-butyl, t-butyl, n-pentyl, n-hexyl, CH.sub.3--CO--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--CH.sub.2--, --OCH.sub.3,
--CR.sub.9--NHR.sub.10, --CH(OR.sub.11)--CH.sub.3 and
--CH.sub.2OR.sub.11; wherein: R.sub.9 is the side chain of an amino
acid; R.sub.10 is --H or an amine protecting group; and R.sub.11 is
--H or an alcohol protecting group.
15. A prodrug represented by the following structural formula:
17and physiologically acceptable salts thereof.
16. A prodrug represented by the following structural formula:
18and physiologically acceptable salts thereof.
17. A method of treating an individual with a viral infection,
comprising administering to the individual a therapeutically
effective amount of a prodrug represented by the following
structural formula: 19and physiologically acceptable salts thereof;
wherein Z is oxygen or sulfur; Y is, together with a hydroxy group,
acyclovir or an acyclovir analog; and A is a substituted benzyl
derivative with one or more protected phenol or protected amine
groups in the ortho or para positions, relative to the
phosphoester, which can be converted in vivo to a hydroxy or amino
group.
18. The method of claim 17 wherein Y is represented by the
following structural formula: 20and physiologically acceptable
salts thereof; wherein: X is sulphur, --NH--, --N(lower alkyl)- or
oxygen; R.sub.a is hydrogen, halogen, hydroxy, (lower alkyl)--O--,
azide, thio, (lower alkyl)thio, amino, (lower alkyl) amino or
di(lower alkyl)amino; R.sub.b is hydrogen, halogen, (lower
alkyl)thio, (lower alkyl)--CO--NH--, amino or azide; R.sub.c is
hydrogen, lower alkyl, substituted lower alkyl, aryl and
substituted aryl; R.sub.d is hydrogen or lower alkyl; R.sub.e is
hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted
aryl, (lower alkyl)O--, (substituted lower alkyl)O--, aryloxy and
substituted aryloxy; and A is a substituted benzyl derivative with
one or more protected phenol or protected amine groups in the ortho
or para positions, relative to the phosphoester, which can be
converted in vivo to a hydroxy or amino group.
19. The method of claim 18 wherein the viral infection is caused by
Herpes Simplex Virus.
20. The method of claim 19 wherein the viral infection is a dermal
viral infection.
21. The method of claim 20 wherein the prodrug is administered
topically to the dermal viral infection.
22. The method of claim 21 wherein Z and X are oxygen, R.sub.a is
--OH and R.sub.b is --NH.sub.2.
23. The method of claim 22 wherein R.sub.c, R.sub.d and R.sub.e are
each --H.
24. The method of claim 23 wherein the one or more groups in the
para or ortho positions of A relative to the phosphoester are
selected from the group consisting of R.sub.8--O--CO--O--,
R.sub.8NH--CO--O--, and R.sub.8CO--O--, wherein R.sub.8 is selected
from the group consisting of lower alkyl, substituted lower alkyl,
aryl, and substituted aryl.
25. The method of claim 24 wherein the benzyl group is
.alpha.-substituted by a moiety such that cleavage of the
phosphoester bond between A and a phosphate oxygen will result in
an elimination reaction to form a carbon-carbon double bond between
the .alpha.-carbon and the moiety.
26. The method of claim 25 wherein the one or more groups in the
para or ortho positions relative to the phosphoester are (lower
alkyl)CO--O--.
27. The method of claim 23 wherein A is of the formula: 21wherein:
R.sub.1, R.sub.3 or R.sub.6 are independently selected from the
group consisting of --H, --O--CO--R.sub.8, --O--CO--OR.sub.8,
--O--C(O)--NHR.sub.8, --O--C(O)--N(R.sub.8).sub.2,
--NH--CO--R.sub.8, --NH--CO--OR.sub.8, --NH--CO--NHR.sub.8 and an
inert group, with the proviso that at least one of R.sub.1, R.sub.3
or R.sub.6 is not an inert group; R.sub.2 and R.sub.7 are hydrogen
--O--CO--R.sub.8, or an inert group and may be the same or
different; R.sub.4 and R.sub.5 are independently selected from the
group consisting of hydrogen, an alkyl group, a substituted alkyl
group and a moiety such that cleavage of the phosphoester bond
between A and a phosphate oxygen results in an elimination reaction
to form a carbon-carbon double bond between the benzylic position
of A and the moiety; and R.sub.8 is selected from the group
consisting of an alkyl group, a substituted alkyl group, an aryl
group, a substituted aryl group, and a group such that the
resulting ester, carbonate, amide, carbamate or urea moiety is
degraded to the free hydroxy group or free amine group in vivo.
28. The method of claim 27 wherein at least one of R.sub.1, R.sub.3
or R.sub.6 is a (--O--CO--R.sub.8) group.
29. The method of claim 28 wherein R.sub.4 is selected from the
group consisting of --CH.sub.2COOR", --CH.sub.2COR",
--CH.sub.2CONH.sub.2, --CH.sub.2CONHR", --CH.sub.2NO.sub.2,
--CH.sub.2SO.sub.2R" and --CH.sub.2CN, wherein R" is selected from
the group consisting of --H, lower alkyl, substituted lower alkyl,
aryl and substituted aryl.
30. The method of claim 29 wherein R.sub.4 is a
(--CH.sub.2--CO.sub.2--R") group.
31. The method of claim 30 wherein R" is selected from the group
consisting of --H, methyl, ethyl, n-propyl, iso-isopropyl, n-butyl,
sec-butyl, t-butyl or --(CH.sub.2).sub.nCH.sub.3, wherein n is 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
32. The method of claim 31 wherein R.sub.1 is a (--O--CO--R.sub.8)
group.
33. The method of claim 32 wherein R.sub.8 is selected from the
group consisting of methyl, ethyl n-propyl, iso-isopropyl, n-butyl,
sec-butyl, t-butyl, n-pentyl, n-hexyl, CH.sub.3--CO--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--CH.sub.2--,
--OCH.sub.3--CR.sub.9--NHR.su- b.10, --CH(OR.sub.11)--CH.sub.3 and
--CH.sub.2OR.sub.11; wherein: R.sub.9 is the side chain of an amino
acid; R.sub.10 is --H or an amine protecting group; and R.sub.11 is
--H or an alcohol protecting group.
34. A method of treating an individual or animal with a viral
infection, comprising administering to the individual a
therapeutically effective amount of a prodrug represented by the
following structural formula: 22and physiologically acceptable
salts thereof.
35. A method of treating an individual or animal with a viral
infection, comprising administering to the individual a
therapeutically effective amount of a prodrug represented by the
following structural formula: 23and physiologically acceptable
salts thereof.
36. A method of preparing a compound represented by the following
structural formula: 24wherein: Y' is, together with a hydroxy
group, a nucleoside analog; and A is a substituted benzyl group
with one or more protected hydroxy or protected amine groups in the
ortho or para positions, relative to the phosphite ester which can
be converted in vivo to a hydroxy or amino group; comprising
reacting Y'--OH, a weak anhydrous organic acid and
(A--O).sub.2--P(NR.sub.21R.sub.22) in an aprotic polar organic
solvent, wherein R.sub.21 and R.sub.22 are each independently a
lower alkyl, a substituted lower alkyl group, an aryl group, a
substituted aryl group or, taken together with the phosphoramide
nitrogen, are a substituted or unsubstituted five, six or seven
membered nitrogen-containing heterocyclic ring.
37. A method of preparing a compound represented by the following
structural formula: 25wherein: Y is, together with a hydroxy group,
acyclovir or an analog of acyclovir; and A is a substituted benzyl
group with one or more protected hydroxy or protected amine groups
in the ortho or para positions, relative to the phosphite ester
which can be converted in vivo to a hydroxy or amino group;
comprising reacting Y--OH, H-tetrazole and (A--O).sub.2--
P(NR.sub.21R.sub.22) in an aprotic polar organic solvent, wherein
R.sub.21 and R.sub.22 are each independently a lower alkyl,
substituted lower alkyl group, an aryl group a substituted aryl
group or, taken together with the phosphoramide nitrogen, are a
substituted or unsubstituted five, six or seven membered
nitrogen-containing heterocyclic ring.
38. The method of claim 37 wherein A is represented by the
following structural formula: 26wherein R.sub.4 is
--CH.sub.2COOCH.sub.3 and R.sub.8 is methyl or t-butyl.
39. The method of claim 38 wherein the aprotic polar solvent is
dimethylformamide and R.sub.21 and R.sub.22 are each ethyl.
40. A method of preparing a prodrug represented by the following
structural formula: 27wherein: Y is, together with a hydroxy group,
acyclovir or an analog of acyclovir; and A is a substituted benzyl
group with one or more protected hydroxy or protected amine groups
in the ortho or para positions, relative to the phosphite ester
which can be converted in vivo to a hydroxy or amino group;
comprising the steps of: a) reacting Y--OH, H-tetrazole and
(A--O).sub.2--P(NR.sub.21R.sub.22)in an aprotic polar solvent,
wherein R.sub.21 and R.sub.22 are each independently a lower alkyl,
a substituted lower alkyl group, an aryl group, a substituted aryl
group or, taken together with the phosphoramide nitrogen, are a
substituted or unsubstituted five, six or seven membered
nitrogen-containing heterocyclic ring, thereby producing an
intermediate represented by the following structural formula: 28b)
reacting the intermediate with hydrogen peroxide thereby forming
the prodrug.
41. The method of claim 40 wherein A is represented by the
following structural formula: 29wherein R.sub.4 is
--CH.sub.2COOCH.sub.3 and R.sub.8 is methyl or t-butyl.
42. The method of claim 41 wherein the aprotic polar is
dimethylformamide, R.sub.21 and R.sub.22 are each ethyl and step b)
is performed without isolating the intermediate.
43. A method of preparing a prodrug represented by the following
structural formula: 30wherein: Y is, together with a hydroxy group,
acyclovir or an analog of acyclovir; A is a substituted benzyl
group with one or more protected hydroxy or protected amine groups
in the ortho or para positions, relative to the phosphate ester
which can be converted in vivo to a hydroxy or amino group;
comprising the steps of: a) reacting Y--OH, a weak anhydrous
organic acid and (A--O).sub.2--P(NR.sub.21R.sub.2- 2) in an aprotic
polar solvent, wherein R.sub.21 and R.sub.22 are each independently
a lower alkyl, substituted lower alkyl group, an aryl group, a
substituted aryl group or, taken together with the phosphoramide
nitrogen, are a substituted or unsubstituted five, six or seven
membered nitrogen-containing heterocyclic ring, thereby producing a
first intermediate represented by the following structural formula:
31b) reacting the first intermediate with a phosphite oxidizing
agent, thereby forming a second intermediate represented by the
following structural formula: 32c) reacting the second intermediate
with a base to form the prodrug.
44. The method of claim 43 wherein A is represented by the
following structural formula: 33wherein: R.sub.8 is selected from
the group consisting of methyl, ethyl, n-propyl, iso-isopropyl,
n-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl,
CH.sub.3--CO--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--CH.sub.2-- and --OCH.sub.3; and R"
is selected from the group consisting of --H, methyl, ethyl
n-propyl, iso-isopropyl, n-butyl, sec-butyl, t-butyl.
45. The method of claim 44 wherein: a) R.sub.4 is --CH.sub.2CCOR";
b) R.sub.8 is methyl or t-butyl; c) the aprotic dipolar solvent is
a dialkyl amide solvent; d) R.sub.21 and R.sub.22 are each ethyl;
and e) the weak anhydrous organic base acid is H-tetrazole.
46. The method of claim 45 wherein the base an amine base and the
dialkylamide solvent is dimethylformamide.
47. A method of preparing a prodrug represented by the following
structural formula: 34wherein: Y is, together with a hydroxy group,
acyclovir or an analog of acyclovir; A is a substituted benzyl
group with one or more protected hydroxy or protected amine groups
in the ortho or para positions, relative to the phosphate ester
which can be converted in vivo to a hydroxy or amino group; said
method comprising the step of reacting a base with a compound
represented by the following structural formula: 35thereby forming
the prodrug.
48. The method of claim 46 wherein A is represented by the
following structural formula: 36wherein: R.sub.8 is selected from
the group consisting of methyl, ethyl, n-propyl, iso-isopropyl,
n-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl,
CH.sub.3--CO--CH.sub.2,--,
CH.sub.3O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2O--CH.sub.2-- and --OCH.sub.3; and R" is
selected from the group consisting of --H, methyl, ethyl n-propyl,
iso-isopropyl, n-butyl, sec-butyl, t-butyl.
49. The method of claim 48 wherein: a) R.sub.4 is --CH.sub.2COOR";
b) R.sub.8 is methyl or t-butyl; and c) R.sub.21 and R.sub.22 are
each ethyl.
50. The method of claim 49 wherein the base is an amine base.
51. A method of preparing a prodrug represented by the following
structural formula: 37wherein: Y' is, together with a hydroxy
group, a nucleoside analog; A is a substituted benzyl group with
one or more protected hydroxy or protected amine groups in the
ortho or para positions, relative to the phosphate ester which can
be converted in vivo to a hydroxy or amino group; said method
comprising the step of reacting a base with a compound represented
by the following structural formula: 38thereby forming the prodrug.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/050,220, filed Mar. 27, 1998, which is a continuation
application of U.S. Ser. No. 08/857,150, filed May 15, 1997, the
entire teachings of which are incorporated herein by reference.
BACKGROUND
[0002] Antiviral agents currently in use are of limited
effectiveness in treating dermal infections caused by viruses. For
example, herpes simplex labialis, commonly referred to as "cold
sores" do not respond to the topical treatment with acyclovir
(Spruance et al., Am. J. Med., 73(1A):315-319 (1982); Shaw et al.,
Br. Med. J. (Clin. Res. Ed.), 291(6487):7-9 (1985); Raborn, et al.,
Oral Surg. Oral Med. Oral Pathol., 67(6):676-679 (1989); Spruance,
et al., Antimicrob. Agents Chemother., 25(5):553-555 (1984);
Raborn, et al., J. Can. Dent. Assoc., 55(2):135-137 (1989)). Oral
administration of acyclovir for the treatment of cold sores is only
partially effective (Spruance et al., J. Infect. Diseases 161:185
(1990)).
[0003] The limited effectiveness of antiviral agents such as
acyclovir applied topically to cold sores and other dermal viral
infections is thought to be a consequence of the limited ability of
most of these agents to penetrate the skin (Parry, et al., J.
Invest. Dermatol., 98(6):856-863 (1992); Spruance, et al.,
Antimicrob. Agents Chemother., 25(1):10-15 (1984)) . Topical
treatments for genital herpes infections are also ineffective for
the same reason. Consequently, there is a need for new antiviral
agents which can penetrate the skin and which are active against
viruses which cause dermal infections.
SUMMARY OF THE INVENTION
[0004] Lipophilic phosphotriester prodrugs of acyclovir, as
disclosed in U.S. Ser. No. 08/635,653, filed Apr. 22, 1996, (the
entire teachings of which are hereby incorporated by reference),
are extremely active topically against herpes virus infection. The
present invention is based on the discovery that the corresponding
phosphodiester prodrugs of acyclovir, which are nonlipophilic and
water soluble, are even more effective antiviral drugs than
phosphotriesters and more effective than acyclovir. For example,
treatment of guinea pigs with dermal herpes simplex virus-1 (HSV-1)
infections with Prodrug 1 and Prodrug 2 resulted in a dramatic
reduction in lesion number, in lesion area and in lesion virus
titer (Examples 1 and 3) when compared to its vehicle. In contrast,
comparable treatment with U.S. ZOVIRAX (acyclovir) provided results
that were statistically the same as with placebo. 2
[0005] In one embodiment, the present invention is a prodrug of
acyclovir, an analog of acyclovir, acyclovir monophosphate or an
acyclovir monophosphate analog. The prodrug is represented by
Structural Formula (I): 3
[0006] and physiologically acceptable salts thereof.
[0007] Z is oxygen or sulfur, preferably oxygen.
[0008] Y is, together with a hydroxy group, acyclovir or an analog
of acyclovir.
[0009] A is a substituted benzyl group with one or more protected
hydroxy or protected amine groups in the ortho or para positions,
relative to the phosphate ester, which can be converted in vivo to
a hydroxy or amino group.
[0010] Another embodiment of the present invention is a method of
treating a viral infection in an individual or animal. The method
comprises administering to the individual or animal a
therapeutically effective amount of a prodrug represented by
Structural Formula (I).
[0011] Another embodiment of the present invention is a method of
preparing the prodrugs of the invention and intermediate used in
the synthesis of phosphate ester prodrugs, including the diester
prodrugs represented by Structural Formula (I), phosphotriester
prodrugs disclosed in U.S. Ser. No. 08/310,972, filed Sep. 23,
1994, and the phosphotriester prodrugs disclosed in U.S. Ser. No.
08/635,553. The intermediate is represented by Structural Formula
(II): 4
[0012] Y' is, together with a hydroxy group, a nucleoside analog,
preferably acyclovir or an acyclovir analog.
[0013] A is a substituted benzyl group with one or more protected
hydroxy or protected amine groups in the ortho or para positions,
relative to the phosphate ester, which can be converted in vivo to
a hydroxy or amino group.
[0014] The method for preparing the intermediate comprises reacting
Y'--OH, a weak anhydrous organic acid such as tetrazole and
(A--O).sub.2--P (NR.sub.21R.sub.22) in an aprotic, polar organic
solvent. Suitable aprotic, polar organic amide solvents include
dialkyl amide solvents (e.g., dimethylformamide (DMF) or
N,N-dimethylacetamide), tetraalkylurea solvents (e.g.,
1,3-dimethylimidazolinone or
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone); dialkyl
sulfoxide solvents (e.g., dimethylsulfoxide and tetramethylene
sulfoxides) and phosphoramide solvents (e.g.,
hexamethylphosphoramide). Preferred solvents are dialkyl amides.
DMF is most preferred. R.sub.21 and R.sub.22 are each independently
a lower alkyl, an aryl group or, taken together with the
phosphoramide nitrogen, can form a five, six or seven membered
nitrogen-containing heterocyclic ring (preferably a saturated
heterocyclic ring). The lower alkyl, aryl or heterocyclic compound
represented by R.sub.12 and R.sub.22 can optionally be substituted
with one or more groups which do not react (or are substantially
inert) with the P--N bond or with the weak acid. Preferred examples
of substituents include non-nucleophilic, non-basic inert groups
such as halides, (lower alkyl)--O--, lower alkyl and aryl.
Preferably, R.sub.21 and R.sub.22 are each a lower alkyl group such
as ethyl.
[0015] The phosphodiester acyclovir prodrugs disclosed herein are
more active against dermal herpes simplex virus 1 infections than
acyclovir or the phosphotriester prodrugs of acyclovir disclosed in
U.S. Ser. No. 08/635,553. In addition, the phosphodiester prodrugs
of acyclovir disclosed herein are chemically more stable, have
longer shelf lives and are generally easier to formulate than the
phosphotriester prodrugs disclosed in U.S. Ser. No. 08/635,553.
BRIEF DESCRIPTION OF THE FIGURE
[0016] FIGS. 1A and 1B represent a schematic showing a synthetic
scheme used to prepare Prodrug 1.
[0017] FIG. 2 is a schematic showing the synthesis of phosphate
triesters by reacting the nucleoside analog Y--OH,
(A--O).sub.2--P--N--(ethyl).sub.- 2 and tetrazole in a
dimethylformamide and oxidizing the resulting phosphite triester
product witn hydrogen peroxide.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Disclosed herein are novel prodrugs of drugs such as
acyclovir, which are nucleoside analogs. The disclosed prodrugs
show increased effectivenes compared with the parent drugs. Also
described are improved methods of preparing phosphate triester
prodrugs of nucleoside analogs. These methods are also useful in
the preparation of the disclosed phosphodiester prodrugs.
[0019] The effectiveness of antiviral agents such as acyclovir and
acyclovir analogs can be enhanced by converting the agent into a
phosphorus containing prodrug represented by Structural Formula
(I). Phosphorus containing prodrugs are described in U.S. Ser. Nos.
08/635,553, 07/714,130, 07/537,332 and 08/310,972, the entire
teachings of which are hereby incorporated into this application by
reference.
[0020] A prodrug, as used herein, is an agent which can be
metabolized in vivo, i.e. undergoes biotransformation, to give the
pharmacologically active agent or a monophosphate of the
pharmacologically active agent. "Metabolized" refers to chemical or
biochemical reactions which the prodrug undergoes in vivo. Examples
include enzyme catalyzed reactions and reactions which occur in
solution such as solvolysis, hydrolysis and elimination
reactions.
[0021] Use of a prodrug for treating an individual can have
advantages over the parent drug, e.g. greater lipophilicity to
enhance delivery of the pharmacologically active agent across cell
membranes or into the stratum corneum of the skin. Accordingly,
lipid solubility is a desirable property for antiviral drugs. The
prodrugs of the present invention have a lipophilic portion that
allows the prodrug to penetrate into the stratum corneum of the
skin and yet are charged molecules which are highly soluble in
water and which can be transformed in vivo into acyclovir, an
analog of acyclovir, acyclovir monophosphate or an analog of
acyclovir monophosphate.
[0022] As used herein, an "acyclovir analog" is an antiviral purine
with a substituted alkyl group (e.g., a C3 to about a C12
substituted alkyl group) bonded to nitrogen nine. As used herein, a
"purine" has a pyrimidine ring fused to an imidazole ring. It will
be understood that tautomeric forms of a purine are also included,
such as in the structure shown for Prodrug 1. Examples of purines
include adanine and guanine.
[0023] The carbon atoms of the purine can be bonded to or
substituted by, for example, a hydrogen, halogen, hydroxy, (lower
alkyl)--O--, thio, (lower alkyl)thio, amino, (lower alkyl)amino,
di(lower alkyl)amino, (lower alkyl)--CO--NH-- or azide.
[0024] The alkyl or substituted alkyl group bonded to nitrogen nine
of the purine (nitrogen nine is indicated in Structural Formula
(III) can optionally have an ether, thioether or amine moiety
linkage within the chain and is straight chained or branched. The
substituted alkyl group can have one or more substituents, such as,
hydroxy, amino, --NH(lower alkyl), (lower alkyl)--O--, (substituted
lower alkyl)--O--, aryl, substituted aryl, aryloxy, substituted
aryloxy, (lower alkyl)NH--SO.sub.2--O--, (substituted lower
alkyl)NH--SO.sub.2--O--, (aryl)NH--SO.sub.2--O--, (substituted
aryl)NH--SO.sub.2--O--, phosphate, --NH--CO--(lower alkyl),
--NH--CO--(substituted lower alkyl), --NH--CO--aryl,
--NH--CO--(substituted aryl), (lower alkyl)--CO--, (substituted
lower alkyl)--CO--, --CO--aryl and --CO--(substituted aryl). Lower
alkyl, substituted lower alkyl, aryl and substituted aryl are
defined hereinbelow.
[0025] In a preferred embodiment, an "acyclovir analog" is
represented by Structural Formula (III): 5
[0026] X is sulphur, --NH--, --N(lower alkyl)--, or oxygen;
[0027] R.sub.a is hydrogen, halogen, hydroxy, (lower alkyl)--O--,
azide, thio, (lower alkyl)thio, amino, (lower alkyl)amino or
di(lower alkyl)amino;
[0028] R.sub.b is hydrogen, halogen, (lower alkyl)thio, (lower
alkyl)--CO--NH-- (referred to herein as "acylamino"), amino or
azide;
[0029] R.sub.c is hydrogen, lower alkyl, substituted lower alkyl,
aryl, substituted aryl. Aryl is preferably phenyl;
[0030] R.sub.d is hydrogen, lower alkyl and substituted lower
alkyl;
[0031] R.sub.e is hydrogen, lower alkyl, substituted lower alkyl,
aryl, substituted aryl, (lower alkyl)O--, (substituted lower
alkyl)O--, aryloxy and substituted aryloxy; and
[0032] R.sub.f is hydroxy.
[0033] In a preferred embodiment, Y is represented by Structural
Formula (IV): 6
[0034] As seen from Structural Formula (IV), when acyclovir or an
acyclovir analog is used to form a prodrug of the present
invention, R.sub.f in Structural Formula (III) is a covalent bond
between acyclovir or the acyclovir analog and an oxygen bonded to a
phosphorus atom of the prodrug. For example, in Structural Formula
(I) R.sub.f is a covalent bond between Y and an oxygen bonded to
phosphorus, referred to herein as a "phosphoester bond". The
covalent bond between A and O is also a phosphoester bond.
[0035] Specific examples of suitable acyclovir analogs are provided
in U.S. Pat. Nos. 4,199,574, 4,294,831 and 4,323,573, the entire
teachings of which are hereby incorporated into this patent
application by reference.
[0036] In a preferred embodiment, R.sub.a is --OH, R.sub.b is
--NH.sub.2 and X is oxygen. It is most preferred that R.sub.c,
R.sub.d, and R.sub.e are each --H and R.sub.f is a phosphoester
bond between Y and an oxygen of the phosphate of the prodrug, i.e.
Y, together with a hydroxy group, is acyclovir.
[0037] The following is a description of the present invention with
respect to prodrugs comprising phosphate groups, e.g., prodrugs
represented by Structural Formula (I).
[0038] A is a group which can be metabolized in vivo to give a
chemically modified A (A'). As a result of the biotransformation,
the phosphoester of the prodrug which comprises modified A (A')
undergoes cleavage in vivo. Cleavage of the phosphoester can
result, for example, from the heterolytic cleavage of the
oxygen-carbon bond of the phosphoester group comprising a modified
A (A'). In this instance, the phosphate acts as a leaving group.
Cleavage in vivo of a phosphoester comprising modified A (A') will
be enhanced relative to a phosphoester comprising A if
biotransformation in vivo results in a greater electron density on
the carbon atom to which the phosphate is bonded in modified A (A')
than on the carbon atom to which the phosphate is bonded in A.
[0039] For example, a compound having a structure represented by
Structural Formula (I) will be cleaved more rapidly in vivo when A
is a benzyl with an electron donating group in the ortho or para
position than when A is an unsubstituted benzyl group. Thus, A can
be, for example, a substituted benzyl group that undergoes
biotransformation in vivo such that groups already present at the
ortho and/or para positions are converted into groups that are more
strongly electron donating in modified A (A') than in A.
[0040] Protected hydroxy groups such as acyloxy groups (e.g., lower
alkyl--CO--O-- and aryl--CO--O--), carbonate groups (e.g.,
--O--CO--O--lower alkyl and --O--CO--O--aryl), carbamate groups
(e.g., --O--CO--NH--lower alkyl and --O--CO--NH--aryl) and
protected aryl amine groups such as acylamine groups (e.g., lower
alkyl--CO--NH-- and aryl--CO--NH--) are only very slightly electron
donating, but can be converted (e.g. unmasked) in vivo into the
strongly electron donating hydroxy or amino groups, respectively.
For example the Hammett para sigma+ constant for the acetoxy group
and the acetyl amino group are -0.06 and -0.60, respectively. In
contrast, the hydroxy group and the amino group are strongly
electron donating. The Hammett para sigma+constant for the hydroxy
and amino groups are -0.92 and -1.7, respectively. The ionized
hydroxy group (--O.sup.-) is even more electron donating with a
Hammett para sigma+constant that has been estimated at -2.3.
Chapman, N. B. and Shorter, J., Correlation Analysis in Chemistry,
Plenum Press, NY, N.Y., page 483-484; Vogel, P., Carbocation
Chemistry, Elsevier, NY, N.Y. (1985) page 243; Hansch, C.,
Comprehensive Medicinal Chemistry, Pergamon Press, NY, N.Y.,
4:235.
[0041] The unmasking of a phenol can be carried out in vivo by
enzymes. For example, nonspecific esterase is ubiquitous within the
cytoplasm of cells and is able to cleave a wide variety of
carboxylate esters. Phenolic carbonates and carbamates are degraded
by cellular enzymes to yield phenols (Ditter et al., J. Pharm. Sci.
57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968); Ditter
et al., J. Pharm. Sci. 58:557 (1969); King et al., Biochemistry
26:2294 (1987); Lindberg et al., Drug Metabolism and Dispositior
17:311 (1989); and Tunek et al., Biochem. Pharm. 37:3867 (1988)).
The unmasking of a phenol can also occur by hydrolysis. For
example, a wide variety of carbonate and carbamate groups are known
which undergo spontaneous cleavage in solution at kinetically
favorable rates (Saari et al., J. Med. Chem. 33:97 (1990) and
Rattie et al., J. Pharm. Sci. 59:1741 (1970)). When A is a
substituted benzyl group, cleavage of, for example, a (lower
alkyl)--CO--O--, --O--CO--O--(lower alkyl) or --O--CO--NH-- (lower
alkyl) group in the ortho or para position to give a modified A
(A') will trigger heterolytic fission of the C--O bond between
modified A (A') and the oxygen of the phosphate. Based on the above
considerations the conversion of, for example, an ortho and/or para
(lower alkyl)--CO--O--, --O--CO--O--(lower alkyl) or
--O--CO--NH--(lower alkyl) group into a hydroxy group will lead to
a rate increase of phosphoester fission of at least 7000 fold. If
the resulting hydroxy group is ionized to an oxyanion, O.sup.-, the
rate of solvolysis can be further increased about 2.times.
10.sup.10 fold. Based on an intracellular pH of 7 and a pKa or for
the phenolic hydroxy group about 0.1% of the hydroxy groups will be
ionized under physiological conditions. The net result is that
overall a rate increase on the order or 2.times.10.sup.7 fold can
occur in the heterolytic cleavage of the C--O bond between modified
A (A') and the oxygen of the phosphoester following cleavage of an
ortho or para acyloxy group in A by nonspecific esterase.
[0042] In a preferred embodiment, the prodrugs of the present
invention are synthesized by replacing one of the hydroxy groups on
the phosphorous atoms of the monophosphate parent drug with a group
"A--O--", wherein the group "A" is a substituted benzyl derivative
with one or more protected hydroxy groups (e.g., (lower
alkyl)--CO--O--, aryl--CO--O, --O--CO--O--(lower alkyl),
--O--CO--O--aryl, --O--CO--NH--(lower alkyl) and --O--CO--NH--aryl)
or protected amine groups (e.g., (lower alkyl)--CO--NH--,
(aryl)--CO--NH--, (lower alkyl)--O--CO--NH--, aryl-- O--CO--NH--,
(lower alkyl)--NH--CO--NH-- and aryl--NH--CO--NH--) in ortho or
para positions relative to the phosphoester. The monophosphate of
the parent drug is liberated following conversion of the protected
hydroxy group or protected amino group into the corresponding
hydroxy group or amino group, respectively. The monophosphate of
the parent drug can be converted to the parent drug by the action
of enzymes such as alkaline phosphatases.
[0043] Preferably, A is a substituted benzyl group which is further
substituted at the benzylic position by a moiety which facilitates
cleavage of the phosphoester bond. Suitable substituents at the
benzylic position include groups which are capable of stabilizing a
carbon cation formed upon cleavage of the phosphoester bond, for
example, a lower alkyl group.
[0044] More preferably, A is substituted at the benzylic position
by a moiety such that cleavage of the phosphoester bond between A
or A' and the phosphate oxygen will result in an elimination
reaction to form a double bond between the benzylic carbon and the
moiety. Suitable moieties generally comprise a methylene or methine
group, wherein said methylene or methine group is 1) bonded to the
benzylic position of A and 2) has an acidic hydrogen. Upon cleavage
of the phosphoester in vivo, A or A' can then undergo an
elimination reaction by loss of the phosphate bonded to the
benzylic carbon and the acidic hydrogen to form a carbon-carbon
double bond at the benzylic position. Alternatively or
additionally, a preferred prodrug of the invention is degraded to
acyclovir monophosphate or an analog of acyclovir monophoschate by
an elimination reaction triggered by the spontaneous or enzymatic
unmasking of a strongly electron donating group, such as a hydroxy
or amino group at the ortho or para positions of a benzyl group
represented by A.
[0045] Suitable moieties at the benzylic carbon of the A include
those having an electron withdrawing group bonded to the methylene
or methine group with the acidic hydrogen (see March, Advanced
Organic Chemistry, John Wiley & Sons, third edition (1985) page
884), for example --CHR'--Z, wherein Z is an electron withdrawing
group such as --COOR", --COR", --CONH.sub.2, --CONHR", --NO.sub.2,
--SO.sub.2R" and --CN.
[0046] R' is --H, a lower alkyl group, substituted lower alkyl
group, aryl or a substituted aryl.
[0047] R" is --H, a lower alkyl group, a substituted lower alkyl
group, an aryl group or a substituted aryl group. In one example, Z
is --COOR", wherein R" is --H, methyl, ethyl, n-propyl, iso-propyl,
n-butyl, sec-butyl or t-butyl. In another example, R" is
--(CH.sub.2).sub.nCH.sub.- 3, wherein n is 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20.
[0048] Preferably, A is represented by the Structural Formula (V):
7
[0049] R.sub.1, R.sub.3, or R.sub.6 are independently selected from
the group consisting of --O--CO--R.sub.8, --O--CO--OR.sub.8,
--O--C(O)--NHR.sub.8, --O--C(O)--N(R.sub.8).sub.2,
--NH--CO--R.sub.8, --NH--CO--OR.sub.8, --NH--CO--NHR.sub.8, and an
inert group, with the proviso that at least one of R.sub.1, R.sub.3
or R.sub.6 is not an inert group.
[0050] R.sub.2 and R.sub.7 are independently an acyloxy group --H,
(--O--COR.sub.8) or an inert group.
[0051] R.sub.4 and R.sub.5 are independently selected from the
group consisting of hydrogen, a lower alkyl group, a substituted
lower alkyl group and a moiety such that cleavage of the
phosphoester bond between A or A' and a phosphate oxygen results in
an elimination reaction to form a carbon-carbon double bond between
the benzylic position of A or A' and the moiety.
[0052] R.sub.8 is selected from the group consisting of a lower
alkyl group, a substituted lower alkyl group, an aryl group, a
substituted aryl group and a group such that the resulting ester
moiety is degraded to the free pherolic hydroxy group in vivo.
Preferably, R.sub.8 is selected from the group consisting of
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, t-butyl,
n-pentyl or n-hexyl. Other examples include
--CHR.sub.9--NHR.sub.10, --CH(OR.sub.11)--CH.sub.3,
CH.sub.3O--(CH.sub.2).sub.2--O-- (CH.sub.2).sub.2--O--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--CH.sub.2--, --OCH.sub.3,
--CH.sub.2--CO--CH.sub.3 and --CH.sub.2(OR.sub.11), wherein R.sub.9
is the side chain of an amino acid, R.sub.10 is H or an amine
protecting group and R.sub.11 is H or an alcohol protecting group.
Suitable protecting protecting groups can be selected by the
skilled artisan and are described in Green and Wuts, "Protecting
Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and
7, 1991, the teachings of which are incorporated herein by
reference.
[0053] The nature of the labile groups at R.sub.1, R.sub.3, and
R.sub.6 determines the rate at which the resulting prodrug is
transformed to the parent phosphorus bearing drug. The solubility
of the prodrug can be varied by changing the nature of the groups
R.sub.1-R.sub.8. Water solubility can be enhanced by selecting
substituents with hydrophilic groups such as --CH.sub.2OH or
--CO.sub.2H. Alternatively, one can select bulky substituents which
can increase lipid solubility.
[0054] In an even more preferred embodiment, the prodrug is
represented by the following structural formula: 8
[0055] or physiologically acceptable salts thereof;
[0056] wherein R.sub.12 and R' are each independently lower alkyl
or substituted lower alkyl. R.sub.12 can also be
CH.sub.3--CO--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--,
CH.sub.3O--(CH.sub.2).sub.2--O--CH.sub.2-- and --OCH.sub.3.
Preferably, R' can also be --H. R.sub.12 and R' are each
independently selected from the group consisting of --H, methyl,
ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl,
n-hexyl. R' is even more preferably methyl.
[0057] The prodrugs of the present invention are degraded in vivo
and in blood to the acyclovir. Without being limited to a
particular reaction the mechanism by which the prodrugs are
believed to undergo transformation to the monophosphate of the
parent drug is shown in the scheme below: 9
[0058] When incubated with pig liver esterase in phosphate buffered
D.sub.2O, NMR results show that Prodrug 1 is degraded to acyclovir
monophosphate and p-hydroxycinnamic, as shown in the Scheme above.
This assay is described in Glazier, WO 91/19721 entitled
"Phosphorus Prodrugs" and can be used to determine suitable
hydroxyl or amino protecting groups which can be cleaved in vivo.
The entire teachings of WO 91/1972 are incorporated herein by
reference. It is to be understood that there are other in vitro
assays known to one of ordinary skill in the art employing
esterases, amidases or enzymes capable of cleaving carbonate,
carbamate, ester and amide groups which can also be used to
determine suitable protecting groups.
[0059] In the above description the term "inert" is used to refer
to groups that are substantially nonreactive and do not influence
in a major fashion the chemistry of the prodrug metabolism or
decay. Examples of inert groups include --H, lower alkyl,
substituted lower alkyl, aryl, substituted aryl, halogen,
--COO(lower alkyl), (lower alkyl)--O--, (substituted lower
alkyl)--O-- (e.g. benzyloxy and substituted benzyloxy),
--CO--(lower alkyl), --CO--(substituted lower alkyl), --CO--(aryl),
--CO--(substituted aryl), --CHO, --CN and --NO.sub.2.
[0060] As used herein, a "lower alkyl" group car have from 1 to
about 20 carbon atoms, preferably 1 to 6 carbon atoms, and can be
straight chained, branched or cyclic. In addition, a "lower alkyl"
group can have one or more double and/or triple bonds. An "aryl"
group includes a heterocyclic or carbocyclic aromatic group such as
phenyl and naphthyl. Heterocyclic atomatic groups include, for
example, imidazolyl, indolyl, thienyl, furanyl, pyrldyl, pyranyl,
pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl,
isoquinolinyl and acridintyl.
[0061] Suitable substituents on a lower alkyl, an aryl group and a
benzyl group include inert substituents, as described above.
[0062] Suitable physiological salts of the prodrugs of acyclovir
and acyclovir analogs disclosed herein are those which are
non-toxic. Examples include, but are not limited to, sodium,
potassium, calcium, magnesium, ammonium monoalkylammonium,
dialkylammonium, trialkylammonium, diethanol ammonium and
triethanol ammonium, salts of the disclosed prodrugs. These salts
can be prepared by an exchange reaction with a salt of the desired
cation with an appropriate anion, including halides,
--ClO.sub.4.sup.-, acetate, or other alkanoates or aryl
carboxylates. These salts can also be prepared with an ion exchange
resin loaded with the desired cation.
[0063] Procedures for preparing phosphate ester prodrugs of
nucleoside analogs (e.g., acyclovir) include using phosphoamidites
in conjunction with H-tetrazole (see Perich and Johns, Synthesis
1988:142). These procedures are described in U.S. Ser. Nos.
07/714,130, 07/537,332 and 08/310,972, the entire teachings of
which have been incorporated into this application by reference.
However, these procedures suffer from a number of shortcomings.
Nucleoslde analogs such as acyclovir are insufficiently soluble in
the organic solvents normally used for this reaction, e.g.,
methylene chloride, chloroform, acetonitrile, tetrahydrofuran and
pyridine. As a result, the nucleoside analog (e.g., acyclovir) must
be protected to enhance its solubility and prevent side reactions,
thereby increasing the cost of preparing phosphate ester prodrugs.
Alternative methods have been reported which use phosphorus
oxychloride in conjunction with trimethyl or triethyl phosphate as
solvent (see U.S. Pat. No. 4,287,188). However, trialkyl phosphates
are toxic carcinogens with alkylating activity. Accordingly, their
use on an industrial scale is problematic.
[0064] Described below are novel synthetic methods for preparing
phosphate esters prodrugs of nucleoside analogs which overcome
these disadvantages. Using these methods, phosphate esters prodrugs
of nucleoside analogs such as acyclovir can be prepared in nearly
quantitative yield without protecting acyclovir or the nucleoside
analog. Acyclovir or a nucleoside analog Y'--OH suspended in an
aprotic polar organic solvent reacts rapidly and quantitatively
with phosphoramidites (A--O).sub.2--P--(NR.sub- .21R.sub.22) in the
presence of a weak anhydrous organic acid to produce the
corresponding phosphite ester, which can be oxidized without
isolation with a phosphite oxidizing agent to the corresponding
phosphate triester. This reaction sequence is shown schematically
in FIG. 2. The phosphate triester can be converted to the diester
prodrug in the presence of a base, for example, an amine base such
as triethyl amine.
[0065] A "nucleoside analog" is a drug whose activity is a
consequence of its structural similarity to a nucleoside. In a
target organism, the drug can be erroneously incorporated into
biological molecules such as DNA or RNA. Biomolecules incorporated
with nucleoside analogs do not undergo the same biochemical
reactions as the corresponding naturally-occurring biomolecules. As
a result, the nucleoside analog toxic to the target organism.
Nucleoside analogs suitable for use in the present invention
(Y'--OH) have a free hydroxyl group. Examples include
3'-azido-thymidine (AZT), 2'3'-didehydro-2'3'-dideoxythymidine
(D4T), 2'3'-dideoxyadenosine and trifluorothymidine.
[0066] Suitable weak anhydrous organic acids include acids suitable
for use in the synthesis of polynucleotides by phosphoramidate
methodology, which are well known in the art. Generally weak
anhydrous organic acids having a pKa from about 2 to about 6 are
preferred, for example, H-tetrazole, 1-H-triazole and
3-nitro-1-H-triazole.
[0067] About a 10-20% molar molar excess of the weak acid and
nucleoside analog with respect to the phosphoramide is generally
used. However, it is also possible to use equimolar amounts of the
three reagents or about a three fold or more excess of any one or
two of these reagents. Suitable concentrations for the reagents
range from about 0.05 M to about 2.0 M and typically range from
about 0.3 M to about 1.3 M. The reaction can be generally carried
out at temperatures ranging from -10.degree. C. to about 80.degree.
C., but typically at room temperature. Reactions can be monitored
by thin layer chromotagraphy to determine reaction times. Polar
co-solvents such as pyridine, acetonitrile, methylene chloride and
chloroform can be added in amounts which do not significantly
decrease the solubility of the nucleoside analog or the reaction
rate.
[0068] The oxidation of the phosphite ester can be carried out with
from about 1.0 to about 3.0 equivalents of the phosphite oxidizing
agent (e.g., hydrogen peroxide) at temperatures ranging from about
0.degree. C. to about 50.degree. C. Preferably, the reaction is
carried out with about a 10-15% excess of phosphite oxidizing agent
at about 10-20.degree. C. Preferably, the oxidation can be
performed without isolating the phosphite ester intermediate.
[0069] A "phosphite oxidizing agent" is a reagent which oxidizes
phosphite esters to phosphate esters. Phosphite oxidizing agents
are known in the art of oligonucleotide synthesis and include
hydrogen peroxide, peracids such as meta chloroperbenzoic acid,
iodine in water and nitrogen tetraoxide. Hydrogen peroxide is
preferred.
[0070] Phosphate triesters containing acyclovir, an analog of
acyclovir or a nucleoside analog can be converted to phosphate
diester prodrugs by reacting the phosphate triester with a suitable
base, e.g., a base which can cause an elimination reaction with an
ester substituted in the beta position with (RO).sub.2PO--O--,
wherein R is a substituted or unsubstituted alkyl, aryl or
heteroaryl group. Examples of suitable bases include, but are not
limited to, hydroxide, amines (e.g., ammonia, an alkyl amine, a
dialkyl amine or a trialkyl amine), alkoxides and hydride bases
(e.g., sodium or potassium hydride). At least one equivalent of
base per mole of phosphate triester is generally used. When an
amine is used as the base, the reaction mixture can contain up to
about 50% amine base by volume in a suitable organic solvent,
preferably between about 20% to about 30%. The reaction can be
carried out between about 0.degree. C. and about 50.degree. C.,
preferably at room temperature. Suitable solvents are those in
which both the base and the phosphate triester are soluble and
compatible (e.g., do not react with the base) and are readily
determined by the person of ordinary skill in the art. For example,
when an amine is used as the base, suitable solvents generally
include polar solvents such as acetonitrile, methylene chloride,
chloroform and nitromethane; alcohols can be used with alkoxide
bases; etheral solvents can be used with hydride or amide
bases.
[0071] Procedures for preparing the disclosed diester prodrugs are
shown in FIG. 1 and described in detail in Example 2. It is to be
understood that certain modifications in these procedures may be
required. For example, changes in the reaction conditions used may
be necessary when different A or Y groups are used. The selection
of suitable reaction conditions is within the ability of one
skilled in the art of organic chemistry.
[0072] The prodrugs of the subject application can be used to treat
infections in individuals (e.g., humans and animals) caused by
various classes of DNA and RNA viruses, including cytomegalovirus,
adenovirus (in particular adenovirus 5), rhino virus, Mengo virus,
Sinbis virus and vaccinia virus. They are especially active against
herpes viruses, including simplex, zoster and varicella, and, in
particular, for dermal herpes simplex virus-1 infection. Animals
which can be treated by the prodrugs of the present invention
include veterinary animals, such as dogs, guinea pigs, cats and the
like, and farm animals, such as cows, horses, pigs, goats, sheep
and the like.
[0073] A "therapeutically effective amount" of a prodrug is an
amount of prodrug which decreases the duration and/or severity of a
viral infection in an individual or animal. Alternatively, a
"therapeutically effective amount" comprises an amount of prodrug
which lowers the virus titer in an individual or animal with a
viral infection or which ameliorates the symptoms and/or discomfort
associated with the viral infection. In the case of dermal viral
infections, including herpes simplex virus-1, a "therapeutically
effective amount" of a prodrug is an amount which decreases lesion
number, lesion area and/or virus titer in the skin of an infected
individual or animal.
[0074] The skilled artisan will be able to determine the precise
amount of prodrug to be administered to an animal or an individual.
The amount of prodrug that is administered to an individual will
depend on a number of factors including the general health, size,
age and sex of the animal or individual and the route of
administration. It will also depend on the degree, severity and
type of viral infection. One of ordinary skill in the art will be
able to determine the precise dosage according to these and other
factors. Typically, between about 0.01 mg/kg body weight per day
and about 200 mg/kg body weight per day are administered to the
individual or animal.
[0075] The prodrug can be administered orally, for example, in
capsules, suspensions or tablets. Other modes of parenteral
administration which can be used include systemic administration,
such as by intramuscular, intravenous, subcutaneous, or
intraperitoneal injection. In the case of a virus infection in the
skin, for example a dermal herpes simplex virus-1 infection, the
prodrug is preferably applied topically directly to skin which
shows symptoms of viral infection.
[0076] The prodrug can be administered to the individual or animal
in conjunction with an acceptable pharmaceutical carrier as part of
a pharmaceutical composition for treating viral infections.
Suitable pharmaceutical carriers may contain inert ingredients
which do not interact with the prodrug. Standard pharmaceutical
formulation techniques may be employed such as those described in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa. Suitable pharmaceutical carriers for parenteral
administration include, for example, sterile water, physiological
saline, bacteriostatic saline (saline containing about 0.9% mg/ml
benzyl alcohol), phosphate-buffered saline, Hank's solution,
Ringer's-lactate and the like. Methods for encapsulating
compositions (such as in a coating of hard gelatin or cyclodextran)
are known in the art (Baker, et al., "Controlled Release of
Biological Active Agents", John Wiley and Sons, 1986).
[0077] For topical administration for the treatment of viral
infections in the skin, the pharmaceutical compositions, in
addition to the prodrug, can additionally comprise an inert,
non-toxic solvent such as water, acetone, an alcohol, or mixtures
thereof, in which the prodrug is dissolved, or, preferably, a
pharmaceutical carrier suitable for local topical administration in
which the prodrug is dissolved or suspended. Examples of
pharmaceutically acceptable carriers include, for example,
commercially available inert gels, or liquids supplemented with
albumin, methyl cellulose or a collagen matrix. Typical of such
formulations are ointments, creams and gels. Ointments are
typically prepared using an oleaginous base, e.g., containing fixed
oils or hydrocarbons, such as white petrolatum or mineral oil, or
an absorbent base, e.g., consisting of an absorbent anhydrous
substance or substances, for example anhydrous lanolin. Following
formation of the base, the active ingredients are added in the
desired concentration. Creams generally comprise an oil phase
(internal phase) containing typically fixed oils, hydrocarbons, and
the like, such as waxes, petrolatum, mineral oil, and the like, and
an aqueous phase (continuous phase), comprising water and any
water-soluble substances, such as added salts. The two phases are
stabilized by use of an emulsifying agent, for example, a surface
active agent, such as sodium lauryl sulfate; hydrophilic colloids,
such as acacia colloidal clays, beegum, and the like. Upon
formation of the emulsion, the active ingredients are added in the
desired concentration. Gels are comprised of a base selected from
an oleaginous base, water, or an emulsion-suspension base, as
previously described. To the base can be added a gelling agent
which forms a matrix in the base, increasing its viscosity to a
semisolid consistency. Examples of gelling agents are hydroxypropyl
cellulose, acrylic acid polymers, and the like. The active
ingredients are added to the formulation at the desired
concentration at a point preceding addition of the gelling
agent.
[0078] The invention is further illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLE 1
Acyclovir Prodrug 1 is More Effective in Reducing Dermal HSV-1
Virus Infection Than Acyclovir
[0079] Formulation
[0080] The formulation for Prodrug 1 contained 5% EL-620 EMULPHOR
(polyoxyethylated castor oil) (Rhone Poluenc), 5% CARBOPOL
(Carboxypolymethylene, sodium salt) (B. F. Goodrich), 10% ethanol
and 2% by weight Prodrug 1. The balance of the formulation
consisted of water.
[0081] EL-620 EMULPHOR and water were mixed and ultrasonicated to
give a clear emulsion, to which the ethanol was added. The prodrug
was then added, and the mixture was ultrasonicated. The sodium
carbopol was then added to the emulsion and mixed until to
homogeneity.
[0082] The placebo was prepared identically to the pharmaceutical
compositions except that the prodrugs were omitted.
[0083] The pharmaceutical compositions were stored at room
temperature.
[0084] Animal Inoculation and Subsequent Treatment with Prodrug
1
[0085] Female Hartley outbred guinea pigs, 400 to 450 grams, were
obtained from Charles River Breeding Labs, Wilmington, Mass.
Animals were anesthetized with 25 mg/kg ketamine and 5 mg/kg
xylazine SQ. Hair on the dorsum from the shoulders to the rump was
removed with electric clippers followed by two 5-10 minute
applications of a chemical depilatory. A grid of four areas was
demarcated with a pen on both sides of the spine at levels
corresponding to mid back and rump.
[0086] Undiluted HSV-1 virus stock (0.035 ml) was applied to each
different area and introduced through the skin at well-spaced sites
at ten activations of a six-pronged spring-loaded vaccination
instrument (Sterneedle, Pan Ray Division, Ormont Drug, Englewood,
N.J.). The day of inoculation is Day 0. Approximately 250 mg of
drug or placebo was applied to the areas according to the dosing
regimen on Days 1, 2 and 3. Eight guinea pigs were used during the
experiments allowing eight comparisons between each drug and its
placebo.
[0087] On Day 4 regrown hair on the dorsum of the guinea pigs was
removed with one 3-4 minute application of a chemical depilatory.
HSV lesions were counted and Polaroid pictures of the animals'
backs were taken. The animals were sacrificed using CO.sub.2 gas
and the full thickness skin of the back was removed by dissection.
The square of the skin from each of the four treatment areas was
placed into 15 mls of tissue culture medium with 2% FBS in an ice
bath. The samples were then homogenized in a stomacher Lab Blender
80 (Tekmar Co.). Debris was pelleted by centrifugation and the
supernatants collected and frozen at -70.degree. C. until assay for
infectivity by plaque formation in VERO cells.
[0088] Results
[0089] The mean and standard deviation for number of lesions, total
lesion area and lesion virus titer were computed. Paired data
(drug/vehicle) were evaluated by the Wilcoxon signed-rank test
using percent differences between log.sub.10 derivatives of mean
lesions severity at drug-treated sites compared to the
vehicle-treated sites.
[0090] U.S. ZOVIRAX ointment applied four times per day is devoid
of activity and is not statistically different from placebo. In
contrast, Prodrug 1 resulted in a 72% reduction in lesion number,
79% reduction in lesion area and 93% reduction in skin viral titres
compared with placebo (p<0.01).
EXAMPLE 2
Synthesis of the Acyclovir Prodrugs
[0091] A. Synthesis of Methyl (4-Hydroxybenzoyl)acetate 10
[0092] Apparatus:
[0093] 3L, 3 neck round bottom flask, mechanical stirrer, addition
funnel thermometer, cooling bath, Argon bubbler.
[0094] Procedure:
[0095] Sodium hydride (60% dispersion in mineral oil, 74.0 grams,
1.85 moles) was suspended under anhydrous argon in petroleum ether
(250 ml), left to settle down and the solvent was removed under
argon. The procedure was repeated with another portion (250 ml) of
petroleum ether. 250 ml of anhydrous DMF was added.
[0096] A solution of 4'-hydroxyacetophenone (50.4 grams, 0,37
moles) in 250 ml dimethylformamide (DMF) was added drop wise to the
suspension of NaH over a 1.5 hour period and the temperature was
kept below 32.degree. C. by intermittent cooling with ice an bath.
When the addition was completed, the reaction mixture was stirred
for 15 minutes until the gas evolution had subsided and the
temperature dropped to 28.degree. C. Dimethylcarbonate (167.0
grams, 1.85 moles) was added drop wise over 1 hour and the
temperature was maintained below 35.degree. C.
[0097] The reaction mixture was stirred at room temperature under
argon for 36 hours. The reaction mixture was cooled with an ice
bath and poured with stirring into a mixture of ice (1.5 L volume),
water (1.0 liter) and concentrated HCl (170 mL) and extracted with
ethylacetate (4.times.250 ml). Combined ethylacetate extracts were
washed with water (1.times. 200 ml), 5% citric acid (2.times.200
ml), 5% NaHCO.sub.3 (2.times.200 ml), water (2.times.200 ml), dried
over Na.sub.2SO.sub.4 and concentrated on rotary evaporator.
[0098] The residual oil was crystallized from toluene/hexane.
[0099] Yield 63 gm -89%.
[0100] The reaction and work up were monitored by TLC in 40%
ethylacetate in hexane.
[0101] B. Synthesis of Methyl
[4-(2,2,2-Trimethylacetoxybenzoyl)acetate] 11
[0102] To a stirred and cooled solution of methyl
(4-hydroxybenzoyl)acetat- e (20 grams, 0.103 moles) in 150 ml
anhydrous chloroform and triethylamine, (15.8 ml, 11.4 grams, 0.113
moles) a solution of trimethylacetyl chloride (13.6 grams, 0.113
moles) in 20 ml chloroform was added drop wise over 15 minutes.
[0103] When the addition was completed, the ice bath was removed
and the reaction mixture was stirred at room temperature for 2
hours.
[0104] The reaction mixture was washed with water (3.times.100 ml),
5% NaHCO.sub.3 (3.times.100 ml), water (1.times.100 ml), dried over
Na.sub.2SO.sub.4 and the solvent removed on rotary evaporator. The
residue was purified by crystallization from diethyl
ether/petroleum ether.
[0105] C. Synthesis of Methyl
3-hydroxy-3-[4-(2,2,2-Trimethylacetoxy)-phen- yl] propionate
(Alcohol 1A) 12
[0106] A Parr apparatus was charged with methyl
[4-(2,2,2-trimethylacetoxy- benzoyl)acetate] (13.0 grams, 46.7
moles), ethanol (100 mL), water (50 mL) and 0.8 grams of 10%
Palladium on activated carbon. The mixture was reacted for eight
hours with stirring under a H.sub.2 atmosphere at 40 psi, filtered
and evaporated to dryness. The residue was recrystallized from
toluene/petroleum ether to give alcohol 1A (12.83 grams, 98%).
[0107] D. Preparation of the Phosphoramide Intermediate 3
[0108] Referring to FIGS. 1A and 1B, alcohol 1A (28.03 g, 0.1 mol)
and a stirring bar were loaded in a 500 mL round bottom flask. The
flask was sealed with a rubber septum and flashed with nitrogen.
Dry THF (150 mL) was added by a syringe followed by triethylamine
(10.12 g, 14.0 mL, 0.1 mol). The mixture was stirred under nitrogen
until alcohol 1A dissolved and then cooled in an ice bath.
Diethylphosphoroamidous dichloride 2 (8.70 g, 7.30 mL, 0.05 mol)
was weighed with a syringe and added dropwise with stirring over
about 5 minutes. After 15 minutes the ice bath was removed and the
reaction mixture stirred for 24 hours. At that point a TLC in
EtOAc/hexane 40:60 showed no starting material (UV detection). The
mixture was filtered under a blanket of nitrogen and the flask and
solid were washed with 3.times.25 mL THF. The combined filtrate and
washings were evaporated on a rotary evaporator equipped with a dry
ice cooled condenser at a bath temperature of 25.degree. C. to
afford Intermediate 3 as an oil.
[0109] E. Preparation of Phosphite Triester Intermediate 6
[0110] The flask with Intermediate 3 was equipped with a stirring
bar and sealed with rubber septum. Dry DMF (45 mL) was added and
the mixture was stirred until the oil dissolved. Acyclovir 4 (9.38
g, 0.0417 mol) and then 1H-tetrazole (3.50 g, 0.05 mol) were added
quickly and the resulting suspension was stirred sealed under
nitrogen for 24 hours. After about 2 hours, acyclovir 4 dissolved
and the reaction mixture became clear. TLC after 24 hours in
chloroform/methanol/water 50:10:1 showed complete consumption of
the acyclovir 4. Comparable results were obtained when
1,3-dimethyl-2-imidozolidinone was used in place of DMF.
[0111] F. Oxidation of Phosphite Triester Intermediate 6 to
Phosphate Triester 7
[0112] The reaction mixture was cooled with an ice bath and
hydrogen peroxide (30% w/w solution in water, 6.80 g, 5.2 mL, 0.06
mole) was added with stirring. The rate of addition was adjusted so
that the temperature remained below 15.degree. C. When the
exothermic reaction subsided the bath was removed and the mixture
was left at room temperature for 1.5 hours.
[0113] The reaction mixture was cooled on an ice bath and
Na.sub.2SO.sub.3 (2.52 g, 0.02 mmol) dissolved in 8 mL of water was
added with stirring. The rate of addition was adjusted so that the
temperature remained below 15.degree. C. The mixture was left at
room temperature for 25 minutes and then evaporated under vacuum
(0.7-0.5 mm Hg) on rotary evaporator equipped with a dry ice cooled
condenser at a bath temperature of 25.degree. C. The residue was
dissolved in 100 mL of methylene chloride, filtered and loaded on a
column of silica gel (420 g, .about.900 mL dry volume) equilibrated
with methylene chloride. The column was eluted with methylene
chloride (2 L) until UV absorption of the eluate returned to the
initial value. This step eluted less polar impurities. The column
was further eluted with methylene chloride/methanol 15:1 (6 L)
until UV absorption returned to the initial value. 200 mL fractions
were collected. Fractions 13-25 were pooled and evaporated to
afford a white foam. This residue was dried overnight under vacuum
(0.5 mm Hg) at room temperature to give 31.82 g (92% yield) of a
white solid.
[0114] G. Preparation of Prodrug 1 from Compound 7
[0115] Compound 7 (8.29 g, 10 mmol) was dissolved in a mixture of
28 mL of triethylamine and 83 mL acetonitile. After 6 hours, the
reaction mixture was evaporated under vacuum and the residue was
dissolved in methylene chloride. This solution was loaded onto a
column of silica gel and the product Prodrug 1 was separated from
cinamate 8 by elution with a gradient from pure methylene chloride
to methylene chloride/methanol/water 15:10:1. The fractions
containing Prodrug 1 were evaporated. The residue was dissolved in
30% iso-propanol and converted to the sodium salt by passing
through a column of Dowex 50WX8 in Na.sup.+ form. The material was
eluted with 30% iso-propanol and evaporated under vacuum to afford
an oil. This oil was crystallized from abs. ethanol, to give 4.77
g., 81% of Sodium 2-((9-Guanylyl)methyl)oxy)ethyl
1-(4-((2,2-dimethylpropionyl)oxy)-phenyl)-2-((methoxy)carbonyl)ethyl
phosphate. .sup.1H NMR (DMSO-d.sub.6, .delta.): 11.40 (1H, s), 7.79
(1H, s), 7.39 (2H, m), 7.00 (3H, m), 5.38 (1H, m) , 5.29 (s, 2H),
3.65 (1H, m), 3.55 (1H, m), 3.47 (3H, s), 3.03 (1H, dd), 2.72 (1H,
dd), 1.28 (9H, s), .sup.31P NMR (DMSO-d.sub.6; 85% H.sub.3PO.sub.4,
external reference): -0.83 (dec. on, s; dec. off, m). MW of the
anion was confirmed by mass spectrometry.
[0116] An analogous synthesis was used to prepare the sodium salt
of Prodrug 2, except that acetyl chloride was used in step B in
place of pivoyl chloride. The structure of Prodrug 2 is shown
below: 13
[0117] Analytical data for Prodrug 2, sodium
2-((9-guanylyl)methyl)oxy)eth-
yl-1-(4-acetoxyphenyl)-2-((methoxy)carbonyl) ethyl phosphate, is as
follows: .sup.1H NMR (DMSO-d.sub.6, .delta.) : 11.09 (1H, s), 7.78
(1H, s), 7.36 (2H, m), 7.04 (2H, m) , 6.82 (2H, broad s), 5.37 (1H,
m) , 5.29 (2H, s), 3.60 (2H, m), 3.49 (3H, s), 3.44 (2H, m), 2.97
(1H, dd), 2.73 (1H, dd), 2.25 (3H, s), .sup.31P MMR (DMSO-d.sub.6;
85% H.sub.3PO.sub.4, external reference): -5.569 (dec. on, s; dec.
off, m). MS ES+ 548 [M+H].sup.+, ES- 524 [M-Na].sup.-.
[0118] An analogous synthesis was used to prepare the sodium salt
of a third prodrug, Prodrug 3, except that diethyl carbonate was
used to prepare ethyl (4-hydroxybenzoyl)acetate in step A. Thus,
Prodrug 3 has the same structure as Prodrug 1, modified so that the
--CH.sub.2COOCH.sub.3 group at the benzylic position is replace
with --CH.sub.2COOCH.sub.2CH.sub.3. Analytical data for Prodrug 3,
Sodium
2-((9-guanylyl)methyl)oxy)ethyl-1(-4-((2,2-dimethylpropyonyl)oxy)phenyl)--
2-((ethoxy)carbonyl)ethyl phosphate, is as follows: .sup.1H NMR
(DMSO-d.sub.6.delta.) : 11.35 (1H, s), 7.78 (1H, s), 7.38 (2H, m),
7.01 (2H, m), 6.96 (2H, broad s), 5.39 (1H, m), 5.31 (2H, s), 3.93
(2H, m), 3.64 (2H, m), 3.45 (2H, m), 3.06 (1H, dd), 2.70 (1H, dd),
1.30 (9H, s), 1.05 (3H, t), .sup.31P NMR (DMSO-d.sub.6; 85%
H.sub.3PO.sub.4, external reference) : -1.511 (dec. on, s; dec.
off, m). MS ES+ 626 [M+Na].sup.+
EXAMPLE 3
Acyclovir Prodrug 2 is More Effective in Reducing Dermal HSV-1
Virus Infection Than Acyclovir
[0119] Prodrug 2 was formulated as described in Example 1. One
Hartley guinea pig was inoculated with HSV-1 as described in
Example 1. The guinea pig was treated with Prodrug 2 according to
the protocol used in Example 1 for Prodrug 1. A comparable
reduction in lesion number, lesion area and in skin viral titres
was obtained with Prodrug 2 as was observed in Example 1 with
Prodrug 1.
EQUIVALENTS
[0120] Those skilled in the art will know, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
* * * * *