WO1994010161A1 - Derives de 1,3-dioxane a activite inhibant la proteine-kinase c - Google Patents

Derives de 1,3-dioxane a activite inhibant la proteine-kinase c Download PDF

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WO1994010161A1
WO1994010161A1 PCT/US1992/009048 US9209048W WO9410161A1 WO 1994010161 A1 WO1994010161 A1 WO 1994010161A1 US 9209048 W US9209048 W US 9209048W WO 9410161 A1 WO9410161 A1 WO 9410161A1
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alkyl
carbon atoms
independently
alkynyl
alkenyl
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PCT/US1992/009048
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English (en)
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Jack B. Jiang
Mary George Johnson
Jeffrey Nichols
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Sphinx Pharmaceuticals Corporation
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Priority to PCT/US1992/009048 priority Critical patent/WO1994010161A1/fr
Priority to AU29244/92A priority patent/AU2924492A/en
Publication of WO1994010161A1 publication Critical patent/WO1994010161A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/04—1,3-Dioxanes; Hydrogenated 1,3-dioxanes
    • C07D319/06—1,3-Dioxanes; Hydrogenated 1,3-dioxanes not condensed with other rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin

Definitions

  • the present invention relates to diagnosis and treatment of inflammatory, cardiovascular and neoplastic diseases. More particularly, the present invention relates to 1,3-dioxane derivatives for inhibiting activity of the enzyme protein kinase C family of enzymes in mammals.
  • PLC protein kinase C
  • PLC protein kinase C
  • Protein kinase C is a family of calcium- and phospholipid-dependent serine/threonine-specific protein kinases which play an important role in cellular growth control, regulation, and differentiation. Protein kinase C is also fundamental to the processes involved in tumorigenicity, since it is the major high-affinity receptor for several classes of tumor promoters as well as for endogenous cellular diacylglycerols.
  • Protein kinase C is activated by diacylglycerol (DAG) , a neutral lipid, and when activated will transfer the ⁇ -phosphate of MgATP to a serine or threonine residue on a substrate protein.
  • DAG diacylglycerol
  • protein kinase C Since the activation of protein kinase C has been implicated in several human disease processes, including cancer tumors, inflammation, and reperfusion injury, inhibition of protein kinase C should be of great therapeutic value in treating these conditions.
  • Certain protein kinase C inhibitors have been reported to potentiate the antitumor activity of cis-platin both in vitro and in vivo . See Grunicke et al.. Adv. Enzyme Regul . 28 : 201, 1989; and German Offenlegungsschrift DE 3827974.
  • protein kinase C would be a potential target for therapeutic design because of its central role in cell growth. See Tritton, T.R. and Hickman, J.A. Cancer Cells 2 : 95-102, 1990.
  • inflammation and reperfusion injury particularly pertaining to cardiac injury, are common conditions for which there exists no definitive treatment despite extensive research, and appropriate treatments for these conditions are needed.
  • Certain protein kinase C inhibitors have been demonstrated to block platelet aggregation and release of neutrophil activating agents such as platelet activating factor, PAF. See Schachtele et al., Biochem . Biophy. Res . Commun . 151 : 542, 1988; Hannun et al., J. Biol. Chem . 262 : 13620, 1987 and Yamada et al. , Biochem . Pharmacol . 37 : 1161, 1988. Protein kinase C inhibitors have also been shown to inhibit neutrophil activation, and chemotactic migration. See Mclntyre et al., J. Biol Chem .
  • inhibitors of protein kinase C have the potential for blocking all three of the most significant mechanisms of pathogenesis associated with myocardial reperfusion injury, and should thus have a decided therapeutic advantage. Additionally, the inhibitory effect of protein kinase C inhibitors on keratinocytes, and on the oxidative burst in neutrophils will lead to an anti- inflammatory effect.
  • German Offenlegungsschrift DE 3827974 Al discloses therapeutic preparations comprising a protein kinase C inhibitor in combination with a lipid, a lipid analog, a cytostatic agent or phospholipase inhibitor useful for cancer therapy.
  • a protein kinase C inhibitor in this publication are 1,3-dioxanes.
  • 1,3-dioxanes have been reported for antifungal, antibacterial and antiviral uses (Houlihan, U.S. patent 3,621,033 issued Nov. 16, 1971, Meiser, et al., U.S. patent 2,882,275 issued April 14, 1959, and Moore, U.S. patent 2,568,555 issued Sept. 18, 1951), and agricultural uses (Hitz, et al., U.S. patent 3,459,771 issued Aug. 5, 1969).
  • 1,3 dioxanes have been reported in research on sphingolipid synthesis and biochemistry. See Stoffel et al., Hoppe- Seyler's Z. Physiol. Chem., 348: 1561-69, 1967; Gigg and Warren, J.
  • Formula I wherein is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms; R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms; and R 4 and R- are independently H, R 6 imino, or amidino, at least one of R 4 and R j being R 6 imino, or amidino, and pharmaceutically acceptable salts thereof.
  • the invention also provides compounds of formula I wherein R 1 is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms; R 2 is alkyl having from 1 to 5 carbon atoms and R 3 is H, or R 3 is alkyl having from 1 to 5 carbon atoms and R 2 is H; R 4 and H- are independently H, R 6 imino, or amidino, and R 6 is independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, and pharmaceutically acceptable salts thereof.
  • the present invention further provides compounds of formula I wherein R, is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms; R 2 is alkyl having from 7 to 20 carbon atoms and R 3 is H, or R 3 is alkyl having from 7 to 20 carbon atoms and R 2 is H; R 4 and g are independently H, R 6 imino, or amidino, and R 6 is independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, and pharmaceutically acceptable salts thereof.
  • the invention additionally provides compounds of formula I wherein R., is alkyl having from 2 to about 20 carbon atoms, or alkenyl or alkynyl having from 2 to 14 carbon atoms; R 2 is phenyl; R 3 and R 6 are independently H, or alkyl having from 1 to about 20 carbon atoms; and R 4 and R j are independently H, R 6 imino, or amidino, and pharmaceutically acceptable salts thereof.
  • the compounds of the invention inhibit protein kinase C and exert anti-inflammatory, anti-cancer, and reperfusion injury protection effects through their anti- proliferative and anti-inflammatory activities in human neutrophils and tumor cells. Also within the scope of the invention are the pharmaceutically acceptable salts and the optically active stereoisomers of the compounds of the invention.
  • the present invention also provides novel methods useful for treating conditions related to, or affected by inhibition of protein kinase C activity, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury.
  • a compound having the formula (formula I) having the formula (formula I)
  • R 1 is alkyl, alkenyl and alkynyl having from 2 to about 20 carbon atoms
  • R 4 and j are independently H, R 6 imino, or amidino is administered to the mammal or cells in amounts effective to inhibit protein kinase C, or ameliorate the condition for which it is administered.
  • compositions comprising a pharmaceutically acceptable carrier or diluent and a compound of formula I wherein R 1 is alkyl, alkenyl and alkynyl having from 2 to about 20 carbon atoms; R 2 , R 3 and R ⁇ are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms; and R 4 and R 5 are independently H, R 6 imino, or amidino, and pharmaceutically acceptable salts thereof.
  • the present invention provides 1,3-dioxanes, and their pharmaceutically acceptable salts that have protein kinase C inhibiting activity, and exert anti-inflammatory, anti-cancer, and reperfusion injury protection effects through their anti-proliferative and anti-inflammatory activities in human neutrophils and tumor cells.
  • the compounds and pharmaceutical compositions of the invention are useful for treating conditions related to, or affected by inhibitions of protein kinase C activity, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury.
  • the compounds useful in the methods of the invention are selective for protein kinase C and have no effect on cyclic AMP (cAMP) dependent protein kinase activity.
  • cAMP cyclic AMP
  • the compounds useful in the invention should thus have no effect on the metabolic pathways associated with stimulation of protein kinase by cAMP.
  • the compounds useful in the invention not only inhibit tumor cell proliferation but are not cross-resistant to the multi-drug-resistant family of agents such as adriamycin.
  • the present invention provides 1,3 dioxanes having the following formula (formula I) :
  • R 1 is preferably alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms, more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms;
  • R 2 , R 3 and R 6 are preferably independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H or alkyl having from 1 to 5 carbon atoms, most preferably independently H, methyl or ethyl; and
  • R 4 and R g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, at least one of R 4 and R g being R 6 imino, or amidino, and pharmaceutically acceptable salts thereof.
  • the invention also provides other 1,3-dioxanes having formula I wherein R, is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms, more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 is alkyl having from 1 to 5 carbon atoms and R 3 is H, or R 3 is alkyl having from 1 to 5 carbon atoms and R 2 is H; R 4 and R g are independently H, R 6 imino, or amidino, and R 6 is independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, and pharmaceutically acceptable salts thereof.
  • a preferred compound of the invention has the structure of formula I wherein R, is alkyl having 15 carbon atoms, R 2 is methyl,R 3 is H, R 4 is H and R g is H.
  • the present invention further provides 1,3-dioxanes having the structure of formula I wherein R, is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms, more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 is alkyl having from 7 to 20 carbon atoms and R 3 is H, or R 3 is alkyl having from 7 to 20 carbon atoms and R 2 is H; R 4 and R g are independently H, R 6 imino, or amidino, and R 6 is H, phenyl or alkyl having from 1 to about 20 carbon atoms, and pharmaceutically acceptable salts thereof.
  • R is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms, more preferably alkyl, alkenyl or alkynyl having from about
  • the invention additionally provides 1,3-dioxanes having the structure of formula I wherein R, is alkyl having from 2 to about 20 carbon atoms, or alkenyl or alkynyl having from 2 to 14 carbon atoms; R 2 is phenyl; R 3 and R 6 are independently H, or alkyl having from 1 to about 20 carbon atoms; and R 4 and R g are independently H, R 6 imino, or amidino, and pharmaceutically acceptable salts thereof.
  • compositions of the invention comprise a pharmaceutically acceptable carrier or diluent and a compound of formula I wherein R 1 is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms, more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H, phenyl or alkyl having from 1 to about 5 carbon atoms, most preferably independently H, methyl or ethyl; and R 4 and g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, or pharmaceutically acceptable salt thereof.
  • R 1 is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms
  • the present invention thus provides methods for inhibiting protein kinase C which comprise contacting protein kinase C with an inhibitory amount of a compound having the formula
  • R 1 is alkyl, alkenyl or alkynyl having from 2 to about 20 carbon atoms, more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms;
  • R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H, phenyl or alkyl having from 1 to about 5 carbon atoms, most preferably independently H, methyl or ethyl; and
  • R 4 and R g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention.
  • Another aspect of the invention provides methods of inhibiting an oxidative burst in neutrophils which comprises contacting a neutrophil with an amount of a compound having the structure of formula I wherein R 1 is alkyl, alkenyl and alkynyl having from 2 to about 20 carbon atoms more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H, phenyl or alkyl having from l to about 5 carbon atoms, most preferably independently H, methyl or ethyl; and R 4 and R g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention effective to inhibit
  • a further aspect of the invention provides methods for treating inflammation which comprises administering to a mammal suffering from inflammation an amount of a compound having the structure of formula I wherein R, is alkyl, alkenyl and alkynyl having from 2 to about 20 carbon atoms more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H, phenyl or alkyl having from 1 to about 5 carbon atoms, most preferably independently H, methyl or ethyl; and R 4 and g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention effective to inhibit inflammation, or administering to the ma
  • Another aspect of the invention provides a method for inhibiting growth of mammalian tumor cells which comprises contacting a mammalian tumor cell with a protein kinase C inhibitory concentration of a compound having the structure of formula I wherein R 1 is alkyl, alkenyl and alkynyl having from 2 to about 20 carbon atoms more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H, phenyl or alkyl having from 1 to about 5 carbon atoms, most preferably independently H, methyl or ethyl; and R 4 and R g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, or pharmaceutically acceptable salt thereof
  • Yet another aspect of the invention provides methods treating mammalian tumors which comprises administering to a mammal having a tumor a protein kinase C inhibitory concentration of a compound having the structure of formula I wherein R 1 is alkyl, alkenyl and alkynyl having from 2 to about 20 carbon atoms more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H, phenyl or alkyl having from 1 to about 5 carbon atoms, most preferably independently H, methyl or ethyl; and R 4 and g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, or pharmaceutically acceptable salt thereof, or a pharmaceutical
  • Still another aspect of the invention provides methods of inhibiting keratinocyte proliferation comprising administering to a keratinocyte a protein kinase C inhibitory amount of a compound having the structure of formula I wherein R 1 is alkyl, alkenyl and alkynyl having from 2 to about 20 carbon atoms more preferably alkyl, alkenyl or alkynyl having from about 10 to about 20 carbon atoms, most preferably alkyl, alkenyl or alkynyl having from about 15 to about 20 carbon atoms; R 2 , R 3 and R 6 are independently H, phenyl or alkyl having from 1 to about 20 carbon atoms, more preferably independently H, phenyl or alkyl having from 1 to about 5 carbon atoms, most preferably independently H, methyl or ethyl; and R 4 and R g are independently H, R 6 imino, or amidino, more preferably independently H or R 6 imino, or pharmaceutically acceptable salt thereof, or a
  • the compounds and pharmaceutical compositions of the invention may be administered by any method that produces contact of the active ingredient with the agent's site of action in the body of a mammal, or in the body fluid or tissue including but not limited to oral, topical, hypodermal, intramuscular, intravenous, and intraparenteral.
  • the compounds may be administered singly, or in combination with other compounds of the invention, other pharmaceutical compounds, such as chemotherapeutic compounds, or in conjunction with therapies, such as radiation treatment.
  • 1,3 dioxane derivatives are preferably administered with a pharmaceutically acceptable carrier selected on the basis of the selected route of administration and standard pharmaceutical practice.
  • the compounds are administered to mammals, preferably humans, in therapeutically effective amounts which are effective to inhibit protein kinase C, or to inhibit tumor cell growth, inhibit inflammation of tissue, inhibit keratinocyte proliferation, inhibit oxidative burst from neutrophils or inhibit platelet aggregation.
  • the dosage administered in any particular instance will depend upon factors such as the pharmacodynamic characteristics of the particular compound, its mode and route of administration, the age, health, and weight of the recipient, the nature and extent of symptoms, kind of concurrent treatment, freguency of treatment, and the effect desired.
  • compositions of the invention are also within the scope of the invention.
  • Such pharmaceutically acceptable salts useful in the invention include hydrochloride, hydrobromide, succinate, fumarate, oxalate, methanesulfonate, sulfate, maleate, malonate, acetate or lactate. It is contemplated that the daily dosage of the compounds will be in the range of from about 0.1 to about 40 mg per kg of body weight, preferably from about 1 to about 20 mg per kg body weight.
  • the pharmaceutical compositions of the invention may be administered in any dosage form, including a single dosage, divided dosages, or in sustained release form. Persons of ordinary skill will be able to determine dosage forms and amounts with only routine experimentation based upon the considerations of the invention. Isomers of the compounds and pharmaceutical compositions, particularly optically active stereoisomers, are also within the scope of the present invention.
  • compositions of the invention may also be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. They may also be administered parenterally in sterile liquid dosage forms or topically in a carrier.
  • the pharmaceutical compositions of the invention may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Remington 's Pharmaceutical Sciences , A. Osol, Mack Publishing Company, Easton, Pennsylvania.
  • the compounds useful in the invention may be mixed with powdered carriers, such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, and stearic acid for insertion into gelatin capsules, or for forming into tablets.
  • powdered carriers such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, and stearic acid for insertion into gelatin capsules, or for forming into tablets.
  • Both tablets and capsules may be manufactured as sustained release products for continuous release of medication over a period of hours.
  • Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere or enteric coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration may contain coloring and flavoring to increase patient acceptance, in addition to a pharmaceutically acceptable diluent such as water, buffer or saline solution.
  • a pharmaceutically acceptable diluent such as water, buffer or saline solution.
  • compounds useful in the invention may be mixed with a suitable carrier or diluent such as water, a oil, saline solution, aqueous dextrose (glucose) , and related sugar solutions, and glycols such as propylene glycol or polyethylene glycols.
  • Solutions for parenteral administration contain preferably a water soluble salt of a compound useful in the invention. Stabilizing agents, antioxidizing agents and preservatives may also be added.
  • Suitable antioxidizing agents include sodium bisulfite, sodium sulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
  • Cancer is a disease characterized in part by uncontrolled cell growth. Protein kinase C is directly involved in cellular growth control and is believed to be involved in tumor formation. Protein kinase C is the major, if not exclusive, intracellular receptor of phorbol esters which are very potent tumor promoters. Phorbol esters and other tumor promoters bind to and activate protein kinase C.
  • DAG diacylglycerol
  • phorbol esters interact at the same site
  • DAG's have been suggested to be the "endogenous phorbol esters" by analogy with the opiate receptor where the conservation of a high affinity receptor implied the existence of an endogenous analogue.
  • DAG has been shown to increase the affinity of protein kinase C for Ca +2 and phospholipid and thus activates protein kinase C at cellular levels of these essential cofactors.
  • Extracellular signals including hormones, growth factors, and neurotransmitters are known to stimulate phosphatidylinositol turnover resulting in the generation of IP 3 and DAG.
  • Structures of 40 distinct oncogenes of viral and cellular origin have revealed that oncogenes encode altered forms of normal cellular proteins.
  • Several of the gene products appear related to growth factors or other elements involved in transmembrane signalling. These oncogene products appear to function by altering the level of critical second messengers.
  • Cells transformed with the oncogenes ras, sis. erbB, abl, and src have been shown to contain elevated levels of DAG which is then believed to activate protein kinase C. Indeed, studies on ras transformed cells have shown protein kinase C activation to be concomitant with elevation of DAG.
  • Phorbol esters such as phorbol myristate acetate
  • PMA protein kinase C
  • oncogenes such as ras. which cause intracellular increases in DAG and concomitant increases in protein kinase C.
  • activation of protein kinase C leads to the expression of c- myc, c-fos. c-cis. c-fms. nuclear protooncogenes important in cell transformation.
  • Overexpression of protein kinase C in NIH 3T3 cells causes altered growth regulation and enhanced tumorigenicity and in rat fibroblasts leads to anchorage- independent growth in soft agar. In these experiments, overexpression of protein kinase C in these cells resulted in tumor formation in animals receiving transplanted cells.
  • Several studies have shown increased expression of protein kinase C in certain tumor types such as breast and lung carcinomas. Activated protein kinase C has also been detected in human colon carcinomas although increased expression on the gene level was not seen.
  • Topoisomerases are directly modulated by protein kinase C as substrates for the enzyme and protein kinase C inhibitors have been shown to potentiate the action of chemotherapy drugs such as cis- platin.
  • Other and more potent compounds which have been identified specifically as inhibitors of protein kinase C have shown early promise as therapeutic agents in inhibiting tumor growth in animal models.
  • ischemic-related myocardial damage can be attributed to polymorphonuclear leukocytes (neutrophils) which accumulate at the site of occlusion. Damage from the accumulated neutrophils may be due to the release of proteolytic enzymes from the activated neutrophils or the release of reactive oxygen intermediates (ROI) .
  • ROI reactive oxygen intermediates
  • Much of the "no reflow" phenomenon associated with myocardial ischemia is attributed to myocardial capillary plugging. The plugging of capillaries has been attributed to both aggregated platelets and aggregated neutrophils. Although both cell types are aggregated during the ischemic event, the relative contribution of each to capillary plugging has not yet been established.
  • protein kinase C inhibitors as therapeutics for reperfusion injury is that they have been demonstrated to 1) block platelet aggregation and release of neutrophil activating agents such as PAF, 2) block neutrophil activation, chemotactic migration, and adherence to activated or damaged endothelium, and 3) block neutrophil release of proteolytic enzymes and reactive oxygen intermediates.
  • neutrophil activating agents such as PAF
  • PAF neutrophil activating agents
  • chemotactic migration chemotactic migration, and adherence to activated or damaged endothelium
  • 3) block neutrophil release of proteolytic enzymes and reactive oxygen intermediates have the capability of blocking all three of the most significant mechanisms of pathogenesis associated with reperfusion injury and should thus have a decided therapeutic advantage.
  • the 2-amino-l,3-diol group of sphingosine and dihydrosphingosine can adopt several stable conformations via intra-molecular hydrogen bonding.
  • One of the hydroxyl oxygen molecules can adopt a
  • the compounds useful in the present invention comprise molecular structures utilizing this added stability of a six-membered ring chair conformation by establishing covalent bonds in place of hydrogen bonding.
  • Such molecules, 1,3 dioxanes are conformationally rigid and should favor interactions with the protein kinase C and thereby improve enzyme inhibitory activities and subsequent biological effects of these compounds.
  • the compounds useful in the present invention may be synthesized by various approaches. Two approaches to the synthesis of the 1,3-dioxanes were investigated. The first approach involved the synthesis of a properly substituted 1,3 dioxane system which is illustrated in Scheme 1.
  • 1,3-dioxane (6_) , (4S, 5S) t ⁇ reo-5-amino-4-pentadecyl-2,2- dimethy1-1,3-dioxane (1,3 . ) was prepared as shown in Scheme 3.
  • the pentultimate compound in the synthesis, (2S, 3S) threo- dihydrosphingosine N-t-butyl carbamate (12.) was prepared from L-serine by the method of Garner et al., J. Org. Chem . , 51 : 2609, 1986.
  • the second synthetic approach to the 1,3-dioxane derivatives of the present invention employed compounds with established 2-amino-l,3-diol functionality as the starting point.
  • One such example is shown in Scheme 4.
  • the 2-monosubstituted 1,3-dioxanes were prepared from racemic t ⁇ reo-dihydrosphingosine, a known sphingolipid, by acetalization of the N-trifluoroacetyl protected derivative (JL4) with acetaldehyde and benzaldehyde, followed by deprotection to give 37 and .18. respectively.
  • the following nonlimiting examples illustrate a preferred embodiment for preparing the compounds of the invention shown in Table 1.
  • the novel 1,3 dioxane derivatives of the invention may be prepared in a number of ways including those described above.
  • the 1,3 dioxane derivatives of the invention may also be prepared by other methods known in the art, including synthetic and semi-synthetic techniques.
  • a 1,3 dioxane derivative structure has the conventional ring numbering as illustrated in the Merck Index, Tenth Edition, Merck & Co., Inc. Rahway, New Jersey, 1983, pp.100-101.
  • a solution comprising 10 g (85 mmole) of 1- hexadecanol in 100 ml of methylene chloride was added to a mixture comprising 10 g (139 mmole) of pyridinium chlorochro ate, 30 g of celite and 12.0 g (146 mmole) of anhydrous sodium acetate in 400 ml of methylene chloride in a dropwise fashion.
  • the reaction mixture was stirred at room temperature for three hours then 300 ml of diethyl ether was added.
  • the reaction mixture was filtered and a precipitate was collected and washed with diethyl ether until clear.
  • Hexadecanal is a white wax having a melting point of approximately 33-34°C.
  • Threo/erythro-2-Nitro-l,3- octadecanediol is a white wax having a melting point of rt 1 approximately 67-71 C.
  • a residue having a mass of approximately 11.7 g was dissolved in 100 ml of anhydrous tetrahydrofuran and added dropwise to a 2.00 g (52.7 mmole) slurry of lithium aluminumhydride in 200 ml of tetrahydrofuran. After the addition was complete the reaction mixture was stirred at room temperature for 12 hours then refluxed for 4 hours. The reaction mixture was cooled to room temperature and quenched slowly and sequentially with 2.0 ml of water, 2.0 ml of a 15% aqueous solution of sodium hydroxide, followed by 6.0 ml of water. The reaction mixture was filtered and the precipitate was washed with diethyl ether.
  • the filtrate was concentrated under reduced pressure and the residue purified by chromatography on silica (230 to 400 mesh) by gradient elution with hexanes and ethyl acetate at a ratio approximately between 9 to 1 and 1 to 1, and with 100% ethyl acetate. Purification yielded erythro-2,2- dimethyl-5-amino-4-pentadecyl-l,3-dioxane having a mass of approximately 1.45 g and of approximately 11% purity. Erythro-2 ,2-dimethyl-5-amino-4-pentadecyl-l,3-dioxane had a n 1 melting point of approximately 42.5 to 44.5 C.
  • a residue was purified by chromatography on silica (230 to 400 mesh) with hexanes and ethyl acetate at a ratio of approximately 1 to 1 yielding erythro-N-Acetyl-5-amino-4-pentadecyl-l,3-dioxane having a mass of 0.22 g and of approximately 98.2% purity _3__ytJiro-N-Acetyl-5-amino-4-pentadecyl-l,3-dioxaneas is a white wax having a melting point of approximately 65 to 66.5 °C.
  • the reaction mixture was refluxed for four hours, cooled to room temperature, then quenched slowly and sequentially with 0.05 ml of water, 0.05 ml of a 15% aqueous solution of sodium hydroxide, and 0.15 ml of water.
  • Elemental analysis calculated for C 23 H 47 N0 2 comprised the following constituents: C: 74.73, H: 12.82, N: 3.79. The values obtained experimentally comprised: C: 74.80, H: 12.75, N: 3.71.
  • EXAMPLE 6 j5__ ⁇ t_iro-5-N,N-Dimethylamino-4-pentadecyl-l,3- dioxane (9.)
  • Elemental analysis calculated for C 23 H 47 N0 2 comprised the following constituents: C: 74.73, H: 12.82, N: 3.79. The values obtained experimentally comprised: C: 74.67, H: 12.99, N: 4.06.
  • EXAMPLE 7 J5 ryt_xro-5-Acetajnidino-2,2-dimethyl-4-pentadecyl- 1,3-dioxane (10)
  • Salts were removed by filtration and the solvent removed under reduced pressure.
  • a residue was purified by flash chromatography on silica (230 to 400 mesh) with hexanes and ethyl acetate at a ratio of approximately 9 to 1 to yield an oil, fc reo-(2S, 3S)-N-tert-Butoxycarbonyl-5-amino-2,2- dimethyl-4-pentadecyl-l,3-dioxane having a mass of approximately 0.287 g and of approximately 52% purity.
  • a solution comprising 0.28 g (0.634 mmole) of threa ⁇ d s , 3S) -N-tert-butoxycarbonyl-5-amino-2,2-dimethyl-4- pentadecy1-1,3-dioxane, 0.25 ml (1.97 mmole) of chlorotrimethylsilane, and 0.3 g (2.0 mmole) of sodium iodide in 20 ml of anhydrous acetonitrile was stirred at room temperature over night. The solvent was removed under reduced pressure and a residue was taken up in 20 ml of methylene chloride.
  • Threo-(2S, 3S)-5-Amino-2,2-dimethyl-4-pentadecyl-l,3-dioxane having a mass of approximately 0.38 mg and of 18% purity.
  • Threo-(2S, 3S)-5-Amino-2,2-dimethyl-4-pentadecyl-l,3-dioxane was a wax having a melting point of approximately 42.5 to 44.5°C.
  • Elemental analysis calculated for C 21 H 43 N0 2 comprised the following constituents: C: 73.84, H: 12.69, N: 4.10. The values obtained experimentally comprised: C: 73.74, H: 12.61, N: 4.12. EXAMPLE11TJireo-N-Trifluoroacety1-2-amino-1,3-octadecanediol (15)
  • a mixture comprising 0.1 (0.331 mmole) of threo-N- trifluoroacetyl-5-amino-2-methyl-4-pentadecyl-l,3-dioxane in 5 ml of a 15% aqueous solution of sodium hydroxide and 5 ml of 1,4-dioxane was refluxed for 8 hours.
  • the solvent was removed under reduced pressure and the residue was taken up in methylene chloride.
  • An organic layer was washed once with water and dried over magnesium sulfate. Salts were removed by filtration and the solvent removed under reduced pressure.
  • Threo-5-Amino-2- methy1-4-pentadecy1-1,3-dioxane having a mass of 70 mg and of 51% purity.
  • Threo-5-Amino-2-methyl-4-pentadecyl-l,3-dioxane is a wax having a melting point of approximately 29 to 31 °C.
  • Example 14 The residue from Example 14 was dissolved in 10 ml of methanol. To the solution was added 0.1 g (2.64 mmole) of sodium borohydride in portions. The reaction mixture was stirred at room temperature over night. The solvent was removed under reduced pressure and the residue taken up in ethyl acetate. An organic layer was washed with water and dried over magnesium sulfate. Salts were removed by filtration and the solvent removed under reduced pressure.
  • Threo-5-Amino-2-phenyl-4-pentadecyl-l,3-dioxane is a wax having a melting point of approximately 41 to 43 C.
  • the infrared spectrum IR (neat) comprised the following peaks: 2952, 2918, 2849, 1470, and 1024 cm "1 .
  • Elemental analysis calculated for C 25 H 43 N0 2 1/4 H 2 0 comprised the following constituents: C: 76.19, H: 11.12, N: 3.55.
  • the values obtained experimentally comprised: C: 76.38, H: 11.05, N: 3.36.
  • Table I illustrates chemical compositions and moieties of 1,3 dioxane derivatives.
  • Moieties of 1,3 dioxane derivatives of Formula I are designated by the symbols ,, R 1 " through “R 6 " and are listed in the columns labeled by these symbols.
  • the approximate melting point of a compound in degrees centigrade is indicated in the column labeled ⁇ mp(°C) .”
  • Each compound is designated by the example in which it is described.
  • a protein kinase C (PKC) assay is designed to duplicate the in vivo conditions required for protein kinase C function. Therefore, pH, salt and cofactor concentrations are similar to physiologic levels.
  • a lysine rich histone, HI was used in the assay as the phosphorylation acceptor-protein because it is readily available and serves as a good substrate for protein kinase C.
  • Enzyme was prepared from rat brain and purified to apparent homogeneity as determined by a single band on silver stained SDS-polyacrylamide.
  • phosphatidylserine (PS) and DAG were co-sonicated to form unilamellar and multilamellar vesicles.
  • concentration of lipids in the assay were suboptimal to maximize the detection potential of the assay for inhibitors.
  • Potential inhibitor compounds were added to the assay in dimethylsulfoxide at three concentrations to give final inhibitor concentrations of 4.3, 43 and 218 ⁇ M, respectively.
  • the assay was started with the addition of enzyme and stopped after 10 min by the addition of 25% trichloroacetic acid (TCA) and 1.0 mg/ml bovine serum albumin (BSA) .
  • TCA trichloroacetic acid
  • BSA bovine serum albumin
  • the radioactive histone product was retained and washed on glass fiber filters that allow the unreacted 3"2" ⁇ P-ATP to pass through.
  • the amount of phosphorylation was determined by the radioactivity measured in a scintillation counter. Controls were included in every assay to measure background activity in the absence of enzyme, activity in the absence of lipids, and the maximum enzyme activity with saturating levels of the activator lipids. Table 2 shows the protein kinase C assay components and their concentrations.
  • Phosphatidylserine 40 ⁇ g/ml Diacylglycerol 1.8 ⁇ g/ml Protein Kinase C 0.6 ⁇ g/ml ⁇ - P-ATP 20 ⁇ M
  • HEPES is N-[2-hydroxyethyl] piperizine-N'-
  • EGTA is Ethylene-bis (oxyethylenenitrilo) tetraacetic acid.
  • Results of the protein kinase C assay are shown in Table 3 in the column labeled PKC. Results are shown as IC 50 , which is the concentration of test compound needed to inhibit 50% of the protein kinase C activity as compared with levels of protein kinase C activity in controls. Compounds of the invention were able to effectively inhibit protein kinase activity.
  • PKA cAMP dependent protein kinase
  • the assay was started by the addition of 32P-ATP and the reaction was allowed to proceed for 10 min before stopping with 25% trichloroacetic acid (TCA) and 1.0 mg/ml bovine serum albumin (BSA) . Phosphorylated protein was then isolated by filtration and the radioactivity was counted in a beta scintillation counter.
  • TCA trichloroacetic acid
  • BSA bovine serum albumin
  • MCF-7 a human breast tumor cell line and MCF-7/ADR an adriamycin resistant line of MCF-7 cells were obtained from the National Cancer Institute, Frederick, Maryland.
  • CEM cells ATCC accession number CCL 119 were obtained from the American Type Culture Collection, Rockville, Maryland.
  • Human tumor cells were trypsinized with 0.05% trypsin (GIBCO) , counted with a hemacytometer and seeded at a concentration of 10,000 cells/well in a 96 well microtiter plate. After allowing cells to attach to the surface overnight, the culture medium was aspirated and replaced with 100 ⁇ l of fresh medium. Test agents were diluted to determine dose response at 2X final concentration and added in quadruplicate at 100 ⁇ l/well to bring the total volume of each well to 200 ⁇ l. The microtiter plate was then incubated at 37°C 5% C0 2 overnight for 18 to 24 hrs before H-thymidine was added at a concentration of 0.5 ⁇ Ci/well in 50 ⁇ l culture medium. The plate was incubated again for 4 hrs under the same conditions as above. Supernatant was then aspirated and 50 ⁇ l of 0.05% trypsin (GIBCO) was added to each well.
  • GEBCO trypsin
  • IC 50 is the concentration of test compound required to inhibit fifty per cent of the incorporation of H-thymidine into proliferating cells not exposed to test agent. Uptake of H-thymidine is a standard test for measuring the metabolism of cells.
  • compounds of the invention were able to inhibit H-thymidine uptake and thus inhibit the proliferation of the tested cell lines.
  • compound 5. had an IC 50 of 7.7 ⁇ M.
  • MCF-7/ADR the IC 50 of the compound was 6.6.
  • the results with the compounds 6_ and ⁇ were similar.
  • the IC 50 was 6.0 ⁇ M and 5.1 ⁇ M respectively, however the IC 50 was 5.5 ⁇ M and 6.3 ⁇ M when compounds 6 and H were tested with cell line MCF-7/ADR.
  • compounds useful in the invention not only inhibit tumor cell proliferation but are not cross-resistant to the multi-drug-resistant family of agents such as adriamycin.
  • Proliferating keratinocytes (NHEK cells purchased from Clonetics, Inc. , San Diego, California) in second passage were grown in Keratinocyte Growth Medium (KGM) (Clonetics, Inc.) Cells were trypsinized (0.025% trypsin, Clonetics), counted with a hemacytometer (Scientific Products) , and seeded at a concentration of 2,500 cells/well in a 96 well microtiter plate. After allowing cells to attach to the surface overnight, the culture medium was aspirated and replaced with 100 ⁇ l of fresh KGM. Test agents were evaluated and IC 50 's were determined according to the H-thymidine incorporation procedures described as in Example 18. IC 50 is the concentration of test compound required to inhibit fifty per cent of the incorporation of H-thymidine into proliferating cells not exposed to test agent.
  • KGM Keratinocyte Growth Medium
  • cytochrome c Tubes were then centrifuged at 900 xg for 10 minutes and 0.5 ml supernatant was removed and added to 0.5 ml H 2 0 in a microcuvette.
  • Optical density (OD) of cytochrome c was read in a spectrophotometer (Shimadzu) at 550 nm. The ⁇ OD of cytochrome c was obtained between PMA-stimulated and non- stimulated tubes, and the dose responses of the test agents were compared to the positive controls which contain HBSS in place of test agents. PMA stimulates 0 2 " production which reduces cytochrome c.
  • Reducing cytochrome c increases its absorbance, and the change in OD of cytochrome c is proportional to the amount of 0 2 " produced by PMA stimulation. Inhibition of the 0 2 " burst by test compounds of the invention is seen as a reduction in the change in optical density. Inhibition is expressed as IC 50 ⁇ M and is the amount of test compound that will inhibit fifty per cent of the PMA- stimulated respiratory outburst, i.e. 0 2 ⁇ production.

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Abstract

Des composés ont la formule (I), dans laquelle R1 représente alkyle, alcényle ou alcynyle ayant entre 2 et environ 20 atomes de carbone; R2, R3 et R6 représentent indépendamment les uns des autres H, phényle ou alkyle ayant entre 1 et environ 20 atomes de carbone; et R4 et R5 représentent indépendamment H, R6imino ou amidino. L'invention concerne également les sels pharmaceutiquement acceptables de ces composés, utiles pour inhiber la protéine-kinase C et pour traiter des états associés à l'inhibition de la protéine-kinase C ou affectés par celle-ci, notamment des tumeurs cancéreuses, des maladies inflammatoires, des séquelles de reperfusion et des dysfonctionnements cardiaques associés aux séquelles de reperfusion.
PCT/US1992/009048 1992-10-23 1992-10-23 Derives de 1,3-dioxane a activite inhibant la proteine-kinase c WO1994010161A1 (fr)

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AU29244/92A AU2924492A (en) 1992-10-23 1992-10-23 1,3-dioxane derivatives having protein kinase c inhibitory activity

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2254876A (en) * 1940-07-18 1941-09-02 Commercial Solvents Corp Substituted-1,3-dioxanes
US3060196A (en) * 1956-06-06 1962-10-23 Ciba Geigy Corp Unsaturated aliphatic amino-diols and process for their manufacture
US4816450A (en) * 1986-09-15 1989-03-28 Duke University Inhibition of protein kinase C by long-chain bases

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2254876A (en) * 1940-07-18 1941-09-02 Commercial Solvents Corp Substituted-1,3-dioxanes
US3060196A (en) * 1956-06-06 1962-10-23 Ciba Geigy Corp Unsaturated aliphatic amino-diols and process for their manufacture
US4816450A (en) * 1986-09-15 1989-03-28 Duke University Inhibition of protein kinase C by long-chain bases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CARBOHYDRATE RESEARCH, 158, 15 December 1986, KISO et al., "A Novel Route to D-Erythro- Sphingusine and Related Compounds from Mono-O- Isopropylidene- D-Xylose or -D- Galactose", 101-111. *

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