Patent Application
20060205692
The present invention relates to 1,1- and 1,2-bisphosphonate compounds, the processes for their preparation, pharmaceutical compositions containing them and their use in therapy, in particular for modulating (increasing and decreasing) apolipoprotein E in plasma and in tissues.
Apolipoprotein E (apoE) is a polymorphic, multifunctional protein synthesized by several cell types and tissues, including liver, kidney, skin, adipose tissue, macrophages and brain. The wide distribution of apoE is associated with the maintenance of key cellular functions such as intracellular cholesterol trafficking, cholesterol distribution between cells, and tissue reparation.
The amino acid sequence of the apoE protein is well conserved throughout species. ApoE can be viewed as a regulator of cholesterol homeostasis in tissues such as the central nervous system (CNS) and peripheral nervous system (PNS) and the arterial wall (cell-cell) or between tissues via the circulating plasma lipoproteins (tissue-tissue).
The major role of plasma apoE containing lipoproteins is to transfer lipids (cholesterol) from peripheral tissues to the liver and to remove excess cholesterol from peripheral tissues via the reverse cholesterol transport system. Dysregulation of this mechanism leads to excess cholesterol deposition in peripheral tissues such as arteries (arteriosclerosis) and skin (xanthomas and xanthelasmas). ApoE has also been shown to have a direct effect on lymphocyte proliferation and thus has an immunomodulatory role.
ApoE is the only lipoprotein synthesized in the brain and has a key role in cholesterol transport between cells of the CNS. Local secretion of apoE by cells such as macrophages or macrophage-derived cells is essential for the uptake of excess tissue cholesterol and the provision of cholesterol for specific needs such as nerve repair and remyelinisation.
Up to the present time, compounds affecting Apo E production in vitro and in vivo have not been extensively investigated. Only hormone-like estrogens and corticoids have been shown to change Apo E levels under various experimental conditions (Srivastava et al., 1997; Stone et al., 1997).
There is currently a need for compounds that modulate apoE synthesis and secretion, such compounds having application in the treatment of diseases such as atherosclerosis, excess lipid deposition in peripheral tissues such as skin (xanthomas), stroke, memory loss, optic nerve and retinal pathologies (i.e., macular degeneration, retinitis pigmentosa), repair of traumatic damage of the central nervous system (brain tissue), repair of traumatic damage of the peripheral nervous system (i.e., nerve section compression or crush), prevention of the degenerative process due to aging (i.e., Alzheimer's disease), prevention of degenerative neuropathies occurring in diseases such as diabetic neuropathies and multiple sclerosis, autoimmune diseases and activation of the innate immune system.
The Applicants have now found that certain 1,1- and 1,2-bisphosphonate compounds modulate (increase or decrease) the production of apoE in vitro and in vivo. One aspect of the current invention are bisphosphonate derivatives of formula (I):
wherein Y is hydrogen, hydroxy, halo, aryl, aryloxy, C1-C6 alkyl, C1-C6 alkoxy or A-L-O; A is hydroxy, aryl, heterocycle or —NR3R4 wherein R3 and R4 are independently hydrogen or C1-C4 alkyl. The bond depicted by represents a single or a double bond. L is —(CH2)m—, —(CH2)pO(CH2)q—, —(CH2)pNR5(CH2)q— or —(CH2)pNHCO(CH2)q—, wherein R5 is hydrogen, C1-C4 alkyl or C1-C3 cyanoalkyl; and subscripts “m,” “p” and “q” are an integer from 0 to 6. R1 and R2 are independently hydrogen or C1-C6 alkyl. M is (CH2)n or (CH═CH)u—CH═ where n is an integer from 0 to 3 and u is 0 or 1, with the proviso that if n is 1, 2, or 3 or M is (CH═CH)u—CH═, then s is 1 and/or Y, Z1 and Z2 are not all independently H, hydroxy, alkyl or alkoxy. B is H or C1-C4 alkyl group and subscript “w” is 0 when
is a double bond and is 1 when
is a single bond, it being understood that the valency of the atoms is respected. Subscript w is also 1 when M is (CH2)n and subscript “n” is 0, wherein there is direct single bond between the substituted phenyl group and the carbon alpha to the PO(OR2) group. The subscript “s” is 0 or 1. Z1 and Z2 are independently hydrogen, C1-C4 alkyl, or C1-C4 alkoxy.
The invention also encompasses pharmaceutically acceptable salts of the compounds of formula (I).
In various embodiments, n is 0 or 1 and Z1 and Z2 are hydrogen. In some embodiments Y is A-O-L-. A may suitably be pyridin-2-yl, pyridin-3-yl, pyrrolidino, succinimido, piperidino, morpholino, phthalimido, phenyl, N, N′-(2-cyanoethyl)phenylamino or p-cyanophenyl. In some embodiments R1 and R2 are methyl, ethyl or isopropyl. In some embodiments the bisphosphonate derivative of formula (I) is tetraethyl 1-[4-(3-N-phthalimido-propoxy)-phenyl]-methylidene-1,1-bisphosphonate, tetraethyl 1-[4-(3-N-phthalimido-propoxy)-phenyl]-ethylidene-1,2-bisphosphonate, tetraethyl 1-{4-[3-(methyl-pyridin-2-yl-amino)-propoxy]-phenyl}-methylidene-1,1-bisphosphonate, or tetraethyl 1-(4-{2-[(2-cyano-ethyl)-phenyl-amino]-ethoxy}-phenyl)-methylidene-1,1-bisphosphonate.
Other aspects of the current invention include methods of modulating the production of apoE comprising contacting an apoE producing cell with an effective amount bisphosphonate derivative of formula(I) and of modulating apoE levels in a patient in need of such treatment, comprising administration of an effective amount of a compound of formula (I). In some embodiments, the levels of apoE are increased and the patient may be suffering from atherosclerosis, Alzheimer's disease, macular degeneration, retinitis pigmentosa, stroke, degenerative neuropathy, xanthoma or xanthelasma. Increasing apoE levels may provide methods for elevating high density cholesterol, preventing and/or treating atherosclerosis, macular degeneration, retinitis pigmentosa, stroke or degenerative neuropathy. Degenerative neuropathy may be associated with diabetic neuropathy or multiple sclerosis. In other embodiments, apoE levels are decreased by administration to a patient of an effective amount of a bisphosphonate derivative of formula (I). The patient may express apoE4, apoE Leiden or a non-functional mutant form of apoE. The patient may be suffering from atherosclerosis or Alzheimer's disease.
A further aspect of the invention, provides for a method for the prevention and/or treatment of Alzheimer's disease or dementia comprising administration to a patient an effective amount of a bisphosphonate derivative of formula (I). The patient may be heterozygous or homozygous for apoE2 and/or apoE3 and the administration of an effective amount of a bisphosphonate derivative of formula (I) increases apoE levels. Alternatively, the patient may be heterozygous or homozygous for apoE4 and the administration of an effective amount of a bisphosphonate derivative of formula (I) decreases apoE levels.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
I. 1,1- and 1,2-bisphosphonate Compounds
The present invention relates to 1,1- and 1,2-bisphosphonate compounds of general formula (I) that modulate (increase or decrease) apoE levels and are useful as agents for the treatment of a number of disorders including cardiovascular and neurological disease states.
As used herein, the term “aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Suitable aryls include phenyl, naphthyl and the like. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of hydroxy, alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, sec-butyl, or tert-butyl), alkoxy (e.g., methoxy, ethoxy, propoxy, tert-butoxy), cyano, amidino, cyanoalkyl (e.g., cyanomethyl, cyanoethyl and cyanopropyl), aryl, halo e.g., (I, Br, Cl, F) or nitro.
As used herein, the term “heterocycle” refers to aromatic and non-aromatic heterocyclic groups and refers to a single ring or fused rings containing up to four heteroatoms in at least one ring, each of which is selected from oxygen, nitrogen and sulphur, which single or fused ring may be unsubstituted or substituted. Each ring suitably has from 4 to 7, preferably 5 or 6 ring atoms. Representative examples of heterocyclic groups include 2-pyridinyl, 3-pyrridinyl, succinimido, pyrrolidino, naphthalimido, phathalimido, morpholino and pipenridino.
Pharmaceutically acceptable salts for use in the present invention include those described by Berge et al. (1997), herein incorporated by reference. Such salts may be formed from inorganic and organic acids. Representative examples thereof include maleic, fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, hydrochloric, hydrobromic, sulfuric, cyclohexylsulfamic, phosphoric and nitric acids.
Since the compounds of the present invention, in particular compounds of formula (I), are intended for use in pharmaceutical compositions, it will be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure, preferably at least 95% pure, and more preferably at least 99% pure (% are on a wt/wt basis). Impure preparations of the compounds of formula (I) may be used for preparing the more pure forms used in the pharmaceutical compositions. Although the purity of intermediate compounds of the present invention is less critical, it will be readily understood that the substantially pure form is preferred as for the compounds of formula (I). Preferably, whenever possible, the compounds of the present invention are obtained in crystalline form.
When some of the compounds of this invention are allowed to crystallise or are recrystallised from organic solvents, solvent of crystallisation may be present in the crystalline product. This invention includes within its scope such solvates. Similarly, some of the compounds of this invention may be crystallised or recrystallised from solvents containing water. In such cases water of hydration may be formed. This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation. In addition, different crystallisation conditions may lead to the formation of different polymorphic forms of crystalline products. This invention includes within its scope all polymorphic forms of the compounds of formula (I).
II. Applications of ApoE Modulators
A. ApoE in Atherosclerosis
As a component of all lipoprotein fractions, apoE plays an important role in cholesterol homeostasis, by mediating their interaction with receptors such as the apoB, low-density lipoprotein (LDL) and other specific receptors. The important role of apoE in cardiovascular diseases is demonstrated by the apoE knock-out mouse model, where the animals rapidly develop hypercholesterolemia and atherosclerosis with pathological features similar to human atherosclerosis (Plump, 1997). In addition, the absence of a functional apoE in humans is associated with abnormally high plasma levels of cholesterol and triglycerides and the rapid development of atherosclerosis, notwithstanding a low fat diet (Richard et al., 1995). In the knock-out mouse model, these changes are prevented by infusion of apoE, transplantation of macrophage producing apoE, or gene therapy by introducing the human apoE gene into apoE knock out mice (Linton et al., 1995). These results indicate a direct beneficial role for apoE and, consequently, a utility for compounds that increase the apoE levels. The 1,1- and 1,2-bisphosphonate compounds of the present invention that increase apoE plasma levels will decrease plasma atherogenic lipoproteins (VLDL, IDL and LDL) by increasing their uptake by the liver. Increasing apoE in HDL will increase the removal of cholesterol from loaded tissues (atherosclerotic arteries) by the reverse cholesterol transport mechanism.
In contrast, hyperlipidemic patients susceptible of developing atherosclerosis due to the expression of a mutated form of apoE, such as apoE Leiden or other variants, should benefit from the treatment with the compounds that decrease apoE production (van Vlijmen et al., 1998; Richard, 1995). Thus, 1,1- and 1,2-bisphosphonate compounds of the present invention that decrease the production of apoE are useful in the prevention and/or treatment of pathological cardiovascular conditions secondary to the presence of non-functional, variants or mutant forms of the apoE molecule.
B. ApoE in the Central Nervous System (CNS)
ApoE also plays a critical role in the CNS. In the brain apoE is synthesized and secreted by astrocytes, its principal role being cholesterol transport between cells. ApoE is considered to redistribute lipids and to participate in the cholesterol homeostasis of the brain.
ApoE is linked to the neuropathological lesions characteristic of Alzheimer's disease. One isoform, ApoE4, is strongly associated with the age of onset of the disease (Poirier, 1994; Rubinsztein, 1995), while another isoform, apoE3, is believed to help maintain healthy microtubules. The increase in both apoE mRNA and the number of astrocytes in the brains of Alzheimer's patients, indicates that increased apoE represents an astrocyte repair-mechanism to ameliorate the damage within the nervous cells. Memory deficit, defective repair of brain injury and deposition of the Alzheimer's associated β-amyloid variant APPV717F have been demonstrated in in the absence of the apoE gene, i.e., apoE knock out mice (Oitzl et al., 1997; Laskowitz et al., 1997; Walker et al., 1997).
Thus, there is a benefit to increasing apoE production in patients bearing the E2 and E3 isoforms of apoE in regard to the occurrence of Alzheimer's or other spontaneous or traumatic neurological diseases. The 1,1- and 1,2-bisphosphonate compounds of the present invention that increase apoE in the brain will prevent the deposition of plaques associated with Alzheimer's disease and increase the repair mechanism of brain injuries due to mechanical traumas or strokes. Through the increase of neurite extension synaptic sprouting the overall brain activity (e.g., memory) should improve.
Conversely, patients at risk of or suffering from Alzheimer's or spontaneous or traumatic neurological diseases who overexpress the pathological isoforms of apoE, such as apoE4, should benefit from the treatment with a compound that decreases apoE. Thus, 1,1- and 1,2-bisphosphonate compounds of the present invention that decrease the production of apoE are useful in the prevention and/or treatment of the symptomatic and neuropathological cardiovascular conditions characteristic of Alzheimer's or other spontaneous or traumatic neurological diseases that are caused or exacerbated by non-functional, variants or mutant forms of the apoE.
C. ApoE in the Peripheral Nervous System (PNS)
The important role of apoE in nerve regeneration in the PNS is demonstrated by the observation that apoE synthesis is dramatically induced when nerves are injured (Poirier, 1994). The maintenance and/or repair of the myelin sheets involves the participation of apoE secreted by support cells such as glial and Schwann cells. Both apoE synthesis and concentration were found to be abnormally low in degenerative diseases of nervous tissues such as in multiple sclerosis (Gaillard, 1996). ApoE is also considered to stabilize the cytoskeleton apparatus and support neurite elongation, thus having a major effect on the development and remodelling following injury of the nervous system occurring late in life. Thus, the compounds of the present invention that increase apoE will support and increase the speed of the healing process of traumatised nerves (nerve section, crush, etc.) and the prevention and/or healing of degenerative nerves (e.g., multiple sclerosis).
D. ApoE as Modulators of the Immune System
ApoE affects the immune system by acting on lymphocyte proliferation. Furthermore apoE knock out mice are highly sensitive to bacterial infection due to a defect in the innate immune system, suggesting that increasing apoE production should augment the immune response (Roselaar & Daugherty, 1998). Increasing apoE production by utilization of compounds of the present invention should augment ameliorate the immune response in patients in need thereof
E. Skin Lipid Metabolism Disorders
Lipid homeostasis is well controlled in epithelial cells such as keratinocytes, wherein exported lipids are important for comeocyte adhesion and for forming the cutaneous barrier to the external environment. Excess cholesterol deposition in skin (xanthomas and xanthelasmas) will be prevented by utilization of 1,1- and 1,2-diphosphonates compounds of the present invention that increase the level of cutaneous apoE.
III. Formulations and Administration
The compounds of formula (I) can be administered by any of a variety of routes. Thus, for example, they can be administered orally, or by delivery across another mucosal surface (for example across the nasal, buccal, bronchial or rectal mucosa), transdermally, or by injection (for example intradermal, intraperitoneal, intravenous or intramuscular injection).
When the compounds are intended for oral administration, they can be formulated, for example, as tablets, capsules, granules, pills, lozenges, powders, solutions, emulsions, syrups, suspensions, or any other pharmaceutical form suitable for oral administration. Oral dosage forms can, if desired, be coated with one or more release delaying coatings to allow the release of the active compound to be controlled or targeted at a particular part of the enteric tract.
Tablets and other solid or liquid oral dosage forms can be prepared (e.g. in standard fashion) from the compounds of formula (I) and a pharmaceutically acceptable solubilizer, diluent or carrier. Examples of solubilizers, diluents or carriers include sugars such as lactose, starches, cellulose and its derivatives, powdered tracaganth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols such as glycerol, propyleneglycol and polyethyleneglycols, alginic acids and alginates, agar, pyrogen free water, isotonic saline, phosphate buffered solutions, and optionally other pharmaceutical excipients such as disintegrants, lubricants, wetting agents such as sodium lauryl sulfate, coloring agents, flavoring agents and preservatives, etc.
Capsules can be of the hard or soft variety and can contain the active compound in solid, liquid or semisolid form. Typically such capsules are formed from gelatine or an equivalent substance and can be coated or uncoated. If it is desired to delay the release of the active compound until the capsule has passed through the stomach and into the intestine, the capsule can be provided with a pH sensitive coating adapted to dissolve at the pH found in the duodenum or ileum. Examples of such coatings include the Eudragits, the uses of which are well known.
Formulations for injection will usually be made up of the appropriate solubilizers such as detergents which may also include compounds and excipients such as buffering agents to provide an isotonic solution having the correct physiological pH. The injectable solutions are typically pyrogen-free and can be provided in scaled vials or ampoules containing a unit dose of compound.
A unit dosage form of the compounds of the invention typically will contain from 0.1% to 99% by weight of the active substance, more usually from 5% to 75% of the active substance. By way of example, a unit dosage form can contain from lmg to lg of the compound, more usually from 10 mg to 500 mg, for example between 50 mg and 400 mg, and typically in doses of 100 mg to 200 mg.
The compounds of the invention will be administered in amounts which are effective to provide the desired therapeutic effect. The concentrations necessary to provide the desired therapeutic effect will vary according to among other things the precise nature of the disease, the size, weight and age of the patient and the severity of the disease. The doses administered will preferably be non-toxic to the patient, although in certain circumstances the severity of the disease under treatment may necessitate administering an amount of compound that causes some signs of toxicity.
Typically, the compounds of the invention will be administered in amounts in the range 0.01 mg/kg to 100 mg/kg body weight, more preferably 0.1 mg/kg to 10 mg/kg body weight and particularly 1 mg/kg to 5 mg/kg body weight. For an average human of 70 kg weight, a typical daily dosage of the compounds of the invention would be in the range of 70 mg to 700 mg. Such a dosage can be administered, for example from two to four times daily. Ultimately however, the size of the doses administered and the frequency of administration will be at the discretion and judgment of the physician treating the patient.
For therapeutic use the compounds of the present invention will generally be administered in a standard pharmaceutical composition obtained by admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsule, ovules or lozenges either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution that may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The choice of form for administration as well as effective dosages will vary depending, inter alia, on the condition being treated. The choice of mode of administration and dosage is within the skill of the art.
The compounds of formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions or as solids for example, tablets, capsules and lozenges. A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agents. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule.
Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
A typical suppository formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
Preferably the composition is in unit dose form such as a tablet or capsule.
Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of a compound of the structure (I) or a pharmaceutically acceptable salt thereof calculated as the free base.
The pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen. For an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between I mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the structure (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered I to 4 times per day.
Disease states which could benefit from increasing plasma and tissue apoE levels include, but are not limited to: atherosclerosis, neurodegenerative disorders such as Alzheimer's disease or dementia. The compounds of this invention modulate apoE and are therefore of value in the treatment of any of these conditions.
Compounds of the present invention may also be of use in preventing and/or treating the above mentioned disease states in combination with anti-hyperlipidaemic, anti-atherosclerotic, anti-diabetic, anti-anginal, anti-inflammatory or anti-hypertension agents. Examples of the above include cholesterol synthesis inhibitors such as statins, for instance atorvastatin, simvastatin, pravastatin, cerivastatin, fluvastatin, lovastatin and ZD 4522 (also referred to as S-4522, Astra Zeneca), anti-oxidants such as probucol, insulin sensitisers such as a PPAR gamma activator, for instance G1262570 (Glaxo Wellcome) and the glitazone class of compounds such as rosiglitazone (Avandia, SmithKline Beecham), troglitazone and pioglitazone, calcium channel antagonists, and anti-inflammatory drugs such as NSAIDs.
IV. Synthesis of Bisphosphonate Compounds of Formula (I)
The present invention also provides methods for the preparation of compounds of formula (I). Such methods include the preparation of methylidene-1,1-bisphosphonates of formula (Ia):
alkylidene-1,1-bisphosphonates of formula (Ib):
alkenylidene-1,1-bisphosphonates of formula (Ic):
alkenylidene phosphonates of formula (Id):
and ethylidene-1,2-bisphosphonates of formula (Ie):
where n, u, Y, Z1, Z2 , R1 and R2 are as described previously.
The first method, shown schematically in
The second method, outlined in
An alternative method of preparing (Ic) consists of derivatizing compound (VII) by a Mitsunobu or a Williamson reaction in the conditions described above for compounds of formula (Ib).
Alkenylidene phosphonates of formula (Id) and ethylidene-1,2-bisphosphonates of formula (Ie) may be prepared as shown in
Alternatively, compounds (Id) and (Ie) can be prepared by reacting the already substituted hydroxybenzaldehyde (XVI) with tetraalkyl methylenebisphosphonate (XI) to give the substituted alkenylidene phosphonate (Id) and reacting this with the dialkyl phosphite (XIV) and sodium hydride.
V. Determination of Biological Activity
The bisphosphonate compounds of the invention can modulate (increase or decrease) apoE levels in plasma and in tissues. The activities of the compounds can be determined in an in vitro cell assay comprising determining the modulatory effect of the test compound on the secretion of apoE by an apoE secreting cell line (e.g., a monocyte-macrophage cell line such as the THP-1 cell line, a liver derived cell line such as the HepG2 cell line, an intestinal derived cell line such as the CaCo2 cell line or a brain derived cell line such as the astrocytoma CCF-STTG1 cell line).
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following specific examples are intended merely to illustrate the invention and not to limit the scope of the disclosure or the scope of the claims in any way whatsoever.
All new products were purified by flash chromatography employing silica gel 60 Fluka 60752 or standard chromatography with silica gel from Chemie Brunschwig CB 09363-25; analytical TLC on silica gel 60 F254 aluminium sheets from Merck. Detection by UV light at 254 nm. The purity of all new products was determined by GC (HP6890 or HP5890, optima5, tR in min.).
NMR spectra were performed using a Bruker AMX400 (1H at 400 MHz) or Bruker AMX-500 (1H at 500 MHz). Chemical shifts 8 in ppm with respect to SiMe4 (δ=0 ppm, internal reference). For 31P-NMR, chemical shifts o are in ppm with respect to H3PO4 (δ=0 ppm, external reference 85% H3PO4 in H2O). Coupling constants are given in Hz. Nonobvious signal assignment were made by comparison with the spectra of the described similar compounds.
MS were carried out with a Varian CH4 or SM1 spectrometer with electron impact or electrospray, m/z (% of base peak).
IR spectra were carried out with a Perkin Elmer Paragon 1000 FT-IR spectrometer. KBr for solids and neat for oil or liquids. Absorption bands in cm−1.
All reactions were conducted under N2. Reaction temperatures refer to that of the heating bath. Reactions carried out with the exclusion of light were performed in flasks completely wrapped in aluminium foil. All reactions were monitored by TLC and/or GC upon total consumption of the starting material.
Diisopropyl amine (24 ml, 169.82 mmol) was added to a stirred mixture of 20.22 g (165.58 mmol) of 4-hydroxybenzaldehyde and 76.0 ml (829.4 mmol) of dimethyl phosphite. The reaction mixture was heated at 110° C. for 5 h, then the excess of phosphite and amine were removed under vacuum. The mixture was poured into water (100 ml) and taken up with CH2Cl2 (6×50 ml). The organic layer was washed with water, saturated NaCl solution and dried over MgSO4. The solvent was evaporated by rotary evaporator under reduced pressure. The oily residue (79.05 g) was purified by silica gel chromatography (AcOEt:MeOH 80:20). Two products were isolated, the wanted 1,1-bisphosphonate (C11H18O7P2, Mw=324.21, Rf 0.24, 33.49 g, 103.30 mmol, yield 41.66%) and a monophosphonate (C10H15O5P, Mw=246.20, Rf 0.49, 29.18 g, 118.50 mmol, yield 47.80%). The monophosphonate was identified as dimethyl 1-(4-hydroxy-phenyl)-1-methoxy-methylidene-1-phosphonate.
1H-NMR (DMSO, 400 MHz)
δ=9.4, s br, 1H; δ=7.26, dxt, 2H, J=8.8, J=2.0; δ=6.69, d, J=8.4; δ=4.29, t, 1H, J=25.2; δ=3.62, d, 3H, J=13.6; δ=3.45, d, 3H, J=13.2.
MS (70 eV)
325 (11), 324 (M+, 82), 231 (7), 230 (18), 218 (18), 216 (12), 215 (50), 187 (8), 183 (9), 137 (7), 121 (14), 120 (13), 109 (11), 107 (32), 105 (7), 93 (100), 81 (11), 79 (12), 66 (15), 65 (23), 63 (16), 51 (8), 47 (24).
Diisopropyl amine (36 ml, 254.01 mmol) was added to a stirred mixture of 30.18 g (247.11 mmol) of 4-hydroxybenzaldehyde and 105.0 ml (815.05 mmol) of diethyl phosphite. The reaction mixture was heated at 110° C. for 2 h, then the excess of phosphite and amine were removed under vacuum. The mixture was poured into water (100 ml) and taken up with CH2Cl2 (4×100 ml). The organic layer was washed with water, saturated NaCl solution and dried over MgSO4. The solvent was evaporated by rotary evaporator under reduced pressure. The oily residue (83.57 g) was purified by repeated flash chromatography (Silica gel 60, AcOEt:MeOH 90:10). Two products were isolated, the wanted 1,1-bisphosphonate (C15H26O7P2, Mw=380.32, Rf 0.25, 56.18 g, 147.72 mmol, yield 59.78%) and a monophosphonate (C13H21O5P, Mw=288.28, Rf 0.56, 4.40 g, 15.26 mmol, yield 6.18%). The monophosphonate was identified as diethyl 1-(4-hydroxy-phenyl)-1-ethoxy-methylidene-1-phosphonate.
1H-NMR (CDCl3, 400 MHz)
δ=8.35, s br, 1H; δ=7.22, dxt, 2H, J=8.4, J=2.0; δ=6.66, d, 2H, J=8.4; δ=4.19-3.84, m, 4×2H; δ=3.64, t, 1H, J=25.2; δ=1.31, t, 2×3H, J=7.2; δ=1.16, t, 2×3H, J=7.2.
MS (70 eV)
381 (27), 380 (M+, 67), 272 (7), 268 (7), 260 (23), 244 (32), 243 (9), 216 (9), 215 (16), 198 (14), 187 (20), 170 (22), 155 (20), 133 (8), 127 (8), 123 (18), 121 (31), 108 (8), 107 (100), 106 (16), 105 (10), 93 (9), 80 (13), 78 (10), 77 (16), 65 (22).
Diisopropyl amine (4.28 g, 42.29 mmol) was added to a stirred mixture of 5.16 g (42.25 mmol) of 4-hydroxybenzaldehyde and 35.13 g (42.29 mmol) of diisopropyl phosphite. The reaction mixture was heated at 110° C. for 2 h, then the excess of phosphite and amine were removed under vacuum. The mixture was poured into water (30 ml) and taken up with CH2Cl2 (4×30 ml). The organic layer was washed with water, saturated NaCl solution and dried over MgSO4. The solvent was evaporated by rotary evaporator under reduced pressure. The oily residue was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.28) to give the wanted 1,1-bisphosphonate (C19H34O7P2, Mw=436.43, 7.66 g, 17.55 mmol, yield 41.54%).
1H-NMR (CDCl3, 500 MHz)
δ=5.6-5.3, s br, 1H; δ=7.20, d, 2H, J=8.4; δ=6.63, d, 2H, J=8.4; δ=4.72, sept, 2×1H, J=1.72; δ=4.53, sept, 2×1H, J=7.0; δ=3.53, t, 1H, J=25.48; ε=1.35-1.30, m, 4×3H; δ=1.27, d, 2×3H, J=6.2; δ=1.03, d, 2×3H, J=6.2.
MS (70 eV)
436 (M+, 15), 310 (8), 272 (20), 269 (11), 268 (100), 251 (14), 230 (13), 188 (55), 187 (33), 170 (13), 135 (9), 125 (9), 123 (22), 121 (8), 109 (21), 107 (41), 106 (10), 99 (11), 83 (26), 77 (89), 45 (18).
A solution of 1.3 ml (14.95 mmol) of 3-bromo-1-propanol in 20 ml 2-butanone was added dropwise to a stirred mixture of 4.98 g (13.09 mmol) of tetraethyl 1-(4-hydroxy-phenyl)-methylidene-1,1-bisphosphonate, 2.77 g (20.03 mmol) of potassium carbonate and 0.05 g (0.144 mmol) of tetrabutyl ammonium bromide in 40 ml of the same solvent. The reaction mixture was warmed under reflux overnight then allowed to cool to room temperature. The final mixture was poured into water and taken up with CH2Cl2 (3×20 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotary evaporator. The residue (6.42 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.31) to give 4.91 g (11.20 mmol, C18H32O8P2, MW=438.40, yield 85.56%) of tetraethyl 1-[4-(3-hydroxy-propoxy)-phenyl]-methylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 400 MHz)
δ=7.39, dxt, 2H, J=8.0, J=2.0; δ=6.87, d, 2H, J=8.0; δ=4.16-3.82, m, 6×2H; δ=3.67, t, 1H, J=25.0; δ=2.04, quint, 2H, J=6.0; δ=1.29, t, 2×3H, J=7.0; δ=1.17, t, 2×3H, J=7.0.
MS (70 eV)
439 (10), 438 (M+, 60), 393 (7), 215 (15), 214 (8), 381 (17), 380 (100), 302 (22), 301 (44), 187 (26), 179 (28), 169 (10), 165 (26), 133 (8), 273 (18), 256 (18), 231 (10), 228 (14), 227 (13), 121 (11), 107 (34).
IR
3400 (s br), 2982 (s), 1740 (m), 1610 (m), 1512 (m), 1249 (s), 1045 (s), 875 (m).
Under nitrogen and with exclusion of light, a solution of 1.2 ml (5.93 mmol) of diisopropyl azodicarboxylate and 2.00 g (4.56 mmol) of tetraethyl 1-[4-(3-hydroxy-propoxy)-phenyl]-methylidene-1,1-bisphosphonate in 30 ml CH2Cl2 was added very slowly (1 drop/2 sec) to the mixture of 1.56 g (5.95 mmol) of triphenyl phosphine and 1.10 g (5.48 mmol) of 1,8-naphthalimide in 40 ml CH2Cl2 at room temperature. The mixture was stirred at room temperature overnight. After solvent removal by rotary evaporator, the residue was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.27) to give 1.1 g (1.73 mmol, C30H37NO9P2, MW=617.58, yield 37.94%) of naphthalimido substituted 1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.61, dxd, 2H, J=7.3, J=1.0; δ=8.23, dxd, 2H, J=8.4, J=1.0; δ=7.77, dxd, 2H, J=8.1, J=7.3; δ=7.34, dxt, 2H, J=8.7, J=2.0; δ=6.80, d, 2H, J=8.7; δ=4.41, t, 2H, J=7.0; δ=4.15-3.88, m, 5×2H; δ=3.66, t, 1H, J=25.2; δ=2.26, quint, 2H, J=7.0; δ=1.28, t, 2×3H, J=7.1; δ=1.16, t, 2×3H, J=7.1.
MS (70 eV)
618 (4), 617 (M+, 7), 393 (15), 380 (5), 245 (7), 239 (51), 238 (100), 211 (8), 210 (47), 203 (10), 187 (5), 180 (10), 161 (9), 107 (7).
A solution of 1.67 g (6.23 mmol) of N-(3-bromopropyl)phthalimide in 10 ml 2-butanone was added dropwise to a stirred mixture of 2.11 g (5.55 mmol) of tetraethyl 1-(4-hydroxy-phenyl)-methylidene-1,1-bisphosphonate, 1.20 g (8.68 mmol) of potassium carbonate and 0.19 g (0.57 mmol) of tetrabutyl ammonium bromide in 20 ml of the same solvent. The reaction mixture was warmed under reflux over 4 h then allowed to cool to room temperature. The final mixture was poured into water and taken up with CH2Cl2 (3×30 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotavapory evaporator. The residue (3.54 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.27) to give 3.13 g (5.52 mmol, C26H35NO9P2, MW=567.52, oil, yield 94.46%) of 1-[4-(3-N-phthalimido-propoxy)-phenyl]-methylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.84, m, 2H, δ=7.72, m, 2H; δ=7.34 txd, 2H, J=8.7, J=1.9; δ=6.77, d, J=8.7; δ=4.15-3.89, m, 6×2H; δ=3.66, t, 1H, J=25.2; δ=2.18, quint, 2H, J=6.6; δ=1.28, t, 3×3H, J=7.1; δ=1.16, t, 2×3H, J=7.1.
13C-NMR (CDCl3, 125 MHz)
δ=168.4, (C); δ=158.28, (C); δ=133.9, (CH); δ=132.2, (C); δ=131.5, (CH), t, J=6; δ=123.2, (CH); δ=122.0, (C), t, J=8; δ=114.5, (CH); δ=65.7, (CH2); δ=63.1, (CH2), d, J=75; δ=44.7, (CH), t, J=133; δ=35.5, (CH2); δ=28.3, (CH2); δ=16.3, (CH3), d, J=11.
MS (70 eV)
568 (1), 567 (M+, 4), 380 (5), 189 (12), 188 (100), 160 (21), 107 (16), 65 (7).
IR
2983 (s), 1770(w), 1713(s), 1611 (m), 1582 (w), 1513 (w), 1469 (w), 1392 (m), 1234(s), 1662 (m), 1045 (s br), 873 (m), 796 (m), 724 (m), 635 (w), 604 (w), 550 (s), 497 (m).
Trimethylsilyl bromide (5.4 ml, 42.3 mmol) was added to a solution of 2.40 g (4.23 mmol) of 1-[4-(3-N-phthalimido-propoxy)-phenyl]-methylidene-l,l-bisphosphonate in 15 ml CCl4 at −10° C. The mixture was allowed to warm to room temperature and stirred for 6 h. The solvent and the excess of trimethylsilyl bromide were removed under vacuum. The residue was taken up with warm CH2Cl2 and the unsoluble bisphosphonic acid was isolated by filtration. The solid was washed with warm CH2Cl2. We obtained 1.40 g (3.08 mmol, C18H19NO9P2; MW=455.30, white solid, mp>250° C., yield 72.81%) of 1-[4-(3-N-phthalimido-propoxy)-phenyl)-methylidene-1,1-bisphosphonic acid.
1H-NMR (DMSO, 500 MHz)
δ=9.4-7.9, s br, 4×1H; δ=7.88-7.82, m, 4×1H; δ=7.33, d, 2H, J=8.6; δ=6.73, d, 2H, J=8.6; δ=3.98, t, 2H, J=5.8; δ=3.76, t, 2H, J=6.8; δ=3.48, t, 1H, J=24.6; δ=2.05; quint, 2H, J=6.2.
MS (70 eV)
188 (17), 187 (100), 169 (45), 160 (29), 158 (10), 130 (21), 105 (11), 104 (21), 77 (15), 76 (33), 51 (13), 50 (18).
A solution of 0.7 g (5.04 mmol) of 3-bromo-1-propanol in 10mi 2-butanone was added dropwise to a stirred mixture of 2.0 g (4.58 mmol) of tetraisopropyl 1-(4-hydroxy-phenyl)-methylidene-1,1-bisphosphonate, 0.95 g (6.87 mmol) of potassium carbonate and 0.22 g (0.68 mmol) of tetrabutyl ammonium bromide in 20 ml of the same solvent. The reaction mixture was warmed under reflux over 4 h then allowed to cool to room temperature. The final mixture was poured into water and taken up with CH2Cl2 (3×20 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotavapory evaporator.
The crude tetraisopropyl 1-[4-(3-hydroxy-propoxy)-phenyl]-methylidene-1,1-bisphosphonate (yield˜97%) was used in the next step without further purification. Under nitrogen and with exclusion of light, a solution of 0.9 ml (5.8 mmol) of diethyl azodicarboxylate and tetraisopropyl 1-[4-(3-hydroxy-propoxy)-phenyl]-methylidene-1,1-bisphosphonate (≦4.58 mmol) in 30 ml CH2Cl2 was added very slowly (1 drop/2 sec) to the mixture of 1.5 g (5.79 mmol) of triphenyl phosphine and 0.8 g (5.34 mmol) of phthalimide in 40 ml CH2Cl2 at room temperature. The mixture was stirred at room temperature over 4 h. After solvent removal by rotary evaporator, the residue was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 98:2, Rf 0.18) to give 1.2 g (1.92 mmol, C30H43NO9P2, MW=623.63, oil, yield 41.92%) of phthalimido substituted 1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.85-7.72, m, 2H; δ=7.74-7.72, m, 2H; δ=7.32, dxt, 2H, J=8.6, J×1.8; δ=6.74, d, 2H, J=8.7; δ=4.70, sept, 2×1H, J=6.2; δ=4.53, sept, 2×1H, J=6.3; δ=4.02, t, 2H, J=6.1; δ=3.91, t, 2H, J=6.7; δ=3.54, t, 1H, J=25.4; δ=2.18, quint, 2H, J=6.5; δ=1.30, d, 2×3H, J=6.2; δ=1.27, d, 2×3H, J=6.2; δ=1.25, d, 2×3H, J=6.4; δ=1.00, d, 2×3H, J=6.2.
MS (70 eV)
624 (1), 623 (M+, 4), 459 (6), 375 (10), 189 (13), 188 (100), 187 (6), 160 (20).
A solution of 1.56 g (5.82 mmol) of N-(3-bromopropyl)phthalimide in 10 ml 2-butanone was added dropwise to a stirred mixture of 2.06 g (4.85 mmol) of tetraethyl 1-(3-methoxy-5-methyl-4-hydroxy-phenyl)-methylidene-1,1-bisphosphonate, 1.0 g (7.28 mmol) of potassium carbonate and 0.15 g (0.45 mmol) of tetrabutyl ammonium bromide in 20 ml of the same solvent. The reaction mixture was warmed under reflux over 4 h then allowed to cool to room temperature. The final mixture was poured into water and taken up with CH2Cl2 (3×30 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotary evaporator. The residue (3.2 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.45) to give 2.0 g (3.27 mmol, C28H39NO10P2, MW=611.57, oil, yield 67.42%) of 1-[4-(3-N-phthalimido-propoxy)-phenyl)-methylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.87-7.85, m, 2H; δ=7.73-7.71, m, 2H; δ=6.97, s, 1H; δ=4.17-3.90, m, 6×2H; δ=3.81, s, 3H; δ=3.61, t, 1H, J=25.1; δ=2.25, s, 3H; δ=2.15, quint, 2H, J=7.2; δ=1.30, t, 2×3H, J=7.1; δ=1.17, t, 2×3H, J=7.1.
MS (70 eV)
612 (2), 611 (M+, 4), 189 (35), 188 (100), 161 (6), 160 (51), 151 (9), 130 (9), 77 (5), 65 (5).
A solution of 1.78 g (6.31 mmol) of N-(3-bromobutyl)phthalimide in 10 ml 2-butanone was added dropwise to a stirred mixture of 2.0 g (5.26 mmol) of tetraethyl 1-(4-hydroxy-phenyl)-methylidene-1,1-bisphosphonate, 1.1 g (7.89 mmol) of potassium carbonate and 0.17 g (0.51 mmol) of tetrabutyl ammonium bromide in 20 ml of the same solvent. The reaction mixture was warmed under reflux over 4 h then allowed to cool to room temperature. The final mixture was poured into water and taken up with CH2Cl2 (3×30 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotary evaporator. The residue (3.54 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, is Rf 0.31) to give 2.45 g (4.21 mmol, C27H37NO9P2, MW=581.54, oil, yield 80.04%) of 1-[4-(3-N-phthalimido-butoxy)-phenyl]-methylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.86-7.84, m, 2H; δ=7.73-7.72, m, 2H; δ=7.36, dxt, 2H, J=8.7; J=1.8; δ=6.84, d, 2H, J=8.7; δ=4.16-4.01 and 3.96-3.89, m, 4×2H; δ=3.98, t, 2H, J=5.8; δ=3.74, t, 2H, J=7.0; δ=3.67, t, 1H, J=25.2; δ=1.92-1.81, m, 2×2H; δ=1.28, t, 2×3H, J=7.1; δ=1.16, t, 2×3H, J=7.1.
MS (70 eV)
582 (6), 581 (M+, 16), 393 (5), 203 (15), 202 (100), 161 (9), 160 (78), 130 (8), 107 (16), 105 (5), 77 (6), 65 (6), 55 (8).
Under nitrogen and with exclusion of light, a solution of 1.1 ml (6.84 mmol) of diethyl azodicarboxylate and 1.2 g (6.31 mmol) of N-(2-cyanoethyl)-N-(2-hydroxyethyl)-aniline in 40 ml CH2Cl2 was added very slowly (1 drop/2 sec) to the mixture of 1.8 g (6.84 mmol) of triphenyl phosphine and 2.0 g (5.26 mmol) of tetraethyl 1-(4-hydroxy-phenyl)-methylidene-1,1-bisphosphonate in 40 ml CH2Cl2 at room temperature. The mixture was stirred at room temperature overnight. After solvent removal by rotary evaporator, the residue was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.20) to give 2.5 g (4.53 mmol, C26H38N2O7P2, MW=552.28, oil, yield 86.12%) of tetraethyl 1-(4-{2-[(2-cyano-ethyl)-phenyl-amino]-ethoxy}-phenyl)-methylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.38, dxt, 2H, J=8.7, J=2.0; δ=7.30-7.27, m, 2H; δ=6.84, d, 2H, J=8.7; δ=6.80, t, 1H, J=7.3; δ=6.72, d, 2H, J=8.2; δ=4.15-3.82, m, 7×2H; δ=3.67, t, 1H, J=25.2; δ=2.69, t, 2H, J=7.1; δ=1.29 and 1.17, t, 4×3H, J=7.1.
MS (70 eV)
552 (M+, 4), 525 (5), 512 (5), 173 (11), 160 (12), 159 (100), 132 (10), 105 (6), 54 (12).
IR
2983 (m), 2932 (m), 1720 (w), 1600 (m), 1508 (s), 1477 (w), 1391 (w), 1367 (w), 1299 (w), 1250(s), 1164 (w), 1029 (s br), 974(s), 877 (m), 798 (w), 750 (m), 696 (w), 553 (m).
A solution of 2.1 g (4.79 mmol) of tetraethyl 1-[4-(3-hydroxy-propoxy)-phenyl]-methylidene-1,1-bisphosphonate in 20 ml CH2Cl2 was added dropwise to a solution of Dess-Martin reagent (3.1 g, 7.31 mmol) in 20 ml CH2Cl2 at 0° C. Stirring was continued at 0° C. for 15 min followed by warming to room temperature. Reaction progress was monitoring by GC. After 3 h the mixture was poured into 100 of saturated bicarbonate solution containing 7.7 g (48.7 mmol) of Na2S2O3. The aqueous solution was extracted with CH2Cl2 (3×30 ml). The organic layers were combined together, washed with water (3×50 ml) and saturated NaCl solution (2×50 ml) and dried over MgSO4. The crude (1.90 g, 4.35 mmol, yield 90.82%) tetraethyl 1-[4-(3-oxo-propoxy)-phenyl]-methylidene-1,1-bisphosphonate was concentred by rotary evaporator and used in the next step without further purification. Acetic acid (2 ml) were added to a solution of 2.35 g (21.88 mmol) of 2-methylaminopyridine and 1.90 g (4.35 mmol) of tetraethyl 1-[4-(3-oxo-propoxy)-phenyl]-methylidene-1,1-bisphosphonate in 15 ml MeOH. The mixture was stirred for 1 h at room temperature then, sodium cyanoborohydride was added portionwise (0.55 g, 8.71 mmol). After addition, stirring was continued overnight. A saturated bicarbonate solution was added and the mixture taken up into CH2Cl2 (3×50 ml) and washed with water (50 ml), saturated NaCl solution (2×50 ml) and dried over MgSO4. The oil residue obtained after solvent removal (3.0 g) was purified by chromatography (Aluminium oxide 90 active neutral, CH2Cl2:MebH 98:2) to give 0.43 g (0.81 mmol, C24H38N2O7P2, MW=528.53, oil, yield 18.6%) of tetraethyl 1-{4-[3-(methyl-pyridin-2-yl-amino)-propoxy]-phenyl}-methylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.14, dxd, 1H, J=5.8, J=2.0; δ=7.41, dxdxd, 1H, J=8.6, J=7.0, J=2.0; δ=7.38, dxt, 2H, J=8.9, J=2.1; δ=6.86, d, 2H, J=8.7; δ=6.53-6.50, m, 2×1H; δ=4.16-4.01 and 3.96-3.91, m, 4×2H; δ=4.00, t, 2H, J=6.1; δ=3.73, t, 2H, J=6.9; δ=3.67, t, 1H, J=25.2; δ=2.09, quint, 2H, J=6.7; δ=1.29 and 1.16, t, 4×3H, J=7.1.
MS (70 eV)
528 (M+, 2), 408 (9), 271 (8), 150 (11), 149 (100), 136 (29), 135 (27), 122 (15), 121 (44), 107 (19), 93 (10), 78 (9), 57 (13).
IR
2982 (m), 2932 (m), 1598 (s), 1560 (w), 1510 (s), 1424 (w), 1389 (w), 1232 (w), 1299 (w), 1251 (s), 1183 (w), 1163 (w), 1028 (s), 973 (s), 876 (m), 711 (w), 552 (m).
Under nitrogen and with exclusion of light, a solution of 0.8 ml (5.07 mmol) of diethyl azodicarboxylate and 0.60 g (5.50 mmol) of 2-hydroxymethyl-pyridine in 30 ml THF was added very slowly (1 drop/2 sec) to the mixture of 1.28 g (4.88 mmol) of triphenyl phosphine and 1.74 g (3.99 mmol) of tetraisopropyl 1-(4-hydroxy-phenyl)-methylidene-1,1-bisphosphonate in 40 ml THF at room temperature. The mixture was stirred at room temperature overnight. After solvent removal by rotary evaporator, the residue (5.11 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.12) to give 1.16 g (2.20 mmol, C25H39NO7P2, MW=527.54, oil, yield 55.00%) of tetraisopropyl 1-[4-(pyridin-2-yl-methoxy)-phenyl]-methylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.60, d, 1H, J=4.6; δ=7.7, t, 1H, J=7.7; δ=7.50, d, 1H, J=7.8; δ=7.38, d, 2H, J=8.4; δ=7.23, dxd, 1H, J=7.2, J=5.2; δ=6.94, d, 2H, J=8.4; δ=5.20, s, 2H; δ=4.74-4.54, m, 4×1H; δ=3.56, t, 1H, J=25.3; δ=1.29, d, 2×3H, J=6.2; δ=1.27-1.23, m, 4×3H; δ=1.01, d, 2×3H, J=6.2.
MS (70 eV)
528 (4), 527 (M+, 14), 226 (7), 187 (6), 94 (8), 93 (100), 92 (30), 65 (8).
Under nitrogen and with exclusion of light, a solution of 3.5 ml (22.17 mmol) of diethyl azodicarboxylate and 2.22 g (20.34 mmol) of 2-hydroxymethyl-pyridine in 10 ml THF was added very slowly (1 drop/2 sec) to the mixture of 5.62 g (21.43 mmol) of triphenyl phosphine and 8.0 g (20.39 mmol) of tetraethyl 2-(4-hydroxy-phenyl)-vinylidene-1,1-bisphosphonate in 50 ml THF at room temperature. The mixture was stirred at room temperature overnight. After solvent removal by rotary evaporator, the residue was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.18) to give 7.43 g (15.37 mmol, C22H31NO7P2, MW=483.44, oil, yield 75.57%) of tetraethyl 2-[4-(pyridin-2-yl-methoxy)-phenyl]-vinylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.61, d, 1H, J=4.9; δ=8.22, dxd, 1H, J=48.0, J=29.4; δ=7.84, d, 2H, J=8.8; δ=7.72, txd, 1H, J=7.7, J=1.6; δ=7.50, d, 1H, J=7.8; δ=7.25, dxd, 1H, J=7.4, J=5.1; δ=7.00, d, 2H, J=8.8; δ=5.25, s, 2H; δ=4.22-4.03, m, 4×2H; δ=1.37 and 1.20, 4×3H, J=7.1.
MS (70 eV)
483 (M+, 10), 391 (20), 374 (12), 347 (22), 346 (100), 293 (9), 199 (16), 93 (71), 92 (46), 65 (18).
Titanium(IV) chloride (9.1 g, 47.97 mmol, was added to 40 ml THF cooled to 0° C. followed by dropwise addition of 3.80 g (17.90 mmol) of 4-benzyloxybenzaldehyde in 20 ml THF. After stirring for 45 min at 0° C., a solution of 6.26 g (21.72 mmol) of tetraethyl methylenediphosphonate in 20 ml THF was added followed, 10 min later, by the addition of 8.69 g (85.9 mmol) of 4-methylmorpholine. The dark resulting mixture was allowed to warm to room temperature. Water was then added and the solution was extracted with diethyl ether (3×30). The organic layer was washed with water and saturated NaCl solution and dried over MgSO4.
The final residue (4.38 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.27) to give 2.07 g (4.29 mmol, C23H32O7P2, MW=482.45, oil, yield 23.97%) of tetraethyl 2-(4-benzyloxy-phenyl)-vinylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz) δ=8.23, dxd, 1H, J=48.4, J=29.5; δ=7.85, d, 2H, J=8.8; δ=7.44-7.32, m, 5H; δ=6.99, d, 2H, J=8.8; δ=5.12, s, 2H; δ=4.23-4.03, m, 4×2H; δ=1.38 and 1.21, t, 2×3H, J=7.1.
MS (70 eV)
483 (1), 482 (M+, 6), 346 (10), 345 (45), 92 (8), 91 (100).
A solution of 12.12 g (43.85 mmol) of N-(3-bromopropyl)phthalimide in 50 ml 2-butanone was added dropwise to a stirred mixture of 5.59 g (43.49 mmol) of 3-hydroxybenzaldehyde, 8.49 g (61.43 mmol) of potassium carbonate and 1.32 g (3.97 mmol) of tetrabutyl ammonium bromide in 150 ml 2-butanone.
The reaction mixture was warmed under reflux overnight then allowed to cool to room temperature. The final mixture was poured into water and taken up with AcOEt (3×50 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotavapory evaporator. The residue (17.27 g) was purified by flash chromatography (Silica gel 60, AcOEt:Hexane 30:70, Rf 0.20) to give 7.29 g (23.57 mmol, C18H15NO4, MW=309.33, oil, yield 54.20%) of 3-(3-N-phthalimido-propoxy)-benzaldehyde.
A solution of 3.5 g (12.14 mmol) of tetraethyl methylenephosphonate in 30 ml 1,4-dioxane was added to a stirred suspension of NaH (1.35 g, 33.75 mmol, 60% dispersion in mineral oil) in 30 ml 1,4-dioxane at 1I0° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of 3.35 g (10.83 mmol) of 3-(3-N-phthalimido-propoxy)-benzaldehyde in 30 ml 1,4-dioxane was added. The reaction mixture was warmed under reflux over 4 h.
The excess of NaH was destroyed by careful treatment with ethyl acetate and the dioxane was removed by rotary evaporator. A saturated ammonium chloride solution was then added and the mixture taken up into AcOEt (4×50 ml) and washed with water and saturated NaCl solution. The organic layer was dried over MgSO4 and concentred by rotary evaporator. The residue (3.66 g) was purified by flash chromatography (Silica gel 60, AcOEt, Rf 0.22) to give 2.48 g (5.59 mmol, C23H26NO6P, MW=443.44, oil, yield 54.20%) of 2-[3-(3-N-phthalimido-propoxy)-phenyl]-vinylidene-1-phosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.85-7.84, m, 2H; δ=7.74-7.72, m, 2H; δ=7.41, dxd, 1H, J=22.4, J=17.5; δ=1.25, t, 1H, J=7.9; δ=7.06, d, 1H, J=7.7; δ=6.89, s br, 1H; δ=6.82, dxd, 1H, J=8.1, J=2.3; δ=6.18, t, 1H, J=17.5; δ=4.18-4.08, m, 2×2H; δ=4.05, t, 2H, J=6.8; δ=3.93, t, 2H, J=6.0; δ=2.20, quint, 2H, J=6.3; δ=1.36, t, 2×3H, J=7.1.
MS (70 eV)
443 (M+, 21), 283 (15), 269 (40), 189 (49), 188 (100), 161 (20), 160 (72), 147 (11), 133 (12), 130 (42), 118 (11), 105 (10), 104 (13), 102 (10), 91 (11), 77 (19), 76 (12).
A solution of 12.41 g (46.29 mmol) of N-(3-bromopropyl)phthalimide in 50 ml 2-butanone was added dropwise to a stirred mixture of 8.99 g (65.05 mmol) of potassium carbonate, 5.59 g (43.49 mmol) of 4-hydroxybenzaldehyde and 1.42 g (4.27 mmol) of tetrabutyl ammonium bromide in 150 ml 2-butanone.
The reaction mixture was warmed under reflux over 5.5 h then allowed to cool to room temperature. The final mixture was poured into water and taken up with AcOEt (3×50 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotary evaporator.
After solvent removal, the residue (GC>99.5%) was used in the next step without fuirther purification. We obtained 12.65 g (40.90 mmol, C18H15NO4, MW=309.33, oil, yield 89.36%) of 4-(3-N-phthalimido-propoxy)-benzaldehyde.
A solution of 7.06 g (24.50 mmol) of tetraethyl methylenephosphonate in 50 ml 1,4-dioxane was added to a stirred suspension of NaH (2.80 g, 70.0 mmol, 60% dispersion in mineral oil) in 50 ml 1,4-dioxane at 10° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of 6.72 g (21.73 mmol) of 4-(3-N-phthalimido-propoxy)-benzaldehyde in 50 ml 1,4-dioxane was added. The reaction mixture was warmed under reflux over 2.5 h.
The excess of NaH was destroyed by careful treatment with ethyl acetate and the dioxane was removed by rotary evaporator. A saturated ammonium chloride solution was then added and the mixture taken up into CH2Cl2 (4×50 ml) and washed with water and saturated NaCl solution. The organic layer was dried over MgSO4 and concentred by rotary evaporator. The residue (10.54 g) was purified by flash chromatography (Silica gel 60, AcOEt, Rf 0.18) to give 5.94 g (13.40 mmol, C23H26NO6P, MW=443.44, solid, mp 87-89° C., yield 61.67%) of 2-[3-(3-N-phthalimido-propoxy)-phenyl]-vinylidene-1-phosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.85-7.83, m, 2H; δ=7.73-7.71, m, 2H; δ=7.43, dxd, 1H, J=22.6, J=17.5; δ=7.40, d, 2H, J=8.6; δ=6.79, d, 2H, J=8.7; δ=6.07, t, 1H, J=17.6; δ=4.15-4.08, m, 2×2H; δ=4.06, t, 2H, J=6.0; δ=3.91, t, 2H, J=6.8; δ=2.20, quint, 2H, J=6.3; δ=1.35, t, 2×3H, J=7.1.
MS (70 eV)
444 (2), 443 (M+, 4), 189 (11), 188 (100), 160 (35), 130 (5).
Under nitrogen and with exclusion of light, a solution of 50.0 ml (0.32 mol) of diethyl azodicarboxylate and 35.7 g (0.29mol) of 3-hydroxybenzaldehyde in 150 ml THF was added very slowly to the mixture of 84.0 g (0.32 mol) of triphenyl phosphine and 36.0 g (0.29 mol) of 2-(2-hydroxyethyl)-pyridine in 400 ml THF at room temperature. The mixture was stirred at room temperature overnight. After solvent removal by rotary evaporator, the residue (146.0 g) was purified by repeated flash chromatography (Silica gel 60, AcOEt:Hexane 40:60, Rf 0.26) to give 25.2 g (0.11 mol, C14H13NO2, MW=227.27, oil, yield 37.93%) of 3-(2-pyridin-2-yl-ethoxy)-benzaldehyde.
A solution of 5.30 g (18.39 mmol) of tetraethyl methylenephosphonate in 50 ml 1,4-dioxane was added to a stirred suspension of NaH (1.49 g, 37.25 mmol, 60% dispersion in mineral oil) in 50 ml 1,4-dioxane at 10° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of 3.50 g (15.44 mmol) of 3-(2-pyridin-2-yl-ethoxy)-benzaldehyde in 50 ml 1,4-dioxane was added. The reaction mixture was warmed under reflux over 2.5 h.
The excess of NaH was destroyed by careful treatment with ethyl acetate and the dioxane was removed by rotary evaporator. A saturated ammonium chloride solution was then added and the mixture taken up into AcOEt (4×100 ml) and washed with water and saturated NaCl solution. The organic layer was dried over MgSO4 and concentred by rotary evaporator. The residue (6.59 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.27) to give 4.03 g (11.15 mmol, C19H24NO4P, MW=361.38, oil, yield 72.22%) of diethyl 2-[3-(2-pyridin-2-yl-ethoxy)-phenyl]-vinylidene-1-phosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.57, d, 1H, J=4.7; δ=7.64, txd, 1H, J=7.7, J=1.8; δ=7.45, dxd, J=22.4, J=17.5; δ=7.29, d, 1H, J=7.5; δ=7.28, t, 2H, J=7.9; δ=7.17, dxd, 1H, J=7.5, J=4.9; δ=7.07, d, 1H, J=7.7; δ=7.03, s, 1H; δ=6.93, dxd, 1H, J=8.1, J=2.3; δ=6.23, t, 1H, J=17.5; δ=4.39, t, 2H, J=6.6; δ=4.16-4.10, m, 2×2H; δ=3.28, t, 2H, J=6.6; δ=1.35, t, 2×3H, J=7.0.
MS (70 eV)
361 (M+, 6), 269 (9),213 (6), 149 (8),125 (6),122 (10), 107 (10), 106 (100), 93 (43), 78 (8).
Under nitrogen and with exclusion of light, a solution of 1.8 ml (1 1.40 mmol) of diethyl azodicarboxylate and 1.12 g (10.26 mmol) of 2-hydroxymethyl-pyridine in 10 ml THF was added very slowly (1 drop/2 sec) to the mixture of 2.86 g (10.90 mmol) of triphenyl phosphine and 4.1 g (10.40 mmol) of tetraethyl 2-(4-hydroxy-phenyl)-ethylidene-1,1-bisphosphonate in 30 ml THF at room temperature. The mixture was stirred at room temperature overnight. After solvent removal by rotary evaporator, the residue was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.18) to give 1.8 g (3.71 mmol, C22H33NO7P2, MW=485.46, oil, yield 36.16%) of tetraethyl 2-[4-(pyridin-2-yl-methoxy)-phenyl]-ethylidene-1,1-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.60, d, 1H, J=4.6; δ=7.71, txd, 1H, J=7.7, J=1.6; δ=7.51, d, 1H, J=7.8; δ=7.22, dxd, 1H, J=7.2, J=5.0; δ=7.20, d, 2H, J=38.6; δ=6.90, d,2H, J=8.6; δ=5.19, s, 2H; δ=4.16-4.04, m, 4×2H; δ=3.19, txd, 2H, J=16.5, J=6.2; δ=2.59, txd, 1H, J=23.9, J=6.3; δ=1.29-1.25, m, 4×3H.
MS (70 eV)
485 (M+, 5), 393 (17), 349 (21), 348 (100), 93 (51), 92 (30), 65 (12).
A solution of 27.12 g (94.09 mmol) of tetraethyl methylenephosphonate in 100 ml 1,4-dioxane was added to a stirred suspension of NaH (8.22 g, 205.50 mmol, 60% dispersion in mineral oil) in 100 ml 1,4-dioxane at 10° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of 17.03 g (80.24 mmol) of 4-benzyloxybenzaldehyde in 100 ml 1,4-dioxane was added. The reaction mixture was warmed under reflux over 2.5 h. The excess of NaH was destroyed by careful treatment with ethyl acetate and the dioxane was removed by rotary evaporator. A saturated ammonium chloride solution was then added and the mixture taken up into AcOEt (4×100 ml) and washed with water and saturated NaCl solution. The organic layer was dried over MgSO4 and concentred by rotary evaporator. The residue (10.58 g) was purified by flash chromatography (Silica gel 60, AcOEt, Rf 0.24) to give 5.55 g (16.0 mmol, C19H23O4P, MW=346.37, oil, yield 19.94%) of diethyl 2-(4-benzyloxyphenyl)-vinylidene-1-phosphonate.
A solution of 7.08 g (51.27 mmol) of diethyl phosphite in 30 ml DME was added to a stirred suspension of NaH (2.78 g, 69.50 mmol, 60% dispersion in mineral oil) in 50 ml DME cooled to 0° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of diethyl 2-(4-benzyloxyphenyl)-vinylidene-1-phosphonate (5.55 g, 16.0 mmol) in 50 ml DME was added. The final mixture was stirred at room temperature overnight. The excess of NaH was destroyed by careful treatment with ethyl acetate. Water was then added and the mixture taken up into CH2Cl2 (3×100 ml) and washed with water, saturated NaCl solution and dried over MgSO4. The residue (5.69 g) obtained after solvent removal was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.21) to give 1.71 g (3.53 mmol, C23H34O7P2, MW=484.47, oil, yield 22.06%) of tetraethyl 1-(4-benzyloxy-phenyl)-ethylidene-1,2-bisphosphonate.
10% Palladium on activated charcoal (1.12 g, 1.05 mmol) was added to a solution of 1.71 g (3.53 mmol) of tetraethyl 1-(4-benzyloxy-phenyl)-ethylidene-1,2-bisphosphonate in 100 ml EtOH. The mixture was then submitted to hydrogenation under pressure (60 p.s.i.) at room temperature for 2 h. After filtration over MgSO4, the solution was concentred by rotary evaporator and the residue (1.52 g) was purified by flash chromatography (Silica gel 60 , AcOEt:MeOH 90:10, Rf 0.24) to give 1.32 g (3.35 mmol, C16H28O7P2, MW=394.34, oil, yield 94.90%) of tetraethyl 1-(4-hydroxy-phenyl)-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=9.0-7.3, s br, 1H; δ=7.13, dxd, 2H, J=8.6, J=2.2; δ=6.61, d, 2H, J=8.5; δ=4.12-3.59, m, 4×2H; δ=3.43-3.32, m, 1H; δ=2.49-2.41, m, 2H; δ=1.31, 1.20, 1.12 and 1.08, t, 4×3H, J=7.1.
MS (70 eV)
395 (5), 394 (M+, 22), 258 (15), 257 (100), 229 (16), 213 (7), 201 (29), 185 (8), 120 (20), 109 (11), 81 (10).
A solution of 20.0 g (69.4 mmol) of tetraethyl methylenephosphonate in 100 ml 1,4-dioxane was added to a stirred suspension of NaH (8.9 g, 222.5 mmol, 60% dispersion in mineral oil) in 10 ml 1,4-dioxane at 10° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of 6.0 g (48.15 mmol) of 3-hydroxybenzaldehyde in 100 ml 1,4-dioxane was added. The reaction mixture was warmed under reflux for 4 h. The excess of NaH was destroyed by careful treatment with ethyl acetate and the dioxane was removed by rotary evaporator. A saturated ammonium chloride solution was then added and the mixture taken up into CH2Cl2 (4×100 ml) and washed with water and saturated NaCl solution. The organic layer was dried over MgSO4 and concentred by rotary evaporator. The residue (21.05 g) was purified by flash chromatography (Silica gel 60, AcOEt, Rf 0.20) to give 11.34 g (44.26 mmol, C19H23O4P, MW=346.37, oil, yield 91.92%) of diethyl 2-(3-hydroxyphenyl)-vinylidene-1-phosphonate.
A solution of 4.5 g (32.59 mmol) of diethyl phosphite in 40 ml DME was added to a stirred suspension of NaH (1.7 g, 42.5 mmol, 60% dispersion in mineral oil) in 40 ml DME cooled to 0° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of diethyl 2-(3-hydroxyphenyl)-vinylidene-1-phosphonate (2.46 g, 9.60 mmol) in 40 ml DME was added. The final mixture was stirred at room temperature overnight. The excess of NaH was destroyed by careful treatment with ethyl acetate. Water was then added and the mixture taken up into CH2Cl2 (3×100 ml) and washed with water, saturated NaCl solution and dried over MgSO4. The residue (5.66 g) obtained after solvent removal was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.18) to give 2.38 g (6.04 mmol, C16H28O7P2, MW=394.34, oil, yield 62.92%) of tetraethyl 1-(3-hydroxy-phenyl)-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.58-8.46, s br, 1H; δ=7.12, t, 1H, J=7.9; δ=7.00, s, 1H; δ=6.84, d, 1H, J=7.5; δ=6.72, d, 1H, J=8.1; δ=4.13-3.48, m, 4×2H; δ=3.43-3.33, m, 1H; δ=2.47-2.39, m, 2H, δ=1.31, t, 3H, J=7.1; δ=1.16, t, 3H, J=7.0; δ=1.04, t, 3H, J=7.1; δ=1.00, t, 3H, J=7.1.
MS (70 eV)
395 (6), 394 (M+, 30), 258 (18), 257 (100), 229 (17), 201 (23), 120 (14), 109 (8), 91 (8), 81 (10).
A solution of 0.98 g (3.66 mmol) of N-(3-bromopropyl)phthalimide in 20 ml 2-butanone was added dropwise to a stirred mixture of 0.73 g (5.28 mmol) of potassium carbonate, 1.18 g (3.0 mmol) of tetraethyl 1-(3-hydroxy-phenyl)-ethylidene-1,2-bisphosphonate and 0.15 g (0.47 mmol) of tetrabutyl ammonium bromide in 40 ml 2-butanone. The reaction mixture was warmed under reflux for 4 h then allowed to cool to room temperature. The final mixture was poured into water and taken up with CH2Cl2 (3×50 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotavapory evaporator. The residue (2.02 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 95:5, Rf 0.20) to give 1.63 g (2.80 mmol, C27H37NO9P2, MW=581.54, oil, yield 93.33%) of tetraethyl 1-[3-(3-N-phthalimido-propoxy)-phenyl]-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.85-7.83, m, 2H; δ=7.74-7.72, m, 2H; δ=7.18, t, 1H, J=7.9; δ=6.97, d, 1H, J=7.6, δ=6.86, s br, 1H; δ=6.70, d, 1H, J=8.2; δ=4.15-3.56, m, 6×2H; δ=3.44-3.33, m, 1H; δ=2.52-2.36, m, 2H; δ=2.18, quint, 2H, J=6.1; δ=1.30, t, 3H, J=7.0; δ=1.14, t, 3H, J=7.0; δ=1.10, t, 3H, J=7.0; δ=1.05, t, 3H, J=7.0.
MS (70 eV)
582 (6), 581 (M+, 18), 445 (13), 444 (49), 394 (21), 285 (13), 258 (15), 257 (83), 229 (19), 201 (26), 189 (13), 188 (100), 160 (32), 120 (16), 109 (12), 91 (12), 81 (14).
Under nitrogen and exclusion of light, a solution of 1.0 ml (6.2 mmol) of diethyl azodicarboxylate and 1.0 g (5.1 mmol) of N-(2-cyanoethyl)-N-(2-hydroxyethyl)-aniline in 30 ml THF was added very slowly (1 drop/2 sec) to the mixture of 1.5 g (5.6 mmol) of triphenyl phosphine and 1.7 g (4.3 mmol) of tetraethyl 1-(4-hydroxy-phenyl)-ethylidene-1,2-bisphosphonate in 30 ml THF at room temperature. The mixture was stirred at room temperature overnight. After solvent removal by rotary evaporator, the residue (5.54 g) was purified by flash chromatography (Aluminium oxide 90 active neutral, CHCl3:hexane 90:10) to give 0.83 g (1.46 mmol, C27H40N2O7P2, MW=566.58, oil, yield 33.96%) of tetraethyl 1-(4-{2-[(2-cyano-ethyl)-phenyl-amino]-ethoxy}-phenyl)-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.31-7.26, m, 4×1H; δ=6.83, 2H, J=8.6; δ=6.79, t, 1H, J=7.3; δ=6.72, d, 2H, J=8.1; δ=4.11, t, 2H, J=5.3; δ=4.09-3.57, m, 6×2H; δ=3.45-3.33, m, 1H; δ=2.68, , t, 2H, J=7.2; δ=2.59-2.34, m, 2H; δ=1.30, t, 3H, J=7.1; δ=1.15, t, 3H, J=7.1; δ=1.10, t, 3H, J=7.1; δ=1.05, t, 3H, J=7.1.
MS (70 eV)
567 (7), 566 (M+, 17), 526 (16), 394 (16), 173 (27), 160 (26), 159 (100), 132 (24), 109 (8), 106 (11), 105 (12), 104 (13), 91 (13), 81 (10), 77 (11).
IR
2963 (m), 2932 (m), 2248 (w), 1600 (m), 1508 (m), 1391 (w), 1367 (w), 1245 (s), 1181 (w), 1098 (w), 1029 (s), 969 (m), 794 (m), 750 (m), 696 (w), 542 (w), 507 (m).
A solution of 1.33 g (4.83 mmol) of N-(3-bromopropyl)phthalimide in 15 ml 2-butanone was added dropwise to a stirred mixture of 0.93 g (6.72 mmol) of potassium carbonate, 1.6 g (4.31 mmol) of tetraethyl 1-(4-hydroxy-phenyl)-ethylidene-1,2-bisphosphonate and 0.18 g (0.56 mmol) of tetrabutyl ammonium bromide in 20 ml 2-butanone.
The reaction mixture was warmed under reflux for 3.5 h then allowed to cool to room temperature. The final mixture was poured into water and taken up with CH2Cl2 (3×100 ml). The organic layer was washed with saturated NaCl solution, dried over MgSO4, filtered and then concentred by rotavapory evaporator. The residue (2.72 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.32) to give 2.2 g (3.78 mmol, C27H37NO9P2, MW=581.54, oil, yield 87.70%) of tetraethyl 1-[3-(3-N-phthalimido-propoxy)-phenyl]-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.85-7.82, m, 2H; δ=7.74-7.72, m, 2H; δ=7.26, dxd, 2H, J=8.8, J=2.2; δ=6.75, d, 2H, J=8.5; δ=4.10-3.54, m, 6×2H; δ=3.44-3.34, m, 1H; δ=2.51-2.34, m, 2H; δ=2.18, quint, 2H, J=6.3; δ=1.30, t, 3H, J=6.9; δ=1.15, t, 3H, J=7.3; δ=1.09, t, 3H, J=7.0; δ=1.05, t, 3H, J=6.6.
MS (70 eV)
582 (12), 581 (M+, 38), 445 (15), 444 (59), 189 (13), 188 (100), 160 (54), 130 (12), 120 (8), 109 (10), 81 (11).
IR
2983 (m), 1773 (w), 1713 (s), 1611 (w), 1513 (m), 1442 (w), 1396 (m), 1372 (w), 1245 (s), 1183 (w), 1029 (s br), 967 (s), 835 (w), 796 (m), 723 (m), 530 (w).
A solution of 7.69 g (50.53 mmol) of 2-(methyl-pyridin-2-yl-amino)-ethanol in 50 ml DMF was added to a stirred suspension of NaH (2.72 g, 68.0 mmol, 60% dispersion in mineral oil) in 150 ml DMF cooled to 0° C. The mixture was allowed to warm to 15° C. and stirred for 1 h then, a solution of 4-fluorobenzaldehyde (7.14 g, 57.52 mmol) in 50 ml DMF was added. The final mixture was stirred at room temperature overnight. The excess of NaH was destroyed by careful treatment with ethyl acetate. A mixture ice-water (500 g) was then added and the mixture taken up into AcOEt (4×500 ml) and washed with water, saturated NaCl solution and dried over MgSO4. The residue (24.00 g) obtained after solvent removal was purified by repeated flash chromatography (Silica gel 60, AcOEt:Hexane 40:60, Rf 0.21) to give 9.89 g (38.59 mmol, C15H16N2O2, MW=256.30, oil, yield 76.37%) of 4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-benzaldehyde.
A solution of 2.85 g (9.89 mmol) of tetraethyl methylenephosphonate in 30 ml 1,4-dioxane was added to a stirred suspension of NaH (0.85 g, 21.25 mmol, 60% dispersion in mineral oil) in 30 ml 1,4-dioxane at 10° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of 2.0 g (7.80 mmol) of 4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-benzaldehyde in 30 ml 1,4-dioxane was added. The reaction mixture was warmed under reflux over 1.5 h.
The excess of NaH was destroyed by careful treatment with ethyl acetate and the dioxane was removed by rotary evaporator. A saturated ammonium chloride solution was then added and the mixture taken up into AcOEt (4×50 ml) and washed with water and saturated NaCl solution. The organic layer was dried over MgSO4 and concentred by rotary evaporator. The residue (2.32 g) was purified by flash chromatography (Silica gel 60 Fluka 60752, AcOEt:MeOH 98:2, Rf 0.27) to give 0.95 g (2.43 mmol, C19H24NO4P, MW=361.38, oil, yield 31.15%) of diethyl 2-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-phenyl}-vinylidene-1-phosphonate.
A solution of 1.10 g (7.96 mmol) of diethyl phosphite in 10 ml DME was added to a stirred suspension of NaH (0.42 g, 10.5 mmol, 60% dispersion in mineral oil) in 20 ml DME cooled to 0° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of diethyl 2-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-phenyl}-vinylidene-1-phosphonate (0.40 g, 1.02 mmol) in 30 ml DME was added. The final mixture was stirred at room temperature overnight. The excess of NaH was destroyed by careful treatment with ethyl acetate. Water was then added and the mixture taken up into CH2Cl2 (3×50 ml) and washed with water, saturated NaCl solution and dried over MgSO4. The residue (1.55 g) obtained after solvent removal was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.24) to give 0.43 g (0.81 mmol, C24H36N2O7P2, MW=528.53, oil, yield 79.41%) of 1-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-phenyl}-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.16, dxd, 1H, J=4.7, J=1.9; δ=7.46, txd, 1H, J=7.8, J=1.9; δ=7.28, dxd, 2H, J=8.6, J=2.2; δ=6.85, d, 2H, J=8.5; δ=6.56, dxd, 1H, J=6.7, J=5.1; δ=6.55, d, 1H, J=8.6; δ=4.16, t, 2H, J=5.8; δ=4.10-3.53, m, 4×2H; δ=3.98, t, 2H, J=5.6; δ=3.45-3.33, m, 1H; δ=3.15, s, 1H; δ=2.50-2.43, m, 2H; δ=1.30, 1.15, 1.09 and 1.04, t, 4×3H, J=7.1.
MS (70 eV)
528 (M+, 11), 422 (8), 421 (22), 136 (21), 135 (27), 135 (41), 122 (17), 121 (100), 108 (14), 81 (18), 79 (15), 78 (35).
A solution of 5.8 g (16.7 mmol) of tetraisopropyl methylenephosphonate in 30 ml 1,4-dioxane was added to a stirred suspension of NaH (1.4 g, 34.8 mmol, 60% dispersion in mineral oil) in 50 ml 1,4-dioxane at 10° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of 3.3 g (12.9 mmol) of 4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-benzaldehyde (for the preparation see synthesis of tetraethyl 1-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-phenyl}-ethylidene-1,2-bisphosphonate, in 50 ml 1,4-dioxane was added. The reaction mixture was warmed under reflux over 2 h. The excess of NaH was destroyed by careful treatment with ethyl acetate and the dioxane was removed by rotary evaporator. A saturated ammonium chloride solution was then added and the mixture taken up into AcOEt (3×100 ml) and washed with water and saturated NaCl solution. The organic layer was dried over MgSO4 and concentred by rotary evaporator. The residue (4.4 g) was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 98:2, Rf 0.34) to give 1.38 g (3.3 mmol, C22H31N2O4P, MW=418.48, oil, yield 25.58%) of diisopropyl 2-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-phenyl}-vinylidene-1-phosphonate.
A solution of 3.6 g (25.9 mmol) of diethyl phosphite in 30 ml DME was added to a stirred suspension of NaH (1.3 g, 32.3 mmol, 60% dispersion in mineral oil) in 50 ml DME cooled to 0° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of diisopropyl 2-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-phenyl}-vinylidene-1-phosphonate (1.3 g, 3.3 mmol) in 40 ml DME was added. The final mixture was stirred at room temperature overnight. The excess of NaH was destroyed by careful treatment with ethyl acetate. Water was then added and the mixture taken up into CH2Cl2 (3×50 ml) and washed with water, saturated NaCl solution and dried over MgSO4. The residue (1.8 g) obtained after solvent removal was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 90:10, Rf 0.26) to give 0.8 g (1.44 mmol, C26H42N2O7P2, MW=556.57, oil, yield 43.64%) of diisopropyl 1-(diethoxy-phosphoryl)-1-{4-[2-(methyl-pyridin-2-yl-amino)-ethoxy]-phenyl}-ethylidene-2-phosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.15, dxd, 1H, J=5.0, J=1.9; δ=7.47, dxdxd, 1H, J=8.5, J=6.9, J=1.9; δ=7.29-7.27, m, 2H; δ=6.86-6.84, m, 2H; δ=6.53, dxd, 1H, J=7.0, J=5.0; δ=6.53, d, 1H, J=8.6; δ=4.16, t, 2H, J=5.8; δ=4.15-3.99 and 3.94-3.30, m, 2×2H and 3×1H; δ=3.97, t, 2H, J=5.8; δ=3.14, s, 3H; δ=2.51-2.33, m, 2H; δ=1.35-0.98, m, 6×3H.
MS (70 eV)
556 (M+, <1), 179 (5), 136 (10), 135 (100), 122 (6), 121 (52), 119 (4), 104 (4), 78 (5).
IR
2981 (m), 2932 (m), 1736 (w), 1654 (w), 1598 (m), 1560 (w), 1511 (s), 1426 (w), 1387 (w), 1324 (w), 1245 (s), 1180 (w), 1162 (w), 1099 (w), 1030 (s br), 853 (w), 772 (m), 507 (m).
A solution of 2.42 g (17.52 mmol) of diethyl phosphite in 10 ml DME was added to a stirred suspension of NaH (0.88 g, 22.00 mmol, 60% dispersion in mineral oil) in 20 ml DME cooled to 0° C. The mixture was allowed to warm to room temperature and stirred for 30 min then, a solution of diethyl 2-[3-(2-pyridin-2-yl-ethoxy)-phenyl]-vinylidene-1-phosphonate (2.00 g, 5.53 mmol) in 20 ml DME was added. The final mixture was stirred at room temperature overnight. The excess of NaH was destroyed by careful treatment with ethyl acetate. Water was then added and the mixture taken up into CH2Cl2 (3×50 ml) and washed with water, saturated NaCl solution and dried over MgSO4. The residue (2.74 g) obtained after solvent removal was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 98:2, Rf 0.34) to give 0.47 g (0.94 mmol, C23H35NO7P2, MW=499.49, oil, yield 17.0%) of tetraethyl 1-[3-(2-pyridin-2-yl-ethoxy)-phenyl]-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=8.56, d, 1H, J=4.8; δ=7.64, txd, 1H, J=7.6, J=1.8; δ=7.28, d, 1H, J=8.0; δ=7.20, t, 1H, J=7.9; δ=7.16, dxd, 1H, J=7.5, J=4.9; δ=6.97, d, 1H, J=7.6; δ=6.95, s, 1H; δ=6.81, d, 1H, J=8.2; δ=4.35, t, 2H, J=6.7; δ=4.12-3.54, m, 4×2H; δ=3.46-3.35, m, 1H; δ=3.26, t, 2H, J=6.7; δ=2.52-2.38, m, 2H; δ=1.30, 1.12, 1.08 and 1.02, 4×3H, J=7.1.
MS (70 eV)
499 (M+, 6), 408 (14), 407 (77), 363 (11), 362 (50), 315 (17), 257 (12), 165 (14), 109 (11), 107 (15), 106 (100), 93 (25), 81 (11), 71 (12), 57 (21).
Triethylphosphite (29.9 g, 180.0 mmol) was added to 15.3 g (60.0 mmol) of 2,6-di-tert-butyl-4-chloromethyl-phenol. The mixture was heated at 120° C. for 3 h then, the excess of reagent and side products were removed under vacuum. The crude diethyl 1-(3,5-di-tert-butyl-4-hydroxy-phenyl)-methylidene-1-phosphonate (22.8 g, yield >99%) was used without further purification (GC>96%). Trifluoromethanesulfonyl chloride was added to a suspension of NaH (2.1 g, 51.2 mmol, 60% dispersion in mineral oil) in 75 ml Et2O at −40° C. (CH3CN/CO2). A solution of 6.62 g (39.4 mmol) of diethyl hydroxymethylphosphonate in 15 ml Et2O was added dropwise at the previous mixture kept at <−30° C. The resulting mixture was stirred at −40° C. for 2 h, then poured into a saturated NaHCO3 solution and extracted with CH2Cl2 and washed with water, saturated NaCl solution and dried over MgSO4. The crude diethyl phosphonomethyltriflate (12.93 g, yield >99%) obtained after solvent removal was used without further purification (GC>99%).
A volume of 35 ml of a 1.6M solution of n-butyl lithium in hexane (55.5 mmol) were added to 75 ml THF kept at −78° C. Diisopropyl amine (5.6 g, 55.5 mmol) was added, the mixture was stirred for 15 min at −78° C., then a solution of 7.9 g (22.2 mmol) of diethyl 1-(3,5-di-tert-butyl-4-hydroxy-phenyl)-methylidene-1-phosphonate in 25 ml THF was added dropwise. After a further 15 min stirring at −78° C. diethyl phosphonomethyltriflate (7.0 g, 23.3 mmol) was added and the resulting mixture was stirred at −78° C. for 1h. The cooling bath was removed, the mixture allowed to warm at room temperature. 100 ml of HCl 10% were then added and the mixture taken up into CH2Cl2 and washed with water, saturated NaCl solution and dried over MgSO4. The residue (17.8 g) obtained after solvent removal was purified by flash chromatography (Silica gel 60, AcOEt:MeOH 50:50, Rf 0.58) to give 8.2 g (16.2 mmol, C24H44O7P2, MW=506.56, oil, yield 72.97%) of tetraethyl 1-(3,5-di-tert-butylhydroxy-phenyl)-ethylidene-1,2-bisphosphonate.
1H-NMR (CDCl3, 500 MHz)
δ=7.16, d, 2H, J=2.2; δ=5.15, s, 1H; δ=4.13-3.47, m, 4×2H; δ=3.41-3.31, m, 1H; δ=2.47-2.36, m, 2H; δ=1.42, s, 6×3H; δ=1.30, t, 3H, J=7.1; δ=1.10, t, 3H, J=7.1; δ=1.04, t, 3H, J=7.0; δ=1.00, t, 3H, J=7.1.
MS (70 eV)
507 (31), 506 (M+, 100), 505 (8), 492 (6), 491 (21), 465 (6), 464 (23), 426 (13), 370 (22), 369 (100), 368 (8), 313 (20), 269 (11), 266 (10), 241 (8), 232 (6), 201 (9), 109 (8), 57 (32).
A. Methods
THP-1 cell line (ATCC TIB-202) are cultured in RPMI 1640 (with 2 mM of L-glutamine and 2 g/l of glucose, Invitrogen) supplemented with 2 g/l of sodium hydrogen carbonate (Fluka), 10 U/ml of penicillin, 10 μg/ml of streptomycin (Penicillin-Streptomycin, Invitrogen), 20 μM of 2-mercaptoethanol (Fluka) and 10% FCS (Amimed). Cells are incubated at 37° C. and 5% CO2.
Cells are seeded in 24 well tissue culture plates (Falcon), 2×105 cells in 500 μl of culture medium per well or 6 well plates for RT-PCR purpose, 1×106 cells in 2.5 ml of medium. PMA (Phorbol 12-myristate 13-acetate, Alexis), (stock in DMSO (Fluka) at 1 mg/ml) and other compounds are diluted in ethanol and added to the cells at an ethanol final concentration not exceeding 1%. Same volume of ethanol is added in controls. Plates are incubated for 3 days at 37° C. and 5% CO2 to allow cell differentiation and apoE secretion.
Medium contained in the wells is harvested and centrifuged 5 min at 1200 rpm. Supernatants are store at −20° C. until apoE quantification. The cell pellets are resuspended in PBS and counted with a ZI Coulter Counter. After a wash with PBS, adherent cells in the well bottoms are detached with trypsin-EDTA solution 10× (Invitrogen) diluted in PBS. The reaction is stopped with the addition of culture medium and the detached cells are counted.
ApoE was quantified by ELISA. 96 well plates (Costar) are coated with 5% of gelatine (from porcine skin, Fluka) in carbonate-bicarbonate buffer (Sigma) for 1 hour at 37° C. After removing the coating solution, 100 μl of THP-1 supernatant are added per well, diluted 5 times in buffer (PBS, 1% Top-Block (Juro), 0.1% Tween 20 (Fluka)). The incubation last 1 hour at 37° C., then the wells are washed 3 times with 200 μl of buffer. Plates are incubated for 1 hour at 37° C. with continuous stirring with the primary antibody (goat anti-human apoE IgG, Calbiochem) at a 10000-time dilution in buffer, 100 μl per well. After 3 washes, plates are incubated with the secondary antibody (rabbit anti-goat-IgG peroxidase conjugated, Sigma) diluted 5000 times, 100 μl per well, at 37° C. with shaking. Then the wells are washed 5 more times and the detection is achieved by adding 100 μl per well of o-phenylenediamine dihydrochloride (Sigma) and incubated for 15-20 minutes with shaking at room temperature. When the appropriated colour is reached, the reaction is stopped by adding 50 μl per well of 3 M sulfuric acid (Fluka) with shaking for 1 minute at room temperature. The absorbance is read at 492 nm versus 620 nm with a microplate photometer (Anthos Reader 2001).
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/14727 | 5/9/2003 | WO | 10/21/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60379835 | May 2002 | US |
