3-deoxy-3-substituted analogs of phosphatidylinositol

The invention provides 3-deoxy-3-substituted analogs of phosphatidylinositol which are useful to inhibit the growth of mammalian cells, i.e., to treat neoplastic conditions and other proliferative disorders of mammalian cells.

BACKGROUND OF THE INVENTION 
For mammalian cells to survive, they must be able to respond rapidly to 
changes in their environment. Furthermore, for cells to reproduce and 
carry out other cooperative functions, they must be able to communicate 
efficiently with each other. Cells most frequently adapt to their 
environment and communicate with one another by means of chemical signals. 
An important feature of these signaling mechanisms is that in almost all 
cases a cell is able to detect a chemical signal without it being 
necessary for the chemical messenger itself to enter the cell. This 
permits the cell to maintain the homeostasis of its internal environment, 
thereby permitting the cell to respond to its external environment without 
being adversely effected by it. 
These sensing functions are carried out by a variety of receptors, which 
are dispersed on the outer surface of the cell and function as molecular 
antennae. These receptors detect an incoming messenger and activate a 
signal pathway that ultimately regulates a cellular process such as 
secretion, contraction, metabolism or growth. In the cell's cellular 
plasma membrane, transduction mechanisms translate external signals into 
internal signals, which are then carried throughout the interior of the 
cell by chemicals known as "second messengers." 
In molecular terms, the process depends on a series of proteins within the 
cellular plasma membrane, each of which transmits information by inducing 
a conformational change in the protein next in line. At some point, the 
information is assigned to small molecules or even to ions within the 
cell's cytoplasm, which serve as the above-mentioned second messengers. 
The diffusion of the second messengers enables a signal to propagate 
rapidly throughout the cell. 
Several major signal pathways are now known, but two seem to be of primary 
importance. One employs cyclic nucleotides as second messengers. These 
cyclic nucleotides activate a number of proteins inside the cell, which 
then cause a specific cellular response. The other major pathway employs a 
combination of second messengers that includes calcium ions as well as two 
substances whose origin is remarkable: myo-inositol-1,4,5-trisphosphate 
(IP.sub.3) and diacylglycerol (DG). These compounds are cannibalized from 
the plasma membrane itself, by enzymes which are activated by specific 
cellular membrane receptors. However, it should be noted that myo-inositol 
in its non-phosphorylated form first must be synthesized by the cell from 
glucose or be obtained from the extracellular environment. The structural 
formula of myo-inositol is shown below: 
##STR1## 
wherein the term "myo" refers to the stereochemical configuration of the 
inositol molecules. Since all known inositol second messengers use the 
D-myo-configuration of inositol, the term "inositol" will herein be 
understood to refer to D-myo-inositol. To form IP.sub.3, a receptor 
molecule on the surface of the cellular plasma membrane transmits 
information through the cellular plasma membrane and into the cell by 
means of a family of G proteins, which are cellular plasma membrane 
proteins that cannot be active unless they bind to guanosine triphosphate 
(GTP). The G proteins activate the so-called "amplifier" enzyme 
phospholipase C, which is on the inner surface of the cellular plasma 
membrane. Phospholipase C cleaves the cellular plasma membrane lipid, 
phosphatidylinositol-4,5-bisphosphate (PIP.sub.2) into DG and IP.sub.3. 
IP.sub.3 is a water-soluble molecule, and therefore, upon being released 
from the inner surface of the cellular plasma membrane, it rapidly 
diffuses into the cytoplasm. IP.sub.3 then releases calcium ions 
(Ca.sup.2+) from non-mitochondrial stores, to increase the cytoplasmic 
free Ca.sup.2+ concentration. DG is an activator of protein kinase C. See 
U. Kikkawa et al., Ann. Rev. Cell Biol., 2, 149 (1986). Taken together, 
the increase in cytoplasmic free Ca.sup.2+ concentration and the increased 
activity of protein kinase C leads to a sequence of events that culminates 
in DNA synthesis and cell proliferation (See M. Whitman et al., Biochim. 
Biophys. Acta, 948, 327 (1988)). Other inositol phosphates, in addition to 
IP.sub.3, are formed in the cell. For example, phosphorylation of IP.sub.3 
by a specific 3-kinase gives inositol-1,3,4,5-tetrakisphosphate (IP.sub.4) 
(R. F. Irvine et al., Nature, 320, 631 (1986)), which may act 
synergistically with IP.sub.3 in the activation of Ca.sup.2+ -mediated 
responses in several systems. 
Recently, another phosphatidylinositol signalling pathway has been 
identified and linked to the action of some growth factors and oncogenes. 
Phosphatidylinositol-3'-kinase (also designated type 1 
phosphatidylinositol kinase) is found associated with a number of protein 
tyrosine kinases including the ligand-activated receptors for insulin, 
platelet derived growth factor (PDGF), epidermal growth factor (EGF), and 
colony-stimulating factor-1 (CSF-1) as well as protooncogene and oncogene 
tyrosine kinases (Y. Fukui et al., Oncogene Res., 4, 283 (1989)). This 
enzyme phosphorylates the D-3 position of the myo-inositol ring of 
phosphatidylinositols to give a class of 
phosphatidylinositol-3'-phosphates that are not substrates for hydrolysis 
by phosphatidylinositol phospholipase C and, therefore, appear to exert 
their signalling action independently of the inositol phosphate pathway. 
Subsequently, DG and IP.sub.3 are recycled. DG is recycled by a series of 
chemical reactions which constitute one component of the lipid cycle, and 
IP.sub.3 is recycled by a series of reactions known as the 
phosphatidylinositol cycle. The two cycles converge at the point when 
inositol is chemically linked to DG. The DG-bound inositol is 
phosphorylated in a series of steps which ultimately results in the 
reformation of phosphatidylinositol diphosphate. 
Previously, A. P. Kozikowski (U.S. Pat. No. 5,053,399) disclosed the 
synthesis of a number of D-3-deoxy-3-substituted-myo-inositols, in the 
expectation that these compounds would act as antimetabolites of 
myo-inositol-derived second messengers. In theory, such myo-inositol 
isosteres could act either by blocking the formation of certain 
phosphatidylinositols and inositol phosphates or by forming fraudulent 
analogs thereof. In fact, certain of these analogs, such as 
3-deoxy-3-fluoro-myo-inositol, were found to exhibit cell growth 
inhibitory activities against normal NIH 3T3 cells in culture and several 
oncogene transformed NIH 3T3 cell lines. However, the 
D-3-deoxy-3-substituted-myo-inositol analogs were only effective 
inhibitors of cell growth in the absence of myo-inositol. In the presence 
of physiological concentrations of myo-inositol in the growth medium, the 
growth inhibitory effect of the analogs was antagonized. It is believed 
that myo-inositol effectively competes with the D-3-deoxy-3-substituted 
myo-inositol analogs either for uptake into the cell and/or for 
incorporation by the cell to phosphatidylinositols. 
Therefore, a continuing need exists for analogs of phosphatidylinositol 
which are effective to inhibit the phosphatidylinositol cycle in a cell, 
e.g., to block cell growth, preferably to inhibit or prevent the growth of 
neoplastic cells and/or neoplastic transformation. 
SUMMARY OF THE INVENTION 
The present invention provides a bioactive 3-deoxy-3-substituted analogs of 
phosphatidylinositol of formula (I): 
##STR2## 
or a pharmaceutically acceptable salt thereof; wherein X is selected from 
the group consisting of halo (Cl, F, Br, I), azido (N.sub.3), CN, NC, OR, 
SR, N(R).sub.2, CO.sub.2 R, C(O)R, P(O)(OR).sub.2, CF.sub.3, S(O)R and 
SO.sub.2 R; wherein each R is H, (C.sub.1 -C.sub.22)alkyl, preferably 
(C.sub.7 -C.sub.20)alkyl (such as n-heptyl, n-deyl, isodecyl, 
n-pentadecyl, n-hexadecyl, n-octadecyl and n-eicosyl); (C.sub.6 
-C.sub.10)aryl, preferably phenyl or naphthyl; (C.sub.3 
-C.sub.8)cycloalkyl, preferably cyclohexyl or cyclopentyl; (C.sub.2 
-C.sub.22)alkenyl, preferably (C.sub.7 -C.sub.20)alkenyl, wherein the 
alkenyl group comprises 1-3 double bonds; (C.sub.5 -C.sub.8)cycloalkenyl, 
preferably cyclohexenyl and cyclopentenyl; (C.sub.7 -C.sub.32)aralkyl, 
(C.sub.7 -C.sub.32)alkylaryl, (C.sub.9 -C.sub.32)aralkenyl and (C.sub.9 
-C.sub.32)alkenylaryl; and wherein the R groups are unsubstituted or are 
substituted by the group X wherein R is unsubstituted; 
n is 0 or 1; 
Y is O, S, NR, CH.sub.2, CF.sub.2, or CHF; and 
Z is 
##STR3## 
wherein R.sup.1 and R.sup.2 are individually R, C(O)R, CO.sub.2 R, 
C(O)NHR, C(O)SR or P(O)(OR).sub.2. 
Preferably, X is halo, most preferably F; N.sub.3, NH.sub.2 or 
P(O)(OR).sub.2, wherein R is H; Y is CH.sub.2, CHF or CF.sub.2 and R.sup.1 
and R.sup.2 are (C.sub.7 -C.sub.22)alkanoyl or (C.sub.7 -C.sub.22)alkenoyl 
groups, i.e., are the alkanoyl or alkenoyl residues of fatty acids such as 
those present in naturally occurring phosphatidylinositols. Representative 
examples of these substituted groups (XR--) are hydroxyethyl, 
3-methoxypropyl, 4-hydroxyphenyl, 3- or 4-chlorobenzyl, 4-trifluorobenzyl, 
2-aminophenethyl, 2-carboxyphenylethenyl, 4-cyanomethylphenyl, 
4-(N,N-dimethylphenyl)cyclohexyl, 2,6-dimethoxyphenyl, 
2-ethoxy-1-naphthyl, 4-amino-4-carboxybutyl, 1-naphthylmethyl, 
1-(N-ethylaminophenyl)-n-butyl 1,2-carbamoylbenzyl, 4-sulfonylbenzyl, 
4-sulfinylbenzyl, 2-methylthiophenyl, 2,4-dinitrobenzyl, 4-phenylbenzyl, 
4-phenoxyphenethyl, and the like. 
The present invention also provides a method for inhibiting cellular growth 
by inhibiting the phosphatidylinositol cycle in mammalian cells, including 
human cells, which comprises administering to said mammal an effective 
phosphatidylinositol cycle-inhibiting amount of a compound of formula I. 
Thus, a method is also provided wherein the compounds of formula I are 
used to treat phosphatidylinositol cycle-dependent conditions in mammals, 
including humans, which comprises administering to said mammal a 
phosphatidylinositol cycle-inhibiting amount of a compound of formula I, 
which is effective to cure or ameliorate said condition or the symptoms 
thereof. 
Inositol phosphate cycle-dependent conditions include normal or abnormal 
cellular growth as found in cancers and in other neoplastic conditions, as 
well as biochemical processes relevant to arthritis, pain, inflammation, 
and platelet aggregation. See Y. Nishizuka, Science, 225, 1365-1370 
(1984); S. K. Fisher et al., J. Neurochem., 48, 999-1017 (1987); Y. 
Sugimoto et al., Molecular and Cellular Biology, 5, 3194-3198 (1985); and 
K. Fukami et al., Proc. Natl. Acad. Sci., USA, 85, 9057-9061 (1988). Solid 
tumors such as sarcomas, melanomas, carcinomas or lymphomas can be treated 
with the present compounds. 
The improved bioactivity of the present compounds is believed to be due, in 
part, to their ability to resist antagonism by endogenous myo-inositol. 
For example, the compounds of formula I can inhibit the growth of normal 
NIH 3T3 cells and v-sis oncogene transformed NIH 3T3 cells and of HT-29 
colon carcinoma cells in culture. The cell growth inhibition occurs at 
physiological concentrations of myo-inositol. Yang et al. (U.S. Pat. No. 
4,515,722) generally disclose phospholipase C inhibitors of the structure 
(II): 
##STR4## 
wherein X is OH (myo-inositol), and R is as defined as, for example, in 
Table I. At Col. 3, line 67 to Col. 4, line 2, it is also disclosed that 
inositol can be "substituted" with N.sub.3, halo and alkyl. An example of 
a simple analog containing a modified inositol ring in the patent is 
2-fluoro-2-deoxy-1-O-octadecylphosphonylscylloinositol (24) (Col. 18, 
lines 34-44). An example of a compound of formula II wherein X is OH is 
given as Example 8. A protected 3-deoxy-3-azido-myo-inositol is disclosed 
in Example 23, but was apparently not incorporated into a compound of 
formula II. 
However, the compounds of formula I are O-(alkyloxyphosphonyl)-substituted 
inositols (or disubstituted phosphates), while the compounds of formula II 
are O-(alkylphosphonyl)inositols (or alkylphosphonates). Also, the Yang et 
al. patent does not disclose that the compounds disclosed therein have 
anticancer or antiproliferative activity, but rather, discloses that they 
are antiinflammatory agents or analgesics.

DETAILED DESCRIPTION OF THE INVENTION 
The phosphatidylinositol analogs of formula I (Y.dbd.O) are generally 
prepared starting with the corresponding 
3-deoxy-3-substituted-myo-inositol, such as 3-deoxy-3-fluoro-myo-inositol 
depicted as compound 1 in FIG. 1, or 3-deoxy-3-azido inositol depicted as 
compound 8 in FIG. 2. Using selective protection/deprotection reactions 
known to the art, a corresponding protected 3-deoxy-3-substituted 
myo-inositol is obtained, wherein the 1-hydroxyl group is protected with a 
group, such as the methoxymethyl moiety shown for compound 4 in FIG. 1 (or 
compound 11 in FIG. 2), which can be removed while retaining the benzyl 
protecting groups on the remaining OH groups. The protecting group is then 
selectively removed from the 1-OH group, i.e., with aqueous acid, and the 
free 1-OH group is phosphitylated to yield the 
1-(O-benzyl-N,N-diisopropyl)phosphoramidite, i.e., compound 5 in FIG. 1. 
This compound can be converted into the pentakisprotected 
(1,2-dialkanoyl-3-propyl)phosphate by reaction of the phosphoramidite with 
1,2-dialkanoylglycerol and tetrazole in an organic solvent, followed by 
oxidation of the protected phosphite to the phosphate with a peroxide, 
i.e., to yield compound 6 in FIG. 1. Removal of the five protecting groups 
(benzyl in compound 6) yields the 3-deoxy-3-substituted-myo-inositol 
phosphatidyl compound of formula I. 
The phosphatidyl inositol analogs of formula I (Y.dbd.CH.sub.2) are 
generally prepared from the 2,4,5,6-tetrakisprotected 
3-deoxy-3-substituted myo-inositols, such as 
2,4,5,6-tetra-O-benzyl-3-deoxy-3-fluoro-myo-inositol shown as compound 40 
in FIG. 4. The inositol-1-OH group of 40 is converted to the moiety 
--CH.sub.2 P(O)(OBn).sub.2 (Bn=benzyl) by the steps outlined for the 
conversion of 40 to 23 on Table I, below. The bis-protected phosphonate is 
mono-deprotected and coupled to a 1,2-dialkanoylglycerol moiety using the 
conditions for the conversion of 23 to 24 (Table I). Removal of the five 
benzyl protecting groups by hydrogenolysis yields the 
1-[O-(alkylphosphonyl)methyl-D-myo-inositol, i.e., 25 on FIG. 4. 
TABLE I 
______________________________________ 
Synthesis of Compound 25 
Starting Material 
Reagents Product 
______________________________________ 
40 a) NaH; CS.sub.2 ; MeI 
21 
b) HC.tbd.C--CH.sub.2 SnBu.sub.3 
AIBN 
21 Ozone, Me.sub.2 S; separate 
22 
axial and equatorial 
isomers; equilibrate 
axial isomer with DBU 
22 a) NaBH.sub.4 23 
b) I.sub.2, PPh.sub.3, imidazole 
c) NaP(O) (OBn).sub.2 
23 a) 1 eq. 2-mercaptobenzo- 
24 
thiazole, i-Pr.sub.2 NEt 
b) 1,2-dipalmitoyl-sn- 
glycerol, mesitylene- 
sulfonyl chloride 
24 H.sub.2, Pd(OH).sub.2 /C, t-BuOH 
25 
______________________________________ 
The phosphatidylinositol analogs of formula I (Y.dbd.CF.sub.2) can also 
generally be prepared starting with a 2,4,5,6-tetrakis-protected 
3-deoxy-3-substituted myo-inositol, such as compound 10 as shown on FIG. 
5. The stereochemistry of the 1-OH group is inverted by sequential 
oxidation, followed by stereoselective reduction of the 1-keto moiety with 
a selective reducing agent such as L-Selectride.RTM. (Aldrich), 
LS-Selectride.RTM. and the like, to yield an inositol of a configuration 
corresponding to that of compound 30 (FIG. 5). The axial 1-OH group is 
then derivatized with a suitable leaving group, such as triflate, and the 
1-(CF.sub.2 -P(O)(OBn).sub.2) group is introduced by reaction of the 
triflate with the organozinc reagent derived from dibenzyl 
bromodifluoromethylphosphonate and zinc metal, catalyzed by CuI. The 
moiety CF.sub.2 P(O)(OBn).sub.2 is then converted into the substituent 
(CF.sub.2 P(O)(OH)--O--CH.sub.2 --CH(OR.sup.1)--CHOR.sup.2)); and the OH 
protecting groups are removed to yield the final product, e.g., 33, by the 
reaction sequence corresponding to that used to convert compound 23 to 
compound 25 (Table I). 
The syntheses of a wide variety of 3-deoxy-3-substituted-myo-inositols, or 
of hydroxyl-protected 3-deoxy-3-substituted myo-inositols which are useful 
as starting materials in the preparation of the compounds of formula I, 
have been reported. For example, A. P. Kozikowski (U.S. Pat. Nos. 
4,988,682 and 5,033,399) discloses the synthesis of 
3-deoxy-3-fluoro-myo-inositol from quebrachitol; as well as the synthesis 
of 3-deoxy-3-mercapto-myo-inositol and 3-cyano-3-deoxy-myo-inositol. S. S. 
Yang (U.S. Pat. No. 4,515,722) discloses the synthesis of 
3-azido-3-deoxy-1,2:4, 5-dicyclohexylidene-myo-inositol. The synthesis of 
3-azido-3-deoxy-myo-inositol has been described by A. P. Kozikowski et 
al., Cancer Chemother. Pharmacol., 29, 95 (1991). 
Conversion of 3-deoxy-3-substituted phosphatidylinositols such as the 
3-amino-, 3-mercapto-, 3-fluoro- or 3-cyano-substituted compounds to 
3-substituted compounds of formula I wherein X is N(R).sub.2, SR, OR, 
chloro, bromo, iodo, CO.sub.2 R, --NC and the like is readily accomplished 
by conventional methodology, for example, when the phosphatidylinositol or 
a precursor thereof is in the fully protected form, e.g., compounds 4, 5 
or 6 in FIG. 1. For example, phenyl- or alkylthio- derivatives can be 
prepared from the corresponding thiols by the procedure of U.S. Pat. No. 
4,383,114 (Ex. 6). Pharmaceutically acceptable salts of the compound of 
formula I can also be prepared as described in U.S. Pat. No. 4,383,114. 
Mode of Administration and Pharmaceutical Compositions 
When the compounds of formula I are utilized in vivo, such compounds can be 
administered orally, topically, parenterally, by inhalation spray or 
rectally in dosage unit formulations containing conventional non-toxic 
pharmaceutically acceptable carriers. 
Accordingly, the present invention also provides pharmaceutical 
compositions, including pharmaceutical unit dosage forms, comprising the 
compounds of formula I in combination with a pharmaceutically acceptable 
carrier. Useful pharmaceutically acceptable carriers include solid or 
liquid diluents, ingestible capsules or microcapsules, and inert matrices, 
such as latexes, pseudolatexes and hydrogels, for the controlled release 
of the compounds of formula I. 
The term parenteral as used herein includes subcutaneous injections, 
intravenous, intramuscular, intrasternal injection or infusion techniques. 
In addition to the treatment of mammals, such as mice, rats, horses, dogs, 
cats, etc., the compounds of the invention are effective in the treatment 
of humans. 
The pharmaceutical compositions containing the active ingredient may be in 
unit dosage forms suitable for oral ingestion, for example, as tablets, 
troches, lozenges, aqueous or oily suspensions, dispersible powders or 
granules, emulsions, hard or soft gelatin capsules, or syrups or elixirs. 
Compositions intended for oral use may be prepared according to any method 
known to the art for the manufacture of pharmaceutical compositions and 
such compositions may contain one or more agents selected from the group 
consisting of sweetening agents, flavoring agents, coloring agents and 
preserving agents in order to provide pharmaceutically elegant and 
palatable preparation. 
Unit dosage forms for oral ingestion include tablets which contain the 
active ingredient in admixture with non-toxic pharmaceutically acceptable 
excipients. These excipients may be, for example, inert diluents, such as 
calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium 
phosphate, granulating and disintegrating agents, for example, maize 
starch, or alginic acid; binding agents, for example, starch, gelatin or 
acacia, and lubricating agents, for example, magnesium stearate, stearic 
acid or talc. The tables may be uncoated or they may be coated by known 
techniques to delay disintegration and absorption in the gastrointestinal 
tract and thereby provide a sustained action over a longer period. For 
example, a time delay material such as glyceryl monostearate or glyceryl 
distearate may be employed. 
Unit dosage forms for oral ingestion may also be presented as hard gelatin 
capsules wherein the active ingredient is mixed with an inert solid 
diluent, for example, calcium carbonate, calcium phosphate or kaolin, or 
as soft gelatin capsules wherein the active ingredient is mixed with water 
or an oil medium, for example, polyoxyalkylene glycols, peanut oil, liquid 
paraffin, or olive oil. 
Aqueous suspensions usually contain the compound of formula I in admixture 
with appropriate excipients. Such excipients are suspending agents, for 
example, sodium carboxymethylcellulose, methylcellulose, 
hydroxypropylmethylcellulose, sodium alginate, polyvinylpryrolidone, gum 
tragacanth and gum acacia; nontoxic dispersing or wetting agents which may 
be a naturally occurring phosphatide, for example, lecithin; a 
condensation product of an alkylene oxide with a fatty acid, for example, 
polyoxyethylene stearate; a condensation product of ethylene oxide with a 
long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol; a 
condensation product of ethylene oxide with a partial ester derived from 
fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate; or 
a condensation product of ethylene oxide with a partial ester derived from 
fatty acids and hexitol anhydrides, for example, polyoxyethylene sorbitan 
monooleate. The aqueous suspensions may also contain one or more 
preservatives, for example, ethyl, n-propyl, or p-hydroxybenzoates; one or 
more coloring agents; one or more flavoring agents; and one or more 
sweetening agents such as sucrose, Nutrasweet.RTM., or saccharin. 
Oil suspensions may be formulated by suspending the compound of formula I 
in a vegetable oil, for example, arachis oil, olive oil, sesame oil or 
coconut oil, or in a mineral oil such as liquid paraffin. The oily 
suspension may contain a thickening agent, for example, beeswax, hard 
paraffin or cetyl alcohol. Sweetening agents such as those set forth 
above, and flavoring agents may be added to provide a palatable oral 
preparation. These compositions may be preserved by the addition of an 
antioxidant such as ascorbic acid. 
Dispersible powders and granules suitable for preparation of an aqueous 
suspension by the addition of water provide the compound of formula I in 
admixture with a dispersing or wetting agent, suspending agent and one or 
more preservatives. Suitable dispersing or wetting agents and suspending 
agents are exemplified by those already mentioned above. Additional 
excipients, for example, sweetening, flavoring and coloring agents, may 
also be present. 
The pharmaceutical compositions of the invention may also be in the form of 
oil-in-water emulsions. The oily phase may be a vegetable oil, for 
example, olive oil or arachis oils, or a mineral oil, for example, liquid 
paraffin or mixtures of these. Suitable emulsifying agents may be 
naturally occurring gums, for example, gum acacia or gum tragacanth, 
naturally occurring phosphatides, for example, soybean lecithin; and 
esters including partial esters derived from fatty acids and hexitol 
anhydrides, for example, sorbitan mono-oleate, and condensation products 
of said partial esters with ethylene oxide, for example, polyoxyethylene 
sorbitan mono-oleate. The emulsions may also contain sweetening and 
flavoring agents. 
Syrups and elixirs may be formulated with sweetening agents, for example 
glycerol, sorbitol or sucrose. Such formulations may also contain a 
demulcent, a preservative and flavoring and coloring agents. The 
pharmaceutical compositions may be in the form of a sterile injectable 
aqueous or oleageous suspension. This suspension may be formulated 
according to the known art using those suitable dispersing or wetting 
agents and suspending agents which have been disclosed above. The sterile 
injectable preparation may be a sterile injectable solution or suspension 
in a non-toxic parenterally acceptable diluent or solvent. Among the 
acceptable vehicles and solvents that may be employed are water, 
1,3-butanediol, Ringer,s solution and isotonic sodium chloride solution. 
In addition, sterile fixed oils are conventionally employed as a solvent 
or suspending medium. For this purpose any bland fixed oil may be employed 
including synthetic mono- or diglycerides. Fatty acids such as oleic acid 
also find use in the preparation of injectables. 
The compounds of formula I can also be administered in the form of 
suppositories for rectal or vaginal administration of the drug. These 
compositions can be prepared by mixing the drug with a suitable 
non-irritating excipient which is solid at ordinary temperatures but 
liquid at the rectal or vaginal temperature and will therefore melt to 
release the drug, for example, cocoa butter and polyethylene glycols. 
When the compounds of formula I are utilized in vivo, dosage levels on the 
order of from about 0.2 mg to about 300 mg, preferably from about 10 mg to 
about 100 mg, per kilogram of body weight per day are useful. 
The amount of active ingredient that may be combined with the carrier 
materials to produce a single dosage form will vary depending upon the 
host treated and the particular mode of administration. For example, a 
formulation intended for the oral administration of humans may contain 
from 5 mg to 5 g of active agent compounded with an appropriate and 
convenient amount of carrier material which may vary from about 5 to about 
95 percent of the total composition. Pharmaceutical unit dosage forms will 
generally contain between from about 25 mg to about 500 mg of active 
ingredient. 
The present invention also provides an article of manufacture comprising 
packaging material, such as an ampoule, vial, bottle, intravenous bag, and 
the like, and at least one compound of formula I contained therein. 
Preferably contained therein is at least one pharmaceutical unit dosage 
form comprising an amount of a compound of formula I in combination with 
at least one pharmaceutically acceptable carrier, as described above. Said 
packaging material further comprises a label or other associated 
instructional material such as a paper package insert or a sound 
recording, which indicates that said compound of formula I can be (a) used 
to treat a neoplastic condition, such as a particular carcinoma or cell 
proliferation disorder, or (b) used as an anti-inflammatory or analgesic 
agent. 
It will be understood, however, that the specific dose level for any 
particular patient will depend upon a variety of factors including the 
activity of the specific compound employed, the age, body weight, general 
health, sex, diet, time of administration, route of administration, rate 
of excretion, drug combination and the severity of the particular disease 
undergoing therapy. 
The invention will be further described by reference to the following 
detailed examples, wherein tetrahydrofuran (THF) was dried over 
sodium/benzophenone. Dimethylformamide (DMF) was distilled in an aspirator 
vacuum over CaH.sub.2. Methylene chloride was distilled over phosphorus 
pentoxide, and for use in phosphoramidite coupling, redistilled over 
CaH.sub.2. Acetonitrile was distilled over phosphorus pentoxide and 
redistilled over calcium hydride. Methanol was refluxed for several hours 
over magnesium turnings, then distilled. Toluene and triethylamine were 
distilled over calcium hydride. Ethyl acetate and hexane were distilled, 
other solvents not referred to as "dry" were used as received. Commercial 
grade tetrazole was purified by vacuum sublimation as described by M. H. 
Caruthers et al., Methods Enzymol., 154, 287 (1987). (Caution, explosion 
hazard!). Diisopropylammonium tetrazolide and 
O-benzyl-N,N,N',N'-tetraisopropylphosphorodiamidite were prepared as 
disclosed by Caruthers et al., ibid., and by W. Bannwarth et al., Helv. 
Chim. Acta, 70, 175 (1987). Other reagents were commercially available and 
were used as received. Column chromatography was performed on EM Science 
No. 7734-7 silica gel 60, particle size 0.063-0.200 mm, thin layer 
chromatography on EM Science No. 5715 silica gel 60 F.sub.254 glass 
plates, layer thickness 0.25 mm. TLC spots were visualized with 
permanganate solution. Melting points were measured in open capillaries 
and are uncorrected. NMR spectra were referenced to internal TMS (.sup.1 
H), CDCl.sub.3 or DMSO-d.sub.6 (.sup.13 C, .delta.=77.09 and 39.5), 
external CFCl.sub.3 (.sup.19 F), and external 85% H.sub.3 PO.sub.4 
(.sup.31 P), respectively. 
EXAMPLE 1 
Preparation of 3-Deoxy-3-fluoro-D-myo-inositol 
[(R)-2,3-bis(hexadecanoyloxy)propyl] hydrogen phosphate (7) 
A. Preparation of 3-Deoxy-3-fluoro-1, 2-O-isopropylidene-D-myo-inositol (2) 
As summarized in FIG. 1, a solution of 3.54 g (19.4 mmol) of 
3-deoxy-3-fluoro-D-myo-inositol, 7.6 ml (78 mmol) of 2-methoxypropene, and 
100 mg of camphorsulfonic acid in 30 ml of dry DMF was stirred in a closed 
flask at 80.degree. C. for 4 hr (Although little pressure buildup is 
observed, it is recommended to use safety shielding.). After cooling, 2 ml 
of triethylamine was added, and volatiles were evaporated in vacuo. The 
residue was taken up in methylene chloride, adsorbed on 20 g of silica 
gel, and chromatographed on silica gel with ethyl acetate/hexane 1:1 
(R.sub.f approx. 0.6 and 0.4, resp.), to yield, after evaporation, 4.24 g 
(84%) of a mixture of diacetonides as a yellowish solid, of which the 
individual components have been previously separated and characterized as 
disclosed by A. P. Kozikowski et al., J. Amer. Chem. Soc., 112, 7403 
(1990). This material was dissolved in a mixture of 140 ml of dry 
methylene chloride and 70 ml of dry methanol, and 40 .mu.l of acetyl 
chloride was added. The mixture was stirred under exclusion of moisture at 
23.degree. C. with close TLC monitoring (silica gel, methylene 
chloride/methanol 5:1; approximate R.sub.f values for fluorodeoxyinositol, 
the monoacetonide, and the diacetonide mixture are 0, 0.4, and 0.75, 
resp.). After 1 hr, most of the diacetonides had reacted while only a 
small amount of the completely deprotected inositol had been formed. The 
reaction was quenched by adding 0.5 ml of triethylamine, 30 g of silica 
gel was added, and the mixture was evaporated and chromatographed on 
silica gel with methylene chloride/methanol mixtures. With a 9:1 ratio of 
eluents, 0.38 g (9%) of the diacetonide mixture was recovered after which 
the ratio was changed to 5:1 to elute 2.89 g (80%) of compound 2 as a 
colourless semisolid of sufficient purity for the following step (Changing 
the eluent further to isopropanol/water 19:1 permitted the recovery of 
0.24 g [8%] of fluorodeoxyinositol which, like the recovered diacetonide, 
could be recycled). The analytical sample was recrystallized from 
methanol/ethyl acetate: colorless needles, mp 147.degree. C.; IR (nujol) 
3397, 2922, 2853, 1374, 1227, 1156, 1105, 1034, 866 cm.sup.-1 ; MS (EI) 
m/z 223 (M+H.sup.+), 207 (100%), 165, 129, 109, 73, 59; HRMS (M.sup.+ 
--CH.sub.3, C.sub.8 H.sub.12 FO.sub.5) calcd 207.0669, found 207.0669; 
[.alpha.].sup.23.sub.D -42.7.degree., [.alpha.].sup.23.sub.578 
-43.7.degree. (c=6.9 gl.sup.-1, methanol). 
B 4,5,6-Tri-O-benzyl-3-deoxy-3-fluoro-1, 2-O-isopropylidene-D-myo-inositol 
Under an argon atmosphere, 1.03 g (25.7 mmol) of NaH (60% dispersion in 
oil) was washed with dry THF and suspended in 10 ml of dry DMF. With ice 
cooling, 3.1 ml (26 mmol) of benzyl bromide was added dropwise, followed 
by a solution of 0.57 g (2.57 mmol) of compound 2 in 2.5 ml of dry DMF. 
The mixture was stirred at ice bath temperature for 3 hr, at 
8.degree.-10.degree. C. for 3.5 hr, and at room temperature for another 3 
hr. After recooling in an ice bath, 1 ml of water was added cautiously, 
and the mixture was directly filtered over silica gel (Larger runs require 
previous removal of the solvent.). Residual benzyl bromide was eluted with 
ethyl acetate/hexane 1:9, then the product was eluted with ethyl 
acetate/hexane 1:6. Evaporation and drying in vacuo left 1.21 g (96%) of 
the tribenzyl ether as a colorless oil: IR (neat film) 3033, 2986, 2932, 
1497, 1455, 1372, 1215, 1073, 866, 737, 696 cm.sup.-1 ; MS (EI) m/z 477 
(M.sup.+ --CH.sub.3), 401, 295, 91 (100%); HRMS (M.sup.+ --CH.sub.3, 
C.sub.29 H.sub.30 FO.sub.5) calcd 477.2077, found 477.2077; 
[.alpha.].sup.23.sub.D -11.7.degree.; [.alpha.].sup.23.sub.578 
-12.0.degree. (c=9.6 gl.sup.-1, CHCl.sub.3). 
C. 4,5,6-Tri-O-benzyl-3-deoxy-3 -fluoro-D-myo-inositol (3) 
A solution of 2.16 g (4.4 mmol) of the above intermediate of Ex. 1(B) in 
100 ml of methanol was stirred with 5 drops of conc. HCl at room 
temperature for 21 hr. After addition of 1 ml of triethylamine, the 
solvent was evaporated. The residue was taken up in methylene chloride and 
adsorbed on 10 g of silica gel. Filtration over silica gel with ethyl 
acetate/hexane 2:3, evaporation, and drying in vacuo yields 1.98 g (100%) 
of compound 3 as a waxy colorless solid: mp 102.degree.-104.degree. C.; IR 
(neat film) 3386, 3031, 2926, 1497, 1455, 1358, 1150, 1129, 1059, 1021, 
729, 696 cm.sup.-1 ; MS (EI) m/z 361 (M.sup.+ --C.sub.7 H.sub.7), 197, 
107, 91 (100%); HRMS (M.sup.+ --C.sub.7 H.sub.7, C.sub.20 H.sub.22 
FO.sub.5) calcd 361.1451, found 361.1451; [.alpha.].sup.23.sub.D 
-29.4.degree.; [.alpha.].sup.23.sub. 578 -30.8.degree. (c=10.2 gl.sup.-1, 
CHCl.sub.3). 
D. 4,5,6-Tri-O-benzyl-3-deoxy-3-fluoro-1-O-(methoxymethyl)-D-myo-inositol 
A solution of 1.05 g (2.32 mmol) of 3 in 80 ml of dry methanol was refluxed 
under argon with 575 mg (2.31 mmol) of di-n-butyltin oxide for 2 hr to 
yield a clear solution. The cooled solution was evaporated to dryness and 
evaporated twice more with 10 ml of toluene each time. The residue was 
taken up in 10 ml of dry DMF and cooled under argon with an external ice 
bath. A solution of 193 .mu.l (2.54 mmol) of chloromethyl methyl ether in 
5 ml of dry toluene was added over a period of 50 min. Stirring in the ice 
bath was continued for 1 hr, then 200 ml of water was added, and the 
product was extracted into 3.times.50 ml of methylene chloride. After 
drying over MgSO.sub.4, 10 g of silica gel was added, and the solvent was 
evaporated. The residue was chromatographed on silica gel with ethyl 
acetate/hexane mixtures, changing the composition from 1:3 (to elute a 
forerun) to 1:2 for the product, finally to 1:1 for unreacted started 
material. The respective solutions, after evaporation and drying in vacuo, 
yielded 139 mg (13%) of starting material 3 and 867 mg (75%) of the title 
compound. The analytical sample was obtained from methylene 
chloride/hexane as cotton-like needles: mp 118.degree.-119.degree. C.; IR 
(neat film) 3476, 3029, 2909, 1453, 1356, 1152, 1090, 1040, 899, 735, 695 
cm.sup.-1 ; MS (EI) m/z 451 (M.sup.+ --CH.sub.2 OCH.sub.3), 405 (M.sup.+ 
--C.sub.7 H.sub.7) 373, 91 (100%); [.alpha.].sup.23.sub.D +37.7.degree., 
[.alpha.].sup.23.sub.578 +39.9.degree. (c=11.4 gl.sup.-1, CHCl.sub.3). 
E. 
2,4,5,6-Tetra-O-benzyl-3-deoxy-3-fluoro-1-O-(methoxymethyl)-D-myo-inositol 
(4) 
Under an argon atmosphere, 94 mg (2.35 mmol) of sodium hydride was washed 
with dry THF. A solution of 584 mg (1.18 mmol) of the above intermediate 
in 5 ml of dry DMF was added dropwise with water cooling, followed by 0.42 
ml (3.5 mmol) of benzyl bromide. After stirring in the water bath for 5 
hr, 5 drops of water were added, and the mixture was directly 
chromatographed on silica gel with ethyl acetate/hexane mixtures (1:7 for 
the forerun, 1:4 for the product). Evaporation and drying in vacuo left 
680 mg (98%) of 4 as a colorless oil: IR (neat film) 3031, 2928, 1497, 
1455, 1358, 1090, 1036, 916, 735, 696 cm.sup.-1 ; MS (EI) m/z 541 
(M.sup.30 --CH.sub.2 OCH.sub.3), 495 (M.sup.30 --C.sub.7 H.sub.7) 463, 
181, 91 (100%); HRMS (M.sup.+ --C.sub.7 H.sub.7, C.sub.29 H.sub.32 
FO.sub.6) calcd 495.2183, found 495.2183; [.alpha.].sup.23.sub.D 
+16.1.degree.; [.alpha.].sup.23.sub.578 +16.3.degree. (c=8.1 gl.sup.-1, 
CHCl.sub.3). 
F. 2,4,5,6-Tetra-O-benzyl-3-deoxy-3-fluoro-D-myo-inositol (40) 
A solution of 874 mg (1.49 mmol) of 4 in 30 ml of methanol, 3 ml of water, 
and 0.3 ml of concentrated HCl was heated under reflux for 5 hr. After 
cooling, the mixture was evaporated, and the residue was chromatographed 
on silica gel with ethyl acetate 1:6 (forerun), then 1:3 (product) to 
leave, after evaporation and drying in vacuo, 57 mg (94%) of a colorless 
solid: mp 49.degree.-50.5.degree. C.; .sup.13 C NMR (CDCl.sub.3) .delta. 
138.30, 138.20, 138.11, 128.47, 128.34, 128.05, 127.99, 127.82, 127.72, 
127.63, 93.74 (d, J=187.5 Hz), 82.11 (d, J=14 Hz), 81.66, 80.38 (d, J=17 
Hz), 77.83 (d, J=16.5 Hz) 75.80, 75.52, 75.36, 74.96, 71.00 (d, J=11.5 
Hz); IR (neat film) 3451, 3033, 1455, 1360, 1069, 737, 698 cm.sup.-1 ; MS 
(EI) m/z 451 (M.sup.+ --C.sub.7 H.sub.7), 181, 91 (100%); 
[.alpha.].sup.23.sub.D -19.9.degree., [.alpha.].sup.23.sub.578 
-20.9.degree. (c=9.3 gl.sup.-1, CHCl.sub.3). 
G. 2,4,5,6-Tetra-O-benzyl-3-deoxy-3-fluoro-D-myo-inositol 
1-(O-benzvl-N,N-diisopropyl) phosphoramidite (5) 
Under an argon atmosphere and with water cooling, 36 mg (0.21 mmol) of 
diisopropylammonium tetrazolide was suspended in 1.5 ml of dry methylene 
chloride, and 0.18 ml (0.51 mmol) of O-benzyl-N,N, 
N',N'-tetraisopropylphosphorodiamidite was added dropwise within 10 min, 
followed by a solution of 228 mg (0.42 mmol) of alcohol 10 in 2.5 ml of 
dry methylene chloride. The mixture was stirred in the water bath for 20 
hr, then 5 ml of saturated NaHCO.sub.3 solution was added. The phases were 
separated, and the aqueous phase was extracted with 2.times.10 ml of 
methylene chloride. The combined organic phases were dried over Na.sub.2 
SO.sub.4 and evaporated, and the residue was rapidly filtered over 30 g of 
silica gel which had previously been deactivated by shaking with 0.5 ml of 
triethylamine, using ethyl acetate/hexane 1:4 as the eluent. Evaporation 
and drying in vacuo afforded 318 mg (97%) of the phosphoramidite as a 
colorless syrup: IR (neat film) 3033, 2967, 2928, 1497, 1455, 1364, 1028, 
801, 733, 696 cm.sup.-1 ; MS (EI) m/z 628 (M.sup.+ --OC.sub.7 H.sub.7 
--C.sub.3 H.sub.8), 234, 219, 83 (100%): HRMS (M.sup.+ --OC.sub.7 H.sub.7 
--C.sub.3 H.sub.8, C.sub.37 H.sub.40 FNO.sub.5 P) calcd 628.2628, found 
628.2628. 
H. 2,4,5,6-Tetra-O-benzyl-3-deoxy-3-fluoro-D-myo-inositol 1-(benzyl)[(R)-2, 
3-bis(hexadecanoyloxy)propyl]phosphite 
To 251 mg (441 .mu.mol) of 1,2-dipalmitoyl-sn-glycerol and 58 mg (0.83 
mmol) of tetrazole in 1.5 ml of dry methylene chloride was added at room 
temperature under argon a solution of 318 mg (408 .mu.mol) of 
phosphoramidite 5 in 1.5 ml of dry acetonitrile. The resulting mixture was 
stirred for 5 hr at room temperature, then for 64 hr at 
35.degree.-40.degree. C. After cooling, 10 ml of saturated NaHCO.sub.3 
solution was added, the phases were separated, and the aqueous phase was 
extracted with 3.times.20 ml of methylene chloride. The combined aqueous 
phases were dried over Na.sub.2 SO.sub.4 and evaporated, and the residue 
was filtered over silica gel with ethyl acetate/hexane 1:8. Evaporation 
and drying in vacuo yielded 404 mg of the title compound (79% rel. to 5) 
of a colorless glass: IR (neat film) 3033, 2924, 2853, 1744, 1456, 1164, 
1024, 735, 696 cm.sup.-1 ; MS (EI) m/z 550, 451, 367, 91 (100%). 
I. 2,4,5,6-Tetra-O-benzyl-3-deoxy-3-fluoro-D-myo-inositol 1-(benzyl)[(R)-2, 
3-bis(hexadecanoyloxy)propyl]phosphate (6) 
To an ice-cooled situation of 353 mg (283 .mu.mol) of the above phosphite 
in 3 ml of dry methylene chloride under argon was added in 4 equal 
portions in 20 min intervals a total of 800 .mu.l (400 .mu.mol) of a 0.5 M 
solution of anhydrous tert-butyl hydroperoxide in methylene chloride. 
Stirring was continued in the ice bath for 90 min, then at room 
temperature for 20 min. The mixture was evaporated and filtered over 
silica gel with ethyl acetate/hexane 1:4 to obtain 351 mg (98%) of the 
phosphate 6 as a colorless syrup: IR (neat film) 2924, 2853, 1744, 1026, 
696 cm.sup.-1 ; MS (EI) m/z 550, 451, 367, 239, 91 (100%); (FAB) m/z 640, 
551, 313, 181. 
J. 3-Deoxy-3-fluoro-D-myo-inositol[(R)-2,3-bis(hexadecanoyloxy)propyl] 
hydrogen phosphate (7) 
A solution of 54.2 mg (42.9 .mu.mol) of 6 in 6 ml of tert-butanol was 
hydrogenated in a Parr shaker under 5 bar of hydrogen for 23.5 hr over 
23.5 mg of 20% Pd(OH).sub.2 /C (Aldrich, containing 50% of water). The 
catalyst was removed by centrifugation and washed with tert-butanol, the 
solution was evaporated, and the residue was dried in vacuo to leave 27.9 
mg (80%; variability of the yield over 6 runs: 71-89%) of phosphate 7 as a 
colorless amorphous solid: mp 132.degree.-133.degree. C. (after 
sintering); .sup.1 H NMR (CDCl.sub.3 /CD.sub.3 OD 2:1) .delta. 5.27 (m, 1 
H, 2-H of glycerol), 4.45-4.35 (m, 2.5 H) 4.27-4.15 (m, 3.5 H), 4.02 (br, 
1 H), 3.97 (dt, 1 H, J =9.5 Hz (t), 12 Hz (d)), 3.86 (br t, 1 H, J=8.5 
Hz), 3.24 (t, 1 H, J=9.5 Hz), 2.36 (t, 2 H, J=7.5 Hz), 2.33 (t, 2 H, J=7.5 
Hz) 1.62 (m, 4 H), 1.27 (m, 48 H), 0.89 (t, 6 H, J=7 Hz); .sup.13 C NMR 
(CDCl.sub.3 /CD.sub.3 OD 2:1) .delta. 173.67, 173.30, 91.30 (d, J=182.5 
Hz), 73.46 (d, J=12.5 Hz), 70.82 (d, J=4 Hz), 70.42 (d, J=18 Hz), 69.52 
(d, J=6 Hz); 69.08 (d, J=17.5 Hz), 64.85, 64.79, 61.86, 33.84, 33.73, 
31.60, 29.34, 29.18, 29.00, 28.79, 24.53, 22.32, 13.57; .sup.19 F NMR 
(CDCl.sub.3 /CD.sub.3 OD 2:1) .delta. -204.51 (ddd, J=47, 11, 10 Hz); 
.sup.31 P NMR (CDCl.sub.3 /CD.sub.3 OD 2:1) .delta. -0.94 (br); IR (KBr) 
3416, 2920, 2851, 1740, 1630, 1468, 1383, 1038 cm.sup.-1 ; 
[.alpha.].sup.23.sub.D -1.3.degree., [.alpha.].sup.23.sub.578 
-1.3.degree., [.alpha.].sup.23.sub.265 -4.2.degree. (c=5.6 gl.sup.-1, 
CHCl.sub.3 /MeOH 2:1). 
EXAMPLE 2 
3-Amino-3-deoxy-D-myo-inositol[(R)-2, 3-bis(hexadecanoyloxy)propyl] 
hydrogen phosphate (13) 
As depicted in FIG. 2, the synthesis of 13 starts from the 
3-azido-3-deoxy-myo-inositol (8). The synthesis of 8 from L-quebrachitol 
has been described by A. P. Kozikowski et al., Cancer Chemother. 
Pharmacol. (in press). After a sequence of routine protection and 
deprotection steps, the MOM group was removed from intermediate 11, and 
the 1-position was then phosphitylated using 
O-benzyl-N,N,N',N'-tetraisopropylphosphorodiamidite. The coupling reaction 
with 1,2-dipalmitoyl-sn-glycerol was carried out and the phosphite 
intermediate oxidized to phosphate. Lastly, all of the benzyl groups were 
removed by hydrogenolysis in the presence of palladium hydroxide on carbon 
in t-butanol as solvent. Under these reaction conditions, the azido group 
was reduced to amine. The preparation of 
3-azido-3-deoxyphosphatidylinositol can also be accomplished from 12. This 
requires an alternative method for debenzylation which can be accomplished 
by using trimethylsilyl iodide as the cleaving reagent. 
More specifically, the conversion of 8 to 13 was accomplished as outlined 
on Table II, below. 
TABLE II 
______________________________________ 
Starting Product 
Material 
Reagents/Reaction Conditions 
(yield) 
______________________________________ 
8 (a) 2-methoxypropene, cat. 
9 (71.3%) 
camphorsulfonic acid 50.degree. C., 18 hr; 
(b) CH.sub.3 COCl, CH.sub.2 Cl.sub.2 /MeOH (1:2), 
25.degree. C., 8 hr 
9 (a) NaH, PhCH.sub.2 Br, DMF, 25.degree. C. 
10 (88%) 
(b) conc. HCl, MeOH, 25.degree. C., 12 hr 
10 (a) Bu.sub.2 SnO, MeOH, reflux, 6 hr; 
11 (81%) 
(b) MeOCH.sub.2 Cl, PhCH.sub.3, 0.degree. C., 1 hr 
(c) NaH, PhCH.sub.2 Br, DMF, 25.degree. C. 
11 (a) conc. HCl, MeOH, H.sub.2 O; 
12 (59%) 
(b) BnOP(Ni--Pr.sub.2).sub.2, diisopropyl- 
ammonium tetrazolide, CH.sub.2 Cl.sub.2, 
25.degree. C.; 
(c) 1,2-dipalmitoyl-sn-glycerol, 
tetrazole, CH.sub.2 Cl.sub.2 /CH.sub.3 CN (1:1); 
(d) t-BuOOH, 0.degree. C., 1.5 hr, 25.degree. C., 3 
hr 
12 5 atm H.sub.2, 20% Pd(OH).sub.2 /C, t-BuOH, 
13 (70%) 
25.degree. C., 1 day 
______________________________________ 
EXAMPLE 3 
Synthesis of 
3-Deoxy-3-phosphonomethyl-D-myo-inositol[(R)-2,3-bis(hexadecanoyloxy)propy 
l] hydrogen phosphate (19) 
As outlined in FIG. 3, the synthesis starts from 11, readily available from 
quebrachitol by the procedure of H. Paulsen et al., Liebige Ann. Chem., 
1073 (1983). This compound has the incorrect stereochemistry at C-3 but is 
preferred as the starting material since it is more readily available than 
its epimer, and the C-3 stereochemistry will be lost in the later steps of 
the synthesis. Tosylation of the free hydroxyl group using TsCl and 
pyridine, followed by exchange of tosylate for iodide using NaI in acetone 
(or alternatively, shorter but in slightly lower yield, direct iodination 
of 11 using iodine, triphenylphosphine, and imidazole) yields the iodide 
which is deprotected by an excess of boron tribromide in methylene 
chloride. The resulting iodopentol (12) is reprotected as a regioisomeric 
mixture of diacetonides 13/14 by warming with 4 eq. of 2-methoxypropene in 
DMF under catalysis by camphorsulfonic acid. The isomer 13 can be 
transformed into 14 by resubjecting it to acid catalysis in warm DMF and 
recycling. Elimination of hydrogen iodide to obtain the olefin 15 is then 
brought about by treatment with DBU in THF at room temperature. The 
C.dbd.C double bond of 15 is cleaved to the ketone by ozonolysis followed 
by in situ reduction of the ozonide with dimethyl sulfide. The phosphonic 
acid side chain is installed by a Wadsworth-Emmons olefination using the 
sodium salt of tetrabenzyl methylenediphosphonate, followed by 
hydrogenation over palladium on carbon, which establishes the desired 
stereochemistry at C-3 and further removes the benzyl protecting groups of 
the phosphonate. Addition of water to the crude reaction mixture and 
stirring at room temperature cleaves the labile trans-acetonide due to the 
acidity of the phosphonate group. The crude triol-phosphonic acid (16) 
which remains after filtration from the catalyst and evaporation is 
perbenzylated using excess sodium hydride and excess benzyl iodide in DMF 
to yield 17. The more nucleophilic alkoxide groups of 16 react before the 
phosphonate so that cyclic phosphonate formation which generally occurs 
when an alkoxide is adjacent to a phosphonate ester is largely avoided. 
The cis-acetonide protecting group of the resulting compound 17 is removed 
by acid treatment (MeOH,HCl) and the free equatorial 1-hydroxyl group 
temporarily protected as its methoxymethyl (MOM) derivative using Bu.sub.2 
SnO and methoxymethyl chloride. The less reactive axial hydroxyl group is 
now benzylated under acidic conditions using O-benzyltrichloroacetimidate 
and catalytic TfOH, and the MOM group is again removed by acidic 
hydrolysis to obtain the intermediate 18. The phosphatidic acid side chain 
is installed using the same technique as for 
3-fluoro-3-deoxy-phosphatidylinositol (6), and finally catalytic 
hydrogenolysis (H.sub.2, Pd(OH).sub.2, t--BuOH) yields the unprotected 
title compound 19. 
EXAMPLE 4 
Synthesis of 1-(O-[(R)-2,3-bis(hexadecanoyloxy)propyl 
]phosphonomethyl)-1,3-dideoxy-3-fluoro-D-myo-inositol (25) 
To avoid problems resulting from the propensity of inosose intermediates 
for elimination, the side chain is instead introduced via a radical 
substitution reaction. As shown in FIG. 4, the starting material (40) is 
first derivatized to its methyl xanthate ester by reaction with CS.sub.2 
and NaH, which is subsequently transformed to a stereoisomeric mixture of 
allenes (21) with propargyltributylstannane under catalysis by 
azobis(isobutyronitrile). Ozonolysis with reductive workup (Me.sub.2 S) 
produces a mixture of aldehydes (22) which is separated by column 
chromatography; the minor axial isomer yields further equatorial product 
on treatment with DBU. 
The equatorial aldehyde is reduced to the alcohol with sodium borohydride, 
and then transformed to the iodide (I.sub.2, PPh.sub.3, imidazole) which 
yields a protected phosphonate (23) on reaction with sodium 
dibenzylphosphite. One of the phosphonate benzyl groups is cleaved by 
treatment with a stoichiometric amount of 2-mercaptobenzotniazole and 
base, and the resulting monoanion is condensed with 
di-O-pal-mitoyl-sn-glycerol in the presence of mesitylenesulfonyl 
chloride, to yield 24. The title compound 25 is obtained by removal of the 
benzyl protective groups through catalytic hydrogenolysis (H.sub.2, 
Pd(OH).sub.2 /C, t--BuOH). 
EXAMPLE 5 
Synthesis of 1-[[O-[(R)-2,3-bis(hexadecanoyloxy) 
propyl]phosphono]difluoromethyl]1,3-dideoxy-3-fluoro-D-myo-inositol (33) 
The key intermediate, 
2,4,5,6-tetra-O-benzyl-3-deoxy-3-fluoro-D-myo-inositol (40), is available 
as outlined earlier. Inversion of the stereochemistry at C-1 is brought 
about by oxidation to the inosose ((COCl).sub.2, DMSO, i-Pr.sub.2 NEt), 
followed by stereoselective reduction of the 1-ketone with 
L-Selectride.RTM. (Aldrich Chem. Co.). The resulting axial alcohol (30) is 
derivatized as its triflate (Tf.sub.2 O, NEt.sub.3), and the 
phosphorus-containing side chain is introduced by the organozinc reagent 
derived from dibenzyl bromodifluoromethylphosphonate and zinc metal, 
catalyzed by cuprous iodide to yield 31. The further reaction sequence to 
yield 33 is the same as for compound 25. 
EXAMPLE 6 
Cell Growth Inhibition Studies 
Wild type NIH 3T3 cells and v-sis oncogene-expressing NIH 3T3 cells were 
maintained in bulk culture in DMEM with 10% heat inactivated calf serum 
and passaged using 0.05% trypsin and 0.5 mM EDTA. For cell growth assays, 
the cells were plated at a density of 5.times.10.sup.3 cells in 1.6 cm 
diameter culture wells in 0.5 ml DMEM containing 10% heat inactivated calf 
serum and allowed to attach to the surface of the well for 24 hr. The 
medium was then replaced with fresh medium containing the myo-inositol 
analogues. In studies where myo-inositol was omitted from the medium, 
myo-inositol-free DMEM and dialyzed, heat inactivated calf serum was used, 
which did not adversely affect cell growth over 3 days. Adherent cells 
were harvested after 3 days and were counted using an automated cell 
counter. Inhibition of cell growth caused by serial concentrations of 
analogues was expressed as a percentage of the number of nontreated cells 
at the end of the 3-day incubation period. Incubations were conducted in 
quadruplicate. The mean concentration of analogue required to cause 50% 
inhibition of cell growth (IC.sub.50).+-.S.E. was calculated from 
nonlinear least-squares regression analysis of the cell-proliferation 
concentration data (P. L. Appel et al., Cancer Chemother. Pharmacol., 17, 
47 (1986)). 
The human colon carcinoma cell line, HT-29, was chosen as being 
representative of a clinically very important, slow growing and 
chemotherapy resistant human solid tumor. Many human colon cancer lines, 
including HT-29, have the v-sis oncogene (D. L. Trainer et al., Int. J. 
Cancer, 41, 287 (1988)). The growth of HT-29 cells was measured by colony 
formation in the soft agar colony forming assay over 7 days, with 
automated colony counting using a Omicon 3600 Image Analysis System, as 
described previously by M. C. Alley et al., 44, 549 (1984). 
All soft-agarose cultures were performed in similar fashion. Each 35-mm 
culture dish contained a base layer consisting of 0.5 ml of standard DMEM 
culture medium (with 40 .mu.M myo-inositol) with 0.5% agarose. On Day 0, 
cells in bulk culture were dissociated with trypsin and EDTA, washed once 
in growth medium, and subcultured by layering 1.times.10.sup.4 viable 
cells in 0.5 ml of growth medium with 0.3% agarose over each base layer. 
Cultures were maintained in cell culture incubators at 37.degree. C., 5% 
CO.sub.2 :95% air, and 100% relative humidity. On Day 1 (24 hr later), an 
upper layer of 1 ml of growth medium containing drug was applied to each 
culture. Cell lines formed a sufficient number of detectable colonies (&gt;60 
.mu.m diameter) for analysis following 7 days of incubation. Viable 
colonies were stained using a metabolizable tetrazolium salt, 
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride. 
The growth inhibitory effects of D-3-deoxy-3-fluorophosphatidylinositol (7) 
compared with those of D-3-deoxy-fluoro-myo-inositol are shown in Table 3. 
The results show the following. First, compound 7 is a more potent 
inhibitor of cell growth than the simple myo-inositol analogue (up to 
1,000 fold with wild type NIH 3T3 cells). Second, growth inhibition by 
compound 7 is not antagonized by myo-inositol at physiological 
concentrations. Third, selectivity for growth inhibition of v-sis compared 
to wild type NIH 3T3 cell is lost. 
TABLE 3 
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Cell growth inhibition by 3-deoxy-3-fluoro-myo- 
inositol and 3-deoxy-fluoro-phosphatidylinositol (7) 
NIH IC.sub.50 (.mu.M) 
v-sis NIH IC.sub.50 (.mu.M) 
______________________________________ 
3-deoxy-3-fluoro- 
myo-inositol 
- myo-inositol 
7,000 .+-. 130 
1,000 .+-. 600 
+ myo-inositol 
NT.sup.a NT 
3-deoxy-3-fluoro- 
phosphatidylinositol (7) 
- myo-inositol 
110 .+-. 20 
107 .+-. 15 
+ myo-inositol 
99 .+-. 13 
81 .+-. 11 
______________________________________ 
.sup.a NT = nontoxic, IC.sub.50 &gt; 33 mM. Values are .+-. S.E. of mean. 
Inhibition of colony formation of HT-29 human colon carcinoma cells by 
D-3-deoxy-3-fluoro-phosphatidylinositol (7) is shown in FIG. 6. The 
calculated IC.sub.50 for inhibition of colony formation was 53.+-.9 .mu.M. 
All cited patents and publications are incorporated by reference herein. 
The invention has been described with reference to various specific and 
preferred embodiments and techniques. However, it should be understood 
that many variations and modifications may be made while remaining within 
the spirit and scope of the invention.