Disclosed are therapeutic compounds having the formula: EQU (R)j - (core moiety), including resolved enantiomers, diastereomers, hydrates, salts, solvates and mixtures thereof. j is an integer from one to three, the core moiety is either non-cyclic or comprises at least one five- to seven-membered ring structure, R may be selected from the group consisting of hydrogen, halogen, hydroxyl, amino, substituted or unsubstituted benzyl, C.sub.1-6 alkyl or C.sub.1-6 alkenyl, and at least one R has the formula I: ##STR1## n is an integer from seven to twenty and at least one of X or Y is --OH. The other of X or Y, which is not --OH, is hydrogen, CH.sub.3 --, CH.sub.3 --CH.sub.2 --, CH.sub.3 --(CH.sub.2).sub.2 -- or (CH.sub.3).sub.2 --CH.sub.2 --, and each W.sub.1, W.sub.2, and W.sub.3 is independently hydrogen, CH.sub.3 --, CH.sub.3 --CH.sub.2 --, CH.sub.3 --(CH.sub.2).sub.2 -- or (CH.sub.3).sub.2 --CH.sub.2 --. The X, Y, W.sub.1, W.sub.2, or W.sub.3 alkyl groups may be unsubstituted or substituted by an hydroxyl, halo or dimethylamino group. The disclosed compounds and therapeutic compositions thereof are useful in treating individuals having a disease or treatment-induced toxicity, mediated by second messenger activity.

TECHNICAL FIELD OF THE INVENTION 
The invention provides a class of substituted hydroxyl-containing compounds 
that are effective agents to inhibit specific intra-cellular signaling 
events often induced by noxious or inflammatory stimuli, or to directly or 
indirectly be anti-microbial to yeast or fungal infections. More 
specifically, the inventive compounds have at least one hyroxyl-containing 
substituent bonded to core moiety. The inventive compounds are useful 
antagonists to control intracellular levels of specific non-arachidonyl 
sn-2 unsaturated phosphatidic acids and corresponding phosphatidic 
acid-derived diacylglycerols which occur in response to cellular 
proliferative stimuli. 
BACKGROUND ART 
Pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine), abbreviated PTX and 
disclosed in U.S. Pat. Nos. 3,422,307 and 3,737,433, is a xanthine 
derivative which has seen widespread medical use for the increase of blood 
flow. Metabolites of PTX were summarized in Davis et al., Applied 
Environment Microbial. 48:327, 1984. One such metabolite, 
1-(5-hydroxyhexyl)-3,7-dimethylxanthine, designated M1 and disclosed in 
U.S. Pat. Nos. 4,515,795 and 4,576,947, increases cerebral blood flow. In 
addition, U.S. Pat. Nos. 4,833,146 and 5,039,666 disclose use of tertiary 
alcohol analogs of xanthine for enhancing cerebral blood flow. 
U.S. Pat. No. 4,636,507 discloses that PTX and M1 stimulate chemotaxis in 
polymorphonuclear leukocytes in response to a chemotaxis stimulator. PTX 
and related tertiary alcohol substituted xanthines inhibit activity of 
certain cytokines to affect chemotaxis (U.S. Pat. No. 4,965,271 and U.S. 
Pat. No. 5,096,906). Administration of PTX and GM-CSF decrease tumor 
necrosis factor (TNF) levels in patients undergoing allogeneic bone marrow 
transplant (Bianco et al., Blood 76: Supplement 1 (522A), 1990). Reduction 
in bone marrow transplant-related complications accompanied reduction in 
assayable levels of TNF. However, in normal volunteers, TNF levels were 
higher among PTX recipients. Therefore, elevated levels of TNF are not the 
primary cause of such complications. 
Therefore, effective therapeutic compounds that are safe and effective for 
human or animal administration and that can maintain cellular homeostasis 
in the face of a variety of inflammatory stimuli are needed. The invention 
is a result of research conducted in looking for such compounds. 
SUMMARY OF THE INVENTION 
We have found a genus of compounds useful in a large variety of therapeutic 
indications for treating or preventing disease mediated by intracellular 
signaling through one or two specific intracellular signaling pathways. In 
addition, the inventive compounds and pharmaceutical compositions are 
suitable for normal routes of therapeutic administration (e.g., 
parenteral, oral, topical, etc.) for providing effective dosages. 
The invention provides a class of compounds containing at least one 
hydroxyl-containing side chain of at least nine carbon atoms in length, 
preferably cyclic compounds. The inventive compounds and pharmaceutical 
compositions thereof have the formula: 
EQU (R)j - (core moiety), 
including resolved enantiomers and/or diastereomers, hydrates, salts, 
solvates and mixtures thereof, wherein j is an integer from one to three, 
the core moiety is either non-cyclic or comprises at least one five- to 
seven-membered ring structure, and R may be selected from the group 
consisting of hydrogen, halogen (preferably bromine, chlorine, fluorine 
and iodine), hydroxyl, amino, substituted or unsubstituted benzyl, alyl 
(C.sub.1-6, preferably methyl) or alkenyl (C.sub.1-6), preferably the 
alkyl or alkenyl groups being substituted by an hydroxy, halogen and 
dimethylamine and/or interrupted by an oxygen atom. Preferred R include, 
but are not limited to, methyl, ethyl, isopropyl, n-propyl, isobutyl, 
n-butyl, t-butyl, 2-hydroxyethyl, 3-hydroxypropyl, 3-hydroxy-n-butyl, 
2-methoxyethyl, 4-methoxy-n-butyl, 5-hydroxyhexyl, 2-bromopropyl, 
3-dimethylaminobutyl, 4-chloropentyl, and the like. Particularly preferred 
R are ethyl, methyl, or H, and most preferably, methyl or H. At least one 
R has the formula I: 
##STR2## 
wherein n is an integer from seven to twenty and at least one of X or Y is 
--OH. If only one of X or Y is --OH, then the other X or Y is hydrogen, 
CH.sub.3 --, CH.sub.3 --CH.sub.2 --, CH.sub.3 --(CH.sub.2).sub.2 --, or 
(CH.sub.3).sub.2 --CH.sub.2 --, and W.sub.1, W.sub.2, and W.sub.3 are 
independently hydrogen, CH.sub.3 --, CH.sub.3 --CH.sub.2 --, CH.sub.3 
--(CH.sub.2).sub.2 --, or (CH.sub.3).sub.2 --CH.sub.2 --, wherein X, Y, 
W.sub.1, W.sub.2, and W.sub.3 alkyl groups may be substituted by an 
hydroxyl, halo or dimethylamino group and/or interrupted by an oxygen 
atom, hydrogen or alkyl (C.sub.1-4). Preferably, n is an integer from 
seven to twelve. Especially preferred compounds have X and Y both being 
--OH and each of W.sub.1, W.sub.2, and W.sub.3 being hydrogen or methyl. 
A non-cyclic core moiety may be, for example, an amino acid (one or two), 
an hydroxyl, carboxyl, sulfoxide, sulfonate, phosphate, amide, amine, or 
ketone group, a simple ionic functional group, or a terminal hydrogen or 
halogen atom. Exemplary core moiety amino acids may include one or more of 
the following: alanine, arginine, asparagine, aspartic acid, cysteine, 
glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, 
methionine, phenylalanine, proline, serine, threonine, tryptophan, 
tyrosine and valine. The non-cyclic core moiety may preferably be a 
dipeptide comprising two amino acids selected from the foregoing exemplary 
list. Exemplary core halogen atoms include bromine, chlorine, fluorine and 
iodine. 
A core moiety may alternatively be at least one five- to seven-membered 
ring, preferably having from one to three, five- to six-membered ring 
structures in a predominantly planar configuration. Preferably, R having 
formula I structure is bonded to a ring nitrogen if one exists. Exemplary, 
cyclic-core moieties may be substituted or unsubstituted: barbituric acid; 
benzamide; benzene; biphenyl; cyclohexane, cyclohexene; cyclohexanedione; 
cyclopentanedione; delta-lactam; flutarimide; glutarimide; 
homophthalimide; imidazole amide; isocarbostyrile; lumazine; napthlalene; 
pteridine; pthalimide; piperidine; pyridine; pyrimidine; pyrrole amide; 
quinazolinedione; quinazolinone; quinolone; recorsinol; stilbene; 
succinimide; theobromine; thymine; triazine; tricyclododecane; uracil; 
xanthine; or derivatives thereof. 
Preferred ring cores include substituted or unsubstituted glutarimide, 
methylthymine, methyluracil, thymine, theobromine, uracil and xanthine. 
Exemplary preferred cores include, but are not limited to: 
1,3-cyclohexanedione, 1,3-cyclopentanedione; 1,3-dihydroxynaphthalene; 
1-methyllumazine; methylbarbituric acid; 3,3-dimethylflutarimide; 
2-hydroxypyridine; methyldihydroxypyrazolopyrimidine (preferably, 
1,3-dimethyldihydroxypyrazolo4,3-d! pyrimidine); methylpyrrolopyrimidine 
(preferably, 1-methylpyrrolo 2,3-d! pyrimidine); 2-pyrrole amides; 
3-pyrrole amides; 1,2,3,4-tetrahydroisoquinolone; 
1-methyl-2,4(1H,3H)-quinazolinedione (1-methylbenzoyleneurea); 
quinazolin-4(3H)-one; alkyl-substituted (C.sub.1-6) thymine; 
methylthymine; alkyl-substituted (C.sub.1-6) uracil; 6-aminouracil; 
1-methyl-5,6-dihydrouracil; 1-methyluracil; 5- and/or 6-position 
substituted uracils; 1,7-dimethylxanthine, 3,7-dimethylxanthine; 
3-methylxanthine; 3-methyl-7-methylpivaloylxanthine; 
8-amino-3-methylxanthine; and 7-methylhypoxanthine. 
Preferably, the ring-core is xanthine or a xanthine derivative. Especially 
preferred xanthine compounds have the following formula II: 
##STR3## 
wherein R is selected from the foregoing members. Most preferably, a 
single R having formula I above is bonded to the N.sub.1 xanthine nitrogen 
in formula II or each of two formula I R are bonded to N.sub.1 and N.sub.7 
xanthine nitrogens, respectively. Remaining R substituents are preferably 
selected from the group consisting of hydrogen, methyl, fluoro, chloro and 
amino. 
The invention provides a pharmaceutical composition comprising an 
inventive-compound and a pharmaceutically acceptable excipient. The 
pharmaceutical composition may be formulated for oral, parenteral, ocular 
or topical administration to a patient. 
The invention includes a method for treating an individual having a variety 
of diseases. The disease is characterized by or can be treated by 
inhibiting an immune response or a cellular response to external or in 
situ primary stimuli, the cellular response being mediated through a 
specific phospholipid-based second messenger described herein. The second 
messenger pathway is activated in response to various noxious, 
proinflammatory or proliferative stimuli characteristic of a variety of 
disease states. More specifically, the invention includes methods for 
treating or preventing clinical symptoms of various disease states or 
reducing toxicity of other treatments by inhibiting cellular signaling 
through a second messenger pathway involving signaling through a 
non-arachidonyl phosphatidic acid intermediate. 
A disease state or treatment-induced toxicity are selected from the group 
consisting of: tumor progression involving tumor stimulation of blood 
supply (angiogenesis) by production of fibroblast growth factor (FGF), 
vascular endothelial growth factor (VEGF) or platelet-derived growth 
factor (PDGF); tumor invasion and formation of metastases through adhesion 
molecule binding, expressed by vascular endothelial cells (VCAM and ICAM); 
tissue invasion through tumor metalloprotease production such as MMP-9; 
autoimmune diseases caused by dysregulation of the T cell or B cell immune 
systems, treatable by suppression of the T cell or B cell responses; acute 
allergic reactions including, but not limited to, asthma and chronic 
inflammatory diseases, mediated by proinflammatory cytokines including 
tumor necrosis factor (TNF) and IL-1, and rheumatoid arthritis, 
osteoarthritis, multiple sclerosis or insulin dependent diabetes mellitus 
(IDDM), associated with enhanced localization of inflammatory cells and 
release of inflammatory cytokines and metalloproteases; smooth muscle 
cell, endothelial cell, fibroblast and other cell type proliferation in 
response to growth factors, such as PDGF-AA, BB, FGF, EGF, etc. (i.e., 
atherosclerosis, restenosis, stroke, and coronary artery disease); 
activation of human immunodeficiency virus infection (AIDS and AIDS 
related complex); HIV-associated dementia; kidney mesengial cell 
proliferation in response to IL-1, MIP-1.alpha., PDGF or FGF; 
inflammation; kidney glomerular or tubular toxicity in response to 
cyclosporin A or amphotericin B treatment; organ toxicity (e.g., 
gastrointestinal or pulmonary epithelial) in response to a cytotoxic 
therapy (e.g., cytotoxic drug or radiation); effects of non-alkylating 
anti-tumor agents; inflammation in response to inflammatory stimuli (e.g., 
TNF, IL-1 and the like) characterized by production of metalloproteases or 
allergies due to degranulation of mast cells and basophils in response to 
IgE or RANTES; bone diseases caused by overproduction of 
osteoclast-activating factor (OAF) by osteoclasts; CNS diseases resulting 
from over-stimulation by proinflammatory neurotransmitters such as , 
acetylcholine, serotonin, leuenkephalin or glutamate; acute inflammatory 
diseases such as septic shock, adult respiratory distress syndrome; 
multi-organ dysfunction associated with inflammatory cytokine cascade; and 
combinations thereof. 
In many cell types, signaling is dependent upon generation of a broad 
variety of non-arachidonyl PA species, some of which are generated from 
lyso-PA by the enzyme lyso-PA acyl transferase (LPAAT). Generation of each 
of these PA species (the predominant forms being: 1-acyl and 1-alkyl 
2-linoleoyl PA compounds, generated by LPAAT) serves to effect both 
proliferative and/or inflammatory signaling in the diseases discussed and 
cell systems described above. 
The inventive compounds are of particular significance for inhibiting 
IL-2-induced proliferative response. IL-2 signaling inhibition is 
potentially useful in the treatment of numerous disease states involving 
T-cell activation and hyperproliferation. Exemplary autoimmune diseases 
are lupus, scleroderma, rheumatoid arthritis, multiple sclerosis, 
glomerula nephritis, insulin dependent diabetes mellitus (IDDM), as well 
as potential malignancies, including but not limited to, chronic 
myelogenous leukemia as well as others.

DETAILED DESCRIPTION OF THE INVENTION 
The invention provides a genus of compounds which can control cellular 
behavior by a particular phase of a secondary messenger pathway system 
(Bursten et al., J. Biol. Chem. 266:20732, 1991). The second messengers 
are lipids or phospholipids and use the following abbreviations: 
PE=phosphatidyl ethanolamine 
LPE=lysophosphoethanolamine 
PA=phosphatidic acid 
LPA=lysophosphatidic acid 
DAG=diacylglycerol 
LPLD=lysophospholipase-D 
LPAAT=lysophosphatidic acid acyl transferase 
PAPH=phosphatidic acid phosphohydrolase 
PLA2=phospholipase A-2. 
PLD=phospholipase D 
PAA=phosphoarachidonic acid 
PLA-2=phospholipase A2 
PC=phosphatidyl choline 
"remodeled" PA, cyclic pathway=PAA, LPA, PA and DAG intermediates 
substituted with 1-saturated, 2-linoleoyl or 1,2-dioleoyl, 
dioleoyl/1,2-sn-dilinoleoyl at the indicated sn-1 and sn-2 positions. 
"Classical PI Pathway"=PI, DAG, PA intermediates substituted with 
1-stearoyl, 2-arachidonoyl fatty acyl side chains. 
"PLD-generated PA"=PE, PC, LPA, PA and DAG intermediates substituted with, 
e.g., 1,2-sn-dioleoyl-, 1-alkyl, 2-linoleoyl-, and 1-alkyl, 
2-docosahexaenoyl-side chains. 
Lysophosphatidic acid transferase (LPAAT) effects the synthesis of 
phosphatidic acid (PA) from lysophosphatidic acid (LPA) by incorporation 
of an acyl group from acyl CoA. Hydrolysis of the phosphate moiety by PA 
phosphohydrolase (PAPH) results in the formation of DAG. These aspects of 
the pathway appear to be activated immediately (within a minute) upon 
stimulation by a primary stimulus (e.g., a cytokine such as IL-1, IL-2 or 
TNF) acting at a receptor on a cellular surface. An immediate detectable 
effect is an elevation of levels of PA and DAG. Administration of the 
compounds of the invention reverse this elevation. 
The compounds and pharmaceutical compositions of the invention include 
inhibitors of subspecies of LPAAT and PAPH enzymes with substrate 
specificity for intermediates with 1,2-diunsaturated and 1-alkyl, 
2-unsaturated subspecies. One representative example of such an inhibitor 
(although not within the genus of inventive compounds) is PTX. PTX blocks 
PAPH in a specific activation pathway that does not involve PI but rather 
derives from a PA that is largely composed of 1,2-diunsaturated and 
1-alkyl, 2-unsaturated subspecies. This was shown, for example, by the 
demonstration that human mesangial cells stimulated with TNF produce DAG 
from PI and regenerate PI in the absence and the presence of PTX. In the 
latter system there is no evidence to suggest that PA or DAG are derived 
from sources other than PI. It should be emphasized that the compounds of 
the invention affect that subset of PAPH and LPAAT that relates to 
substrates with unsaturated fatty acids other than arachidonate in the 
sn-2 position, not the housekeeping forms of these enzymes that serve the 
PI pathway. 
Each membrane phospholipid subclass (e.g., PA, PI, PE, PC and PS) reaches a 
stable content of characteristic fatty acyl side chains due to cyclic 
remodeling of the plasma membrane as well as turnover for each subclass. 
PA is often stable, but present in relatively small quantities. PA in 
resting cells consists mostly of saturated acyl chains, usually consisting 
of myristate, stearate and palmitate. In resting cells, PC's acyl side 
chains consist mostly of acyl palmitate in the sn-1 position and oleate in 
the sn-2 position. PE and PI are predominantly composed of sn-1 stearate 
and sn-2 arachidonate. 
Due to this characteristic content of acyl groups in the sn-1 and sn-2 
positions, the origin of any PA species may be deduced from the chemical 
nature of its acyl groups in the sn-1 and sn-2 positions. For example, if 
PA is derived from PC through action of the enzyme PLD, the PA will 
contain the characteristic acyl side chains of PC substrate passed through 
the second messenger pathway. Further, the origin of any 1,2 sn-substrate 
species may be differentiated as to its origin. However, it is important 
to know whether or not each phospholipid species passes through a PA form 
previous to hydrolysis to DAG. The lyso-PA that is converted to PA and 
thence to DAG may be shown. The complexities of this second messenger 
pathway can be sorted by suitable analyses by fatty acyl side chain 
chemistry (i.e., by thin layer chromatography, gas-liquid chromatography, 
or high pressure liquid chromatography) of intermediates in cells at 
various time points after stimulation of the second messenger pathway. 
In certain meseachymal cells, such as neutrophils and rat or human 
mesangial cells, several signaling pathways may be activated in tandem, 
simultaneously or both. For example, in neutrophils, F-Met-Leu-Phe 
stimulates formation of PA through the action of PLD, followed in time by 
formation of DAG through the action of PAPH. Several minutes later, DAG is 
generated from PI through the classical phosphoinositide pathway. In many 
cells, DAG is derived from both PA that is being remodeled through a cycle 
whereby PA is sn-2 hydrolyzed by PLA-2, followed by sn-2-transacylation by 
LPAAT, and a PLD-pathway from PA that is generated from either PE or PC or 
both substrates by PLD. 
The present second messenger pathway involves substrates with unsaturated 
fatty acids in the sn-2 position other than arachidonate and those sub 
species of PAPH and LPAAT that are not involved in normal cellular 
housekeeping functions that are part of the classical PI pathway. The PAPH 
and LPAAT enzymes involved in the present second messenger pathway are 
exquisitely stereo specific for different acyl side chains and isomeric 
forms of substrates. Therefore, the inventive compounds are preferably, 
substantially enantiomerically pure, and preferably are the R enantiomer 
at the chiral carbon atom bonded to the hydroxyl group. 
PTX (in vitro) blocks formation of remodeled PA through the PA/DAG pathway 
at high PTX concentrations (greater than those that could be achieved in 
patients without dose-limiting side effects) by blocking formation of PA 
subspecies at LPAAT. Even in the presence of PTX, cells continue to form 
PA through the action of PLD, and DAG is also formed through the action of 
phospholipase C on PC and PI. The latter pathway are not inhibited by the 
inventive compounds or PTX. In PTX-treated cells, DAG derived from 
remodeled and PLA-generated PA is diminished (e.g., 1,2-sn-dioleoyl DAG, 
1-alkyl, 2-linoleoyl DAG and 1-alkyl, 2-docosahexaneolyl DAG). Therefore, 
the inventive compounds and PTX inhibit the formation of only a certain 
species of PA and DAG by selectively inhibiting a specific second 
messenger pathway that is only activated in cells by noxious stimuli, but 
is not used to signal normal cellular housekeeping functions. 
Therapeutic Uses of the Inventive Compounds 
The specific activation inhibition of the second messenger pathway, as 
described above and activated primarily by various noxious stimuli, 
suggests that the inventive compounds are useful in treating a wide 
variety of clinical indications, mediated at the cellular level by a 
common mechanism of action. Moreover, in vitro and in vivo data, presented 
herein, provides predictive data that a wide variety of clinical 
indications, having similar effects on the specific second messenger 
pathway, may be treated by the inventive compounds, which specifically 
inhibit the pathway, activated by noxious stimuli and mediated through, 
for example, inflammatory cytokines. In fact, the mechanism of action for 
the inventive compounds explains why these compounds have a multifarious 
clinical indications. 
Activation of the second messenger pathway is a major mediator of response 
to noxious stimuli and results in cellular signals that lead to, for 
example, acute and chronic inflammation, immune response and cancer cell 
growth. Although the inventive compounds may desirably inhibit many other 
unmentioned, noxious stimuli, they most effectively mediate the above 
conditions. Signals mediated by-the present second messenger pathway 
include, for example, those cellular responses of LPS directly, T cell 
activation by antigen, B cell activation by antigen, cellular responses to 
IL-1 mediated through the IL-1 Type 1 receptor (but not the IL-1 Type 2 
receptor), the TNF Type 1 receptor, growth stimulated by transformations 
including, but not limited to, activated oncogenes (e.g., ras, abl, her 
2-neu and the like), smooth muscle cell proliferation stimulated by PDGF, 
b-FGF and IL-1; T cell and B cell growth stimulation by IL-2, IL-4 or IL-7 
and IL-4 or IL-6, respectively; and more generally, T cell receptor 
signaling. 
In vitro, the inventive compounds: (1) block IL-1 signal transduction 
through the Type 1 receptor as shown, for example, by preventing IL-1 and 
IL-1 plus PDGF (platelet derived growth factor) induction of proliferation 
of smooth muscle, endothelial and kidney mesengial cells; (2) suppress 
up-regulation of adhesion molecules as shown, for example, by blocking 
VCAM in endothelial cells; (3) inhibit TNF, LPS and IL-1 induced 
metalloproteases (an inflammation model); (4) block LPS, TNF or IL-1 
induced metalloprotease and secondary cytokine production (for prevention 
and treatment of septic shock); (5) suppress T cell and B cell activation 
by antigen, for example, IL-2 and IL-4; (6) inhibit mast cell activation 
by IgE; (7) are cytotoxic for transformed cells and tumor cell lines, yet 
not for normal cells; and (8) block signaling by IL-2, IL-4, IL-6 and IL-7 
on T and B cells. 
The foregoing in vitro effects give rise to the following in vivo biologic 
effects, including, but not limited to, protection and treatment of 
endotoxic shock and sepsis induced by gram positive or gram negative 
bacteria, inhibition of tumor cell growth, synergistic immunosuppression, 
active in autoimmune diseases and in suppressing allograft reactions, and 
stimulation of hair grow through reversal of an apoptotic process. The 
inventive compounds are most potent when used to prevent and treat septic 
shock, treat acute and chronic inflammatory disease, treat or prevent an 
autoimmune disease and stimulate hair growth (when applied topically). 
The inventive compounds also are useful as an adjuvant to inhibit toxic 
side effects of drugs whose side effects are mediated through the present 
second messenger pathway. 
Metalloproteases mediate tissue damage such as glomerular diseases of the 
kidney, joint destruction in arthritis, and lung destruction in emphysema, 
and play a role in tumor metastases. Three examples of metalloproteases 
include a 92 kD type V gelatinase induced by TNF, IL-1 and PDGF plus bFGF, 
a 72 kD type IV collagenase that is usually constitutive and induced by 
TNF or IL-1, and a stromelysin/PUMP-1 induced by TNF and IL-1. The 
inventive compounds can inhibit TNF or IL-1 induction of the 92 kD type V 
gelatinase inducable metalloprotease. Moreover, the inventive compounds 
can reduce PUMP-1 activity induced by 100 U/ml of IL-1. Accordingly, the 
inventive compounds prevent induction of certain metalloproteases induced 
by IL-1 or TNF and are not involved with constitutively produced proteases 
(e.g., 72 kD type IV collagenase) involved in normal tissue remodeling. 
The inventive compounds inhibit signal transduction mediated through the 
Type I IL-1 receptor, and are therefore considered as IL-1 antagonists. A 
recent review article entitled "The Role of Interleukin-1 in Disease" 
(Dinarello and Wolff N. Engl. J. Med. 328, 106, Jan. 14, 1993) described 
the role of IL-1 as "an important rapid and direct determinant of 
disease." "In septic shock, for example, IL-1 acts directly on the blood 
vessels to induce vasodilatation through the rapid production of platelet 
activating factor and nitric oxide, whereas in autoimmune disease it acts 
by stimulating other cells to produce cytokines or enzymes that then act 
on the target tissue." The article describes a group of diseases that are 
mediated by IL-1, including sepsis syndrome, rheumatoid arthritis, 
inflammatory bowel disease, acute and myelogenous leukemia, 
insulin-dependent diabetes mellitus, atherosclerosis and other diseases 
including transplant rejection, graft versus host disease (GVHD), 
psoriasis, asthma, osteoporosis, periodontal disease, autoimmune 
thyroiditis, alcoholic hepatitis, premature labor secondary to uterine 
infection and even sleep disorders. Since the inventive compounds inhibit 
cellular signaling through the IL-1 Type I receptor and are IL-1 
antagonists, the inventive compounds are useful for treating all of the 
above-mentioned diseases. 
For example, for sepsis syndrome, the mechanism of IL-1-induced shock 
appears to be the ability of IL-1 to increase the plasma concentrations of 
small mediator molecules such as platelet activating factor, prostaglandin 
and nitric oxide. These substances are potent vasodilators and induce 
shock in laboratory animals. Blocking the action of IL-1 prevents the 
synthesis and release of these mediators. In animals, a single intravenous 
injection of IL-1 decreases mean arterial pressure, lowers systemic 
vascular resistance, and induces leukopenia and thrombocytopenia. In 
humans, the intravenous administration of IL-1 also rapidly decreases 
blood pressure, and doses of 300 ng or more per kilogram of body weight 
may cause severe hypotension. The therapeutic advantage of blocking the 
action of IL-1 resides in preventing its deleterious biologic effects 
without interfering with the production of molecules that have a role in 
homeostasis. The present inventive compounds address the need, identified 
by Dinarello and Wolff, by inhibiting cellular signaling only through the 
IL-1 Type I receptor and not through the IL-1 Type II receptor. 
With regard to rheumatoid arthritis, Dinarello and Wolff state: 
"Interleukin-1 is present in synovial lining and synovial fluid of 
patients with rheumatoid arthritis, and explants of synovial tissue from 
such patients produce IL-1 in vitro. Intraarticular injections of 
interleukin-1 induce leukocyte infiltration, cartilage breakdown, and 
periarticular bone remodeling in animals. In isolated cartilage and bone 
cells in vitro, interleukin-1 triggers the expression of genes for 
collagenases as well as phospholipases and cyclodxygenase, and blocking 
its action reduces bacterial-cell-wall-induced arthritis in rats." 
Therefore, the inventive compounds, as IL-1 antagonists, are useful to 
treat and prevent rheumatoid arthritis. 
With regard to inflammatory bowel disease, ulcerative colitis and Crohn's 
disease are characterized by infiltrative lesions of the bowel that 
contain activated neutrophils and macrophages. IL-1 can stimulate 
production of inflammatory eicosanoids such as prostaglandin E.sub.2 
(PGE.sub.2) and leukotriene B.sub.4 (LTB.sub.4) and IL-8, an inflammatory 
cytokine with neutrophil-chemoattractant and neutrophil-stimulating 
properties. Tissue concentrations of PGE2 and LTB4 correlate with the 
severity of disease in patients with ulcerative colitis, and tissue 
concentrations of IL-1 and IL-8 are high in patients with inflammatory 
bowel disease. Therefore, an IL-1 antagonist, such as the inventive 
compounds, would be effective to treat inflammatory bowel disease. 
With regard to acute and chronic myelogenous leukemia, there is increasing 
evidence that IL-1 acts as a growth factor for such tumor cells. 
Therefore, the inventive compounds should be effective to prevent the 
growth of worsening of disease for acute and chronic myelogenous 
leukemias. 
Insulin-dependent diabetes mellitus (EDDM) is considered to be an 
autoimmune disease with destruction of beta cells in the islets of 
Lagerhans mediated by immunocoinpetent cells. Islets of animals with 
spontaneously occurring IDDM (e.g., BB rats or NOD mice) have inflammatory 
cells that contain IL-1. Therefore, the inventive compounds should be 
useful for the prevention of and treatment of IDDM. 
IL-1 also plays a role in the development of atherosclerosis. Endothelial 
cells are a target of IL-1. IL-1 stimulates proliferation of vascular 
smooth muscle cells. Foam cells isolated from fatty arterial plaques from 
hypercholesterolemic rabbits contain IL-1.beta. and IL-1.beta. messenger 
RNA. The uptake of peripheral blood monocytes results in initiation of 
IL-1 production by these cells. IL-1 also stimulates production of PDGF. 
Taken together, IL-1 plays a part in the development of atherosclerotic 
lesions. Therefore, an IL-1 antagonist, such as the inventive compounds 
should be useful in preventing and treating atherosclerosis. 
IL-1 activates (through the Type I IL-1 receptor) a lyso-PA acyltransferase 
(LPAAT) and phosphatidate phosphohydrolase within 5 seconds of cell (for 
example, human mesangial cells, HMC) exposure to this cytokine. Activation 
of both enzymes results in production of PA species with sn-1 and sn-2 
unsaturated acyl groups, with the majority of sn-2 acyl chains being 
polyunsaturated. Both IL-1 and a product of LPAAT, 1,2-sn-dilinoleoyl PA, 
activate a signaling pathway involving hydrolysis of PE to PA. This 
reaction is followed by dephosphorylation of PA to produce both 
1,2-sn-diacylglycerol, and 1-o-alkyl or 1-o-alkenyl acylglycerol (AAG) 
species. The inventive compounds exert their activity by inhibiting one or 
both enzymes at the inner leaflet of the plasma membrane. Therefore, 
appropriate in vitro models for drug activity is to measure inhibition of 
stimulation caused by a pro-inflammatory cytokine or other inflammatory 
cellular signal. 
The generation of the sn-2 unsaturated PA fraction by LPAAT serves to 
activate either G-proteins, or acts directly upon PLD through alteration 
of its lipid microenvironment. Activation of LPAAT and generation of the 
sn-2-unsaturated PA species is an energy sensitive pathway of PLD. This 
provides a mechanism for a limited-receptor system to amplify a signal and 
generate a cellular response by rapid synthesis of small amounts of PA. 
Uptake of di-unsaturated PA, which is about &lt;0.1% of total membrane lipid 
mass, is sufficient to activate PLD activity. This quantity of PA is 
similar to that endogeneously synthesized by LPAAT. The PA-stimulated PLD 
acts upon PE, which should be localized to the inner leaflet of the cell 
membrane, which is enriched in PE relative to the outer leaflet. 
Therefore, the cellular inflammatory response to IL-1 is mediated by the 
pathway: IL-1R.fwdarw.PA.fwdarw.(PLD).fwdarw.PE. Whereas a localized 
tissue response is: lysoPA.fwdarw.PI.fwdarw.PKC.fwdarw.(PLD).fwdarw.PC. 
The PLD species are likely to be different isozymes. The second messenger 
pathway whose activation is inhibited by the inventive compounds is not a 
PI-derived pathway and does not involve PKC in the time courses of 
inhibition. PKC is acutely activated by PI-derived DAG, but chronic 
activation (i.e., &gt;30 min) is maintained by PC-derived PA generated by 
PC-directed PLD. Therefore, the pathway inhibited by the inventive 
compounds is PE-directed and not PC-directed. Moreover, the PE-directed 
PLD favors substrates with sn-2 long-chain unsaturation. 
DAG and PA are upregulated in oncogenically transformed cells. For example, 
activating ras mutations result in increased generation of DAG on 
stimulation with mitogens, although the sources of DAG have differed 
between experimental systems. In nontransformed renal mesangial cells, 
IL-1 stimulation increased PLA2 and LPAAT activation, resulting in 
generation of sn-2 unsaturated PA and subsequent hydrolysis to DAG by 
phosphatidate phosphohydrolase. The ras transformation in NIH/3T3 cells 
upregulates serum-stimulated generation of DAG and PA. The specific 
species of DAG that is stimulated by serum is dioleoyl and for PA are 
dilinoleoyl and dioleoyl. This upregulation occurs over 4-12 hours and 
pretreatment of cells with an inventive compound, or PTX, blocks 
generation of these phospholipid second messengers. The inhibition occurs 
either through suppressing the generation of PA de novo from lysoPA, or 
through inhibition of one or both arms of the Lands cycle. The coordinate 
increase of lysoPA in the setting of diminished PA/DAG production suggests 
inhibition of transacylation of a precursor lipid. Therefore, the ras 
transformation mediates an upregulation of PA through indirect stimulation 
of PLA2 and/or LPAAT activity. The inventive compounds inhibit the 
conversion of the upregulated lysoPA to PA and subsequently block the 
phenotypic changes induced by PA/DAG in the membrane. 
The ability of the inventive compounds to inhibit generation of unsaturated 
phospholipids is mirrored by the ability of inventive compounds to inhibit 
proliferation and tumorogenicity of ras-transformed cells in vitro and in 
vivo. PTX inhibits ras-transformed NIH/3T3 cells more than parental cells. 
This inhibition is reversible and is not associated with significant 
cytotoxicity. 
Excessive or unregulated TNF (tumor necrosis factor) production is 
implicated in mediating or exacerbating a number of diseases including 
rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty 
arthritis and other arthritic conditions; sepsis, septic shock, endotoxic 
shock, gram negative sepsis, toxic shock syndrome, adult respiratory 
distress syndrome, cerebral malaria, chronic pulmonary inflamrnmatory 
disease, silicosis, pulmonary sarcoidosis, bone resorption diseases, 
reperfusion injury, graft versus host reaction, allograft rejections, 
fever, myalgias due to infection such as influenza, cachexia secondary to 
infection, AIDS or malignancy, AIDS, other viral infections (e.g., CMV, 
influenza, adenovirus, herpes family), keloid formation, scar tissue 
formation, Crohn's disease, ulcerative colitis, or pyresis. The inventive 
compounds or pharmaceutically acceptable salts thereof can be used in the 
manufacture of a medicament for the prophylactic or therapeutic treatment 
of any disease state in a human or other mammal, which is exacerbated or 
signaled through the present second messenger cellular phospholipid-based 
signaling pathway and by excessive or unregulated production of "first 
messenger" inflammatory cytokines such as TNF or IL-1. With regard to TNF 
first messenger signaling, there are several disease states in which 
excessive or unregulated TNF production by monocytes/macrophages is 
implicated in exacerbating or causing the disease. These include, for 
example, neurodegenerative diseases such as Alzheimers disease, 
endotoxemia or toxic shock syndrome (Tracey et al., Nature 330:662, 1987 
and Hinshaw et al., Circ. Shock 30:279, 1990); cachexia (Dezube et al., 
Lancet 355:662, 1990), and adult respiratory distress syndrome (Miller et 
al., Lancet 2(8665):712, 1989). The inventive compounds may be used 
topically in the treatment of prophylaxis of topical disease states 
mediated or exacerbated by excessive TNF or IL-1, such as viral infections 
(herpes or viral conjunctivitis), psoriasis, fungal or yeast infections 
(ringworm, athletes foot, vaginitis, dandruff, etc.) or other dermatologic 
hyperproliferative disorders. High TNF levels have been implicated in 
acute malaria attacks (Grau et al., N. Engl. J. Med. 320:1585, 1989), 
chronic pulmonary inflammatory diseases such as silicosis and asbestosis 
(Piguet et al., Nature 344:245, 1990, and Bissonnette et al., Inflammation 
13:329, 1989), and reperfusion injury (Vedder et al., Proc. Natl. Acad. 
Sci. USA 87:2643, 1990). 
The compounds of the invention can inhibit certain VEGF (vascular 
endothelial growth factor), FGF (fibroblast growth factor) and PDGF 
(platelet derived growth factor) effects in vivo, such as inhibition of 
angiogenesis or restenosis. For example, Ferns et al. (Science 253:1129, 
1991) have shown that neointimal smooth muscle chemotaxis and angioplasty 
are inhibited in rats using a neutralizing antibody to PDGF. Also, Jawien 
et al. (J. Clin Invest. 89:507, 1992) have shown that PDGF promotes smooth 
muscle migration and intimal thickening in a rat model of balloon 
angioplasty. Inhibition of the PDGF-mediated effects following balloon 
angioplasty by the inventive compounds is the pharmacological rationale 
for using the inventive compounds as therapeutic agents to prevent 
restenosis. The inventive compounds also inhibit atherogenesis because 
increased levels of PDGF expressed by macrophages are associated with all 
phases of atherogenesis (Ross et al., Science 248:1009, 1990). Further, 
many human tumors express elevated levels of either PDGF, FGF, receptors 
for FGF or PDGF, or mutated cellular oncogenes highly homologous to these 
growth factors or their receptors. For example, such tumor cell lines 
include sarcoma cell lines (Leveen et al., Int. J. Cancer 46:1066, 1990), 
metastatic melanoma cells (Yamanishi et al., Cancer Res. 52:5024, 1992), 
and glial tumors (Fleming et al., Cancer Res. 52:4550, 1992). 
Thus, the drugs of the invention are also useful to raise the seizure 
threshold, to stabilize synapses against neurotoxins such as strychnine, 
to potentiate the effect of anti-Parkinson drugs such as L-dopa, to 
potentiate the effects of soporific compounds, to relieve motion disorders 
resulting from administration of tranquilizers, and to diminish or prevent 
neuron overfiring associated with progressive neural death following 
cerebral vascular events such as stroke. In addition, the compounds of the 
invention are useful in the treatment of norepinephrine-deficient 
depression and depressions associated with the release of endogenous 
glucocorticoids, to prevent the toxicity to the central nervous system of 
dexamethasone or methylprednisolone, and to treat chronic pain without 
addiction to the drug. Further, the compounds of the invention are useful 
in the treatment of children with learning and attention deficits and 
generally improve memory in subjects with organic deficits, including 
Alzheimer's patients. 
In Vitro Assays for Physiologic and Pharmacological Effects of the 
Inventive Compounds 
Various in vitro assays can be used to measure effects of the inventive 
compounds to module immune activity and have antitumor activity using a 
variety of cellular types. For example, a mixed lymphocyte reaction (MLR) 
provides a valuable screening tool to determine biological activity of 
each inventive compound. In the MLR, PBMCs (peripheral blood mononuclear 
cells) are obtained by drawing whole blood from healthy volunteers in a 
heparinized container and diluted with an equal volume of hanks balanced 
salt solution (HBSS). This mixture is layered on a sucrose density 
gradient, such as a Ficoll-Hypaque.RTM. gradient (specific gravity 1.08), 
and centrifuged at 1000.times.g for 25 minutes at room temperature or 
cooler. PBMC are obtained from a band at a plasma-Ficoll interface, 
separated and washed at least twice in a saline solution, such as HBSS. 
Contaminating red cells are lysed, such as by ACK lysis for 10 min at 
37.degree. C., and the PBMCs are washed twice in HBSS. The pellet of 
purified PBMCs is resuspended in complete medium, such as RPMI 1640 plus 
20% human inactivated serum. Proliferative response of PBMC to allogeneic 
stimulation is determined in a two-way MLR performed in a 96-well 
microtiter plate. Briefly, approximately 105 test purified PBMC cells in 
200 .mu.l) complete medium are co-cultured with approximately 10.sup.5 
autologous (control culture) or allogeneic (stimulated culture) PBMC 
cells, wherein the aliogeneic cells are from HLA disparate individuals. 
Varying doses of compounds (drug) are added at the time of addition of 
cells to the rnicrotiter plate. The cultures are incubated for 6 days at 
37.degree. C. in a 5% CO.sub.2 atmosphere. At the conclusion of the 
incubation tritiated thymidine is added (for example, 1 .mu.Ci/well of 40 
to 60 Ci/mmole) and proliferation determined by liquid scintillation 
counting. 
Another assay for measuring activity of the inventive compounds involves 
determining PDGF, FGF or VEGF proliferative response using either mouse 
NIH-3T3 (Balb) cells or human-derived stromal cells. Human stromal cells 
are plated (e.g., about 2000 cells per well) in defined media (e.g., 69% 
McCoy's, 12.5% fetal calf serum, 12.5% horse serum, 1% antibiotics, 1% 
glutamine, 1% vitamin supplement, 0.8% essential amino acids, 1% sodium 
pyruvate, 1% sodium bicarbonate, 0.4% non-essential amino acids and 0.36% 
hydrocortisone). Two to three days later, the stromal cells are starved in 
serum-free media. Twenty four hours later, the cells are treated with a 
stimulating agent, such as PDGF-AA, PDGF-BB or basic FGF (fibroblast 
growth factor) with or without IL-1.alpha. or TNF, and tritiated 
thymidine. Cell proliferation is determined by liquid scintillation 
counting. 
A B-cell proliferation assay determines the effect of the inventive 
compounds on inhibiting proliferation of stimulated B-cells, stimulated by 
an anti-mu antibody (40 .mu.g/ml), IL-4 or PMA (2.5 nM). Ramos B-cell 
tumor cells or murine splenocytes can be incubated with a stimulating 
agent, an inventive compound and tritiated thymidine to measure inhibition 
of cell proliferation caused by the stimulating agent. 
Compounds of the Invention 
The inventive compounds contain at least one hydroxyl-containing side chain 
of at least nine carbon atoms in length and are preferably cyclic 
compounds. The inventive compounds and pharmaceutical compositions thereof 
have the formula: 
EQU (R)j - (core moiety), 
including resolved enantiomers and/or diastereomers, hydrates, salts, 
solvates and mixtures thereof, wherein j is an integer from one to three, 
the core moiety is either non-cyclic or comprises at least one five- to 
seven-membered ring structure, and R may be selected from the group 
consisting of hydrogen, halogen (preferably bromine, chlorine, fluorine 
and iodine), hydroxyl, amino, substituted or unsubstituted benzyl, alkyl 
(C.sub.1-6, preferably methyl) or alkenyl (C.sub.1-6), preferably the 
alkyl or alkenyl groups being substituted by an hydroxy, halogen and 
dimethylamine and/or interrupted by an oxygen atom. Preferred R include, 
but are not limited to, methyl, ethyl, isopropyl, n-propyl, isobutyl, 
n-butyl, t-butyl, 2-hydroxyethyl, 3-hydroxypropyl, 3-hydroxy-n-butyl, 
2-methoxyethyl, 4-methoxy-n-butyl, 5-hydroxyhexyl, 2-bromopropyl, 
3-dimethylaminobutyl, 4-chloropentyl, and the like. Particularly preferred 
R are ethyl, methyl, or H, and most preferably, methyl or H. At least one 
R has the formula I: 
##STR4## 
wherein n is an integer from seven to twenty and at least one of X or Y is 
--OH. If only one of X or Y is --OH, then the other X or Y is hydrogen, 
CH.sub.3 --, CH.sub.3 --CH.sub.2 --, CH.sub.3 --(CH.sub.2).sub.2 --, or 
(CH.sub.3).sub.2 --CH.sub.2 --, and W.sub.1, W.sub.2, and W.sub.3 are 
independently hydrogen, CH.sub.3 --, CH.sub.3 --CH.sub.2 --, CH.sub.3 
--(CH.sub.2).sub.2 --, or (CH.sub.3).sub.2 --CH.sub.2 --, wherein X, Y, 
W.sub.1, W.sub.2, and W.sub.3 alkyl groups may be substituted by an 
hydroxyl, halo or dimethylamino group and/or interrupted by an oxygen 
atom, hydrogen or alkyl (C.sub.1-4). Preferably, n is an integer from 
seven to twelve. Especially preferred compounds have X and Y both being 
--OH and each of W.sub.1, W.sub.2, and W.sub.3 being hydrogen or methyl. 
A non-cyclic core moiety may be, for example, an amino acid (one or two), 
an hydroxyl, carboxyl, sulfoxide, sulfonate, phosphate, amide, amine, or 
ketone group, a simple ionic functional group, or a terminal hydrogen or 
halogen atom. Exemplary core moiety amino acids may include one or more of 
the following: alanine, arginine, asparagine, aspartic acid, cysteine, 
glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, 
methionine, phenylalanine, proline, serine, threonine, tryptophan, 
tyrosine and valine. The non-cyclic core moiety may preferably be a 
dipeptide comprising two amino acids selected from the foregoing exemplary 
list. Exemplary core halogen atoms include bromine, chlorine, fluorine and 
iodine. 
A core moiety may alternatively be at least one five- to seven-membered 
ring, preferably having from one to three, five- to six-membered ring 
structures in a predominantly planar configuration. Preferably, R having 
formula I structure is bonded to a ring nitrogen if one exists. Exemplary, 
cyclic-core moieties may be substituted or unsubstituted: barbituric acid; 
benzamide; benzene; biphenyl; cyclohexane, cyclohexene; cyclohexanedione; 
cyclopentanedione; delta-lactam; flutarimide; glutarimide; 
homophthalimide; imidazole amide; isocarbostyrile; lumazine; napthlalene; 
pteridine; pthalimide; piperidine; pyridine; pyrimidine; pyrrole amide; 
quinazolinedione; quinazolinone; quinolone; recorsinol; stilbene; 
succinimide; theobromine; thymine; triazine; tricyclododecane; uracil; 
xanthine; or derivatives thereof. 
Preferred ring cores include substituted or unsubstituted glutarimide, 
methylthymine, methyluracil, thymine, theobromine, uracil and xanthine. 
Exemplary preferred cores include, but are not limited to: 
1,3-cyclohexanedione, 1,3-cyclopentanedione; 1,3-dihydroxynaphthalene; 
1-methyllumazine; methylbarbituric acid; 3,3-dimethylflutarimide; 
2-hydroxypyridine; methyldihydroxypyrazolopyrimidine (preferably, 
1,3-dimethyldihydroxypyrazolo4,3-d! pyrimidine); methylpyrrolopyrimidine 
(preferably, 1-methylpyrrolo 2,3-d! pyrimidine); 2-pyrrole amides; 
3-pyrrole amides; 1,2,3,4-tetrahydroisoquinolone; 
1-methyl-2,4(1H,3H)-quinazolinedione (1-methylbenzoyleneurea); 
quinazolin-4(3H)-one; alkyl-substituted (C.sub.1-6) thymine; 
methylthymine; alkyl-substituted (C.sub.1-6) uracil; 6-aminouracil; 
1-methyl-5,6-dihydrouracil; 1-methyluracil; 5- and/or 6-position 
substituted uracils; 1,7-dimethylxanthine, 3,7-dimethylxanthine; 
3-methylxanthine; 3-methyl-7-methylpivaloylxanthine; 
8-amino-3-methylxanthine; and 7-methylhypoxanthine. 
Preferably, the ring-core is xanthine or a xanthine derivative. Especially 
preferred xanthine compounds have the following formula II: 
##STR5## 
wherein R is selected from the foregoing members. Preferably, a single R 
having formula I above is bonded to the N.sub.1 xanthine nitrogen in 
formula II or each of two formula I R are bonded to N.sub.1 and N.sub.7 
xanthine nitrogens, respectively. Remaining R substituents are preferably 
selected from the group consisting of hydrogen, methyl, fluoro, chloro and 
amino. 
Synthesis of the Inventive Compounds 
The invention includes a method for preparing compounds according to the 
invention. Exemplary methods for preparing the inventive compounds are 
discussed below and in the following examples. 
In a method according to the invention, a compound containing a desired 
core (intended as a "core moiety" in compounds of the invention) undergoes 
a reaction to produce an anion. The anion is then subsequently reacted 
with a substituted olefin to displace a targeted functional group on the 
olefin, resulting in an intermediate product. In a preliminary reaction, a 
predetermined amount of a core-containing compound is reacted with a 
suitable base, a solvent and a substituted olefin, the substituted olefin 
having at least one functional group which may be substituted in a 
displacement reaction by the desired core-containing compound. 
Preferred bases include, but are not limited to, sodium hydride, sodium 
amide, sodium alkoxide, lithium hydride, potassium hydride, lithium amide, 
sodium amide and potassium amide. An especially preferred base is is 
sodium hydride. Preferred solvents may be dimethylsulfoxide, 
dimethylformamide, or an alcohol. Exemplary preferred alcohols include, 
but are not limited to, methanol, ethanol or isopropanol. Any substituted 
olefin comprising a chain structure of the inventive compounds may be used 
in the preliminary reaction according to the invention. Preferred olefins 
may be .omega.-substituted olefins. Preferred substituted olefins include, 
but are not limited to halo-substituted olefins. 
The intermediate product, having a composite structure of the 
core-containing compound and substituted olefin may subsequently be 
converted to a corresponding compound having an hydroxyl functional group. 
Primary and other, less-substituted compounds are within the scope of the 
inventive compounds and methods. The intermediate product is reacted with 
a hydroborating agent to obtain a desired borane derivative. The borane 
derivative is subsequently reacted in an oxidative hydrolysis reaction 
with an oxidative-hydrolyzing agent to obtain the corresponding compound 
having the desired hydroxyl functional group. Exemplary hydroborating 
agents include, but are not limited to, diborane, borane-methyl sulfide 
complex, borane-pyridine complex, thexylborane, disiamylborane, and 
9-borabicyclo3.3.1!nonane, most preferably, borane-tetrahydrofuran 
complex. Exemplary oxidative-hydrolyzing agents include strong oxidizers 
such as a hydrogen peroxide solution and the like. 
Alternatively, the inventive compounds may be prepared by reacting a 
compound having at least one hydroxyl group with a predetermined amount of 
a compound containing a desired core (intended as a "core moiety" in 
compounds of the invention) with a suitable base and a solvent. The 
compound having at least one hydroxyl group has at least one other 
functional group which may be substituted in a displacement reaction by 
the core-containing compound. Other functional group may be, for example, 
halogen atoms. 
In another process for preparing the inventive compounds, the intermediate 
product, which may be prepared in the above-discussed procedure, may be 
converted to a corresponding diol by reacting the intermediate product 
with a suitable oxidizing agent. Preferred oxidixing agents include, but 
are not limited to, osmium tetroxide. Preferred oxidizing agents, such as 
osmium tetroxide may require a catalytic amount of the oxidizing agent in 
the presence of a regenerating agent Exemplary, regenerating agents may be 
4-methylmorpholine-N-oxide and trimethylainine-N-oxide. An especially 
preferred regenerating agent is 4-methylmorpholine-N-oxide. 
The inventive method is also directed to a process for subsequently 
converting the intermediate product, having a composite structure of the 
core-containing compound and substituted olefin, to a corresponding 
epoxide. In the method according to the invention, the intermediate 
product may be reacted with an organic peracid to obtain a desired 
epoxide. Preferred exemplary organic peracids include 
3-chloroperoxybenzoic acid, peracetic acid and trifluoroperacetic acid. An 
especially preferred peracid is 3-chloroperoxybenzoic acid. 
Subsequently, the corresponding epoxide is reacted with a reducing agent to 
convert the correponding epoxide to an inventive compound. Exemplary 
reducing agents may be selected from the non-exhaustive group of hydride 
reducing agent (preferably sodium borohydride or lithium aluminum hydride) 
or hydrogenating agent (such as, for example, hydrogen gas in the presence 
of a metal catalyst). Preferred metal catalysts may be, for example, 
palladium, platinum, or Raney nickel. 
The compounds of the invention may be provided as enantiomeric or 
diastereomeric mixtures or in resolved or partially resolved forms. 
Standard procedures are used for resolving optical isomers. Different 
enantiomeric variants (e.g., stereoisomers and chiral forms) of the 
inventive compound may have different drug activities, based upon their 
differential ability to inhibit PAPH and LPAAT. An optical isomer, 
substantially free of the corresponding enantiomer and/or diastereomers, 
is at least about 85% of a relevant optical isomer, preferably at least 
about 95% relevant optical isomer and especially at least about 99% or 
higher relevant optical isomer. Most preferably an amount of other optical 
forms is undetectable. 
Chain length appears to exhibit some significance in predicting degree of 
activity of the compounds. For example, when n is 2 or less, the compounds 
show little activity in exemplary assays used herein. When n is 3 or 4, 
more activity is observed, particularly inhibitive activity in 
proliferation assays described herein. When n is 6 there is moderate 
activity. Activity increases significantly (on a potency basis) when n is 
7 or greater. A steep-rising curve is apparent for compounds having n 
equal to 7, 8 or more. 
The invention provides a pharmaceutical composition comprising an inventive 
compound and a pharmaceutically acceptable excipient. The pharmaceutical 
composition may be formulated for oral, parenteral or topical 
administration to a patient. 
The invention further provides a pharmaceutical composition comprising an 
inventive compound and a pharmaceutically acceptable excipient, the 
pharmaceutical composition being formulated for oral, parenteral or 
topical administration to a patient. A pharmaceutical composition may 
alternatively comprise one or a plurality of inventive compounds and a 
pharmaceutically acceptable carrier or excipient. Treatment of individuals 
with an inventive compound or pharmaceutical composition may include 
contacting with the inventive compound in vitro culture, in an 
extracorporeal treatment, or by administering (oral, parenteral or 
topical) the inventive compound or pharmaceutical composition to a subject 
whose cells are to be treated. 
Exemplary, preferred compounds of the invention include both R and S 
nantiomers and racemic mixtures of the following compounds: 
__________________________________________________________________________ 
1104 
N-(5,6-Dihy- droxyhexyl)- phthalimide 
##STR6## 
1106 
N-(8,9-Dihy- droxynonyl)- phthalimide 
##STR7## 
1108 
N-(10,11-Dihy- droxyundecyl)- phthalimide 
##STR8## 
1113 
N-(10,11-Dihy- droxyundecyl)- homophthali- mide 
##STR9## 
1118 
(N-(9-Phthali- midononyl)- phthalimide 
##STR10## 
1204 
1-(5,6-Dihy- droxyhexyl)-3- methylbenzoyl- eneurea 
##STR11## 
1207 
1-(5-Hydroxy- hexyl)-3- methylbenzoyl- eneurea 
##STR12## 
1215 
3-(11,10-Dihy- droxyundecyl)- quinazoline-4- (3H)-one 
##STR13## 
1320 
N-(11,10-Dihy- droxyundecyl)- diacetamide 
##STR14## 
1401 
1-(5-Hydroxy- 5-methyl- hexyl)-3- methylxanthine 
##STR15## 
1402 
1-(5-Hydroxy- 5-methyl- hexyl)-3- methyl-7- ethoxymethyl- xanthine 
##STR16## 
1407 
1-(10,11-Dihy- droxyundecyl)- 3-methyl-7- methylpival- oylxanthine 
##STR17## 
1408 
1-(10,11-Dihy- droxyundecyl)- 3-methyl- xanthine 
##STR18## 
1417 
1-(10-Hydroxy- undecyl)-3- methylxanthine 
##STR19## 
1420 
7-(10,11-Dihy- droxyundecyl)- 1,3-dimethyl- xanthine 
##STR20## 
1428 
3-(11,10-Dihy- droxyundecyl)- 1-methyl-2,4- dioxotetra- hydropteridine 
##STR21## 
1429 
3-(10-Hydroxy- undecyl)-1- methyl-2,4- dioxotetra- hydropteridine 
##STR22## 
1440 
1-(5,6-Dihy- droxyhexyl)-3- methylxanthine 
##STR23## 
1444 
1-(10-Hydroxy- undecyl)-3- methyl-7- methylpival- oylxanthine 
##STR24## 
1528 
1-(6,7-Dihy- droxynonyl)- 3,7-dimethyl- xanthine 
##STR25## 
1536 
1-(7-Hydroxy- octyl)-3,7- dimethyl- xanthine 
##STR26## 
1538 
1-(7,8-Dihy- droxyoctyl)- 3,7-dimethyl- xanthine 
##STR27## 
1540 
1-(2,3-Dihy- droxypropyl)- 3,7-dimethyl- xanthine 
##STR28## 
1542 
1-(4-Hydroxy- pentyl)-3,7- dimethyl- xanthine 
##STR29## 
1544 
1-(4-Hydroxy- butyl)-3,7- dimethyl- xanthine 
##STR30## 
1545 
1-(7-Hydroxy- heptyl)-3,7- dimethyl- xanthine 
##STR31## 
1546 
1-(8-Hydroxy- octyl)-3,7- dimethyl- xanthine 
##STR32## 
1551 
1-(8-Hydroxy- nonyl)-3,7- dimethyl- xanthine 
##STR33## 
1552 
1-(9-Hydroxy- decyl)-3,7- dimethyl- xanthine 
##STR34## 
1556 
1-(6-Hydroxy- hexyl)-3,7- dimethyl- xanthine 
##STR35## 
1559 
1-(10-Hydroxy- decyl)-3,7- dimethyl- xanthine 
##STR36## 
1561 
1-(8,9-Dihy- droxynonyl)- 3,7-dimethyl- xanthine 
##STR37## 
1564 
1-(9,10-Dihy- droxydecyl)- 3,7-dimethyl- xanthine 
##STR38## 
1566 
1-(5-Hydroxy- 5-methyl- hexyl)-3,7- dimethyl- xanthine 
##STR39## 
1584 
1-(4,5-Dihy- droxypentyl)- 3,7-dimethyl- xanthine 
##STR40## 
1585 
1-(6,7-Dihy- droxyheptyl)- 3,7-dimethyl- xanthine 
##STR41## 
1587 
1-(10-Hydroxy- undecyl)-3,7- dimethyl- xanthine 
##STR42## 
1592 
1-(10,11-Dihy- droxyundecyl)- 3,7-dimethyl- xanthine 
##STR43## 
1597 
1-(3-(R)- Methyl-7- methyl-6,7- dihydroxy- octyl)-3,7- dimethyl- 
xanthine 
##STR44## 
1597 
1-(3-(S)- Methyl-7- methyl-6,7- dihydroxy- octyl)-3,7- dimethyl- 
xanthine 
##STR45## 
1598 
1-(5-Hydroxy- pentyl)-3,7- dimethyl- xanthine 
##STR46## 
1599 
1-(6-Hydroxy- heptyl)-3,7- dimethyl- xanthine 
##STR47## 
1601 
N-(5-Hydroxy- hexyl)- glutarimide 
##STR48## 
1603 
N-(5,6-Dihy- droxyhexyl)- glutarimide 
##STR49## 
1609 
N-(8,9-Dihy- droxynonyl)- glutarimide 
##STR50## 
1612 
N-(10-Hy- droxyundecyl)- glutarimide 
##STR51## 
1617 
N-(10,11-Dihy- droxyundecyl)- 2-piperidone 
##STR52## 
1621 
N-(10-Hy droxyundecyl)- 2-piperidone 
##STR53## 
1622 
N-(10,11-Dihy- droxyundecyl)- piperidine 
##STR54## 
1806 
1-(5-Hydroxy- hexyl)uracil 
##STR55## 
1807 
1,3-Bis-(5-Hy- droxyhexyl)- uracil 
##STR56## 
1811 
3-(5,6-Dihy- droxyhexyl)-1- methyluracil 
##STR57## 
1818 
3-(8,9-Dihy- droxynonyl)-1- methyluracil 
##STR58## 
1821 
3-(5,6-Dihy- droxyhexyl)-1- methyldihy- drouracil 
##STR59## 
1824 
3-(10-Hydroxy- undecyl)-1- methyldihy- drouracil 
##STR60## 
1825 
3-(10,11-Dihy- droxyundecyl)- 1-methyldihy- drouracil 
##STR61## 
1903 
1-(5-Hydroxy- hexyl)thymine 
##STR62## 
1904 
Bis-1,3-(5-hy- droxyhexyl)- thymine 
##STR63## 
1907 
3-(5,6-Dihy- droxyhexyl)-1- methylthymine 
##STR64## 
1911 
3-(5-Hydroxy- hexyl)-1- methylthymine 
##STR65## 
1915 
3-(8-Hydroxy- nonyl)-1- methylthymine 
##STR66## 
1918 
3-(8,9-Dihy- droxynonyl)-1- methylthymine 
##STR67## 
2101 
5-(Hydroxy- hexyl)phenyl- sulfone 
##STR68## 
2509 
1-(3,4-Dihy- droxybutyl)- 3,7-dimethyl- xanthine 
##STR69## 
2520 
1-(11-Hydroxy- dodecenyl)- 3,7-dimethyl- xanthine 
##STR70## 
2517 
1-(11,12-Dihy- droxydodecyl)- 3,7-dimethyl- xanthine 
##STR71## 
2537 
(1-(4-(R)- Methyl-7,8-di- hydroxy-8- methylnonyl)- 3,7-dimethyl- 
xanthine 
##STR72## 
2537 
1-(4-(S)- Methyl-7,8-di- hydroxy-8- methylnonyl)- 3,7-dimethyl- 
xanthine 
##STR73## 
2540 
1-(9,10-Dihy- droxyoctadec- yl)-3,7-di- methylxanthine 
##STR74## 
2546 
1-(3,7-Di- methyl-2,3,6,7- tetrahydroxy- octyl)-3,7- dimethyl- 
xanthine 
##STR75## 
2556 
1-(12,13-Di- hydroxytri- decyl)-3,7-di- methylxanthine 
##STR76## 
2568 
1-(7,8-Dihy- droxydecyl)- 3,7-dimethyl- xanthine 
##STR77## 
2569 
1-(12-Hydroxy- tridecyl)-3,7- dimethyl- xanthine 
##STR78## 
2595 
1-(13,14-Dihy- droxytetra- decyl)-3,7- dimethyl- xanthine 
##STR79## 
3504 
1-(13-Hydroxy- tetradecyl)- 3,7-dimethyl- xanthine 
##STR80## 
3514 
1-(16,17-Dihy- droxyheptadec- yl)-3,7-di- methylxanthine 
##STR81## 
3515 
1-(5-Hydroxy- heptyl)-3,7- dimethyl- xanthine 
##STR82## 
3518 
1-(16-Hydroxy- heptadecyl)- 3,7-dimethyl- xanthine 
##STR83## 
3520 
1-(10-Hydroxy- eicosyl)-3,7- dimethyl- xanthine 
##STR84## 
3524 
1-(5-Hydroxy- 4-methyl- pentyl)-3,7- dimethyl- xanthine 
##STR85## 
3539 
1-(9-Hydroxy- nonyl)-3,7- dimethyl- xanthine 
##STR86## 
3540 
1-(11-Hydroxy- undecyl)-3,7- dimethyl- xanthine 
##STR87## 
3553 
1(4-Hydroxy- hexyl)-3,7- dimethyl- xanthine 
##STR88## 
__________________________________________________________________________ 
The compounds of the invention further are able to decrease enhanced levels 
of a relevant PA and DAG resulting from stimulation of synaptosomes with 
acetylcholine and/or epinephrine. This suggests that the effects of the 
compounds of the invention are to both enhance the release of inhibitory 
neural transmitters such as dopamine, and to modulate the distal "slow 
current" effects of such neurotransmitters. 
While dosage values will vary, therapeutic efficacy is achieved when the 
compounds of the invention are administered to a human subject requiring 
such treatment as an effective oral, parenteral, or intravenous sublethal 
dose of about 50 mg to about 5000 mg per day, depending upon the weight of 
the patient. A particularly preferred regimen for use in treating leukemia 
is 4-50 mg/kg body weight. It is to be understood, however, that for any 
particular subject, specific dosage regimens should be adjusted to the 
individual's need and to the professional judgment of the person 
administering or supervising the administration of the inventive 
compounds. 
Coadministration With a P-450 Inhibitor 
The coadministration in vivo of the compounds of the invention along with 
an inhibitor of P-450 results in an enhanced effect due to a longer half 
life of the inventive compounds. This in vivo effect is due to the 
inhibition of a degradation pathway for the compounds of the invention; in 
particular with respect to dealkylation at the N7 position of the xanthine 
core. For example, NIH3T3-D5C3 cells can be used to compare effects of a 
compound of Formula 1 alone or in combination with a P-450 inhibitor by 
comparing transformation phenotype among control, incubation with a 
compound of Formula 1 alone, and coincubation of a compound of Formula 1 
with the P-450 enzyme inhibitor. 
Compounds that inhibit P-450 include, for example, (mg range daily dosage) 
propranolol (20-100), metaprolol (20-100); verapamil (100-400), diltiazem 
(100-400), nifedipine (60-100); cimetidine (400-2,400); ciprofloxacin 
(500-2000), enoxacin (500-2,000), norfloxacin (500-2000), ofloxacin 
(500-2,000), pefloxacin (500-2,000); erythromycin (100-1,000), 
troleandomycin (100-1,000); ketoconizole (100-2,000), thiabenzadole 
(100-1,000); isoniazid (100-1000); mexiletine (100-1,000); and 
dexamethasone (1-100 mg). 
Pharmaceutical Formulations 
A suitable formulation will depend on the nature of the disorder to be 
treated, the nature of the medicament chosen, and the judgment of the 
attending physician. In general, the inventive compounds are formulated 
either for injection or oral administration, although other modes of 
administration such as transmucosal or transdermal routes may be employed. 
Suitable formulations for these compounds can be found, for example, in 
Remington's Pharmaceutical Sciences (latest edition), Mack Publishing 
Company, Easton, Pa. 
The inventive compounds and their pharmaceutically acceptable salts can be 
employed in a wide variety of pharmaceutical forms. The preparation of a 
pharmaceutically acceptable salt will be determined by the chemical nature 
of the compound itself, and can be prepared by conventional techniques 
readily available. Thus, if a solid carrier is used, the preparation can 
be tableted, placed in a hard gelatin capsule in powder or pellet form or 
in the form of a troche or lozenge. The amount of solid carrier will vary 
widely but preferably will be from about 25 mg to about 1 gram, wherein 
the amount of inventive compound per dose will vary from about 25 mg to 
about 1 gram for an adult. When a liquid carrier is used, the preparation 
will be in the form of a syrup, emulsion, soft gelatin capsule, sterile 
injectable liquid such as an ampule or nonaqueous liquid suspension. Where 
the inventive composition is in the form of a capsule, any routine 
encapsulation is suitable, for example, using the aforementioned carriers 
in a hard gelatin capsule shell. Where the composition is in the form of a 
soft gelatin shell capsule, any pharmaceutical carrier routinely used for 
preparing dispersions of suspensions may be considered, for example, 
aqueous gums, celluloses, silicates or oils and are incorporated in a soft 
gelatin capsule shell. A syrup formulation will generally consist of a 
suspension or solution of the compound or salt thereof in a liquid carrier 
(e.g., ethanol, polyethylene glycol, coconut oil, glycerine or water) with 
a flavor or coloring agent. 
The amount of inventive compound required for therapeutic effect on topical 
administration will, of course, vary with the compound chosen, the nature 
and severity of the disease and the discretion of the treatment provider. 
Parenteral includes intravenous, intramuscular, subcutaneous, intranasal, 
intrarectal, intravaginal or intraperitoneal administration. Appropriate 
dosage forms for such administration may be prepared by conventional 
techniques. A typical parenteral composition consists of a solution or 
suspension of the inventive compound or a salt thereof in a sterile or 
non-aqueous carrier optionally containing a parenterally acceptable oil, 
for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis 
oil, or sesame oil. The daily dosage for treatment of sepsis or another 
severe inflammatory condition via parenteral administration is suitable 
from about 0.001 mg/kg to about 40 mg/kg, preferably from about 0.01 mg/kg 
to about 20 mg/kg of an inventive compound or a pharmaceutically 
acceptable salt thereof calculated as the free base. 
The inventive compounds may be administered orally. The daily dosage 
regimen for oral administration is suitably from about 0.1 mg/kg to about. 
1000 mg/kg per day. For administration the dosage is suitably form about 
0.001 mg/kg to about 40 mg/kg of the inventive compound or a 
pharmaceutically acceptable salt thereof calculated as the free base. The 
active ingredient may be administered from 1 to 6 times a day, sufficient 
to exhibit activity. 
The inventive compounds may be administered by inhalation (e.g., intranasal 
or oral). Appropriate dosage forms include an aerosol or a metered dose 
inhaler, as prepared by conventional techniques. The daily dosage is 
suitably form about 0.001 mg/kg to about 40 mg/kg of the inventive 
compound or a pharmaceutically acceptable salt thereof calculated as the 
free base. Typical compounds for inhalation are in the form of a solution, 
suspension or emulsion that may be administered as a dry powder or in the 
form of an aerosol using a conventional propellant. 
The following examples, which should not be regarded as limiting in any 
way, illustrate the invention. In these examples PTX means Pentoxifylline. 
EXAMPLE 1 
This example illustrates a method for synthesis of compound no. 1551. The 
synthesis began with a solution of 8-nonene-1-ol (3.52 mmol, 0.5 g) in 30 
ml of dichloromethane. Methanesulfonyl chloride (3.52 mmol, 0.4 g, 270 
.mu.l) was added with stirring at 0.degree. C., followed by an addition of 
triethylamine (5.28 mmol, 0.534 g, 736 .mu.l). The mixture was warmed to 
room temperature over an hour and then was poured into 50 ml of saturated 
aqueous sodium bicarbonate solution. The organic layer was washed with an 
equal volume of brine, dried over magnesium sulfate, filtered and the 
solvent evaporated to give a mesylate, which was taken up in 10 ml of DMSO 
(dimethylsulfoxide). 
A mixture of theobromine (3.52 mmol, 0.63 g), stirring in 20 ml DMSO, was 
added to sodium hydride (3.87 mmol, 93 mg). After 1 hour of vigorous 
stirring, the mesylate in 10 ml of DMSO was added to this viscous mixture. 
The mixture became less viscous as the reaction proceeded. After 54 hours 
of stirring, the mixture was poured into water (50 ml) and extracted with 
diethylether (3.times.50 ml) followed by dichloromethane (4.times.40 ml). 
After the dichloromethane was evaporated, a remaining brown oil residue 
was purified using chromatography on silica with ethylacetate, yielding 
530 mg of 1-(8-nonene)-3,7-dimethylxanthine as an off-white powder (50% 
yield). 
1-(8-nonene)-3,7-dimethylxanthine (380 mg, 1.25 mmol) was dissolved in 1 ml 
water and then 1 ml of concentrated sulfuric acid was added at once. This 
mixture was stirred for 24 hours. The reaction mixture was poured over 50 
ml water and extracted with dichloromethane (3.times.50 ml). The 
dichloromethane extractions were combined and dried over magnesium 
sulfate, and evaporated to yield a viscous oil. Recrystalization from 
minimal dichloromethane/excess diethyl ether yielded 110 mg of 
1-(8-hydroxynonyl)-3,7-dimethylxanthine (0.34 mmol, 27% yield). However, 
this .omega.-1 alcohol preparation also contained a significant 
concentration of a contaminating .omega.-2 alcohol, 
(1-(7-hydroxynonyl)-3,7-dimethylxanthine), which is also a compound within 
the scope of Formula II. 
EXAMPLE 2 
This example illustrates a synthesis procedure for compound no. 1564 (see 
compound names and structures above). The synthesis began with a solution 
of 9-decene-1-ol (3.0 g, 19.2 mmol) in dichloromethane (100 ml) at 
0.degree. C. To this solution was added methanesulfonyl chloride (2.2 g, 
1.5 ml, 19.2 mmol), followed by triethylarnine (2.91 g, 28.8 mmol). After 
stirring for 15 minutes at 0.degree. C., the reaction mixture was allowed 
to warm to room temperature. After 2 hours, the reaction mixture was 
poured into 100 ml of water and extracted with dichloromethane (3.times.60 
ml). The organic portions were combined, dried in sodium sulfate, and 
evaporated to give 9-decene-1-mesylate as a yellow oil (4.52 g, 100% 
yield). The mesylate was used without further purification. 
Theobromine (3.45 g, 19.2 mmol) was added to a suspension of NaH (461 mg, 
19.2 mmol) in DMSO (30 ml). After 15 minutes, 9-decene-1-methanesulfonate 
(2.25 g, 11 mmol) was added and the reaction mixture was stirred for 18 
hours at 25.degree. C., and then at 100.degree. C. for 40 minutes. The 
reaction mixture was poured into 100 ml of water and extracted with 
dichloromethane (3.times.50 ml). The organic portions were combined, 
washed with brine (60 ml), dried with magnesium sulfate, and evaporated to 
provide a white solid. Recrystalization of this solid (in 
dichloromethanelpetroleum ether) provided 3.40 g of a colorless oil, 
1-(9-decenyl)-3,7-dimethylxanthine (56% yield). 
A solution of 1-(9-decenyl)-3,7-dimethylxanthine, prepared according to the 
foregoing procedure (3.2 g, 10.1 mmol), 4-methylmorpholine-N-oxide (1.41 
g, 12 mmol) and OsO.sub.4 (3 drops of a 2.5% solution by weight in tBuOH) 
in acetone (40 ml) and water (10 ml) was stirred for 24 hours. A saturated 
solution of sodium dithionite (5 ml) was added to the reaction mixture 
which was then stirred for 15 minutes. The reaction mixture was extracted 
with 25% EtOH/dichloromethane (4.times.50 ml). The organic layers were 
combined, dried with sodium sulfate and evaporated, leaving a white solid 
which was recrystalized in ethanol, resulting in 3.3 g of compound no. 
1564 (93% yield). 
EXAMPLE 3 
This example illustrates a synthesis for compound no. 1552. The synthesis 
begins with a solution of compound no. 1564 
1-(9,10-dihydroxydecyl-3,7-dimethylxanthine (2.11 g, 6.0 mmol)! from 
Example 2. Compound no. 1564 was stirred with HBr (5.38 ml, 4.85 g of a 
30% solution in acetic acid, 18 mmol) for 90 minutes. The mixture was 
added to a flask containing saturated aqueous sodium bicarbonate solution 
(40 ml) and 50 ml of dichloromethane. After 10 minutes of vigorous 
stirring, the layers were separated and the aqueous layers washed with 
dichloromethane (2.times.50 ml). The organic portions were combined, dried 
with sodium sulfate, and evaporated to give 
l-(9'-acetoxy-10'-bromodecyl)-3,7-dimethylxanthine as a yellow oil (2.72 
g, 100% yield). Without further purification, the oil was taken up in 
methanol (30 ml), and treated with a solution of sodium methoxide 
(prepared from 151 mg, 6.6 mmol sodium and 6 ml methanol). After 30 
minutes, the reaction mixture was added to water (30 ml) and extracted 
with dichloromethane (3.times.50 ml). The organic layers were combined and 
dried with sodium sulfate to give an off-white solid which was 
recrystalized (in dichloromethane/petroleum ether) to yield 
1-(9,10-oxidodecyl)-3.7-dimethylxanthine racemic mixture. 
A solution of 1-(9,10-oxidodecyl)-3,7-dimethylxanthine (200 mg, 0.6 mmol) 
and sodium borohydride (61 mg, 1.6 mmol) was stirred in ethanol (10 ml) at 
80.degree. C. for 4 hours. After cooling, the reaction mixture was poured 
into 10 ml of saturated aqueous ammonium chloride. Water (10 ml) was added 
to dissolve any solids that were formed and the mixture was extracted with 
dichloromethane (3.times.50 ml). The organic extracts were combined, dried 
with sodium sulfate, and evaporated to an off-white solid. The solid was 
recrystalized (in dichloromethane/petroleum ether), resulting in 180 mg of 
a racemic mixture of compound no. 1552, a white solid (89% yield). 
EXAMPLE 4 
This example illustrates a synthesis procedure for compound no. 1561 
(chemical name and structure above). The synthesis began by adding a 
solution of 8-nonene-1-ol (1.50 g, 10.5 mmol) in dichloromethane (100 ml) 
at 0.degree. C. to methanesulfonyl chloride (1.20 g, 813 .mu.l, 10.5 
mmol), followed by triethylamine (1.59 g, 15.8 mmol). After stirring for 1 
hour at 0.degree. C., the reaction mixture was allowed to warm to room 
temperature. The reaction mixture was poured into 100 ml of water and 
extracted with dichloromethane (3.times.50 ml). The organic portions were 
combined, dried with sodium sulfate, and evaporated, resulting in 2.25 g 
of 9-methanesulfonyl-1-nonene, a yellow oil (97% yield), which was used 
without further purification. 
Theobromine (1.98 g, 11 mmol) was added to a suspension of NaH (600 mg of a 
50% mineral oil slurry, 12 mmol) in DMSO (15 ml). After 15 minutes, 
9-methanesulfonyl-1-nonene, prepared according to the foregoing procedure 
(2.25 g, 11 mmol), was added and the reaction mixture stirred for 6 days 
at 25.degree. C. The reaction mixture was poured into 60 ml of water and 
extracted with dichloromethane (3.times.50 ml). The organic portions were 
combined, dried with magnesium sulfate, and evaporated to give a dark oil. 
Chromatography over silica gel using an ethyl acetate eluant produced 810 
mg of 1-(8-nonenyl)-3,7-dimethylxahthine, a colorless oil (26% yield). 
A solution of 1-(8-nonenyl)-3,7-dimethylxanthine (810 mg, 2.9 mmol), 
4-methylmorpholine-N-oxide (340 mg, 2.9 mmol) and OsO.sub.4 (3 drops of a 
2.5% solution by weight in tBuOH) in acetone (20 ml) and water (20 ml) was 
stirred for 24 hours. A saturated solution of sodium dithionite (5 ml) was 
added to the reaction mixture, which was subsequently stirred for 15 
minutes. The reaction mixture was extracted with 25% EtOH/dichloromethane 
(4.times.50 ml). The organic layers were combined, dried with sodium 
sulfate and evaporated, leaving a white solid. Recrystalized of the white 
solid in ethanol/chloroform resulted in 490 mg of compound no. 1561 (54% 
yield). 
EXAMPLE 5 
This example illustrates another method for synthesizing compound no. 1551 
(in addition to the method described in Example 1). 
1-(8,9-dihydroxynonyl)-3,7-dimethylxanthine (compound no. 1561, 428 mg, 
1.3 mmol) was stirred with HBr (777 .mu.l, 1.05 g of a 30% solution in 
acetic acid, 3.9 mmol) for 90 minutes. The mixture was then added to a 
flask containing aqueous sodium bicarbonate solution (10 ml, 1.35 g) and 
dichloromethane (10 ml) and stirred vigorously for 10 minutes. The layers 
were separated and the aqueous portion was washed with dichloromethane 
(3.times.15 ml). The organic portions were combined, dried with sodium 
sulfate and evaporated to give 
1-(8-acetoxy-9-bromononyl)-3,7-dimethylxanthine as a yellow oil (550 mg, 
96% yield). Without further purification, the oil was taken up in methanol 
(5 ml) and treated with a solution of sodium methoxide (prepared from 33 
mg, 1.4 mmol sodium and 1.4 ml methanol). After 30 minutes, the reaction 
mixture was added to water (30 ml) and extracted with dichloromethane 
(3.times.40 ml). The organic portions were combined and dried to give an 
off-white solid. Recrystalization of the remaining solid was recrystalized 
in dichloromethane/petroleum ether, resulting in 380 mg of 
1-(8,9-oxidononyl)-3,7-dimethylxanthine (91% yield). 100 mg of 
1-(8,9-oxidononyl)-3,7-dimethylxanthine (0.3 mmol) was dissolved in 
methanol (20 ml). Palladium catalyst (10% on carbon, 100 mg) was added and 
the slurry was placed under hydrogen (50-55 psi) on a Parr reactor for 16 
hours. The slurry was filtered through celite, evaporated to a yellow oil 
and purified using chromatography over silica gel using 10% ethanol/ethyl 
acetate eluant, producing 53 mg of compound no. 1551, a white solid (55% 
yield). 
EXAMPLE 6 
This example illustrates a synthesis for compound no. 1559. A mixture of 
theobromine (1.0 g, 5.5 mmol) and 50% NaH in oil (264 mg, 5.5 mmol) in 
DMSO (35 ml) was stirred for 5 minutes and then 10-bromodecane-1-ol (1.3 
g, 5.5 mmol) was added and stirred for 14 hours. The solution was treated 
with water (100 ml) and extracted with ether (2.times.50 ml). The 
heterogeneous aqueous phase was extracted with dichloromethane (3.times.30 
ml). The combined organic layers were washed with water (2.times.100 ml), 
dried with magnesium sulfate, and the dichloromethane was evaporated under 
vacuum, resulting in 1.6 g of compound no. 1559, a white powder (87% 
yield). 
EXAMPLE 7 
This example illustrates a synthesis of compound no. 1545 (see above for 
chemical name and structure). Sodium hydride (95%; 840 mg, 35 mmol) was 
added to a solution of theobromine (2.88 g, 16 mmol) in dimethylsulfoxide 
(50 ml). After 20 minutes of stirring, 7-bromoheptanol (2.92 g, 15 mmol) 
was added. The reaction mixture was warmed to 60.degree. C. and stirred 
for 16 hours at 60.degree. C. The reaction was poured into a separatory 
funnel containing 100 ml of saturated NH.sub.4 Cl solution and extracted 
with dichloromethane (3.times.100 ml). The organic portions were combined, 
washed with water (2.times.100 ml) and brine (100 ml), dried over 
anhydrous magnesium sulfate and concentrated under reduced pressure. The 
resulting crude product was purified by flash chromatography over silica 
gel using an ethyl acetate eluent, producing 2.58 g of compound no. 1545 
(58% yield). 
EXAMPLE 8 
This example illustrates a synthesis for compound no. 1592. Sodium 
hydride(95%, 1.26 g, 50 mmol) was added to a solution of theobromine (7.2 
g, 40 mmol) in dimethylsulfoxide (300 ml). After 20 minutes of stirring, 
undecenylmesylate (7.95 g, 30 mmol) was added and the resulting mixture 
stirred for 12 hours at room temperature. The reaction was warmed to 
70.degree.-80.degree. C. and stirred for 4 hours. The reaction mixture was 
then poured into a separatory funnel containing 1 L of water and extracted 
with dichloromethane (5.times.200 ml). The organic extracts were combined, 
washed with water (100 ml) and brine (100 ml), dried over anhydrous 
magnesium sulfate and concentrated under reduced pressure. The crude 
product obtained was further purified by flash chromatography over silica 
gel using 20% hexane and dichloromethane eluent, producing 4.6 g of 
1-(10-undecenyl)-3,7-dimethylxanthine (46.3% yield). 
A solution of 1-(10-undecenyl)-3,7-dimethylxanthine, prepared in the 
foregoing procedure (4.3 g, 13 mmol), 4-methylmorpholine-N-oxide (1.942 g, 
16.6 mmol), and potassium osmate dihydrate (9.5 mg; 0.026 mmol) in acetone 
(45 ml) and water (10 ml) was stirred for 6 hours. A solution of 20% 
aqueous sodium sulphite (12 ml) was added and the resulting mixture 
stirred for 30 minutes. The reaction mixture was extracted with 25% 
ethanol/dichloromethane (4.times.100 ml). The combined organic extracts 
were dried over anhydrous magnesium sulfate, concentrated under reduced 
pressure and purified by flash chromatography over silica gel using a 
methanol (5%)/dichloromethane eluant, producing 3.6 g of compound no. 1592 
(76% yield). 
EXAMPLE 9 
This example illustrates a synthesis for compound no. 1587 (see above for 
chemical name and structure). Compound no. 1592 
(10,11-dihydroxyundecanyl)-3,7-dimethylxanthine! as prepared above (3.6 
g, 10 mmol) was stirred with hydrogen bromide (6.2 ml, 8.4 g of a 30% 
solution in acetic acid, 31.1 mmol) for 90 minutes. The mixture was then 
added to a flask containing 100 ml aqueous sodium bicarbonate solution and 
75 ml dichloromethane. After 10 minutes of vigorous stirring the layers 
were separated and the aqueous portion washed with dichloromethane 
(3.times.75 ml). The organic portions were combined, dried over magnesium 
sulfate, and evaporated, yielding 3.6 g of 
1-(10-acetoxy-11-bromoundecanyl)-3,7-dimethylxanthine. Without further 
purification, the bromoacetate was taken up in methanol (25 ml) and 
treated with a solution of sodium methoxide (prepared from 0.28 g, 12.2 
mmol sodium, and 25 ml methanol). After 30 minutes, most of the solvent 
was removed under reduced pressure and the residue extracted with 
dichloromethane (3.times.75 ml). The organic portions were combined, dried 
over magnesium sulfate and concentrated under reduced pressure, resulting 
in an off-white solid. The solid was purified by column chromatography 
over silica gel using dichloromethane/(3%) methanol eluant, producing 2.0 
g of 1-(10,11-oxidoundecanyl)-3,7-dimethylxanthine (57.5% yield) 
348 mg (1 mmol) 1-(10,11-oxidoundecanyl)-3,7-dimethylxanthine, prepared 
according to the forgoing procedure was added to a suspension of sodium 
borohydride (115.6 mg; 3 mmol) in 10 ml of ethanol. The reaction was 
warmed to 60.degree. C. and stirred overnight. Most of the ethanol was 
removed under reduced pressure, at standard ambient temperature. 20 ml of 
NH4Cl solution was added and extracted with ethyl acetate (3.times.75 ml). 
The combined organic extracts were dried over anhydrous magnesium sulfate 
and concentrated under reduced pressure. The resulting crude product was 
purified by flash chromatography over silica gel using 3% 
methanol/dichloromethane eluant, producing 237 mg of compound no. 1587 
(68% yield). 
EXAMPLE 10 
This example illustrates a process for making compound no. 1596. Sodium 
hydride(95%, 631 mg, 25 mmol) was added to a solution of theobromine (4.14 
g, 23 mmol) in dimethylsulfoxide (75 ml). After 20 minutes of stirring, 
(R)(-)citronellyl bromide (5.0 g, 22.8 mmol) was added. After 16 hours of 
stirring the resulting mixture at room temperature, the reaction was 
poured into a separatory funnel containing 500 ml of water and extracted 
with dichloromethane (3.times.100 ml). The organic extracts were combined, 
washed with water (100 ml) and brine (100 ml), dried over anhydrous 
magnesium sulfate and concentrated under reduced pressure. The resulting 
crude product was purified by flash chromatography over silica gel using 
30% petroleum ether/ethyl acetate eluant, producing 5.9 g of compound no. 
1596, a yellowish oil (81.5% yield). 
EXAMPLE 11 
This example illustrates a synthesis of compound no. 1824. Sodium hydride 
(288 mg, 12 mmol) was added to a solution of N-methylhydrouracil (1.54 g, 
12 mmol) and 1-bromo-10-undecene (2.33 g, 10 mmol) in 20 ml of dimethyl 
sulfoxide at room temperature and stirred for 12 hours. The reaction 
mixture was then quenched with water (80 ml) and extracted with 
dichloromethane (3.times.100 ml). The combined organic extract was washed 
with saturated aqueous saturated salt solution solution (50 ml) , dried 
over anhydrous magnesium sulfate and concentrated under reduced pressure. 
The resulting crude product was purified by flash chromatography over 
silica gel using 20% acetone/hexane eluant, producing 2.04 
g3-(10-undecenyl)-1-methylhydrouracil (61.8% yield). 
A solution of 0.28 g (1 mmol) 3-(10-undecenyl)-1-methylhydrouracil, 
prepared as above, and 0.517 g (1.5 mmol) m-chloroperoxybenzoic acid (50% 
by wt.) in dichloromethane (6 ml) was stirred for 5 hours. The reaction 
mixture was diluted with 75 ml of dichloromethane and successively washed 
with 20% aqueous sodium sulphite solution(25 ml), saturated sodium 
bicarbonate solution (25 ml), water (25 ml) and aqueous saturated salt 
solution (25 ml), dried over anhydrous magnesium sulfate and concentrated 
under reduced pressure. The resulting crude product was purified by flash 
chromatography over silica gel using 20% acetone/hexane eluant, producing 
0.22 g of 3-(10,11-oxidoundecanyl)-1-methylhydrouracil (74.3% yield). 
600 mg (2.03 mmol) of 3-(10,11-oxidoundecanyl)-1-methylhydrouracil was 
added to a suspension of sodium borohydride (230 mg, 6.1 mmol) in 10 ml of 
ethanol. The reaction was warmed to 60.degree. C. and stirred overnight. 
Most of the ethanol was removed under reduced pressure, at standard 
ambient temperature. Ammonium chloride solution (20 ml) was added and the 
reaction mixture extracted with ethyl acetate (3.times.75 ml). The 
combined organic extracts were dried over anhydrous magnesium sulfate and 
concentrated under reduced pressure. The resulting crude product was 
further purified by flash chromatography over silica gel using 30% 
acetone/hexane eluant, producing 594 mg of compound no. 1824 (92.8% 
yield). 
EXAMPLE 12 
This example illustrates the effect of compounds nos. 1551 and 1559 as an 
immune modulator. FIG. 1 shows a mixed lymphocyte reaction of PTX and two 
inventive compounds nos. 1551 and 1559 (see above for chemical names and 
structures). The mixed lymphocyte reaction shows a proliferative response 
of PBMC (peripheral blood mononuclear cells) to allogeneic stimulation 
determined in a two-way mixed lymphocyte reaction, described above. Each 
of the inventive compounds tested was more effective and more potent than 
PTX in this immune modulating activity assay procedure. 
EXAMPLE 13 
This example illustrates a comparison of compounds nos. 1551 and 1559 on 
PDGF-induced (platelet derived growth factor) proliferation of human 
stromal cells. Human stromal cells were starved in serum-free media for 24 
hours and then stimulated with 50 ng/ml of PDGF-BB. The drugs were added 
at various indicated concentrations one hour prior to PDGF stimulation. 
Tritiated thymidine was added at the time of PDGF stimulation. The cells 
were harvested and counted by liquid scintillation 24 hours after 
stimulation with PDGF. As shown in FIG. 2, both compound nos. 1551 and 
1559 inhibited PDGF-induced stimulation. Background counts (i.e., starved 
cells) were approximately 10% of control levels. 
EXAMPLE 14 
This example provide data from an experiment measuring compound no. 1559 
cytotoxicity on LD-2 cells, a human malignant melanoma cell line. The 
cells were treated with various concentrations of compound no. 1559 and 
later stained for cell viability with a fluorescence stain (BCECF) and 
analyzed using a Milipore fluorescence plate reader. As shown in FIG. 3, 
compound no. 1559 is cytotoxic at higher concentrations, and thus shows 
antitumor activity. 
EXAMPLE 15 
This example provides data from an experiment measuring compound no. 1559 
cytotoxicity on NIH-3T3 cells and their Ras transformed counterpart, 
NIH-3T3 Ras cells. The cells were treated with various concentrations of 
compound no. 1559 and later stained for cell viability with a fluorescence 
stain (BCECF) and analyzed using a Milipore fluorescence plate reader. As 
shown in FIG. 4, compound no. 1559 is cytotoxic at higher concentrations, 
and thus shows antitumor activity. 
EXAMPLE 16 
This example illustrates the effect of compound no. 1559 on inhibiting cell 
surface expression of VCAM in human umbilical vein endothelial cells 
(HUEC). The HUVEC cells were stimulated with 20 ng/ml TNF-a for 20 hours 
and then stained for immunofluorescence using a monoclonal antibody 
recognizing VCAM, followed by a goat anti-mouse antibody conjugated to 
phycoerythrin. The cells were analyzed for antibody binding using flow 
cytometry. FIG. 5 shows the flow cytometric frequency histograms plotting 
cell number versus relative fluorescence intensity. The top left histogram 
is non-TNF induced expression of VCAM (% of cells in gate A is 0.4%). The 
top right shows cells treated with TNF (% of cells in gate B is 34.5%). 
The lower left shows cells treated with compound no. 1559 (0.25 mM), one 
hour prior to TNF addition (% of cells in gate C is 24%). In the lower 
left, cells treated have been treated with PTX for comparison (% of cells 
in gate D is 36.8%). 
EXAMPLE 17 
This example illustrates the effect of compound no. 1559 on inhibiting cell 
surface expression of VCAM in HUVEC cells. The cells were stimulated with 
TNF-A (20 ng/ml) for 20 hours and then stained for immunofluorescence 
using a monoclonal antibody recognizing VCAM, followed by a goat 
anti-mouse antibody conjugated to phycoerythrin. The cells were analyzed 
for antibody binding using flow cytometry. FIG. 6 shows an analysis of 
mean fluorescence intensity of cells analyzed by flow cytometry. The mean 
fluorescence levels were decreased by compound no; 1559 treatment (1.7 
fold decrease) when compared with control levels (TNF treatment, no drug). 
EXAMPLE 18 
This example illustrates a comparison of MLR (mixed lymphocyte reaction) 
data for inventive comopounds of varying chain lengths to show a 
comparison of biological activity as a function of chain length (the 
number of carbon atoms between the hydroxyl carbon and the core moiety. A 
mixed lymphocyte reaction was run with a series of inventive compounds and 
other compounds. IC50 values for each compound tested was determined and 
the results listed in Table I below: 
TABLE I 
______________________________________ 
Cpnd Chain Alcohol 
no. Length Mean IC50 (.mu.M) 
Formula II 
type 
______________________________________ 
1551 9 120 Y secondary 
1559 10 150 Y primary 
1561 9 185 Y diol 
1564 10 210 Y diol 
1501 6 &gt;500 N primary 
1502 6 &gt;500 N diol 
1536 8 250 N secondary 
1538 8 &gt;500 N diol 
1540 3 &gt;500 N diol 
1542 5 &gt;500 N secondary 
1545 7 300 N primary 
1546 8 320 N primary 
1556 6 &gt;500 N primary 
______________________________________ 
Accordingly, these data show the importance of chain length for immune 
modulating activity in the MLR assay. 
EXAMPLE 19 
This example illustrates dose response curves used to generate 50% 
inhibition concentrations (ICS50) of inventive compounds nos. 1551 and 
1564 for murine thymocyte proliferation, co-stimulated by Concanavalin A 
(ConA) and interleukin-2 alpha (IL-2). ConA, used to activate CD3, along 
with IL-2 co-stimulation, induces T-cell proliferation and 
differentiation. Thymuses, obtained from normal, female Balb/C mice, were 
dissociated and plated into 96-well plates at a density of 
2.times.10.sup.5 cells/well. ConA (0.25 mg/ml) were added to the wells. 
The cells were incubated for 4 days at 37.degree. C. On day 4, the cells 
were pulsed with tritiated thymidine and incubated for an additional 4 
hours. The amount of tritiated thymidine dye incorporated by the harvested 
cells was determined in a liquid scintillation counter. Drug doses (shown 
in FIGS. 7A and 7B) were added two hours prior to CoaA and IL-2 
activation. Background counts were less than 200 cpm. Both the inventive 
compounds inhibit thymocyte proliferation and activation and reported IC50 
values for compounds nos. 1551 and 1564 are 28 and 49 .mu.M. 
EXAMPLE 20 
This example illustrates inhibitive and cytotoxic effects of inventive 
compounds nos. 2556 and 3504 on Balb/3T3 cell proliferation in response to 
PDGF-BB stimulation. 
The inventive compounds possess inhibitory effects on PDGF-induced 
proliferation of Balb/3T3 cells. Balb/3T3 cells respond vigorously to PDGF 
stimulation, and are useful in vitro models for further study of 
PDGF-induced proliferation. Disregulated PDGF-proliferative response has 
been linked to a variety of diseases, including, e.g., restenosis, 
atherosclerosis, fibrosis, and tumor cell angiogenesis. Cells were plated 
in low serum-containing medium for 24 hours prior to stimulation with 
various concentrations (as reported in FIGS. 8A and 9A) of inventive 
compounds nos.2556 and 3504 (FIGS. 8A and 9A, respectively). PDGF-BB was 
added at a constant concentration in each assay. Tritiated thymidine was 
added and cells harvested for scintillation counting 24 hours later. FIGS. 
8A and 9A are dose response curves from this assay for compound nos. 2556 
and 3504, respectively. FIGS. 8B and 9B report cytotoxicity results for 
compounds nos. 2556 and 3504 in the Balb/3T3 cell line. Both inventive 
compounds tested inhibited PDGF-induced proliferation in Balb/3T3 cells.