Preparation and use of thiophosphonates and thio-analogues of phosphonoformic acid

Methods for converting phosphonates into thiophosphonates and specific thiophosphonate compounds so produced are disclosed and claimed. The methods start with a reaction mixture formed of a phosphonate compound, including one or more strong electron-withdrawing groups located adjacent to the phosphorus in the compound, a slight excess of Lawesson's reagent, and a polar aprotic solvent. The reaction mixture is heated until reaction is complete and may be followed with separation or hydrolyzation steps to produce thiophosphonic acids and their addition salts. One of these thio-analogues, thiophosphonoformic acid (TPFA) is particularly effective at inhibiting HIV replication and in treating mammals infected with HIV.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention is directed to new and useful processes for the large 
scale production of thio.analogues of phosphonoformic acid (PFA) and to 
the conversion of phosphonates into thiophosphonates in general as well as 
to the thio-PFA (TPFA) compounds produced by these procedures. An 
additional aspect of the present invention relates to the use of these 
thio-PFA compounds as antiviral agents which are particularly effective 
against HIV. 
2. Description of the Prior Art 
Organic compounds of the general structure 
##STR1## 
wherein X is oxygen (O) or sulphur (S) are known, respectively, as 
phosphonates and thiophosphonates. These compounds are implicated in a 
variety of biological processes and show promise in basic research for 
medical and agricultural uses including pesticides and antiviral 
compounds. Unfortunately, research involvingthiophosphonates is often 
hindered by the extreme difficulty in producing even small quantities of 
these phosphonate analogues. Moreover, economic methods for the large 
scale production of thiophosphonates are virtually unknown. 
For example, of particular interest to the present invention are the 
thio-analogues of phosphonoformic acid. Phosphonoformic acid (PFA) and its 
thio-analogue. thiophosphonoformic acid (TPFA) have the following general 
formulae and structures: 
##STR2## 
Early efforts reportedly producing TPFA utilized the Michaelis-Becker 
reaction between the sodio-derivative of diethyl thiophosphite and ethyl 
chloroformate or chloroacetate, followed by the removal of the P-OEt 
groups with iodotrimethylsilane (ITMS) at high temperature over 48 hours. 
However, recent research has indicated that this method is not 
reproducible and, because of difficulties including the removal of the 
ethyl groups, produces mixtures of a variety of compounds rather than the 
desired TPFA. Other proposed methods for the synthesis of TPFA are equally 
difficult and expensive utilizing numerous steps with exceptionally 
lowproduct yields. Similar difficulties and expense are associated with 
the production of the thio-analogues of other phosphonates as well. 
Difficulties in producing usable quantities of thiophosphonates are not 
restricted to commercial applications requiring large quantities of 
product. Basic research involving these compounds also requires readily 
available, pure materials. For example, as proposed and claimed by the 
present invention, TPFA shows great promise as an antiviral agent for use 
in combatting HIV infection and AIDS in mammals. These properties could 
not be determined in the past due to the inability of the prior art 
methods to produce usable quantities of essentially pure TPFA. However, 
before discussing these antiviral properties in detail, a general 
understanding of antiviral therapy will be of assistance. 
Unlike infectiousbacteria, which are functionally and physically distinct 
and can reproduce outside the cells of their host organisms, the 
simplicity of viruses makes them able to replicate only by physically 
invading a host cell and co.opting its biochemical mechanisms to make new 
viral components. As a result of this intimate connection with the 
replication cycle of the host cell, viruses present few unique biochemical 
features which can be selectively attacked without poisoning the host 
cell. As recently as the 1960's, it was believed that the only strategy 
for controlling viral infections was the development of vaccines against 
specific viruses to forestall infection by stimulating the immune system 
of uninfected individuals in advance. 
In spite of these problems, recent developments in the understanding of the 
details of viral functions have brought to light unique aspects of viral 
activities which may provide targets for attack. This accumulating body of 
knowledge has made it possible to identify compounds that may selectively 
interfere with these viral activities without poisoning the host cells of 
the infected organism. For example, in both lytic viral infections (those 
that spread rapidly throughout the population of vulnerable cells, 
destroying them early in the illness) and persistent viral infections 
(those that do not always kill an infected cell) the viral agents complete 
their replication cycles through a number of unique steps that an 
antiviral drug may interrupt. 
Unfortunately, the present state of the art is such that antiviral drugs 
are only capable of attacking such viruses when they are replicating. 
Attacking a latent virus such as HIV which does not reproduce itself 
following infection until reactivated by presently unknown factors 
wouldrequire distinguishing the viral genetic material from the 
surrounding host genetic material and selectively destroying it. Thus. the 
current generation of antiviral drugs is only effective against 
replicating viruses. 
Nonetheless, there are notable successes in the field of antiviral drug 
therapy. An exemplary antiviral compound is acyclovir, a nucleoside 
analogue which mimics the structure of a precursor of DNA Acyclovir has 
been found to interfere with the viral enzymes thymidine kinase and DNA 
polymerase specific to some herpes viruses, thereby inhibiting the 
synthesis of the viral DNA and ultimately viral replication itself. 
Similar antiviral effectiveness has been produced with a different 
nucleoside analogue. ribavirin which interferes with a viral enzyme 
crucial to the synthesis of DNA and RNA as well as selectively inhibiting 
viral mRNA and thus the production of viral proteins. Though far more 
effective against viral functions, ribavirin, like many antiviral 
compounds, may also affect human cells and thus may be toxic to rapidly 
metabolizing cells such as blood cells, limiting its applicability and 
usefulness. 
In spite of these and other antiviral success stories, the most important 
current challenge for the development of antiviral compounds is the need 
for an effective treatment against HIV, the viral cause of the AIDS 
pandemic. In contrast to the bleak epidemiological picture of AIDS wherein 
potentially millions of people are believed to be infected, the 
accumulation of knowledge aboutHIV and its functions has been 
unprecedentedly rapid. Though only identified in 1983, HIV is known to be 
a retrovirus whose main target is the T4 lymphocyte, a white blood cell 
which marshals the immune defenses of the infected host. Additionally, the 
virus also infects cells in the central nervous system. 
After binding to a host cell, HIV penetrates the cell and exposes its viral 
genetic material: a single strand of RNA. Accompanying the viral RNA is a 
viral enzyme known as reverse transcriptase which converts the viral 
genetic material into DNA which becomes integrated into the chromosomes of 
the infected host cell. The integrated viral genome or "provirus" remains 
latent until the host cell is stimulated and then directs the synthesis of 
viral proteins andRNA which assemble to form new HIV particles which burst 
from and destroy the host cell. 
The current target for antiviral drug therapy against HIV replication is 
the reverse transcription step which is crucial to the viral replication 
yet irrelevant to the infected host cells. A variety of antiviral drugs 
have been shown to reduce the activity of HIV reverse transcription in 
vitro to varying degrees. These compounds include azidothymidine (AZT), 
suramin, antimoniotungstate, dideoxynucleotides, and phosphonoformate AZT, 
has shown significant positive effects in large.scale clinical trials 
though major concerns remain about its considerable toxicity to 
bone.marrow cells. 
Several researchers have indicated that the pyrophosphate analogues. 
phosphonoacetic acid (PAA) and phosphonoformic acid (PFA) possess 
antiviral properties in that they inhibit the replication of several 
viruses including influenza virus A and herpes virus HSV.I. Research has 
shown that these compounds have an inhibitory activity on the reverse 
transcriptase of influenza virus A and the DNA polymerase of HSV.I as well 
as on the DNA polymerase of mammalian cells. (D. W. Hutchinson, G. Semple. 
and D. M. Thornton. Synthesis and Biochemical Properties of Some 
Pyrophosphate Analogues, Biophosphates and Their Analogues Synthesis, 
Structure, Metabolism and Activity, K. S. Bruzik and W. J. Stec (Eds.). 
Elsevier Science Publishers, B. V., 1987, 441.450.) 
Additionally, it has also been suggested in the art that the thio-analogues 
of phosphonoacetic acid (PAA) and phosphonoformic acid (PFA) may have 
potential as antiviral agents. (D. W. Hutchinson and S. Masson. The 
antiviral potential of compounds containing the thiophosphoryl group. 
I.R.C S. Medical Science 14 (1986) 176.177.) However, recent research by 
the inventor has raised significant questions as to the veracity of such 
reports. It is believed that the reported activities of the alleged 
thio-PFA compounds discussed in these prior art references are deceivingly 
incorrect as the prior art methods for preparing these compounds do not 
produce TPFA but, instead, produce mixtures of different, unidentified 
compounds. 
Moreover, as those skilled in the art will appreciate, further questions as 
to the accuracy and basis of such unsupported speculation with respect to 
the proposed properties of TPFA results from the fact that the inhibition 
of viral enzymes by such compounds in general is uniquely specific to the 
viral enzymes involved. Thus, it is impossible to predict the antiviral 
activity of a particular compound as that compound may or may not be 
effective against a particular virus. For example. acyclovir has proven to 
be beneficial in infection by Herpes virus, yet acyclovir.resistent 
strains of Herpes virus have been located. Similarly, Epstein-Barr virus 
(EBV) is relatively insensitive to acyclovir. Thus, it is clear that early 
signs of some antiviral activity are not indicative of a compound's 
effectiveness as an antiviral drug. 
Further complicating matters, a compound which may inhibit viral activity 
may also inhibit critical functions of the host cell and thus prove to be 
toxic to the host. As a result, antiviral compounds which may be effective 
in vitro may not be effective as antiviral agents in vivo due to a lack of 
significant differences in their relative inhibitory activities with 
respect to viral and host cell mechanisms. 
Accordingly. it is a principal object of the present invention to disclose 
methods for the effective production of large quantities of thio-analogues 
of PFA in order to facilitate the research and utilization of such 
compounds. 
It is an additional object of the present invention to disclose Processes 
for inexpensively producing large quantities of relatively pure TPFA and 
its analogues. 
It is a further object of the present invention to disclose novel 
thio-analogues of PFA. 
As those skilled in the art will also appreciate, it is also an object of 
the present invention to disclose novel methods for converting the general 
class of phosphonate compounds into thiophosphonates in a simple and 
economical manner. 
It is yet another object of the present invention to disclose methods for 
inhibiting viral and viral enzyme activities, including those of HIV, 
utilizing TPFA. 
Lastly, it is a further additional object of the present invention to 
disclose methods for treating HIV infection in mammalian cells utilizing 
TPFA or its addition salts as effective antiviral compounds. 
SUMMARY OF THE INVENTION 
Generally stated, the present invention accomplishes the above-described 
objectives by providing methods for readily converting phosphonates such 
as PFA into thiophosphonate analogues such as trimethyl-TPFA in a single 
step reaction which provides unusually high product yields. Moreover, 
following hydrolysis the TPFA and TPFA analogues produced through the 
methods of the present invention have unexpectedly high antiviral 
activities against HIV while exhibiting unexpectedly low DNA polymerase 
inhibiting activity against mammalian enzymes making them particularly 
well suited for use as effective anti-HIV agents. 
What is more, the processes of the present invention have wide 
applicability in converting phosphonates into thiophosphonates for the 
economical production of a wide variety of compounds including 
insecticides incorporating thiophosphonate units. Because the methods of 
the present invention produce desirable thio-analogues of phosphonates 
with a synthesis that is short, simple, efficient and which utilizes 
inexpensive starting materials, the present invention also produces such 
compounds in large quantity at relatively low cost. 
More particularly, the methods of the present invention convert 
phosphonates of the general formula: 
##STR3## 
where R.sub.1, R.sub.2, and R.sub.3 when present as substitutents are each 
independently hydrogen, hydroxy, methyl, alkyl, aryl, saturated 
unsaturated. substituted or unsubstituted organic compounds, through the 
following steps. 
First, the phosphonate is modified by substituting one or more strong 
electron withdrawing groups such as a halogen or doubly-bonded oxygen for 
x and y on the alpha carbon adjacent to the phosphorus in the general 
formula. Those skilled in the art will appreciate that it is unnecessary 
to modify phosphonate compounds in accordance with the teachings of the 
present invention where the alpha carbon is already bonded to a 
sufficiently strong electron withdrawing group. It should be noted that, 
without limiting the scope of the present invention, it is believed that 
the electron withdrawing group or groups must be directly adjacent to the 
phosphorus atom of the molecule in order for the reaction to proceed and 
must be sufficiently strong to drive the following unexpected reactiuon 
sequence. 
The next phase of the method of the present invention involves forming a 
reaction mixture of the modified phosphonate, an effective amount of one 
or more forms of Lawesson's reagent (for example, approximately one 
equivalent of a dimer of P-methoxyphenylthionophosphine sulfide or other 
suitable phosphetane ring containing compounds) and polar, aprotic 
solvent. An exemplary solvent is acetonitrile or toluene, though those 
skilled in the art will appreciate that any suitable polar aprotic solvent 
may be utilized. Following the formation of the reaction mixture the 
mixture is heated until conversion of the phosphonate into its 
thiophosphonate analogue is substantially complete. 
Preferably, the heating will take place under an inert, anhydrous 
atmosphere to prevent interference with the conversion reaction. Exemplary 
heating temperatures can range from approximately 66.degree. C. to 
110.degree. C. depending on the solvent utilized and may include reflux 
conditions. Additionally. heating times may be 1 hour or less, though 
preferably will be on the order of 2 to 6 hours. In practice, upon heating 
the reaction mixture, the Lawesson's reagent will be observed to gradually 
disappear and dissolve into the mixture conveniently signaling that the 
reaction is progressing to completion. 
Once reaction is substantially complete, if desired, the reaction product 
can be separated from the reaction mixture in a variety of manners. For 
example, the solvent can be evaporated and any side product (for example 
modified Lawesson's reagent, a trimer of P-methoxyphenylthionophosphine 
oxide) can be precipitated out of the solution. Conversely, it is possible 
to distill the thiophosphonate analogue from the mixture directly. Using 
the foregoing methodology and distilling the product directly from the 
reaction mixture will produce a pure product with yields up to and 
possibly exceeding 87%. In that the reagents utilized for this essentially 
one-step reaction are relatively inexpensive. and the yields of pure 
product are so high, the economies of the present invention become readily 
apparent. 
An exemplary phosphonate conversion in accordance with the teachings of the 
present invention utilizes trimethyl phosphonoformate as a starting 
material to produce trimethyl thiophosphonoformate (O,O-dimethyl 
carboxymethylphosphonothioate). The trimethyl phosphonoformate is mixed 
with approximately one equivalent (.+-.2.5%), of Lawesson's reagent in a 
generally two-to-one stoichiometric relationship such that there are two 
moles of trimethyl phosphonoformate for every mole of Lawesson's reagent. 
The aprotic, polar solvent used is, preferably, either acetonitrile or 
toluene and the mixture is heated under argon for two to six hours at a 
preferred temperature of 82.degree. C. for acetonitrile until the 
Lawesson's reagent is observed to dissolve into the mixture. 
The trimethyl thiophosphonoformate so produced may be separated from the 
reaction mixture if desired through precipitation or distillation and, in 
accordance with the teachings of the present invention, may be further 
modified through hydrolysis to produce thiophosphonoformic acid (TPFA) and 
its addition salts. Preferably, when desired, hydrolysis will take place 
under basic conditions such as the utilization of sodiumhydroxide (NaOH) 
to directly hydrolyze the trimethyl-TPFA to TPFA. Conversely, though 
ITMS-H.sub.2 O will not hydrolyze the ethyl-ester of TPFA, it was 
surprisingly discovered to be effective at hydrolysing the methyl ester. 
The TPFA so produced may then be utilized in accordance with the teachings 
ofthe present invention as a antiviral inhibitor against HIV virus and in 
a method for treating mammals infected with HIV. As will be discussed in 
detail below. this unique and unexpected antiviral activity against HIV is 
a product of the high therapeutic index of this compound with respect to 
HIV. More specifically, recent studies made possible by the method of the 
present invention show TPFA to be surprisingly effective at inhibiting HIV 
and HIV reverse transcriptase while being surprisingly less toxic with 
respect to inhibition of mammalian DNA polymerase. 
Moreover, as will be appreciated by those skilled in the art, the 
conversion of PFA to TPFA gives the sulfur analogue a lower polarity thus 
providing enhanced cell-penetration and higher water solubility. As a 
result, it is believed that the TPFA antiviral compounds of the present 
invention willbe significantly less toxic than PFA in treating mammalian 
cases of HIV infection and inhibit HIV in general. 
The above discussed and many other features and attendant advantages of the 
present invention will become apparent to those skilled in the art from a 
consideration of the following detailed description. 
DETAILED DESCRIPTION OF THE INVENTION 
In a broad aspect, the methods of the present invention are based upon two 
surprising discoveries. First, while it is known in the art that 
Lawesson's reagent (LR) is effective at converting oxygen to sulfur in 
carbonyl groups such as those found in ketones or in converting phosphites 
to thio-phosphites, it was completely unexpected that LR would prove to be 
so effective at converting phosphonates to thiophosphonates in accordance 
with the techniques of the present invention. Secondly, the large 
quantities of pure TPFA so produced made it possible to determine the 
relatively high therapeutic index of TPFA with respect to the inhibition 
of HIV versus mammalian enzymes; a result which was also completely 
unexpected in view of the teachings of the prior art. Thus, the methods of 
the present invention provide new, uniquely effective procedures for 
rapidly, simply. and inexpensively producing large quantities of 
essentially pure thiophosphonates. Of equal or greater significance, the 
methods of the present invention make it possible to efficiently produce 
TPFA and other thio-analogues of PFA in sufficient purity and quantity for 
use as new, effective antiviral agents against HIV. 
Turning first to the general process of converting phosphonates into their 
thiophosphonate analogues and additions salts thereof, a more detailed 
understanding of the limitations of the prior art methodologies for 
producing such compounds is in order. The most widely-known method in the 
art for reportedly synthesizing thiophosphonates is that currently 
reported by Hutchinson (D. W. Hutchinson and Masson, The antiviral 
potential of compounds containing the thiophosphoryl group, I.R.C.S. 
Medical Science, 14 (1986) 176-177). Briefly, Hutchinson, et al. report 
the preparation of alkylthiophosphonate intermediate compounds utilizing 
the Michaelis-Becker reaction followed by removal of the alkyl groups 
utilizing iodotrimethylsilane (ITMS). Generally stated, the prior art 
Hutchison et al. reaction mechanics are reported as follows: 
##STR4## 
Additionally, a more limited, alternative prior art synthesis of TPFA was 
proposed in a verbal presentation by Dr. William Egan. Though few details 
are available on this proposed synthesis. it is believed to include at 
least eight to ten steps and may require unique and expensive starting 
materials. Thus, as will be appreciated by those skilled in the art, any 
such synthesis would be a very long, complicated and expensive process 
having very low overall product yields. 
Similarly. the synthesis proposed by Hutchinson et al. is not without its 
own problems. In addition to very low yields, the proposed reaction is 
incomplete and produces mixtures of unidentified products. Though 
apparently successful at producing some of the intermediate tri.ester 
compounds, the Hutchinson et al. method apparently is not successful at 
ultimately removing the alkyl groups with ITMS. Elaboration of these 
distinguishing features of the prior art methodology will be provided 
following a more detailed explanation and understanding of the present 
invention. 
As discussed in the foregoing summary section it is known in the art that 
Lawesson's reagent (LR) will convert doubly-bonded oxygen to sulfur on 
carbonyl groups such as those in ketones and will convert phosphites into 
thio-phosphites. There the relative bond energies drive the reaction to 
completion. However, in the case of phosphonates where oxygen is 
doubly-bonded to phosphorus the favorable bond energies are not present to 
drive the reaction as LR also includes phosphorus double bonds. 
Nevertheless, as disclosed by the present invention. it was surprisingly 
discovered that LR would successfully convert phosphonates into their 
thiophosphonate analogues if a sufficiently strong electron-withdrawing 
group was located adjacent to the phosphorus in the phosphonate as 
evidenced by the following non-limiting experimental reactions. 
All reactions were performed in scrupulously dried glassware under N.sub.2. 
Lawesson's reagent (LR) and Me.sub.3 PFA were used without purification: 
CH.sub.3 CN was distilled from CaH.sub.2 under N.sub.2. All reactions were 
performed in acetonitrile unless otherwise noted. 
REACTION BETWEEN ME.sub.3 PFA and LR 
1.66 g (4.11 mmol) LR and 1.38 g (8.22 mmol) Me.sub.3 PFA were suspended in 
25 ml CH.sub.3 CN and stirred for 2 hours with no apparent reaction. After 
12 hours, still no reaction had occurred. After heating for 2 hours, the 
LR dissolved, and NMR indicated that reaction had occurred. Major product, 
.sup.31 P NMR: .delta.=64 ppm. and .sup.31 C NMR indicated that the ester 
group was intact. Impurities in the .sup.31 P NMR spectrum could be 
removed by evaporating the mixture, and extracting residue between 
saturated aq. NaHCO.sub.3 and ether, with the product found in the organic 
layer. In a reaction between 1 g ester and 1.3 g LR in refluxing THF for 2 
hours. NMR suggested that partial reaction had occurred. 
REACTION BETWEEN (tmso).sub.2 p(O)CO.sub.2 ME AND LR 
0.5 g ester and 0.35 g LR were reacted as above for 24 hours; .sup.31 P NMR 
suggested that reaction had occurred: .delta.=46 ppm. with impurities. The 
same reaction was performed with 0.5 g ester and 0.7 g LR, and the same 
product was obtained in a cleaner reaction. Thus, the reaction was 
repeated using 6.5 g ester and 6 g LR, and Proton.coupled .sup.31 P NMR 
showed that the phosphorus was coupled to a methyl group. (q, .sup.J PH - 
11 Hz.) CN test was negative. Evaporation of the mixture was done: 
attempted Kugelrohr distillation failed. 
REACTION BETWEEN LR AND OTHER SUBSTRATES 
Further experiments conducted as above indicated that LR was inert to 
Et.sub.3 PFA, Et.sub.3 FPAA, 1PR.sub.4 MDP, 1PR.sub.4 N.sub.2 MDP under 
the foregoing conditions. However, slightreaction occurred with Et.sub.3 
F.sub.2 PAA indicating that substituting strong electron.withdrawing 
groups such as fluorine adjacent to the phosphorus in the phosphonate 
compound would drive the reaction to the point that LR would be successful 
at converting such modified compounds into their thiophosphonate 
analogues. However, it should be noted that decomposition took place with 
dibromo and monobromo compounds under the above conditions. 
Further demonstrating the utility of the present invention, a variety of 
experiments were conducted utilizing trimethyl phosphonoformate (Me.sub.3 
PFA) as a starting material for use in accordance with the teachings of 
the present invention. Those skilled in the art will appreciate that 
Me.sub.3 PFA incorporates a strongly electron.withdrawing doubly bonded 
oxygen directly adjacent to the phosphorus in the compound which, in 
accordance with the present invention, will enable the thio-conversion 
reaction to proceed utilizing LR. The following non-limiting experiments 
were conducted utilizing larger amounts of reagents and under varying 
conditions to further illustrate the scope of the present invention.

GENERAL EXPERIMENTAL PROTOCOL 
All glassware was scrupu-lously oven, or flame-dried. All reactions were 
performed under dry, pre-purified argon (passed successively through 
columns of drierite; activated Linde Type 4A molecular sieves; and BASF 
catalyst). Lawesson's reagent (LR) was purchased from Aldrich Chemical 
Company (97%) and was used without further purification. Trimethyl 
phosphonoformate (Me.sub.3 PFA) was also purchased from Aldrich Chemical 
Company and was purified by vacuum distillation prior to use (60.degree. 
C., 15 .mu.m). 
Solvents were purified using standard methods. Acetonitrile was distilled 
from P.sub.2 O.sub.5, then from CaH.sub.2 ; tetrahydrofuran was distilled 
from benezophenone/sodium ketal, thenfrom lithium aluminum hydride; 
toluene was distilled from benezophenone/sodium ketal. Hexane and ethyl 
acetate for chromatography were reagent grade and were used directly. 
Reactions were monitored by thin layer chromatography using silica gel 
60F-254 (Kieselgel) and detected by an ultraviolet lamp (Mineralight 
UVS-12). Flash column chromatography was performed as described in the 
literature (Still, W. C.; Kahn, M.; Mitka, A. J. Org. Chem. 1978, 43, 
2923.) 
NMR spectra were obtained on a Bruker IBM WP-270SY spectrometer operated at 
frequencies of 270.02 MHz (.sup.1 H), 109.35 MHz (.sup.31 P) and 67.92 MHz 
(.sup.13 C). NMR spectra for isolated compounds were obtained in 5 mm 
tubes using 10% solutions (CDCl.sub.3 for esters. D.sub.2 O for salts). 
Chemical shifts are reported relative to TMS (.sup.1 H: internal 
CHCl.sub.3, .delta.=7.24 ppm; .sup.13 C: using internal CDCL.sub.3, 
.delta.=77.0 ppm); or external 85% H.sub.3 PO.sub.4 (.sup.31 P). Vacuum 
distillations were performed on a vacuum line equipped with an all glass 
oil diffusion pump; pressures were measured on a MacLeod gauge. 
Infrared spectra of esters were obtained on a Perkin.Elmer 281 infrared 
spectrophotometer. Neat samples were run as thin films between NaCl 
plates. Spectra of salts were taken measured on a FT-IR/32, infrared 
spectrophotometer 
High resolution mass spectra were obtained at the Mass Spectral Facility, 
University of California, Riverside, Calif. 
Elemental analyses were performed by Galbraith Laboratories, Knoxville, 
Tenn. 
EXPERIMENT 1 
A 500 mL three.necked round.bottomed flash equipped with reflux condenser, 
magnetic stirrer and thermometer was connected to an Ar bubbler, flushed 
with Ar and charged with 375 mL acetonitrile. Me.sub.3 PFA (22.0 g, 130.9 
mmol) was added(syringe) and dissolved (magnetic stirring). Lawesson's 
reagent (LR) (dimer of p-methoxyphenylthionophosphinesulfide)(26.40 
g,65.45 mmol)wasadded as a pale yellow, chalky powder to the flask (glove 
bag, Ar). After stirring for 2 h at room temperature, no reaction or 
solubilization was evident. The mixture was then refluxed at 82.degree. C. 
for 6 h, during which time the LR appeared to gradually dissolve, giving a 
dark-yellow solution. The reaction mixture was cooled to room temperature 
causing precipitation of a creamy white powder. This was removed by 
suction filtration (9.186 g, fluted filter paper, Eatman Diechmann Grade 
515, 2.5 cm) giving a pale yellow solution which was washed and filtered 
three times with 20 mL diethyl ether. Excess ether and acetonitrile were 
removed by evaporation, leaving a pale yellow oil, whose .sup.31 P NMR 
(.sup.1 H) showed signals at .delta.=71.9-83.8 ppm and at .delta.=65 
ppm(s); no signal was observed at .delta.=2 ppm (trimethyl PFA). After 1 
day at room temperature, a white crystalline solid precipitated from the 
crude residue. TLC analysis of the supernatant (TLC - I), (hexane:ethyl 
acetate, 6:1) revealed a single migrating spot at R.sub.f 0.35, in 
addition to a spot at the sample origin. The following summary of 
analytical TLC results (TLC - II) evaluating different solvents were 
performed using silica gel 60.degree. F. (Eastman Chromagram), detected by 
an ultraviolet lamp. In all cases, the mixture resolved into two very 
intense blue mobile spots with no detection of Me.sub.3 PFA present. In a 
comparison between Me.sub.3 PFA (R.sub.f 0.06) and Me.sub.3 TPFA (R.sub.f 
0.35), the latter was much more intense under ultraviolet detection. TLC 
(hexane:EtOAC, 7:3): Rf 0.07, 0.62, 0.81. (hexane): too weak. (ether): 
R.sub.5 0.66, 0.10. (chloroform): no separation. EtOAC: R.sub.f 0.10, 
0.90. (methylene chloride): R.sub.f 0.06, 0.64, 0.90, no strong 
separation. 
The reaction mixture (29 g of total 44 g of crude product) was separated by 
flash column chromatography (TLC - I, same solvent system). Like fractions 
(TLC) were combined and evaporated to yield (.sup.31 P NMR) virtually pure 
Me.sub.3 TPFA (O,O - Dimethyl Carboxymethylphosphonothioate) (13.29 g), 
(contained a trace of impurity with .delta.=72 ppm). Pure product was 
obtained by fractional distillation in vacuo: very pale mobile oil, bp 
37.39.degree. C. (10 .mu.m), 10.83 g (87% yield). 
More conveniently. the crude supernatant (10 g of 44 g crude product) was 
directly distilled, giving 4.75 g (87% yield) distillate, pure by TLC, IR, 
.sup.31 P and .sup.1 H NMR. Bp: 37.degree.-39.degree. C. (10 .mu.m) TLC 
(hexane:EtOAC, 6:1): R.sub.f 0.35. .sup.1 H NMR: .delta.=3.81 ppm (d, 
.sup.3 J.sub.PH =14 Hz, C(O)OCH.sub.3); 3.85 ppm (d, .sup.3 J.sub.PH =1 
Hz, P(S)OCH.sub.3). .sup.13 C NMR: .delta.=54.3 ppm (qd, .sup.1 J.sub.CH 
=150 Hz, .sup.3 J.sub.CP =7 Hz, CH).sub.3); .delta.=52.7 ppm (qd, .sup.1 
J.sub.CP =149 Hz, .sup.3 J.sub.CP =5 Hz). .sup.31 P NMR: .delta.=64.8 ppm 
(septet, .sup.3 .sup.3 J.sub.PH =14 Hz). IR: 1722 cm.sup.-1 (s, v.sub.CO), 
1035 cm.sup.-1 (m, .sup.V POC) no peak at 1290 cm.sup.-1 (v.sub.PO, 
Me.sub.3 PFA). MS: Parent ion m/e 1984, major fragments m/e 125 [(CH.sub.3 
O).sub.2 PS.sup.+ ], m/e 93,79. Parent ion m/e calculated: 183.9959; 
Found: 183.9956. Anal. calculated for C.sub.4 H.sub.9 O.sub.4 PS: C, 
26.09; H, 4.93; S, 17.41. Found: C, 26.23; H, 5.00., S, 17.82. 
It was shownby TLC that the second fraction collected from flash column 
chromatography (R.sub.f 0.14) upon evaporation crystallized from 
hexane:EtOAC, 4:1. These crystals were very soluble in chloroform and had 
the same R.sub.f values as the creamy white precipitate that came out of 
solution and the crystalline solid found on the bottom of the crude flask. 
IR and NMR spectra indicate aromatic rings present. A white diamond-shaped 
crystal suited for x-ray diffraction crystallography was mounted on a 0.3 
mm glass capillary. Data was collected on a four.circle.Nicolet/Syntex 
P.sub.21 diffractometer employing Cuo radiation. A large triclinic cell 
with a volume of 2438 As was revealed by carefully machine-centering 
fifteen strong reflections, a procedure which also yielded the orientation 
matrix needed for data collection. The crystal data is currently being 
solved. .sup.31 P NMR: .delta..about.7 ppm. .sup.1 H NMR: .delta.=72 ppm 
(m). 
Attemqpts to remove byproducts by extractions between saturated aqueous 
NaHCO.sub.3 and ether were unsuccessful. 
EXPERIMENT 2 
The same reaction was repeated under identical conditions LR gradually went 
into solution and thereaction was refluxed at 82.degree. C. for 6 h. 
However, upon cooling to room temperature quickly followed by evaporation 
of acetonitrile, no precipitation was seen. The reaction mixture (57.7 g) 
was separated by flash column chromatography (TLC I, solvent system) with 
10-12 g of crude residue loaded per column. One column was done with 
Aldrich silica gel (TLC standard grade, 2-25 .mu.). Routine TLC - I 
analysis indicated that in addition to MeTPFAs (R.sub.f 0.36), another 
nonpolar intense blue spot was observed (R.sub.f 0.46). Like fractions and 
fractions with mixtures of these two spots were combined, evaporated and 
vacuum distilled (bp 38.5.degree. C., 10 .mu.m) yielding Me.sub.3 TPFA 
contaminated with impurities (7.84 g, 33% yield). .sup.31 P NMR {.sup.1 H} 
of the distillate indicated only pure Me.sub.3 TPFA (.delta.=64.8 ppm) 
however TLC analysis still showed two spots. A further TLC - I analysis of 
the impurity (R.sub.f 0.56) was made by varying the amount of sample 
spotted on the plate. The plate was observed under an ultraviolet lamp and 
then treated with phosphomolybdic acid (spray, 7% solution in ethanol). 
Examination of the TLC plate revealed that this compound consisted of a 
light blue spot (R.sub.f 0.56) in addition to a dark violet-blue spot 
(R.sub.f 0.39), the latter of which has a similar R.sub.f value to 
Me.sub.3 TPFA (R.sub.f 0.35) which appeared to be a faint blue color. 
.sup.31 P NMR {.sup.1 H} showed a signal at .delta.=99.99 ppm. This 
impurity was carefully removed by flash column chromatography (TLC - I, 
solvent system). A long column vacuum distillation yielded pure Me.sub.3 
TPFA (6.50 g, 27% yield, bp 39.5.degree. C., 10 .mu.m). 
EXPERIMENT 3 
Several reactions wererun changing the reactants addition order. In a 100 
mL round-bottomed flask, LR (5.3 g, 13.09 mmol) was added under Ar and 
charged with 75 mL acetonitrile (glove bag, Ar). No immediate reaction or 
solubilization was evidentwith stirring. Me.sub.3 PFA (4.4 g, 26 18 mmol) 
was added (syringe) and the mixture was refluxed at 82 .degree. C. for 6 h 
at which time LR appeared to gradually dissolve giving a dark yellow 
solution. When the reaction mixture was cooled to room temperature, no 
precipitation was seen. Only after evaporation of acetonitrile, a 
crystalline solid precipitated from the crude residue which was identical 
to thesolid described in the experiment previously (TLC - I). Flash column 
chromatography (TLC - I. solvent system) and vacuum distillation yielded 
pure product, Me.sub.3 TPFA (2.78 g, 57% yield). 
EXPERIMENT 4 
In a similar reaction, LR (5.3 g, 13.09 mmol) was added under Ar and 
charged with 75 mL THF (glove bag, Ar). Again, no immediate reaction or 
solubilization was evident with stirring. Me.sub.3 PFA (4.4 g, 26.18 mmol) 
was added (syringe) and the mixture was refluxed at 66.degree. C. LR went 
into solution within 2 h. Refluxing was continued for 4 h more. NMR 
indicated no starting material left. Again, the crystalline solid 
precipitated after 1 day standing at room temperature. Pure product was 
obtained after flash column chromatography (TLC - I, solvent system) and 
distillation in vacuo, Me.sub.3 TPFA (2.20 g, 48% yield). 
EXPERIMENT 5 
In a similar reaction, excess LR (22.62 g, 55.03 mmol) was added under Ar 
to a 500 mL round-bottomed flask and charged with 375 mL acetonitrile 
(glove bag, Ar). Again, no immediate reaction or solubilization was 
evident with stirring Me.sub.3 PFA (17.56 g, 104.4 mmol) was added 
(syringe) and the mixture refluxed at 82.degree. C. for 6 h at which time 
the LR appeared to gradually dissolve giving a yellow solution. The 
reaction mixture was cooled to room temperature and excess acetonitrile 
was removed by evaporation leaving a pale yellow oil with slight 
precipitation of a creamy white powder. After one day standing at room 
temperature, a large amount precipitated (39.00 g) containing a small 
amount of yellow oil. The oil was found to be very soluble in hot hexane 
leaving the solid residue behind. To the crude residue, 200 mL hexane was 
added and heated with steam until reflux and gravity filtered while hot 
(steam filtration, Whatman filter paper Grade #1). Excess hexane was 
removed by evaporation giving a pale yellow oil (13.48 g). TLC analysis 
ofthe yellow oil (hexane:ETOAC, 4:1) revealed intense blue spots (R.sub.f 
0.46, 0.26, 0.04) under an ultraviolet lamp Analysis of the precipitate 
showed two intense blue spots (R.sub.f 0.2, 0.04) and two spots which were 
lighter blue (R.sub.f 0.56, 0.44). Direct distillation of the crude yellow 
oil gave 11.63 g (61% yield) distillate (bp 38.degree.-39.degree. C., 10 
.mu.m). TLC and .sup.31 P NMR revealed this to be Me.sub.3 TPFA 
(.delta.=64.8 ppm) with a little Me.sub.3 PFA starting material (6%). The 
crude residue left in the pot was a thick lemon yellow gum (bp&gt;100.degree. 
C.) having the same properties as the LR side product discussed earlier 
(TLC). 
EXPERIMENT 6 
Three reactions were performed simultaneously using toluene as the solvent 
varying the relative amounts of LR used. A 100 mL, and two 250 mL 
round.bottomed flasks were flushed with Ar and charged with 5 mL toluene. 
To each flask, Me.sub.3 PFA (4.4 g. 26.18 mmol) was added (syringe) and 
dissolved (magnetic stirring). Consecutively, LR (5.6 g, 3.72 mmol) was 
added to the 100 mL flask (I), LR (11.1 g, 27.4 mmol) was added to one 250 
mL flask (II), and LR (16.7 g, 41.2 mmol) was added to the other 250 mL 
flask (III), (glove bag, Ar). Again. no immediate reaction or 
solubilization was evident with stirring. The mixtures were refluxed at 
100.degree. C. and LR gradually dissolved giving yellow solutions. 
All reactions were stopped after 1 h. I was a pale yellow solution with no 
precipitate present even after cooling to room temperature (similar to the 
THF reaction). II was a clarified lemon-yellow solution which precipitated 
a pale yellow crystalline residue at room temperature. III was a darker 
lemon-yellow solution with a large amount of lemon-yellow precipitate. A 
summary of TLC results (hexane:ETOAC, 7:3) monitoring the reactions were 
detected by an ultraviolet lamp. I showed very little of both Me.sub.3 PFA 
(R.sub.f 0.08) and MeTPFAs (R.sub.f 0.5) but an intense blue spot similar 
to the LR crystalline side product previously described (R.sub.f 0.32). II 
and III showed similar results, however, more Me.sub.3 PFA was present for 
these two, I, II and III had similar .sup.31 P NMR (.sup.1 H) showing 
signals for MeTPFAs .delta.=65 ppm(s) and signals at .delta.=4, 71.8-75.1, 
and 92.3 ppm. 
Refluxing was continued for 1 h and after standing at room temperature 
overnight I was a pale yellow solution, II was a clarified lemon-yellow 
solution with a pale yellow crystalline residue and III was a darker 
clarified lemon-yellow solution with a large amount of crystalline 
precipitate. TLC analysis (hexane:ETOAC, 7:3) showed an intense blue spot 
for all three (R.sub.f 0.34) similar to the LR crystalline side product, a 
light blue spot (R.sub.f 0 56) similar to MesTPFA. in addition to two 
lightblue nonpolar spots (R.sub.f 0.62, 0.96). Both II and III still 
showed some Me.sub.3 PFA present (R.sub.f 0.5). .sup.31 P NMR (.sup.1 H) 
of I, II and III revealed the peaks at .delta.&gt;71 ppm. Analysis of I 
showed the singlet at .delta.=65 ppm now was a multiplet 
(.delta.=65.4-65.6 ppm). Similarly, analysis of II showed a doublet 
(.delta.=65.3-65.4 ppm) III showed no change (.delta.=65.5 ppm). 
From the foregoing experiments, it will be apparent to those skilled in the 
art that the methods of the present invention are particularly effective 
at producing the thio analogue of Me.sub.3 PFA, namely trimethyl 
thiophosphonoformate (O,O-Dimethyl Carboxymethylphosphonothioate). 
Moreover, the methods of the present invention produce this compound with 
exceedingly high yields in a very simply, essentially one.step reaction 
utilizing inexpensive starting materials. As detailed above, the trimethyl 
thiophosphonoformate can be readily separated from the reaction mixture 
through distillation or precipitation or chromatographic methodologies. 
It is also contemplated as being within the scope of the present invention 
to utilize the additional step of hydrolyzing the trimethyl 
thiophosphonoformate to produce thiophosphonoformic acid and/or its 
addition sales. Preferably, hydrolyzation will take place under basic 
conditions as illustrated by the following non-limiting examples detailing 
the production of TPFA and its sodium addition salt. However, those 
skilled in the art will appreciate that other hydrolyzation methods 
including the correct usage of ITMS are contemplated as being within the 
scope of the present invention 
EXPERIMENT 7 
2.75 ml of 10 N sodium hydroxide solution were added to 1.0 g (5.43 mmol) 
of Me.sub.3 TPFA with vigorous stirring at room temperature. After 3.5 
minutes, the mixture became hot and the methanol produced evaporated. 
Stirring was continued for ca. 15 min. and the mixture cooled in an ice 
bath. The pH was adjusted to 10.5 with 1N HCl. The solvent was evaporated 
by dry.ice freeze pumping. Distilled water (2 mL) and excess methanol were 
added. The precipitate formed was centrifuged and over.dried in vacuo, 
neutralized to pH 6 with 1N HCl then readjusted to pH 10.5 with 1N NaOH. 
The solvent was again evaporated by dry-ice freeze pumping. Then 1.5 ml 
water and excess methanol were added. The precipitate formed was 
centrifuged and oven. dried in vacuo. The process was repeated, 207.5 mg 
of pure desired salt were obtained (18.4% yield) .sup.31 P NMR: 
.delta.=37.7 ppm (s). .sup.1 H NMR: .delta.=4.63 ppm (H.sub.2 O) in 
D.sub.2 O :.sup.13 C NMR: .delta.=183.2 ppm (d, .sup.1 J.sub.CP =181 Hz, 
CO). IR. Anal. Calculated for: C 5.77; H, 0.00; S, 15.41. Found: C, 5.83; 
H, 0.12; S, 14.91. 
Those skilled in the art will appreciate that while the cleavage of ester 
compounds through basic hydrolysis is known in the art, whether or not a 
particular di.functional ester will be cleaved under such conditions 
cannot be predetermined. Thus, as further evidence of the scope of the 
present invention, it is possible to substitute the di.ethyl ester of TPFA 
for Me.sub.3 TPFA in the above experiment to produce TPFA. As expected, 
when utilizing Et.sub.3 TPFA as a starting material, some of the foregoing 
reaction conditions for base hydrolysis must be changed. For example, the 
reaction time must be increased to approximately thirty minutes and it is 
also anticipated that the production of side products may be increased. 
EXPERIMENT 8 
An improved yield of pure Na.sub.3 TPFA was obtained utilizing the 
following protocol. As before 2.75 ml of 10N sodium hydroxide solution 
were added to 1.0 g (5.43 mmol) of Me.sub.3 TPFA with vigorous stirring at 
room temperature. After 3.5 minutes, the mixture become hot and cloudy, 
and most of the methanol produced evaporated. Stirring was continued for 
approximately 10 min., and the mixture was cooled in an ice bath. 
Distilled water (3 mL) and 30 mL methanol were added. The precipitate 
formed was centrifuged and oven-dried in vacuo (&lt;1 mm Hg, 50.degree. C.) 
10 min., neutralized to pH 4.5 to remove CO.sub.2 (from Na.sub.2 CO.sub.3 
formed during the reaction), with 3N HCl (approximately 4-5 mL) then 
readjusted to pH 10.5 with 3N NaOH (approximately 0.5 mL). The solvent was 
evaporatedby lyophilization. Water (2.5 mL) and methanol (30 mL) were then 
added. The precipitate formed was centrifuged as before and oven-dried in 
vacuo (&lt;1 mm Hg 55.degree. C. for 6 h). The process was repeated. 231.5 mg 
of pure Na.sub.3 TPFA (white powder) was obtained (20.5% yield). .sup.31 P 
NMR: .delta.=37.7 ppm (s). .sup.1 H NMR: no resonances other than HDO. 
.sup.13 C NMR: .delta.=183.2 ppm (d, .sup.1 J.sub.CP =181 Hz. CO). IR: 
1680 cm.sup.-1 (m), 1095 cm.sup.-1 (shoulder), 1580 cm.sup.-1 (s), 1375 
cm.sup.-1 (s), 1140 cm.sup.-1 (s), 1030 cm.sup.-1 (s). UV: .sup..delta. 
254 nm=1.05.times.10.sup.3, .sup..epsilon. 233=2.44.times. 10.sup.3, 
.sup..epsilon. 205=6.0.times.10.sup.3. Analytical calculated for Na.sub.3 
TPFA: C, 5.77; H, 0.00; S, 15.41. Found: C, 5.83; H, 0.12; S, 14.91. 
It should be noted that by using Et.sub.3 TPFA as starting material, the 
same product can be obtained, but in low yield and accompanied by 
impurities. Additionally, when using Et.sub.3 TPFA as a starting material 
the reaction time needed to be increased to approximately 30 minutes. 
resulting in increased formation of side product. 
Nonetheless, as will be appreciated by those skilled in the art, the 
reagents utilized in the foregoing basic hydrolysis experiments are very 
inexpensive relative to compounds such as ITMS, further contributing to 
the superior economics of the methods of the present invention. Moreover, 
as detailed in the following examples, the prior art methodologies 
utilizing expensive reagents such as ITMS are not successful at producing 
thiophosphonates such as TPFA. 
In the following examples, a variety of phosphonate starting materials were 
synthesized utilizing either the method of the present invention or that 
of Hutchinson et al. where possible. These compounds were then subjected 
to ITMS hydrolysis as disclosed in the prior art to illustrate the 
difficulties of this prior art methodology and to further distinguish the 
novel methods of the present invention. 
PREATION OF O,O-DIETHYL HYDROGEN PHOSPHOROTHIOIITE [EtO).sub.2 P(S)H] 
(Michaelis-Becker Reaction) 
A mixture of diethyl dithiophosphate and triphenylphosphine was stirred 
vigorously at 65.degree. C. for 7 h. After fractional distillation in 
vacuo, the product was obtained in 59% yield. Bp: 61.degree.-62.degree. C. 
(4 mm). .sup.31 p NMR: .delta.=69.3 ppm (dp, .sup.1 J.sub.PH - 647 Hz, 
.sup.2 J.sub.PH - 11 Hz). .sup.1 H NMR: .delta.=1.16 ppm (t, .sup.3 
J.sub.HH - 7 Hz, CH.sub.3 CH.sub.2); .delta.=3.98 ppm, (m, .sup.3 J.sub.HH 
- 7 Hz, CH.sub.3 CH.sub.2); .delta.=7.57 ppm (d, .sup.1 J.sub.HP - 647 Hz, 
P(S)H). .sup.13 C NMR: .delta.=15.69 ppm (qd, .sup.1 J.sub.CH - 204 Hz, 
.sup.3 J.sub.CP - 12 Hz, CH.sub.3 [PO]); .delta.=61.78 ppm (t, .sup.1 
J.sub.CH -238 Hz). 
PREATION OF Et.sub.3 TPFA cl (Michaelis-Becker Reaction) 
Finely divided sodium was suspended in dry benzene. The suspension was 
added to the solution of diethyl thiophosphite also dissolved in benzene. 
The mixture was heated to 50.degree. C. for 2 h, then cooled to 5.degree. 
C. with ice bath. Ethyl chloroformate dissolved in benzene was added 
drop.wise to the above mixture at room temperature. The mixture was heated 
to 50.degree. C. for 3 h, then cooled to room temperature, and 
centrifuged. The clear resulting solution was washed with water, dried 
with MgSO., evaporated, and separated by fractional distillation in vacuo. 
The yield was 43%. Bp: 95.degree.-96.degree. C. (1.22 mm). IR. .sup.31 P 
NMR: .delta.=61.4 ppm, (P, .sup.3 J.sub.PH - 10 Hz). .sup.1 H NMR: 
.delta.=1.25 ppm (m, .sup.3 J.sub.HH -7 Hz, CH.sub.3), 9H; .delta.=4.17 
ppm (m, .sup.3 J.sub.HH - 7 Hz, CH.sub.2), 6H. .sup.13 C NMR: .delta.=13.7 
ppm (q, .sup.1 J.sub.CH - 128 Hz, CH.sub.3 [CO]); .delta.=15.8 (qd, 
.sup.1 J.sub.CH - 128 Hz, .sup.3 J.sub.CP - 7 Hz, CH.sub.3 [PS]); 
.delta.=62.1 ppm (td, .sup.1 J.sub.CH - 145 Hz, .sup.3 J.sub.CP - 4 Hz, 
CH.sub.2 [CO]); .delta.=64.2 ppm (td, .sup.1 J.sub.CH - 149 Hz, .sup.3 
J.sub.CP - 7 Hz, CH.sub.2 [PS]); .delta.=167.3 ppm (d, .sup.1 J.sub.CP - 
225 Hz, CO). Anal. Calculated for: C, 37.16; H, 6.68; S, 14.17. Found: C, 
36.9; H, 6,62; S, 14.06 
PREATION OF METHYL (O,O-DIMETHYL) THIOPHOSPHONOFORMATE (ME.sub.3 TPFA) 
(Present Invention Methodology) 
LR and Me.sub.3 PFA were suspended in CH.sub.3 CN or THF and heated ro 
78.degree. C. for 6 h and reaction was followed to TLC. The reaction 
mixture was then evaporated in vacuo; the components were separated by 
silica gel column using a mixture of hexane and ethyl acetate (6:1) as 
eluting solvents. The progress of the separation followed by TLC. The 
fractions were collected, combined, evaporated, and distilledin vacuo. The 
yield was 87%. Bp: 37.degree.-38.degree. C. (10 .mu.m). IR. .sup.31 P NMR: 
.delta.=64.8 ppm (p, .sup.3 J.sub.PH =14 Hz). .sup.1 H NMR: .delta.=3.81 
ppm (d, .sup.4 J.sub.HP =1, Hz.sup.6, CH.sub.3 [CO]); .delta.=3.83 ppm (d, 
.sup.3 JHP =14 Hz, CH.sub.3 [PO]). .sup.13 C NMR: .delta.=54.3 ppm (qd, 
.sup.1 J.sub.CH =150 Hz, .sup.3 J.sub.CP =7 Hz, CH.sub.3 [PS]); .delta. 
=52.7 ppm (qd, .sup.1 J.sub.CH =149 Hz, .sup.3 J.sub.CP =5 Hz, CH.sub.3 
[CO]); .delta.=167.2 ppm (d, .sup.1 J.sub.CP =226 Hz, CO). Mass. M/e: 184 
(FW: 184.15). Anal Calculated for: C, 26.09; H, 4.93; S, 17.41. Found: C, 
26.23., H, 5.00., S, 17.82. 
PREATION OF BENZYL (O,O-DIETHYL) THIOPHOSPHONOFORMATE 
(Michaelis-Becker Reaction) 
##STR5## 
A finely divided sodium was suspended in dry benzene. The suspension was 
then added to a solution of diethyl thiophosphite in benzene. The mixture 
was heated to 50.degree. C. for 1.5 h, then cooled to 5.degree. C. in ice 
bath. Benzyl chloroformate dissolved in benzene was added drop.wise to the 
above mixture at room temperature. The resulting mixture was heated to 
50.degree. C. for 2.5 h, washed with H.sub.2 O, dried with MgSO.sub.4, 
evaporated, and distilled in high vacuo. The yield was 48%..sup.1 Bp: 
130.degree.-132.degree.0 C. (4 .mu.m). IR. .sup.31 P NMR: .delta.=61.2 ppm 
(p, .sup.3 J.sub.PH =10 Hz). .sup.1 H NMR: .delta.=1.196 ppm (t, .sup.3J 
HH =7 Hz), CH.sub.3 [PS]; .delta.=4.132 ppm (m, .sup.3 J.sub.HH =7 Hz), 
CH.sub.2 [PS]; .delta.=5.025 ppm (s), CH.sub.2 [CO]; .delta.=7.230 ppm, 
C.sub.6 H.sub.5 [CO]. .sup.13 C NMR: .delta.=16.0 ppm (qd, .sup.1 J.sub.CH 
=128 Hz, .sup.3 J.sub.CP =7 Hz, CH.sub.3 [PS]; .delta.=64.6 ppm (td, 
.sup.1 J.sub.CH =149 Hz, .sup.3 J.sub.CP =7 Hz, CH.sub.2 [PS]); 
.delta.=69.5 ppm (t, .sup.1 J.sub.CH =148 Hz, CH.sub.2 [CO]); 
.delta.=128.4 ppm (d, .sup.1 J.sub.CH =159 Hz, C.sub.6 H.sub.5 [CO]); 
.delta.=167.3 ppm (d, .sup.1 J.sub.CP =224 Hz, CO). 
The compounds so produced were then subjected to ITMS hydrolysis as 
disclosed by Hutchinson et al. with the following results: 
USING ME.sub.3 TPFA AS STARTING MATERIAL 
On a small scale, the prior art method was successful at obtaining the 
desired product though it was expensive and time consuming. Utilizing 
classical aqueous TMS ester quenching conditions xcess ITMS was added to 
Me.sub.3 TPFA at room temperature with stirring under N.sub.2. The mixture 
was heated to 110.degree. C. (oil bath) for 5 h and the reaction progress 
followed by .sup.31 P NMR Excess ITMS was removed under high vacuum and 
.sup.31 P and .sup.1 H NMR showed that the reaction was complete. 
##STR6## 
The residue was cooled in an ice bath. Cold water was added and the mixture 
stirred for 10 min. It was then titrated to pH 10.50 with NaOH solution. 
Excess solvent was evaporated by lyophilization. Methanol was added, and 
the mixture centrifuged. The product was oven.dried under vacuum producing 
a low yield of less than 10%. 
When the hydrolyzed crude product was carefully analyzed, a decarboxilation 
reaction was also discovered: 
##STR7## 
However, when increased amounts of Me.sub.3 TPFA (2g) were used in the 
reaction, thereby necessitatinglonger reaction times (e.g., refluxing for 
15 h) the reaction was not complete and the decomposition product quickly 
increased. 
USING Et.sub.3 TPFA AS STARTING MATERIAL 
Dealkylation of Et.sub.3 TPFA by treatment with ITMS was more difficult 
than that of MeTPFA.s A mixture of Et.sub.3 TPFA and excess ITMS was 
refluxed at 120.degree. C. (oil bath) and the reaction progress was 
followed by .sup.31 P NMR. 
After 3 h, an intermediate was obtained: 
##STR8## 
After 7 h, the didealkylation produced was obtained: 
##STR9## 
Accordingly, the reaction mechanism was presumed to be as follows: 
##STR10## 
It should be noted that it was very difficult to remove the C.sub.2 H.sub.5 
from the carbonyl group because continued reflux with ITMS caused the side 
products to increase quickly. The reaction was repeated in the Presence of 
an alkyl iodide in an attempt to use a Pishchimuka thiono-thiolo 
rearrangement to obtain the thiolo analogue prior to 
dealkylation.silylation with ITMS. Unfortunately, even after refluxing 
Et.sub.3 TPFA with 1.iodobutane in nitromethane for 44 h, only about 35% 
of thiolo analogue could be obtained (.sup.31 P,.delta.=26.9 ppm). 
Continued refluxing caused decomposition. 
Further demonstrating the distinguishing features of the present invention 
over the teachings of the prior art, the following hydrolyzation 
methodology was developed in accordance with the teachings of the present 
invention in order to hydrolyze Me.sub.3 TPFA using ITMS while avoiding 
the problems of decarboxylation and decomposition associated with the 
prior art methodologies. 
USING Me.sub.3 TPFA AS STARTING MATERIAL 
2.5 mL (ca. 16 mmol) ITMS was added to 500 mg (2.72 mmol) of Me.sub.3 TPFA 
at room temperature with stirring under N.sub.2. The mixture was heated to 
115.degree.-120.degree. C. for 7 h and the reaction progress followed by 
.sup.31 P NMR. Excess ITMS was then removed under high vacuum (0.001 mm 
Hg) at room temperature. The residue was cooled in an ice bath and was 
then added to 318 mg (3 mmol) of Na.sub.2 CO.sub.3 in 2 mL water and 
dissolved with stirring. The mixture was adjusted to pH 10.5 using 3N NaOH 
solution (approximately 0.5 mL) On addition of 35 mL of methanol, a 
precipitate formed which was centrifuged 10 min. and dried in a vacuum 
oven (&lt;1 mm Hg at 50.degree. C.), neutralized to pH 4.5 with 3N HCI 
(approximately 4 mL), then readjusted to pH 10.5 with 3N NaOH (ca. 0.5 
mL). Reprecipitation with 35 mL of methanol, centrifugation as above, and 
oven drying in vacuo (&lt;1 mm Hg, 50.degree. C.) gave a white precipitate. 
This was redissolved in 3 mL of water, and the same procedure repeated 
three times. After drying (&lt;1 mm Hg, 55.degree. C., 6h), 243.9 mg (43% 
yield) NasTPFA, (white powder) was obtained. .sup.31 P NMR: .delta.=37.7 
ppm (s). .sup.1 H NMR: no resonances except HDO. .sup.13 C NMR: 
.delta.=183.3 ppm (d, .sup.1 J.sub.CP =181 Hz, CO). 
Accordingly, from the foregoing it will be readily apparent to those 
skilled in the art that the prior art method of Hutchinson et al., while 
marginally successful at producing some intermediate compounds, does not 
produced TPFA or its addition salts. Apparently, the conversion of the 
phosphonate to the thiophosphonate compound interferes with the reactivity 
of ITMS. As a result, mixtures are produced by the prior art rather than 
pure compounds. As those skilled in the art will also appreciate, the 
significant questions raised as to the results of the reported synthesis 
by Hutchinson et al. also raise doubts as to the reproducability or 
veracity of any antiviral data that may have been published with respect 
to PFA or TPFA. 
In contrast, the method of the present invention is highly successful at 
inexpensivelyproducing large yieldsof pure TPFA thereby enabling its 
antiviral properties to be properly ascertained. More particularly, as 
shown in the following, non-limiting examples, in accordance with the 
teachings of the present invention, TPFA is unexpectedly effective against 
both HIV and HIV reverse transcriptase. 
Inhibition of a variety of viral enzymes was measured in order to determine 
the IDss or Inhibitory Dosage 50 of TPFA versus its phosphonate analogue 
PFA utilizing the following protocol. 
PREATION OF DNA POLYMERASES 
Viral DNA polymerases (HSV-1, HSV-2, EBV) were purified by previously 
published methods (Derce, D. K. F. Bastow, and Y. C. Cheng (1982) J. Boil. 
Chem. 257:10251-10260; Ostrander, M., and Y. C. Cheng (1980) Biochim. 
Biophys. Acta 609:232-245; Tan, R. S., A. Datta. and Y. C. Cheng (1982) J. 
Virol. 44:893-899). These procedures generally included sequential 
chromatography on DEAE-cellulose. HIV reverse transcriptase was purified 
by antibody affinity column chromatography as descrbed (Starnes. M. C. and 
Y. C. Cheng (1989) J. Biol. Chem. 264:7073-7077). The purified enzymes 
were dialyzed against and stored in 50 mM Tris-HCl (pH 7.5) containing 1 
mM each of DTT, EDTA and PMSF plus 30% glycerol. Mammalian DNA polymerases 
alpha, beta, gamma, and delta were partially purified from K562 celles 
(chronic myelogenous leukemia tissue culture line). Briefly, washed cell 
pellets were extracted and passed throgh DEAE-cellulose in the presence of 
300 mM KPO.sub.4 (pH 7.5) as previously described (Starnes, M. C., and Y. 
C. Cheng (1987) J. Biol. Chem 282:988-981) to remove DNA. The column 
flow-through fractions were dialyzed against buffer which contained 50 mM 
Tris-HCl (pH 7.5), and fractionated on a single-stranded DNA-cellulose 
column with a 0-1M KCl gradient. Mammalian polymerases (peak fractions) 
were completely separated from each other and exhibited the typical 
inhibitor profile and associated enzyme activities for each enzyme (effect 
of aphidicolin, dideoxynucleotides, butyiphenyl-dGTP, and ionic strength, 
presence of primase and reverse transciptase activity). 
HIV-1 REVERSE TRANSCRIPTASE ASSAYS 
Standard assays were run at 37.degree. C. and contained: 50 mM Tris, pH b 
8.0, 0.5 mM DTT, 8 mM MgCl.sub.2, 100 .mu.g/mL BSA, 150 .mu.g/mL gapped 
calf thymus DNA, 100 .mu.M each dATP, dCTP, dGTP, 10 .parallel.M [.sup.3 
H]-dTTP, and 1-5 .parallel.L enzyme in a final volume of 50 .parallel.L. 
Modified assays for pH dependence inhibition studies with PFA (1) and 
.alpha.--oxo phosphonates (4 and 5) contained 50 mM Hepes, pH 8.2-6.5, 8 
mM MgCl.sub.2, 100 mM KCl, 100 .mu.g/ml BSA, 0.5 A.sub.260 units/ml of 
poly(rA).(dT).sub.10, 100 .mu.M [.sup.3 H]dTTP, and 1.5 .parallel.L enzyme 
in a final volume of 50 .mu.L. Samples were processed as described above. 
The results of these assays were tabulated as follows: 
TABLE I 
______________________________________ 
Viral Polymerase Inhibition 
ID.sub.50 (.mu.M)* 
Virus TPFA PFA 
______________________________________ 
HIV-1 1 0.7 
HSV-1 11.8 0.7 
HSV-2 11.3 0.7 
EBV 70 1 
HV-6 70 1 
______________________________________ 
*All assays with "activated" DNA. 
As those skilled in the art will appreciate, from the foregoing the 
ID.sub.50 with respect to HIV for TPFA is essentially identical to that 
for PFA. This is in startling contrast to the effectiveness of TPFA 
relative to PFA with respect to the other virus enzymes tested. As shown 
in Table I TPFA is fifteen to twenty times less effective than PFA with 
respect to Herpes Simplex virus Type I and II and more than seventy times 
less effective against Epstein-Barr virus and Herpes Virus 6. Yet TPFA is 
equally effective against HIV. Thus, as will be appreciated by those 
skilled in the art, while the antiviral inhibitory activity of TPFA is 
completely unpredictable it is also surprisingly effective against HIV. 
Moreover, the test results shown in Table I indicate that the previously 
reported inhibitory effects of TPFA and PFA are incorrect. More 
specifically, Hutchinson et al. reported relative ID.sub.50 values for PFA 
and TPFA with respect to HSV-1 of, respectively, 12 and 9. In that the 
pure compounds of the present invention tested in Table I show a 
difference in inhibition activity with respect to HSV-1 on the order of a 
factor of 12 between TPFA and PFA, it would appear that Hutchinson et al. 
who report on essentially identical activity between the two compounds 
were most likely measuring mixtures rather than pure compounds. Thus, 
because of the prior art difficulties in producing TPFA, it is clear that 
the previously reported antiviral activities of TPFA are incorrect. 
In accordance with the teaching of the present invention similar enzyme 
inhibition assays were also conducted with respect to mammalian enzymes, 
more particularly, human DNA polymerase. The results of these tests are 
tabulated as follows: 
TABLE II 
______________________________________ 
Human DNA Polymerase Inhibition 
% Control at 100 .mu.M .+-. S.D. (ID.sub.50, .mu.M).sup.a 
Pol TPFA PFA 
______________________________________ 
.alpha. 72 .+-. 3 (&gt;100) 
16 .+-. 2 (31) 
.beta. 91 .+-. 4 (&gt;100) 
89 .+-. 8 (&gt;100) 
.gamma. 75 .+-. 3 (&gt;100) 
55 .+-. 5 (&gt;100) 
.delta. 69 .+-. 6 (&gt;100) 
36 .+-. 3 (71) 
______________________________________ 
.sup.a All assays with "activated" DNA. 
As shown in Table II the estimated ID.sub.50 of TPFA with respect to these 
mammalian enzymes is significantly higher than that for PFA, especially 
with respect to human DNA polymerase-Alpha, the most important DNA 
polymerase in this comparison. Even more significantly, TPFA is greater 
than 100 fold less active against the human enzyme than it is against the 
HIB enzyme tested in Table I. Accordingly, the therapeutic index for TPFA 
is relatively high. In fact, as shown in Tables i and II the therapeutic 
index for TPFA with respect to HIV enzyme and human enzyme is such that 
TPFA is, at a minimum, three times less toxic to the human enzyme than PFA 
yet is equally effective against the viral enzyme. 
In addition to testing the activity of TPFA against HIV enzyme, the 
activity against the virus itself was also determined in accordance with 
the following protocol. The experimental protocol involved incubatino of 
H9 lymphocytes (3.5.times.10.sup.6 cells/ml) in the presence or absence of 
HIV-1 (HTLV-IIIB) for one hour at 37.degree. C. Cells were washed 
thoroughly to remove unabsorbed virions and re-suspended at 
4.times.10.sup.5 cells/ml in culture medium. Aliquots (1 ml) were placed 
in wells of 24 well culture plates containing any equal volume of test 
compound (diluted in culture medium). After incubation for three days at 
37.degree. C., cell density of uninfected cultures was determined to 
assess toxicity of the test compound. A p24 antigen capture was used to 
determine the level of HIV infection in HIV treated cultures. The ability 
of test compounds to inhibit HIV replication was measured at different 
concentrations relative to infected, untreated cultures. Test compounds 
were considered to be active if p24 levels were &lt;70% of infected, 
untreated cultures. Cytotoxicity in uninfect4ed H9 cells was not detected. 
The results from this experimental data were tabulated in the following 
table: 
TABLE III 
______________________________________ 
HIV Inhibition In Cell Culture.sup.a 
Cell 
Drug Drug Conc. Survival p24 Ave 
Inhibition 
Type (.mu.g/mL) (%) Toxicity 
(%) Score.sup.b 
______________________________________ 
PFA 120 102 non-T -2 *** 
30 92 non-T 10 *** 
7.5 96 non-T 35 ** 
1.9 98 non-T 88 
TPFA 200 84 non-T -3 *** 
50 93 non-T 0 *** 
10 104 non-T 35 ** 
2 110 non-T 90 
0.5 113 non-T 117 
0.1 113 non-T 139 
______________________________________ 
.sup.a p24 antigen capture assay. 
.sup.b ***, strong; **, moderate; *, weak. 
REMARKS: 
Control untreated H9 cell count 1.07 million 
Control infected H9 p24 = 357 .mu.g/mL 
Drug concentration .mu.g/mL 
Toxic: 
"nonT" &gt;70% cell survival 
"T" &lt;70% cell survival 
Score: 
*50-69% control p24 
**25-49% control p24 
***&lt;25% control p24 
As those skilled in the art will appreciate, in Table III the lower the 
value of p24 the more active the compound is at inhibiting HIV. As shown 
in Table III, TPFA is as effective at inhibiting HIV at 50 .mu.g/mL as PFA 
is at 120 .mu.g/mL. Coupling these results with the previously discussed 
therapeutic index of TFPA, it becomes abundantly clear that TPFA is 
unexpectedly superior to its phosphonate analogue PFA with respect to 
inhibition of HIV. What is more, as those skilled in the art will also 
appreciate, there is good reason to believe that TPFA will be 
significantly less toxic in treating mammals as well as more effective. 
For example, TPFA exhibits a lower molecular polarity enhancing its water 
colubility and cell penetration properties. Thus, unlike PFA which may 
crytalize in the kidneys causing a toxic reaction, the higher solubility 
compound TPFA is less likely to crystalize and cause such side effects. 
Additionally, because the thiophosphonate compound is less like a 
phosphate compound it should exhibit a reduced tendency to deposit in 
bone. 
Moreover, as those skilled in the art will also appreciate, TPFA and/or its 
addition salts can be administered to mammals including humans in an 
effective amount as determined by clinical trials. The compound may be 
administered orally, parenterally, topically or by other standard 
administration routes. Additionally, the compound can include a 
pharmaceutically acceptable carrier such as the normally acceptable 
additives, excipients, and the like and may also be combined with other 
bioreactive compounds such as AZT, DDC, DDI and antibiotics. 
An additional advantage of TPFA over PFA is that the ultraviolet spectrum 
of TPFA is about ten times more intense than that of PFA and is easier to 
detect. The presence of sulfur in TPFA tends to shift the ultraviolet 
absorption towards the red-end of the spectrum making it more convenient 
to measure. As a result, the TPFA produced in accordance with the 
teachings of the present invention has additional analytical benefits for 
chemical research. 
Having thus described exemplary embodiments of the present invention, it 
should be noted by those skilled in the art that the within disclosures 
are exemplary only and that various other alternatives, adaptations and 
modifications may be made within the scope of the present invention. For 
example, solvents other than acetonitrile or toluene may be utilized as 
well as other inert gases in place of the argon disclosed and claimed. 
Additionally, other phosphonate starting materials may be utilized than 
those disclosed in the foregoing non-limiting examples. Accordingly, the 
present invention is not limited to the specific embodiments as 
illustrated herein, but is only limited by the following claims.