Note: Descriptions are shown in the official language in which they were submitted.
10695Z6
P,ack&ro~nd of the Invention
Herpes virus infections, though kno~lg are
difficult to treat because of the lack of effective drugs.
An effective anti-herpes drug could be used in the treatment
or prevention of herpes dermatitis, herpes genitalis, herpes
keratitis, herpes encephalitis and as provided by the presen~
invention, herpes simplex virus. Although herpes simplex
is a very common, though minor disease, the only basic
treatment presently available is the application of
5-iodo-2'-deoxuridine (idoxuridine).
Detailed Description of the Invention
This invention relates to a method of treating
herpes simplex infections in warm-blooded animals comprising
administering to such infected animal, a phosphorus ester of
phosphonoacetic acid, or its inorganic salts, of the formula
O O
Il 11
RO- P - CH2- C -OH
OH ~ (I)
wherein R is an alkyl of 1 to 8 carbon atoms
The ester compounds are active against herpes
simplex viru~ in tissue culture. They are also active in vivo
when administered either as the acid or the alkali metal salts,
particularly the mono or di sodium, and calcium salts. The
compounds are preferably administered topically, but can also
be given by the oral or intraperitoneally (i p ) route.
--2--
~, .
~0 6~ Sz ~
Since the herpes viruses depend for replication
upon a unique DNA polymerase independent from the DNA
polymerase of the mammalian host, growth of the virus
may be stopped by inhibiting this necessary enzyme. These
phosphorus esters of phosphonoacetic acid are potent inhibit-
ors of the enzyme
The present phosphorus esters of phosphonoacetic
acid (PA) may be prepared by two methods. In the first
method, Method (A), there is employed dimethylphenyl phosphite
whereas in the second method, Method (B)~ there is employed ~-
an alkylbis(trimethylsilyl)phosphite. The phosphites are
reacted in each procedure with benzyl haloacetate to give
tri-esters of phosphonoacetic acid which are converted by
hydrolysis and hydrogenolysis to mono esters of phosphono-
acetic acid. The esters are converted to crystalline
ammonium salts and characterized by nmr and carbon, hydrogen
and nitrogen combustion values. The two methods that may
be used to produce the phosphorus esters o phosphonoacetic
acid are illustrated below:
'
.~ .
~o6~526
THOD (A)
O
PhOP (OCH3) 2 + BrCH2C02CH2Ph , ~ Pho lpcH2co2cH2ph
CH30
Pt-H2 ¦¦
HOAc >CH30 1PCH2C02H
HO
METHOD (B)
Me3SiCl
ROPC12 Pyridine > ROP (OH) 2 Et3N ROP (OTMS) 2
H2 Pyridine>
O O :--
ClcN2co2cH2ph~ ROPCH2C02CH2Ph 1 H20 ~ ROICH2C02H
OTMS OH
: .
R = propyl,
hexyl
!
lOG!35;Z6
The phosphorus esters of phosphonoacetic acid
of the present invention that may be prepared according to
the Procedures (A) or (B) as illustrated above i.nclude the
following: :
(II) P-Propylphosphonoacetic acid
O O
Il il
HO- C- CH2- P -O(CH2)2CH3
OH
(III) P-Hexylphosphonoacetic acid
O O
Il 11
HO- C -CH2- P- O(CH2)sCH3
OH
(IV) P-Methylphosphonoacetic acid
O O
Il 11
HO- C -CH2-,P- OCH3
OH
The following examples will further illustrate
the present invention. The terms and symbols used in
the examples stand for:
Ph = phenyl;
TMS = tetramethylsilane;
Pt = platinum;
MW = molecular weight;
HQAc = acetic acid,
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10695~6
Example 1
P-Methylphosphonoacetic Acid
A solution of 11.40 g. (0.05 mole) of benzyl
bromoacetate and 10.23 g. (0,055 mole) of dimethylphenyl
phosphite was heated at 130-140C. (oil bath). A stream of
nitrogen was passed through the solution to remove the methyl
bromide formed. The residue was evaporatively distilled and
chromatographed to give 9.00 g. of benzyl methylphenyl-
phosphonoacetate. Debenzylation with hydrogen on a palladium
catalyst followed by hydrogenolysis with a platinum catalyst
in acetic acid gave P-methylphosphonoacetic acid. This
compound was treated with ammonium hydroxide and the salt
crystallized from ethanol to give 2.74 g. (57% for the last
three steps) of mono-ammonium salt, m.p. 183-6 (decomp).
Analysis Calcd. for C, 21.06; H, 6.31; N, 8.19 `
Found: C~ 21.13; H~ 6.03; NJ 7.85
Example 2
P~Propylphosphonoacetic Acid
A solution of 80 g. (0.50 mole) of propylphosphoro-
dichloridite in 60 ml. of ether was added dropwiseto a solution of 79 g. (1.00 mole) of pyridine and 18 g.
(1.00 mole) of water in 500 ml. of ether cooled in an ice
bath. The mixture was allowed to warm gradually to room
temperature and stirred for two hours. The mixture was
filtered and the filtrate dried over MgS04 and evaporated
at reduced pressure to give propyl phosphite. This
compound was dissolved in 500 ml. of dry pyridine and cooled
--6--
106~5Z6
in an ice bath, To this solution was added 208 ml, (1.5 mole)
of dry triethylamine followed by the dropwise addition of
189 ml, (1,5 mole) of trimethylsilyl chloride, The mixture
was allowed to warm gradually to room temperature and stirred
overnight. The mixture was diluted with an equal volume of
ether, fil~ered and the filtrate concentrated. The residue
was distilled to ~ive 50,34 g. of propyl~bis(trimethylsilyl)~
phosphite, b.p, 81,5-83,0C,/ca 20 mm,
A solution of 9.2 g, ~0,05 mole) benzyl chloroacetate
and 13,4 g, (0,05 mole) of propylLbis~trimethylsilyl)~phos-
phite was heated at 160-5C, (oil bath) for 1 hour, The
trimethylsilyl chloride formed ln the reaction was allowed to
distill from the reaction mixture, The residue was evapor-
atively distilled and the fraction distilling at 140-160C,
(air bath temperature/0,5 mm) collected, The trimethylsilyl
group was removed by hydrolysis and the resulting di-ester
debenzylated with hydrogen over Pd/C catalyst, The acid was
converted to the ammonium salt to give 5,35 g, (38% yield
for the last three steps) of ammonium P-propylphos-
phonoacetate, m,p, 155-156,5C,
Analysis Calcd, for: C, 30,15; H, 7,08; N, 7,03
Found: C, 30,26; H, 7,37; N, 7,03
Ammonium P-hexylphosphonoacetate was prepared
` in a similar manner, The salt was crystallized from ethanol
and had a m,p, of 178-190C.
Analysis Calcd, for: C, 39,83; H, 8,36; N, 5,81
Found: C, 39,90; H, 8.68; N, 5.78
_7_
~695Z6
Example 3
Benzyl P-Meth~l-P-phenylphosphonoacetate
A solution of 11 40 g. (0.05 mole) of benzyl
bromoacetate, and 10 23 g. (0 055 mole) of dimethylphenyl
phosphite was heated at 130-140C. (oil bath) for 5-1/4
hours A stream of N2 was bubbled through the mixture to
help eliminate the CH3Br formed. The residue was evapor-
atively distilled to remove the volatile material to give
14.82 g. of residue. This material was chromatographed on
300 g. of Florisil, and elution with 2% MeOH-PhH gave 9.00
g. of product.
Example 4
P-Methyl-P-phenYlphosphonoacetic Acid
Benzyl P-methyl-P-phenylphosphonoacetate, 9,00 g.
(0.0281 mole), was debenzylated with hydrogen on a palladium
catalyst to give 6.168 g. (0.0268 mole, 95% of theory) of
P-methyl-P-phenylphosphonoacetic acid.
Example 5
P-Methylphosphonoacetic Acid
P-Methyl-P-phenylphosphonoacetic acid, 6.168 g.
(0 268 mole), was dephenylated with PtO2-HOAC-H2 The
residue was azeotroped with toluene to remove the HOAc The
residue was dissolved in H20, made basic with concentrated
NH40H The solvent was evaporated and the residue
crystallized from 95% EtOH to give 2.74 g. (60%) of
product, m p. 183-6~C (decomposed, bubbles formed)
--8--
1c)69~'~6
Analysis Calcd. for C3HloN05P: C, 21 06; H, 6.31; N, 8.19
Found: C, 21.13; H, 6.03; N, 7.85
Example 6
Hexylbis(trimethylsilyl~ Phosphite
Hexylphosphorodichloridite, 40.40 g. (0.20 mole),
in 50 ml. of (C2Hs)2O was added dropwise to a mixture of
34.76 g. (0.44 mole) of H20 in 300 ml of (C2Hs)20, cooled
in an ice bath. The mixture was allowed to warm to room
temperature, filtered and the filtrate dried over MgSO4,
and evaporated to give 31 0 g. of a clear colorless liquid.
This material, CH3(CH2)50P(OH)2, was dissolved in
250 ml. of dry pyridine and 83 ml. (0.60 mole) of dry
(C2Hs)3N was added and the solution was cooled in an ice
bath. Chlorotrimethylsilane, 26 ml. (0.60 mole) was added ~ ~
dropwise. After the addition was complete, the mixture was ;-
allowed to war~ to room temperature and stirred overnight.
The mixture was dilutéd with (C2H5)20, filtered, and the
filtrate concentrated on a rotary evaporator. The residue
was distilled to 38.24 g. of product, b.p. 85-8C.I'0.5 mm.
Example 7
BenzYl P-Hexyl-P-trimethYlsilylphosphonoacetate
A solution of 16.80 g. (.0541 mole) of hexylbis-
(trimethylsilyl) phosphite and 11.04 g. (0.06 mole) of benzyl
chloroacetate was heated in a 165-170 oil bath for 1,5 hours,
Chlorotrimethylsilane be~an to distill at 165. The
_g_
`
106~5Z6
volatile material was evaporatively distilled at temperatures
up to 150 at 0.5 mm. The product was distilled at
175/0 5 mm to give 13.88 g (66% yield, 0.036 mole).
Example 8
P-HexYlphosphonoacetic Acid Monoammonium Salt
Benzyl P-hexyl-P-trimethylsilylphosphonoacetate,
13.0 g., was hydrolyzed with NH40H, and the diester debenzyl-
ated with H2-Pd. The product was crystallized from EtOH-
EtOAc to give 4 07 g. (50%) of product, m.p. 178-190.
Analysis Calcd. for C8H20NO5P: C, 39.83; H, 8.36; N, 5.81
Found: C, 39.90; H, 8.68; N, 5.78
Example 9
PropYlbis(trimethylsilyl) Phosphite
A solution of 80 g (0.50 mole) of propylphosphoro-
dichloridite in 60 ml. of (C2H5)2O was added dropwise to a
solution of 79 g. (1 00 mole) of pyridine and 18 g. (1.00
mole)of H2O in 500 ml. of (C2Hs)2O, and cooled in an ice bath.
After the addition was complete, the mixture was stirred at
room temperature for approximately 2 hours. The mixture was
filtered, and the filtrate dried over MgSO4 and evaporated
to give a clear colorless liquid.
The liquid was dissolved in 500 ml. of dry pyridine
and the solution was cooled in an ice bath. Dry (C2H5)3N,
208 ml. (1.5 mole) was added to the solution followed by the
dropwise addition of 189 ml. (1.5 mole) of chlorotrimethyl-
silane. The mixture was stirred overnight at room temperature,
diluted with (C2H5)2O, filtered and concentrated at reduced
- 10 -
:., : . , ~,. :. . .
695~Z6
pressure. The mixture was refiltered and the residue
distilled to give 50.34 g (37%) of product, b.p. 81.5-83.0/
10 mm.
Example 10
Benzyl-P-Propyl-P-trimethylsilylphosphonoacetate
A solution of 13.4 g. (0.05 mole) of propylbis- -
(trimethylsilyl) phosphite and 9.2 g. (0.05 mole) of benzyl
chloroacetate was heated at 160-5C for 1 hour. The
yellow-green residue was evaporatively distilled. The
fraction distilling up to 130/0.5 mm was discarded. The
fraction, temperature 140-160C/0.5 mm was collected;
yield 12,77 g. (74%).
Example 11
P-Propylphosphonoacetic Acid Monoammonium Salt
Benzyl P-(propyl)-P-(trimethylsilyl)phosphono-
acetate, 24.20 g. (0.0703 mole), was hydrolyzed with H20
and concentrated at reduced pressure. The solution was
chilled in an ice bath and made basic with concentrated
NH40H. Hydrogenolysis with H2-Pd gave the desired product.
; 20 The product was crystallized from C2H5OH to give 5 35 g
(38%) of the title compound, m.p. 155-156.5.
Analysis Calcd. for: C5H14NO5P: C, 30.15; H, 7.08; N, 7.03
Found: C, 30.26; H, 7.37; N, 7.03
-11-
~o695Z6 .
In Table I below, there is listed the characteristic
nuclear magnetic resonance (NMR) signals of compounds II,
III and IV as illustrated and defined above. Table I
~ncluc1es the solvent in which each compound was dissolved
in order to record the NMR spectra. The NMR spectra given :~
in Table I are for the ammonium salts of II, III and IV.
~ ,
-12-
10695ZG
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-13-
~0695Z6
Example 12
Herpes Sim~lex Viruses
Isolation and Purification of Herpes Simplex Type 2
_ _ Deoxyribonucleic Acid (DNA~ PolYmerase
Herpes virus infected Wi-38 cells were grown and
harvested when 25% of the cells showed cytopathic effect of
the virus. The DNA polymerase was isolated according to
the procedure of Smith and Gallo (1972) which involved
column chromatography on DEAE-cellulose and phosphocellulose,
However, buffer connaining 20% glyceryl instead of 10% was
used. The final enzyme preparation has a specific activity
of 313 units/mg. for herpes simplex virus type 2.
Viral Deoxyribonucleic Acid (DNA) Polymerase Assay
The reaction mixture (0,2 ml.) contains 10 ~M
of 2'-deoxyadenosine-5'-triphosphate~2'-deoxycytidine-5'-
triphosphateJ 2'-deoxyguanosine-5'-triphosphate~and 2,5
tritium labeled thymidine-5'-triphosphate which was
appropriately diluted with unlabeled dTTP to give 880 counts
per minute per pico-mole, 10 ~g of activated calf thymus DNA
50 mM Tris-HCl buffer (pH 8,0), 3mM MgC12, 100 mM KCl and 1
mM dithiothritol, The amounts of enzyme used in each r~action
was chosen to give a linear rate for at least 30 minutes at
37C, The reaction was terminated by the addition of 3 ml,
of cold 5% trichloroacetic acid - 0,01 M sodium pyrophosphate,
; 25 The acid-insoluble material was collected, washed twice on
glass filter discs (Reeve Angel 984-H) and the incorporated
3H-dTMP was determined by a liquid scintillation counter,
-14-
., . ~
10695Z~
The effect of each of the compounds in the
inhibition of Herpes simplex type 2 deoxyribonucleic
acid (DNA) is recorded below in Table II
Table II
INHIBITION OF HERPES SIMPLEX TYPE 2
Percent
Compound Concentration (u~L~l) Inhibition
II' 8.0 50%
III 166 0 50%
IV 19 9 50%
Example 13
The effectiveness of the phosphorus ester of
phosphonoacetic acid against herpes simplex infections in
mice was determined as follows: -
' 15 Mice were infected with herpes simplex virus,
type 2 and treated with a phosphorus ester of phosphonoacetic
;
acid two hours post infection and each of the five days
thereafter. Virus inoculation was accomplished by plucking
the fur from the flank and back of anesthetized mice and
'placing a drop (0.05 ml.) of herpes virus on the surface of
the pluc'ked s'kin. Using a needle, the skin of the mouse was
pricked through a drop of virus. The mice utilized for
control purposes were not treated in any manner.
In Table III below, there is listed the survival
time of untreated, infected mice and infected mice treated
1069526
with compounds of the present invention, The treated mice
had topically applied to them~ 2% aqueous solutions of
compounds II and III.
Table III
Average Survival Average Survival
Time of Untreated Time of Treated
Compound Mice (days) Mice (days)
II 9,1 11.8 (P = 0.05)*
III 8.7 9.5 (P = 0.05)*
*Statistical analysis using the Mann-Whitney U test.
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