CN1216921A - Method of inhibiting vascular cell adhesion molecule-1 and treating chronic inflammatory diseases with 2,6-di-alkyl-4-silyl-phenols - Google Patents

Abstract

Disclosed is a methods useful for inhibiting VCAM-1 and for treating chronic inflammatory conditions in a patient in need thereof comprising administering to the patient effective amounts of a compound of formula (1) wherein R1, R2, R3 and R4 are each independently a C1-C6 alkyl group; Z is a thio, oxy or methylene group; A is a C1-C4 alkylene group; R5 is a C1-C6 alkyl or -(CH2)n-(Ar) wherein n is an integer 0, 1, 2 or 3; and Ar is phenyl or naphthyl unsubstituted or substituted with one to three substituents selected from the group consisting of hydroxy, methoxy, ethoxy, chloro, fluoro or C1-C6 alkyl.

Description

Induction of vascular cell adhesion molecule-1 by 2,6-dialkyl-4-silyl-phenol and method of treating chronic inflammatory diseases

Background of the invention

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules in the immunoglobulin superfamily, which are regulated by the following cytokines in the inner wall of blood vessels and smooth muscle cells Upregulates, for example, interleukin-1 (IL-1), interleukin-4 (IL-4) and tumor necrosis factor- _α (TNF- _α ). Through interactions with integrin counter-receptors, VCAM-1 and ICAM-1 mediate leukocyte adhesion and transinterior migration during inflammatory responses. VCAM-I and/or ICAM-I inhibitors may be useful in the treatment of many types of chronic inflammatory disorders, including asthma, rheumatoid arthritis, and autoimmune diabetes. For example, increased expression of VCAM-1 and ICAM-1 is known in asthmatic patients. Pilewski, JM et al., Am. Respiratory Cell Mol. Biol., 12, 1-3 (1995); Ohkawara, Y. et al., Am. Respiratory Cell Mol. Biol., 12, 4-12 (1995). In addition, blockade of integrin receptors for VCAM-1 and ICAM-1 (VLA-4 and LFA-1, respectively) simultaneously inhibited the early stages of allergic airway response in an ovalbumin-sensitized rat model and late reactions. Rabb, HA, et al., American Respiratory Care Medicine, 149, 1186-1191 (1994).

Furthermore, VCAM-1 as a mediator has also been implicated in other chronic inflammatory disorders, including, for example, rheumatoid arthritis and autoimmune diabetes. For example, in the microvessels of patients with rheumatoid arthritis, increased expression of inner wall adhesion molecules, including VCAM-1, was found. Koch, A.E. et al., Experimental Research 64, 313-322 (1991); Morales-Ducret, J. et al., Immunology 149, 1421-1431 (1992). Neutralizing antibodies against VCAM-1 or its counter-receptor, VLA-4 delayed the onset of diabetes in a naturally occurring mouse model (NOD mice). Yang, X.D. et al., Proceedings of the National Academy of Sciences USA 90, 10494-10498 (1993); Burkly, L.C., et al., Diabetes 43, 523-534 (1994); Raron, J.L., et al., J. Clin. Invest. 93, 1700-1708 (1994). Monoclonal antibodies to VCAM-1 also had a beneficial effect in animal models of allograft rejection, suggesting that inhibitors of VCAM-1 expression could be useful in preventing graft rejection. Orocz, C.G. et al., Immunology Letters 32, 7-12 (1992).

VCAM-1 is expressed by cells in a membrane-bound and soluble form. A soluble form of VCAM-1 has been shown to induce chemotaxis of vascular endothelial cells in vivo, as well as stimulate angiogenic effects in rat cornea. Koch, A.E. et al., Nature 376, 517-519 (1995). Inhibitors of soluble VCAM-1 expression have potential therapeutic value in the treatment of diseases with a strong angiogenic component, including tumor growth and metastasis. Folkman, J. and Shing, Y., J. Biol. Chem. 10931-10934 (1992).

The promoters of VCAM-1 and ICAM-1 have been cloned and characterized. For example, both promoters contain various DNA sequence elements that bind the transcription factor NF-kB. Iademarco, M.F. et al., J. Biol. Chem. 267, 16323-16329 (1992); Voraberger et al., J. Immunol. 147, 2777-2786 (1991). The NF-kB family of transcription factors plays a major role in the regulation of several genes that are upregulated in inflammatory sites. The activity of NF-kB as a transcription factor is associated with dissociation from the inhibitory subunit IkB in the cytoplasm. The NF-kB subunit translocates to the nucleus, binds to specific DNA sequence elements, and reactivates the transcription of several genes, including VCAM-1 and ICAM-1. Collins T. et al., Experimental Research 68, 499-508 (1993).

It has been postulated that regulation of the expression of the VCAM-1 gene can be coupled to oxidative stress through specific attenuation-oxidation (redox)-sensitive transcriptional or post-transcriptional regulators. The antioxidants pyrrollidine dithiocarbamate and N-acetylcysteine could inhibit the expression of the induced cytokine VCAM-1, but not the expression of ICAM-1 in vascular endothelial cells. Mauri, N. et al., J. Clin. Invest. 92, 1866-1874 (1993). This suggests that VCAM-1 expression can be inhibited without modulating ICAM-1 expression by antioxidants including certain additional factors.

Parker et al. (US Patent No. 5,155,250, issued October 13, 1992) disclose that 2,6-dialkyl-4-silyl-phenols are useful as antiatherogenic agents. Furthermore, 2,6-dialkyl-4-silyl-phenols are disclosed in PCT International Publication (WO95/15760, published June 15, 1995) as serum cholesterol lowering agents.

Controlling VCAM-1 release and treating VCAM-1 mediated effects would be beneficial. It would also be beneficial to control or treat VCAM-1 inflammation without the attendant side effects associated with the known use of anti-inflammatory steroids and anti-inflammatory non-steroidal agents.

Summary of the invention

Compounds corresponding to formula (1) have now been found:

in

R ₁ , R ₂ , R ₃ and R ₄ groups are each C ₁ -C ₆ alkyl;

Z is sulfur, oxygen or methylene;

A is C ₁ -C ₄ alkylene;

R ₅ is C ₁ -C ₆ alkyl or -(CH ₂ ) _n -(Ar) group, where n represents an integer of 0, 1, 2 or 3; and Ar represents unsubstituted phenyl or naphthyl , or phenyl or naphthyl substituted by one to three of the following groups: hydroxyl, methoxy, ethoxy, chlorine, fluorine, or C ₁ -C ₆ alkyl, which can be used to inhibit Cytokine-induced VCAM-1 expression. Such compounds can be administered to a patient to inhibit VCAM-1; to inhibit or treat VCAM-1-mediated effects; and to inhibit or treat VCAM-1-mediated inflammation. The compounds can be administered to inhibit or treat VCAM-1 mediated effects in conditions such as chronic inflammation, asthma, rheumatoid arthritis and autoimmune diabetes.

Brief description of the drawings

Figure 1 illustrates the effect of 2,6-di-tert-butyl-4-[(xylylsilyl)methylthio]phenol (MDL29,353) on VCAM-1 expression in LPS-induced rabbit aorta in vivo effects. Data are expressed as percentage of aortic surface endothelium expressing VCAM-1, as measured by immunostaining with anti-rabbit VCAM-1 antibody.

Detailed description of the invention

As used herein, the term "C ₁ -C ₆ alkyl" refers to a saturated hydrocarbon group consisting of one to six carbon atoms in linear, branched and cyclic configurations. Included within the scope of this term are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclo Hexyl and its analogs.

Likewise, the term "C ₁ -C ₄ alkylene" refers to a saturated hydrocarbon group consisting of one to four carbon atoms in straight chain, branched chain and cyclic configuration. Included within the scope of this term are methylene, 1,2-ethane-diyl, 1,1-ethane-diyl, 1,3-propane-diyl, 1,2-propane-diyl, 1,3-butane-diyl, 1,4-butane-diyl and the like.

In those cases where R ₅ is a -(CH ₂ ) _n -(Ar) group, the "-(CH ₂ ) _n -" moiety represents a saturated straight-chain configuration of a hydrocarbon group. The term "n" is defined as the integer 0, 1, 2 or 3. Thus, the "-(CH ₂ ) _n -" moiety represents a bond, methylene, 1,2-ethanediyl or 1,3-propanediyl. And the "-(Ar)" moiety represents an aromatic group defined by substituted or unsubstituted phenyl or naphthyl. In those cases where the -(Ar) group is a substituted aromatic group, any position of its phenyl or naphthyl group can bear 1 to 3 substituents unless its position is substituted by a hydrogen atom. The substituents are selected from the following groups: hydroxy, methoxy, ethoxy, chloro, fluoro and C ₁ -C ₆ alkyl. Specifically included within the term "-(CH ₂ ) _n -(Ar)" are phenyl; naphthyl; benzyl; phenethyl; 3,4,5-trihydroxyphenyl; Oxyphenyl; 3,4,5-triethoxyphenyl; 4-chlorophenyl; 4-methylphenyl; 3,5-di-tert-butyl-4-hydroxyphenyl; 4-fluorobenzene Base; 4-chloro-1-naphthyl; 2-methyl-1-naphthylmethyl; 2-naphthylmethyl; 4-chlorobenzyl; 4-tert-butylphenyl; 4-tert-butyl Benzyl and its analogs.

Compounds of formula (1) can be prepared by employing known methods and techniques and will be apparent to those of ordinary skill in the art. A general synthetic scheme for the preparation of compounds of formula (1) is given in Scheme A, wherein Z represents sulfur or oxygen; and unless otherwise indicated, all substituents are as previously defined.

Option A

2 3 3 1a

Z'=S or O

x=chlorine, bromine, or iodine,

In general, phenols of structure 1a can be synthesized by combining an appropriate 2,6-dialkyl-4-mercaptophenol or 2,6-dialkylhydroquinone (or a suitable protecting group-bearing derivative) of structure 2 with A non-nucleophilic base such as sodium hydride, potassium carbonate or cesium carbonate, and a suitable haloalkylene silane (such as a suitable chloroalkylene silane) of structure 3 is prepared by reacting in a suitable aprotic in a solvent such as acetonitrile, N,N-dimethylformamide or N,N-dimethylacetamide, or in an aqueous solution such as water/2-butanone.

The starting materials employed in the general synthetic methods outlined in Scheme A are relatively readily available to those of ordinary skill in the art. For example, certain phenolic starters (where Z is sulfur, such as 2,6-di-tert-butyl-4-mercaptophenol) of various compounds of formula (1) are disclosed in U.S. Patent No. 3,576,883, No. 3,952,064, No. Both are described in Patent No. 3,479,407 and Japanese Patent Application No. 73-28425. Also, silyl starting materials of various compounds of formula (1) such as (trimethylsilyl)-methyl iodide, (trimethylsilyl)methyl bromide, (trimethylsilyl) ) methyl chloride, (1-chloropropyl)trimethylsilane, as described in Synthesis 4, 318-19 (1988) and J. Amhem. Societies 105, 5665-75 (1983). Other methods for preparing suitable silanes include Grignard reactions, such as the reaction of 4-bromoanisole with magnesium metal to form a Grignard reagent, which is then reacted with chlorodimethylchloromethylsilane to give chlorine Methyldimethyl-4-methoxyphenylsilane.

In addition, anisole can be lithiated by reacting with Δ-butyllithium, and the formed lithium compound is then reacted with chlorodimethylsilane to obtain chloromethyldimethyl-2-methoxyphenylsilane.

In those instances where the 1-phenol function of a compound of structure 2 can be reacted with a compound of structure 3 under the conditions specified for this reaction, the compound of structure 2 can be protected with standard phenol protecting agents well known and commonly used in the art. 1-phenol functional group. The selection and utilization of particular protecting groups is well known to those of ordinary skill in the art. In general, the selected protecting group should sufficiently protect the mentioned phenol during subsequent synthetic steps and be easily removed under conditions that do not degrade the desired product.

Examples of suitable phenol protecting groups are ethers such as methoxymethyl, 2-methoxyethoxymethyl, tetrahydropyranyl, tert-butyl and benzyl; silyl ethers such as tris Methylsilyl and tert-butyldimethylsilyl; esters, such as acetate and benzoate; carbonates, such as methylcarbonate and benzylcarbonate; and sulfonic acids Salts such as methanesulfonate and toluenesulfonate.

In those instances where R ₁ , R ₂ are each t-butyl, the reaction of Scheme A can be conveniently carried out without protection of the 1-phenol function.

The following examples represent typical syntheses described in Scheme A. These examples are to be understood as illustrative only and are not intended to limit the scope of the invention in any way. As used herein, the following terms have specific meanings: "g" means gram; "mmol" means millimole; "mL" means milliliter; "bp" means boiling point; "mmHg" refers to millimeters of mercury; "mp" refers to melting point; "mg" refers to milligrams; "μM" refers to micromoles; "μg" refers to micrograms.

Example 1

2,6-Di-tert-butyl-4-[(xylylsilyl)methylthio]phenol (MDL29,353)

Mix 2,6-di-tert-butyl-4-mercaptophenol (2.4 g, 10 mmol), potassium carbonate (1.4 g, 10 mmol) and chloromethylxylylsilane (1.9 g, 10 mmol ) and N,N-dimethylformamide (50 mL), and stirred overnight at room temperature under argon. Dilute with ice-water and extract the mixture with ether. The ether layer was washed with water then brine, filtered through flourosil-sodium sulfate and evaporated to an orange oil (3.5 g). The product was purified by a first distillation (boiling point 160-170° C. @ 0.1 mmHg), followed by silica gel chromatography (CCl ₄ :CHCl ₃ /1:1) to give the title compound as a light yellow oil, which Will slowly crystallize to form a white waxy solid (2.3 g, 59%)

Analysis _{for C23H34OSSi} : Calcd: C, 71.44; H, 8.86; S, 8.29;

Found: C, 71.14; H, 8.86; S, 7.98.

Example 2

2,6-Di-tert-butyl-4-[(trimethylsilyl)methylthio]phenol (MDL28,235)

Mix 2,6-di-tert-butyl-4-mercaptophenol (2.4 g, 10 mmol), potassium carbonate (1.4 g, 10 mmol), and N,N-dimethylacetamide (50 mL) , and stirred at room temperature under argon. Chloromethyltrimethylsilane (1.3 g, 10 mmol) was added and stirred overnight. Heat on a steam bath for 2 hours, cool and dilute with water. Extraction with ether, drying and evaporation gave a pale yellow solid (2.8 g), which was finally recrystallized ( _CH3CN ) to give 1.1 g (34%) of the title compound, mp 100-101°C.

Analysis _{for C18H32OSSi} : Calcd: C, 66.60; H, 9.88; S, 9.88;

Found values: C, 66.83; H, 10.05; S: 9.91.

Example 3

2,6-Dimethyl-4-[(trimethylsilyl)methoxy]phenol

Mix 2,6-dimethylhydroquinone (1.4 g, 10 mmol), potassium carbonate (1.4 g, 10 mmol), chloromethyltrimethylsilane (1.9 g, 10 mmol) and N,N- Dimethylformamide (50ml). Stir at room temperature under an inert atmosphere until the reaction is complete. Dilute with ice-water and extract the mixture with ether. The ether layer was washed with water then brine and filtered through flourosil-sodium sulfate. Then, evaporate to give the title compound, which is purified by silica gel chromatography.

Example 4

2,6-Di-tert-butyl-4-[(4-chlorophenyldimethylsilyl)methoxy]phenol (MDL104,280)

2,6-di-tert-butylbenzohydroquinone (13.7 g, 61.6 mmol), potassium carbonate (9.4 g, 68 mmol), chloromethyl (4- Chlorophenyl)dimethylsilane (14.9 g, 68 mmol) and catalytic amount of potassium iodide for three days. The solid was filtered and evaporated. Redissolved in ethyl acetate and washed with water and brine, then dried over anhydrous magnesium sulfate, filtered and evaporated. The resulting orange oil was purified by distillation to 135°C@0.1 mmHg to remove low-boiling impurities, followed by distillation of the product (boiling point: °C@0.1 mmHg). The product which crystallized upon standing was recrystallized from hexane to give fine white needles (7.4 g, 27% yield). Melting point: 102-105 degrees Celsius.

Analysis _{for C23H33ClO2Si} : Calcd.: _C , 68.20; H, 8.21

Found values: C, 68.39; H, 8.13

NMR (CDCl ₃ ): 7.53 (d, 2H, J8.3), 7.34 (d, 2H, J8.3), 6.79 (s, 2H), 4.73 (s, 1H), 3.71 (s, 2H), 1.42 ( s,18H), 0.41(s,6H).

Example 5

2,6-Di-tert-butyl-4-[(dimethyl-4-fluorophenylsilyl)methoxy]phenol (MDL104,389)

2,6-di-tert-butylbenzohydroquinone (10.0 g, 45 mmol), potassium carbonate (6.2 g, 45 mmol) and dimethyl (4- Fluorophenyl)iodomethylsilane (13.2 g, 45 mmol) for three days. The solid was filtered and evaporated. Redissolved in ethyl acetate and washed with water and brine, then dried over anhydrous magnesium sulfate, filtered and evaporated to a very pale yellow oil which crystallized on standing. This material was recrystallized from methanol to give a white crystalline solid (5.9 g, 34% yield). Melting point: 90-93 degrees Celsius.

Analysis for _C23H33FOSi : Calcd _: C, 71.09; H, 8.86

Measured value: C, 70.96; H, 8.58

NMR(CDCl ₃ ): 7.58(dd,2H,J8.5,6.2),7.10-7.04(m,2H),6.80(s,2H),4.73(s,1H),3.71(si,2H),1.43 (s,18H),0.41(s,6H).

Example 6

2,6-Di-tert-butyl-4-[(xylylsilyl)methoxy]phenol (MDL103,902)

2,6-Di-tert-butylbenzohydroquinone (5.43 g, 24.4 mmol), potassium carbonate (3.7 g, 26.8 mmol) and dimethyl(iodo Methyl)phenylsilane (7.4 g, 26.8 mmol) overnight. The solid was filtered and evaporated. Redissolved in ethyl acetate and washed with water and brine, then dried over anhydrous magnesium sulfate, filtered and evaporated. The resulting orange oil was purified by distillation to 135°C @ 0.1 mmHg to remove low-boiling impurities, followed by distillation of the product (boiling point: 155-165°C @ 0.1 mmHg). The product which crystallized upon standing could be recrystallized from methanol to give a white solid (5.8 g, 64% yield). Melting point: 82-83 degrees Celsius.

Analysis _{for C23H34O2Si} : Calcd: _C , 74.54; H, 9.25

Found values: C, 74.51; H, 9.28

NMR(CDCl ₃ ):7.64-7.58(m,2H),7.42-7.32(m,2H),6.80(s,2H),4.72(s,1H),3.73(s,2H),1.42(s,18H ), 0.42(s,6H).

Example 7

2,6-Di-tert-butyl-4-[(dimethyl-4-methoxyphenylsilyl)methoxy]phenol (MDL105,074)

2,6-Di-tert-butylbenzohydroquinone (13.7 g, 61.1 mmol), potassium carbonate (9.4 g, 68 mmol), chloromethyl (dimethyl yl)-4-methoxyphenylsilane (14.6 g, 68 mmol) and a catalytic amount of potassium iodide for three days. The solid was filtered and evaporated. Redissolved in ethyl acetate and washed with water then brine, then dried over anhydrous magnesium sulfate, filtered and evaporated. The resulting orange oil was purified by distillation to 135°C @ 0.1 mmHg to remove low-boiling impurities, followed by distillation of the product (boiling point: 155-165°C @ 0.1 mmHg). The product which crystallized upon standing was recrystallized from hexane to give a white solid (4.9 g, 19% yield). Melting point: 122-123 degrees Celsius.

Analysis _{for C24H36O3Si} : Calcd: C, 71.95; H, _9.06

Measured value: C, 71.80; H, 9.00

NMR(CDCl ₃ ): 7.53(d,2H,J8.6),6.93(d,2H,J8.6),6.80(s,2H),4.71(s,1H),3.81(s,3H),3.70 (s,2H), 1.42(s,18H), 0.39(s,6H).

Example 8

2,6-Dimethyl-4-[(xylylsilyl)methoxy]phenol (MDL103,719)

2,6-Dimethylhydroquinone (10.0 g, 72.4 mmol), potassium carbonate (10.0 g, 72.4 mmol), and dimethyl(chloromethyl)benzene were refluxed under argon in acetonitrile (150 mL). oxysilane (13.4 g, 72.4 mmol) for 72 hours. After cooling the mixture was diluted with water and extracted with ether. The resulting oil was distilled at 145 to 160°C @ 0.1 mmHg to obtain 4.9 g of a pale yellow oil.

Analysis _{for C17H22O2Si} : Calcd: _C , 71.28; H, 7.74

Found: C, 71.27; H, 7.74.

Example 9

2-tert-Butyl-6-methyl-4-[(xylylsilyl)methylthio]phenol (MDL104,518)

2-tert-butyl-6-methyl-4-mercaptophenol (11.8 g, 60.1 mmol), potassium bicarbonate (6.0 g, 11.8 mmol) and Dimethyl(chloromethyl)phenylsilane (11.1 g, 60.1 mmol) 24 hours. After cooling the mixture was diluted with water and extracted with ether. Evaporation of the ether layer to dryness gave 21.9 g of an oil. The oil was distilled at 145-160°C (0.1 mmHg) to obtain 5.5 g of light yellow oil.

Analysis _{for C20H28OSSi} : Calcd: C, 69.71; H, 8.19

Found values: C, 69.76, H, 8.20.

Example 10

2,6-Di-tert-butyl-4-[(dimethyl-2-methoxyphenylsilyl)-methoxy]phenol (MDL104,036)

A mixture of chloromethyldimethyl(2-methoxy)phenylsilane (27.2 g, 0.127 mol), sodium iodide (19 g, 0.127 mol) and acetonitrile (350 mL) was heated to reflux for 28 hours. The mixture was cooled to ambient temperature and 2,6-di-tert-butyl-1,4-hydroquinone (31.5 g, 0.14 mol) and potassium carbonate (20.8 g, 0.15 mol) were added. The mixture was refluxed under nitrogen for 7 days. The mixture was cooled, poured into water (400 mL) and ethyl acetate (400 mL) and the organic layer was separated. The organic layer was evaporated and the residue was chromatographed on silica gel (hexane/ethyl acetate: 9/1). Recrystallization (methanol) of the chromatographic product gave the product as a white solid (15.6 g, 31%). Melting point: 89-90 degrees Celsius.

Analysis for _C24H36O3Si : Calcd: _C , 71.95; H, _9.06

Found: C, 71.84; H, 9.05.

Example 11

2,6-Di-tert-butyl-4-[(dimethyl-2,5-dimethoxyphenylsilyl)-methoxy]phenol (MDL103,016)

Preparation was carried out as in the previous compound using chloromethyldimethyl-2,5-dimethoxy-phenylsilane (14 g, 57 mmol) as the silane to give a white solid. Melting point: 103-104 degrees Celsius.

Analysis _{for C25H38O4Si} : Calculated: C, 69.72; H, _8.89

Found: C, 69.71; H, 8.72.

Example 12

2,6-Di-tert-butyl-4-[(dimethyl-2,3-dimethoxyphenylsilyl)methoxy]phenol (MDL108,750)

Prepared as above using chloromethyl(dimethyl)-2,3-dimethoxyphenylsilane (11.3 g, 46 mmol) as the silane to give a white solid. Melting point: 94.5-96 degrees Celsius.

Analysis _{for C25H38O4Si} : Calculated: C, 69.72; H, _8.89

Found: C, 69.84; H, 8.91.

Example 13

2,6-Di-tert-butyl-4-[(dimethyl-4-tert-butylphenylsilyl)-methoxy]phenol (MDL106,630)

Prepared as above using 4-tert-butylphenylchloromethyldimethylsilane (6.2 g, 25.7 mmol) as the silane to give a white solid. Melting point: 114-115 degrees Celsius.

Analysis _{for C27H42O2Si} : Calculated: _C , 76.00; H, 9.92

Found: C, 75.94; H, 10.13.

Example 14

2,6-Di-tert-butyl-4-[(benzyldimethylsilyl)methoxy]phenol (MDL107,411)

Prepared as above using benzylchloromethyldimethylsilane (7.13 g, 35.9 mmol) as the silane to give a white solid. Melting point: 76-77 degrees Celsius.

Analysis _{for C24H36O2Si} : Calcd: C, 74.95; H, _9.43

Found: C, 74.94; H, 9.36.

Example 15

2,6-Di-tert-butyl-4-[(dimethyl-p-methoxybenzylsilyl)methoxy]phenol (MDL108,816)

Step a; Preparation of Dimethyl-p-Methoxybenzyl-Chloromethylsilane: Magnesium paraffin (9.7 g, 0.4 gram atom) was stirred overnight in a Teflon(R) stirrer under nitrogen. The "activated" magnesium was suspended in dry THF (100 mL), and iodine crystals were added. A solution of p-methoxy-benzyl bromide (80.8 g, 0.4 mol) in THF (400 ml) was added at a rate to keep the solution under gentle reflux. Once the addition is complete, continue stirring (1-2 hours) until almost all the magnesium is consumed. A solution of dimethylchloromethylchlorosilane (52.7 mL, 0.4 mol) in THF (200 mL) was added dropwise, and the mixture was stirred at room temperature overnight. The reaction mixture was quenched with saturated aqueous ammonium chloride (500 mL), then stirred at room temperature (-2 hours). The precipitated magnesium salt was filtered and diluted with ether (300 mL). The organic phase was separated, washed with water (3 x 250 mL), saturated aqueous sodium chloride (3 x 250 mL), then dried over anhydrous magnesium sulfate, filtered and evaporated. The resulting brown oil was purified by distillation to obtain the title compound. Yield: 55%, Boiling point: 110-115 degrees Celsius at 5 mm Hg.

Analysis _{for C10H15ClOSi} : Calculated: C, 55.93, H7.15.

Found values: C, 55.40, H, 7.15.

Step b; Preparation of 2,6-di-tert-butyl-4-[(dimethyl-p-methoxybenzyl-silyl)methoxy]phenol (MDL108,816): in acetonitrile ( 250 mL), dimethyl-p-methoxybenzyl-chloromethylsilane (28 g, 0.13 mol), sodium iodide (0.5 g, catalytic amount) and 2,6-di-tert-butyl A mixture of benzylhydroquinone (23 g, 0.1 mol) and cesium carbonate (32 g, 0.1 mol) was used for 6 days. After cooling, a mixture of water and ethyl acetate (400 ml each) was injected. The organic layer was then separated, dried, evaporated and the residue was heated on a Kugelrohr apparatus at 110°C (0.1 mm) for 3 hours and at 140-160°C for a further 2 hours. The residue solidified on standing to give the product as a waxy solid (20.9 g, 39%). Melting point: 58-60 degrees Celsius.

Analysis _{for C25H38O3Si} : Calculated: C, 72.41, _H , 8.87.

Found values: C, 70.29, H, 8.96.

The following compounds were prepared by methods similar to those described above in Examples 1-14:

2,6-Di-tert-butyl-4-[(triethylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(diethylphenylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(tripropylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(dipropylphenylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(triisopropylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(diisopropylphenylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(tributylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(dibutylphenylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(triisobutylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(diisobutylphenylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(tri-tert-butylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(di-tert-butylphenylsilyl)methylthio]phenol

2,6-Di-methyl-4-[(trimethylsilyl)methylthio]phenol

2,6-Di-methyl-4-[(xylylsilyl)methylthio]phenol

2,6-Di-methyl-4-[(dibutylphenylsilyl)methylthio]phenol

2,6-Di-methyl-4-[(tri-tert-butylsilyl)methylthio]phenol

2,6-Di-methyl-4-[(di-tert-butylphenylsilyl)methylthio]phenol

2,6-Di-ethyl-4-[(trimethylsilyl)methylthio]phenol

2,6-Di-ethyl-4-[(xylylsilyl)methylthio]phenol

2,6-Di-ethyl-4-[(tri-tert-butylsilyl)methylthio]phenol

2,6-Di-ethyl-4-[(di-tert-butylphenylsilyl)methylthio]phenol

2,6-Di-propyl-4-[(trimethylsilyl)methylthio]phenol

2,6-Di-propyl-4-[(xylylsilyl)methylthio]phenol

2,6-Di-isopropyl-4-[(trimethylsilyl)methylthio]phenol

2,6-Di-isopropyl-4-[(xylylsilyl)methylthio]phenol

2,6-Di-butyl-4-[(trimethylsilyl)methylthio]phenol

2,6-Di-butyl-4-[(xylylsilyl)methylthio]phenol

2,6-Dimethyl-4-[(trimethylsilyl)methoxy]phenol

2,6-Dimethyl-4-[(xylylsilyl)methoxy]phenol

2,6-Dibutyl-4-[(triethylsilyl)methoxy]phenol

2,6-Dibutyl-4-[(diethylphenylsilyl)methoxy]phenol

2,6-Di-tert-butyl-4-[(trimethylsilyl)methoxy]phenol

2,6-Di-tert-butyl-4-[(xylylsilyl)methoxy]phenol.

A general synthetic scheme for the preparation of compounds of formula 1 wherein Z is methylene is set forth in Scheme B, where all substituents are as previously defined unless otherwise indicated.

Option B

                              1b

In general, phenols of structure 1b can be prepared according to the two-step process of Scheme B. In step a, a suitable haloalkylenesilane of structure 3 is reacted with metallic magnesium in a suitable aprotic solvent such as diethyl ether to form a magnesium halide. Magnesium halide (Grignard reagent) is then reacted with the appropriate 3,5-dialkyl-4-hydroxy-benzaldehyde of structure 4 (or a suitable protected derivative) to give the alcohol of structure 5. In step b, the alcohol of structure 5 is reduced to the desired phenol of structure 1b by a series of reduction techniques and methods well known and widely used in the art. For example, alcohols of structure 5 can be reduced by reacting them with sodium in liquid ammonia using Birch reduction.

The general synthetic procedures outlined in Scheme B employ starting materials that are readily available or can be readily prepared according to standard techniques and procedures. To prevent undesired side reactions from occurring, the 1-phenol function of the 3,5-dialkyl-4-hydroxy-benzaldehyde of structure 4 in Scheme B can be used as previously described in Scheme A, using standard The phenol protectant is protected before the Grignard reaction.

The following examples represent typical syntheses described in Scheme B. These examples are to be understood as illustrative only and are not intended to limit the scope of the invention in any way.

Example 16

2,6-Dimethyl-4-[2-(trimethylsilyl)ethyl]phenol

Step a: Magnesium paraffin (240 mg, 10 mmol) and anhydrous diethyl ether were mixed under an inert atmosphere. A solution of chloromethyltrimethylsilane (1.9 g, 10 mmol) in anhydrous ether was added. Stir until the magnesium metal dissolves. A solution of 3,5-dimethyl-4-hydroxybenzaldehyde (1.5 g, 10 mmol) in anhydrous ether was then added. Stir until reaction is complete. After cooling the reaction mixture to 0°C, saturated ammonium chloride solution was added. The ether layer was separated, washed with water and dried ( _MgSO4 ). Evaporation gives 4-hydroxy-3,5-dimethyl-a-[(trimethylsilyl)-methyl]benzyl alcohol, which is then purified by chromatography on silica gel.

Step b: Sodium metal (520 mg, 22.6 mmol) and liquid ammonia (13 mL) were mixed. To this solution was added dropwise 4-hydroxy-3,5-dimethyl-α-[(trimethylsilyl)-methyl]phenylmethanol (2.22 g, 10 mmol) Ethanol (0.5 g) and ether (5 ml) solution. After the blue color of the solution had disappeared, water (13 mL) was carefully added, extracted with ether, dried ( _MgSO4 ), and the solvent was evaporated. Purification of the residue by silica gel chromatography afforded the title compound.

Additionally, compounds of formula 1 wherein Z is methylene can be prepared according to the procedure outlined in Scheme C, wherein all substituents, unless otherwise indicated, are as previously defined.

Plan C

In general, phenols of structure 1b can be prepared by first reacting metallic magnesium with an appropriate silane of structure 3 in a suitable aprotic solvent such as diethyl ether to form a magnesium halide. This magnesium halide (Grignard reagent) is then reacted with the appropriate 3,5-dialkyl-4-hydroxy-benzyl halide (or a suitable protecting derivative) of structure 6 to give the desired structure Phenols of 1b.

The general synthetic procedures outlined in Scheme C employ starting materials that are readily available or can be readily prepared according to standard techniques and procedures. For example, the preparation of 3,5-dimethyl-4-acetoxy-benzyl bromide has been described in tetrahedron 33, 3097-103 (1977). 3,5-Dimethyl-4-acetoxy-benzyl bromide can be converted to the corresponding phenolic starting material by standard hydrolysis procedures.

To prevent undesired side reactions from occurring, the 1-phenol function of the 3,5-dialkyl-4-hydroxy-benzyl halide of structure 6 in Scheme C can be as previously described in Scheme A, Protect with standard phenol protectants prior to the Grignard reaction.

The following examples represent typical syntheses described in Scheme C. These examples are to be understood as illustrative only and are not intended to limit the scope of the invention in any way.

Example 17

2,6-Diethyl-4-[2-(trimethylsilyl)ethyl]phenol

Magnesium paraffin (240 mg, 10 mmol) and anhydrous diethyl ether were mixed under an inert atmosphere. A solution of chloromethyltrimethylsilane (1.9 g, 10 mmol) in anhydrous ether was added. Stir until the magnesium metal dissolves. A solution of 4-bromomethyl-2,6-diethylphenol (2.43 g, 10 mmol) in anhydrous ether was added, and the mixture was refluxed until the reaction was complete. A mixture of ice/hydrochloric acid was poured and the layers were separated. The ether layer was washed, dried ( _MgSO4 ) and evaporated to give the title compound which was purified by silica gel chromatography.

The following compounds can be prepared by similar procedures as described in Example 16 above:

2,6-Dipropyl-4-[2-(trimethylsilyl)ethyl]phenol

2,6-Dipropyl-4-[2-(xylylsilyl)ethyl]phenol

2,6-Diisopropyl-4-[2-(trimethylsilyl)ethyl]phenol

2,6-Diisopropyl-4-[2-(xylylsilyl)ethyl]phenol

2,6-Diisobutyl-4-[2-(trimethylsilyl)ethyl]phenol

2,6-Diisobutyl-4-[2-(xylylsilyl)ethyl]phenol

2,6-Dibutyl-4-[2-(trimethylsilyl)ethyl]phenol

2,6-Dibutyl-4-[2-(xylylsilyl)ethyl]phenol

2,6-Di-tert-butyl-4-[2-(trimethylsilyl)ethyl]phenol

2,6-Di-tert-butyl-4-[2-(xylylsilyl)ethyl]phenol

2,6-Di-tert-butyl-4-[2-(tri-tert-butylsilyl)ethyl]phenol

2,6-Di-tert-butyl-4-[2-(di-tert-butylphenylsilyl)ethyl]phenol

2,6-Dimethyl-4-[2-(trimethylsilyl)ethyl]phenol

2,6-Dimethyl-4-[2-(xylylsilyl)ethyl]phenol.

It will be appreciated that compounds of formula (1) may exist in various stereoisomeric forms. All stereoisomeric forms corresponding to the above formulas (interpreted according to standard conventions for representing stereoisomeric structures) are considered to be within the scope of the present invention.

Compounds of formula (1 ) such as 2,6-di-alkyl-4-silylphenols are well known in the art. Specifically, compounds of formula (1) have been described in US Patent No. 5,155,250. Preferred compounds of formula (1) are those wherein R ₁ and R ₂ are C ₄ alkyl, R ₃ and R ₄ are C ₁ alkyl, A is C ₁ alkylene, and R ₅ is -(CH ₂ ) _n -(Ar) group, where n represents 0 and Ar represents phenyl not substituted by substituents, or phenyl substituted by one to three groups in the following groups: hydroxyl, methoxy , ethoxy, chlorine, fluorine or C ₁ -C ₆ alkyl. More preferred is the compound 2,6-di-tert-butyl-4-[(xylyl-silyl)methyl]-thio-phenol.

As used herein, the term "patient" refers to a warm-blooded animal or mammal suffering from a particular VCAM-1 mediated inflammatory disease. It is understood that guinea pigs, dogs, cats, rats, mice, hamsters, rabbits and primates (including humans) are examples of patients within the scope of the term.

The term "chronic inflammatory disease" refers to a disease or condition characterized by persistent inflammation in the absence of an identifiable stimulus or microbial pathogen. Inflammatory diseases treated with compounds of formula (1) will be particularly useful in diseases including: asthma, chronic inflammation, rheumatoid arthritis, autoimmune diabetes, transplant rejection and tumor angiogenesis. Particularly preferred compounds of formula (1) in the treatment of inflammatory diseases in patients in need of such treatment include:

2,6-Di-tert-butyl-4-[(xylylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(trimethylsilyl)methylthio]phenol

2,6-di-tert-butyl-4-[(4-chlorophenyldimethylsilyl)methoxy]phenol;

2,6-Di-tert-butyl-4-[(dimethyl-4-fluorophenylsilyl)methoxy]phenol

2,6-Di-tert-butyl-4-[(xylylsilyl)methoxy]phenol

2,6-Di-tert-butyl-4-[(dimethyl-4-methoxyphenylsilyl)methoxy]phenol;

2,6-Dimethyl-4-[(xylylsilyl)methoxy]phenol

2-tert-butyl-6-methyl-4-[(xylylsilyl)methylthio]phenol

2,6-Di-tert-butyl-4-[(dimethyl-2-methoxyphenylsilyl)-methoxy]phenol

2,6-di-tert-butyl-4-[(dimethyl-2,5-dimethoxyphenylsilyl)-methoxy]phenol

2,6-Di-tert-butyl-4-[(dimethyl-2,3-dimethoxyphenylsilyl)-methoxy]phenol

2,6-Di-tert-butyl-4-[(dimethyl-4-tert-butylphenylsilyl)-methoxy]phenol

2,6-Di-tert-butyl-4-[(benzyldimethylsilyl)methoxy]phenol.

The "therapeutically effective dose" of the compound of formula (1) refers to the dose that can alleviate the symptoms associated with inflammatory diseases after single or multiple doses are administered to patients. The "effective vascular cell adhesion molecule-1 inhibitory amount" of the compound of formula (1) refers to the amount that can alleviate the symptoms related to the disease mediated by vascular cell adhesion molecule-1 after being administered to a patient in single or multiple doses. quantity. As used herein, "remission of symptoms" of an inflammatory disease or a disease mediated by vascular cell adhesion molecule-1 refers to lessening of symptom manifestations than expected without treatment and does not necessarily exhibit a cure or symptoms of the disease completely disappeared. Relief of symptoms also includes prophylaxis.

In determining a therapeutically effective amount or dosage, a number of factors need to be considered by the diagnostician, including but not limited to: the species of mammal; its size, age, and general health; the specific disease involved; the extent or Severity; individual patient response; administration of particular compounds; mode of administration; bioavailability characteristics of administered formulations; selected treatment regimens; use of concomitant medications; and other relevant circumstances.

A therapeutically effective amount of a compound of formula (1) generally varies from one milligram per kilogram of body weight per day (mg/kg/day) to five milligrams per kilogram of body weight per day (mg/kg/day). A preferred dosage is between about 1 mg/kg and 500 mg/kg per day. Likewise, effective VCAM-1 inhibitory amounts of compounds of formula (1) generally range from one milligram per kilogram of body weight per day (mg/kg/day) to five milligrams per kilogram of body weight per day (mg/kg/day). days) vary. A preferred dosage is between about 1 mg/kg and 500 mg/kg per day.

The compounds of the present invention are inhibitors of VCAM-1 expression. We believe that the compounds of the present invention exert inhibitory effects on VCAM-1 up-regulation through their cytokines, thereby preventing or alleviating the symptoms of inflammatory diseases, including asthma, chronic inflammation, rheumatoid arthritis, autoimmune diabetes and the like disease. It is to be understood, however, that the invention is not bound by any particular theory nor is it intended as a known mechanism to explain its effectiveness in an end use.

In the treatment of a patient, compounds of formula (1) may be administered in any form or manner that renders the compound bioavailable in effective amounts, including oral and parenteral routes. For example, the compounds can be administered orally, subcutaneously, intramuscularly, intravenously, intradermally, intranasally, rectally, and the like. Oral administration is generally preferred. Those skilled in the pharmaceutical arts will readily select the appropriate mode or form of administration depending on the disease state desired to be treated, the stage of the disease and other relevant circumstances. Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company (1990).

Administration of the compound of formula (1) can be carried out in the form of a pharmaceutical composition or medicament, and these medicines are made by combining the compound of formula (1) with acceptable pharmaceutically acceptable carriers or excipients, the ratio and characteristics of which are determined by the selected route of administration As well as standard pharmaceutical practice determinations.

The pharmaceutical composition or medicament is prepared in a manner well known in the field of pharmacy. Its carrier or excipient may be solid, semi-solid or liquid material (which may serve as a carrier or medium for the active ingredient). Suitable carriers or excipients are well known in the art. The pharmaceutical composition can be adapted for oral or parenteral use, and administered to patients in the form of tablets, capsules, suppositories, solutions, suspensions or the like.

The pharmaceutical composition can be taken orally, for example, with an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, compounds of formula (1) may be combined with excipients and formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, lozenges, chewable gums and the like form of use. These preparations should contain at least 4% of the compound of formula (1) (active ingredient), but this may vary according to the particular form and may conveniently vary between 4% and about 70% per unit weight. The active ingredient is present in the compositions in such an amount that a unit dosage form suitable for its administration will be obtained.

The tablets, pills, capsules, lozenges and the like may also contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or Lactose; disintegrants such as alginic acid, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; slip agents such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin may be added, or Flavoring agents, such as peppermint, methyl salicylate, or orange flavoring. If the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coatings. A syrup may contain (in addition to the active ingredients) sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. The materials used in preparing these various compositions should be of pharmaceutical purity and nontoxic in the amounts employed.

For the purpose of parenteral administration, compounds of formula (1) can be incorporated into solutions or suspensions. These preparations should contain at least 0.1% of the compound of the invention, but the amount incorporated can vary from 0.1% to 50%. The amount of active ingredient present in the composition should be appropriate.

The solution or suspension should also include one or more of the following adjuvants (depending on the solubility and other properties of the compound of formula (1): sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol Glycols, glycerin, propylene glycol or other synthetic solvents; antimicrobials such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, lemon salt or phosphate and agents such as sodium chloride or dextrose for toxicity adjustment. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Example 18

Cell Surface ELISA Assay for VCAM-1 and ICAM-1

Proliferating human umbilical vein endothelial cells (HUVEC) or human aortic smooth muscle cells (HASMC) (Clonetics, San Diego, CA) were plated at a density of 20,000 cells per square centimeter in 96-well plates containing 100 μL of medium per well. The cultures were maintained on growth medium (EGM or SMGM2, Clonetics, San Diego, CA) for two days before addition of cytokines or drugs. Cytokine plus or minus compounds were added 20 to 24 hours before the analysis of adhesion molecule levels. Tumor necrosis factor (Genzyme, Cambridge, MA) was added to the cultures at a concentration of 500-1000 units per milliliter. Interleukin-4 (GIBCO-BRL, Gaithersburg, MD) was added to the culture at a concentration of 100-200 pg per ml. [Additions were made by transferring 100 [mu]L of serially diluted cytokine plus compounds in separate 96-well plates to the plate containing the cells. The medium of the culture was not changed before addition of the effector]. The medium was removed while the monolayer was washed twice with Hanks buffered saline solution (HBSS) at room temperature. Add the primary antibody (anti-human VCAM-1 from Upstate Biotech, LakePlacid, NY or anti-human ICAM-1 from lmmunotech, Westbrook, ME) to each well (1 μg/ml of HBSS plus 5% Neonatal calf serum, GIBCO-BRL, Gaithersburg, MD) and incubated at 37°C for one hour. Wells were washed twice with HBSS, and 100 μL of a 1/1000 goat anti-mouse IgG diluted in HBSS and 5% newborn calf serum conjugated to horseradish peroxidase (BioRad, Hercules, CA) was added to each well solution and incubated at 37°C for one hour. Wells were washed three times with HBSS, followed by the addition of 100 μL of TMB substrate (BioRad, Hercules, CA) to each well. Add 50 μL of 1N sulfuric acid to end the reaction after turning blue. Absorbance was measured at 450 nm with a plate reader. _IC50 values were determined from absorbance curves obtained from serially diluted compounds (dissolved in dimethylsulfoxide).

The _IC50 value is defined as the concentration of drug that inhibits cytokine-induced expression of adhesion molecules by 50%. The maximum expression of adhesion molecules in cytokine-induced cultures was subtracted from the basal level of adhesion molecule expression in the cultures to determine the level of induction. Typically VCAM-1 is induced about 5-7 fold. ICAM-1 is typically induced about 5-10 fold. The concentration of each drug was measured in quadruplicate wells. Single point assays of compounds at 50 [mu]M were determined as for _IC50 determinations, except that data represent levels of inhibition uncorrected for basal expression. (Basal adhesion molecule expression accounts for 10-20% of total induced expression).

Table 1 summarizes the ability of various compounds of the invention to inhibit VCAM-1 [by using human aortic smooth muscle cells (HASMC)]. In these experiments, cells were incubated with interleukin-4 and the listed compounds for approximately 20 hours prior to measuring cell surface VCAM-1 levels. Each column represents an individual experiment.

Table 1 Compound number (MDL number) HSMC-1 (% inhibition 50μM) HSMC-2 (% inhibition 50μM) HSMC-3 (% inhibition 50μM) VCAM-1 (average) 28,235 7.8 18.0 42.0 22.6 29,353 58.0 60.0 46.0 54.7 103,719 49.0 42.0 45.5 103,902 49.0 60.0 43.0 50.7 104,280 54.7 63.0 44.0 53.9 104,518 4.4 47.0 26.0 25.8 105,074 28.3 52.0 47.0 42.4

Table 2 summarizes the ability of various compounds of the invention to selectively inhibit VCAM-1 or both VCAM-1 and ICAM-1 [by using proliferating human umbilical vein endothelial cells (HUVEC)]. In these experiments, cells were incubated with tumor necrosis factor-alpha and the indicated compounds for approximately 20-24 hours prior to measuring cell surface adhesion molecule expression.

Table 2 Compound number (MDL number) VCAM-1 (% inhibition 50 μM) ^* ICAM-1 (% inhibition 50μM)@ 28,235 4.3 (3.0) 29,353 8.7 6.0 103,719 17.0 77.0 103,902 25.3 39.0 104,280 27.3 22.0 104,518 (1.0) 78.0 105,074 20.0 (8.0)

*: Refers to the average of three determinations

@: Refers to the average value of the second measurement

Table 3 describes the activity of selected compounds tested in eight serial dilutions for cytokine-induced VCAM-1 expression in vascular endothelial and smooth muscle cells. Subtraction of basal VCAM-1 expression was also used in the calculation of _IC50 values. The inhibitory effect of each compound on ICAM-1 was examined in the same manner. No inhibitory effect (up to 100 [mu]M) was found in vascular smooth muscle cells. In vascular endothelial cells, only MDL 103,902 showed a significant inhibitory effect on ICAM-1 expression at 50 and 100 μM, which was demonstrated by a decrease in the number of cells adhered to the surface of tissue culture by microscopy.

table 3 Compound number (MDL number) VCAM-1, HUVEC (IC ₅₀ , μM) ICAM-1, HSMC (IC ₅₀ , μM) 29,353103,902105,074 191112 10540

Example 19

In vivo inhibition of VCAM-1 upregulation by MDL29,353 in rabbit aorta

New Zealand white rabbits were fed a diet with or without 0.4% MDL29,353 for three weeks before challenge with lipopolysaccharide (LPS, 40 μg per animal via ear vein). The aorta was removed from each animal 4 hours after LPS injection and flushed briefly in phosphate buffered saline. Immunohistochemistry was performed on VCAM-1 by co-incubating the tissue with RB1/9 antibody (mouse anti-rabbit VCAM-1) overnight at 4°C. Bound RB1/9 was detected with biotinylated goat anti-mouse secondary antibody (60 min, RT). Staining of VCAM-1 was accomplished by adding streptavidin-alkaline phosphatase conjugate (30 min, RT), followed by incubation with BT red (Biotechnology, 10, RT). Images of the stained tissue were acquired using an image analysis system, from which the percentage of aortic endothelium showing immunoreaction with anti-VCAM antibodies was determined.

The in vivo activity of these compounds can also be determined in other models of inflammation predicted to be associated with increased VCAM-1 levels. One such model of respiratory disease (eg, asthma) is the ovalbumin-sensitization model. Kung, T.T. et al., Int. Arch. Allergy Immunol. 105, 83-90 (1994). This model of lung inflammation is IgE-mediated with eosinophilia (also seen in asthmatic patients). Several parameters of bronchoalveolar lavage (BAL) flow obtained from experimental animals can be assessed, including soluble adhesion molecule expression and accumulation of leukocytes. Determination of adhesion molecule expression can be performed by immunohistochemistry in animal tissues, especially the lung. The desired compound (eg, MDL29,353) should act to inhibit the upregulation of VCAM-1 expression and prevent the accumulation of eosinophils in the BAL stream. The inhibitor was tested in a rat model of rheumatoid arthritis and had previously been shown to respond to monoclonal antibodies against ICAM-1. Iigo, Y. et al., J. Immunol. 147, 4167-4171 (1991). In this model, adhesion molecule expression will be determined in the limbs (joints) of experimental animals. For autoimmune diabetes, one can test the ability of compounds to delay onset or prevent adoptive transfer of disease in the NOD mouse model. Heinke, E.W. et al., Diabetes 42, 1721-1730 (1993); Baron, J.L. et al., J. Clin. Invest. 93, 1700-1708 (1994). In addition, one can monitor the expression level of VCAM-1 in tissues such as the pancreas and monitor the development of diabetes in experimental animals. The therapeutic potential of graft rejection was assessed by monitoring cardiac allograft survival (Balb/c hearts transplanted into C3H/He recipients). Isobe, M. et al., J. Immunol. 153, 5810-5818 (1994). In vivo administration of anti-VCAM-1 and anti-VLA-4 monoclonal antibodies induces immunosuppression against cardiac allografts and soluble antigens. The effect of compounds on tumor metastasis and angiogenesis can be evaluated in several models. These models include experimental metastasis models of B16 (mouse) and M24met (human) melanoma. Fidler, I.J., Cancer Res. 35, 218-224 (1975); Meuller, B.M. et al., Cancer Res. 51, 2193-2198. The activity of the compounds can be assessed by their effect on the number of lung metastases that develop and their effect on the expression of VCAM-1 in the lung as described for the mouse respiratory model. The model used to evaluate anti-angiogenic compounds can be used to test these compounds in relation to monitoring the vascular response to a mixture of angiogenic factors mixed with basement membrane proteins (injected subcutaneously in mice). Passaniti, A. et al., Experimental Research 67, 519-528 (1992). Angiogenesis was assessed by the number of blood vessels supplemented in matrigel and by the hemoglobin content of glia. Adhesion molecule expression and accumulation by leukocytes was determined immunohistochemically as in all the above examples.

result

The claimed compounds inhibit the cytokine-induced up-regulation of VCAM-1 gene expression in vivo in vascular cells. Selective inhibition of VCAM-1 expression compared to another cytokine-induced adhesion molecule (ICAM-1) has been shown in certain members of the claimed compound (Table 3). In vivo experiments showed that when sufficiently accumulated, MDL29,353 could inhibit LPS-induced VCAM-1 expression levels in rabbit aortic endothelium (Figure 1). This experiment also shows that such compounds are orally active.

Claims (46)

1. method that in the patient, suppresses the cytokine-abduction delivering of vascular cell adhesion molecule-1, this inhibitory action of said needs of patients, this method comprise the chemical compound of said patient being used the following formula of effective vascular cell adhesion molecule-1 amount of suppression:

Wherein

R ₁, R ₂, R ₃And R ₄Group is respectively C separately ₁-C ₆Alkyl;

Z is a sulfur, oxygen or methylene;

A is C ₁-C ₄Alkylidene;

R ₅Be C ₁-C ₆Alkyl or-(CH ₂) _n-(Ar) group, wherein n represents integer 0,1, and 2 or 3; And Ar representative is not substituted the phenyl or naphthyl that base replaces, or the phenyl or naphthyl that is replaced by one to three group in the group in following group: hydroxyl, methoxyl group, ethyoxyl, chlorine, fluorine or C ₁-C ₆Alkyl.

2. according to the process of claim 1 wherein R ₁And R ₂It is the tert-butyl group.

3. according to the method for claim 2, R wherein ₃And R ₄It is methyl.

4. according to the method for claim 3, wherein A is a methylene.

5. according to the method for claim 4, wherein Z is a thio group.

6. according to the method for claim 4, wherein Z is an oxygen.

7. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(xylyl silicyl) methyl mercapto] phenol.

8. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(trimethyl silyl) methyl mercapto] phenol.

9. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(4-chlorphenyl dimetylsilyl) methoxyl group] phenol.

10. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-4-fluorophenyl silicyl) methoxyl group] phenol.

11. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(xylyl silicyl)-methoxyl group] phenol.

12. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-4-methoxyphenyl silicyl) methoxyl group] phenol.

13. according to the process of claim 1 wherein that said chemical compound is 2,6-dimethyl-4-[(xylyl silicyl)-methoxyl group] phenol.

14. according to the process of claim 1 wherein that said chemical compound is 2-tert-butyl-6-methyl-4-[(xylyl silicyl) methyl mercapto] phenol.

15. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-2-methoxyphenyl silicyl) methoxyl group] phenol.

16. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-2,5-Dimethoxyphenyl silicyl) methoxyl group] phenol.

17. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-2,3-Dimethoxyphenyl silicyl) methoxyl group] phenol.

18. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-4-tert-butyl phenyl silicyl) methoxyl group] phenol.

19. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(benzyl dimethyl silicyl)-methoxyl group] phenol.

20. according to the process of claim 1 wherein that said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-right-methoxy-benzyl silicyl) methoxyl group] phenol.

21. a treatment suffers from the patient's of chronic inflammation disease method, this method comprises the chemical compound to the following formula of said patient's administering therapeutic effective dose:

Wherein

R ₁, R ₂, R ₃And R ₄Group is respectively C separately ₁-C ₆Alkyl;

Z is a sulfur, oxygen or methylene;

A is C ₁-C ₄Alkylidene;

R ₅Be C ₁-C ₆Alkyl or-(CH ₂) _n-(Ar) group, wherein n represents integer 0,1, and 2 or 3; And Ar representative is not substituted the phenyl or naphthyl that base replaces, or the phenyl or naphthyl that is replaced by one to three group in the group in following group: hydroxyl, methoxyl group, ethyoxyl, chlorine, fluorine or C ₁-C ₆Alkyl.

22. according to the method for claim 21, wherein R ₁And R ₂It is the tert-butyl group.

23. according to the method for claim 22, wherein R ₃And R ₄It is methyl.

24. according to the method for claim 23, wherein A is a methylene.

25. according to the method for claim 24, wherein Z is a thio group.

26. according to the method for claim 24, wherein Z is an oxygen.

27. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(xylyl silicyl) methyl mercapto] phenol.

28. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(trimethyl silyl) methyl mercapto] phenol.

29. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(4-chlorphenyl dimetylsilyl) methoxyl group] phenol.

30. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-4-fluorophenyl silicyl) methoxyl group] phenol.

31. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(xylyl silicyl)-methoxyl group] phenol.

32. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-4-methoxyphenyl silicyl) methoxyl group] phenol.

33. according to the method for claim 21, wherein said chemical compound is 2,6-dimethyl-4-[(xylyl silicyl)-methoxyl group] phenol.

34. according to the method for claim 21, wherein said chemical compound is 2-tert-butyl-6-methyl-4-[(xylyl silicyl) methyl mercapto] phenol.

35. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-2-methoxyphenyl silicyl) methoxyl group] phenol.

36. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-2,5-Dimethoxyphenyl silicyl) methoxyl group] phenol.

37. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-2,3-Dimethoxyphenyl silicyl) methoxyl group] phenol.

38. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-4-tert-butyl phenyl silicyl) methoxyl group] phenol.

39. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(benzyl dimethyl silicyl)-methoxyl group] phenol.

40. according to the method for claim 21, wherein said chemical compound is 2,6-two-tert-butyl-4-[(dimethyl-right-methoxy-benzyl silicyl) methoxyl group] phenol.

41. according to the method for claim 21, wherein said diseases associated with inflammation is asthma.

42. according to the method for claim 21, wherein said diseases associated with inflammation is a chronic inflammatory disease.

43. according to the method for claim 21, wherein said diseases associated with inflammation is a class pathogenic wind-warm arthritis.

44. according to the method for claim 21, wherein said diseases associated with inflammation is an Autoimmune Diabetes.

45. according to the method for claim 21, wherein said diseases associated with inflammation is a transplant rejection.

46. according to the method for claim 21, wherein said diseases associated with inflammation is a tumor-blood-vessel growth.