HK1026211B - Substituted benzamides - Google Patents

Description

Substituted benzamides

Technical Field

The invention relates to substituted benzamides of general formula I and their use in medicine.

Background

Autoimmune diseases are the result of the immune system's response to endogenous structures. During this process, tolerance to the normal presence of endogenous tissues is discontinued. Antibodies, particularly T-lymphocytes and monocytes/macrophages, play an important role in the pathological process of various autoimmune diseases. Activated monocytes/macrophages secrete a number of different inflammatory mediators which directly or indirectly cause damage to tissues affected by autoimmune diseases. Whereas activation of monocytes/macrophages is influenced by interaction with T-lymphocytes or by bacterial products such as Lipopolysaccharide (LPS). Activation of monocytes/macrophages and granulocytes induced by various cellular products is often generally characteristic of an inflammatory response.

Due to numerous experiments in animals and initial clinical trials, the importance of the balance between inflammatory cytokines (e.g., interleukin IL-12) and anti-inflammatory cytokines (e.g., interleukin IL-10) in the development and progression of inflammatory and autoimmune diseases has been well documented. The pathophysiological role of IL-12 is evident in various animal models of disease such as rheumatoid arthritis, multiple sclerosis, diabetes, and inflammatory diseases of the skin and mucosa (immunological. today 16/8: 383-387, 1995; J.immunological.155: 4661-4668, 1995; J.exp.Med.182: 1281-1290, 1995; J.exp.Med.187/4: 537-546, 1998). Each of these diseases can be initiated by the use of IL-12, and after endogenous IL-12 is inhibited, the disease process is significantly arrested and the animal subsequently heals.

In inflammatory bowel disorders, such as those affected animals and patients with Crohn's disease, there is a significant increase in T-cell activity at the inflamed portion of the bowel. This T-cell activity is characterized by increased expression of IL-12 and IFN- γ in the site of injury. On the other hand, the immunosuppressive cytokine IL-10 is also significantly reduced at the site of injury (Immunity 3: 171-174, 1995; J.exp.Med.182: 1281-1290, 1995; Eur.J.Immunol.26: 1156-1163; EurJ.Immunol.28: 379-389, 1998), and the significance of the immunosuppressive cytokine IL-10 for the development of inflammatory intestinal disorders is apparently seen from the fact that IL-10 loses its potency and mice develop idiopathic colitis (Immunity 3: 171-174, 1995), and the activation of IFN-. gamma. -producing T-cells in the gut's natural membrane essentially depends on locally produced IL-12. In an animal model of allergen-induced colitis, it has been shown that antibodies can be used to remove IL-12 in the presence of severe colitis. Inhibition of IL-12 with the antibody resulted in normalization of clinical and pathological tissues within several days. No further production of IFN-. gamma.was detectable in T-cells of the innate membrane of mice receiving anti-IL-12 treatment. (J.Exp.Med.182: 1281-1290).

Initial application of recombinant IL-10 in humans demonstrated anti-inflammatory properties. After IL-10 administration to healthy subjects, monocytes in vivo were activated with LPS and the inflammatory cytokines TNF-. alpha.and IL-1 formed were reduced by 65 to 95% (J.Immunol.154: 5492-5499, 1995). The use of IL-10 in patients with steroid-refractory Crohn's disease results in an improvement in clinical symptoms (Gastroenterology, 113: 383-. More recently, it has also been reported that the subcutaneous application of IL-10 to three patients with psoriasis results in an improvement in the symptoms of the disease. Furthermore, the expression of IL-12 and TNF formed and molecules on the surface of monocytes was also reduced (J.Clin.Invest.101: 783-794). The use of antibodies against IL-12 in humans is now about to proceed.

In short, it can be said that the absence of IL-10 or the excess of IL-12 determines the pathophysiology of a number of inflammatory diseases. Attempts to normalize the IL-10/IL-12 balance have tremendous therapeutic potential.

Disclosure of Invention

Therefore, the aim of the invention is to develop new immune modulators, they do not lead to general immunosuppression, but rather influence IL-10/IL-12 balance normalization.

It has now been found that these requirements for the substances to be developed are met by specific substituted benzamides.

Accordingly, the present invention provides substituted benzamides of formula I,

wherein R is¹Represents a radical corresponding to the formula COOR⁴Where R is⁴Represents a compound having 1 to 6C atoms

A linear or branched alkyl group; or correspond to the formula CONR⁵R⁶Where R is⁵And R⁶Is composed of

Identical or different and represent an alkyl radical having 1 to 6C atoms (linear or branched), or

Or together with the N atom, represents a pyrrolidine, piperidine, hexamethyleneimine or morpholine ring; r²Represents chlorine, fluorine, CF₃And alkyl having 1 to 3C atoms or hydrogen, and R³Represents a hydroxyl group, an alkyl or alkoxy group having 1 to 6C atoms (linear or branched and may be

Optionally substituted by OH-, alkoxy, ester groups or open-chain or cyclic acyl groups having 1 to 6C atoms

Amino substituted), or CH₂-NR⁵R⁶Where R is⁵And R⁶As previously defined.

The compounds of the invention may be in racemic form or in the form of pure enantiomers or in the form of salts with pharmaceutically compatible acids.

Preferred substituted benzamides are those wherein R is¹The radicals representing radicals corresponding to the formula COOR⁴Where R is⁴Represents a linear or branched alkyl group having 1 to 6C atoms; or correspond to the formula CONR⁵R⁶Where R is⁵And R⁶Together with the N atom represents a morpholine ring; r²Is H and R³Represented by hydroxy or a group CH₂-NR⁵R⁶Where R is⁵And R⁶Together with the N atom, is represented by a morpholine ring.

Particularly preferred are 2- (morpholino-4-carbonyl) -N- (1-morpholino-4-ylmethyl-2, 6-dioxopiperidin-3-yl) benzamide and N- (1-hydroxy-2, 6-dioxopiperidin-3-yl) anthranilic acid methyl ester.

The compounds corresponding to formula I can be prepared as follows: first, the carboxylic acids corresponding to the formula IIa or IIb are converted into esters (R) in a known manner¹＝COOR⁴) Or amides (R)¹＝CONR⁵R⁶). The compounds of the invention can thus be obtained from carboxylic acids IIb. Then, according to known methods, the group R not representing a hydroxyl group³Into these compounds, which are correspondingly derived from carboxylic acids IIa, for example with paraformaldehyde and corresponding to the formula HNR⁵R⁶(ii) a Mannich reaction of a secondary amine.

The invention also provides the use of substituted benzamides corresponding to formula I as medicaments, in particular as immunomodulators. The substances of the invention significantly inhibit the inflammatory cytokine IL-12 produced by LPS-activated human monocytes. On the other hand, this group of substances increases the production of the anti-inflammatory cytokine IL-10 by LPS-activated human monocytes. This distinguishes the novel substances of the invention from known immunomodulators, such as steroids and phosphodiesterase inhibitors (which inhibit the synthesis of IL-12 and also of IL-10). Owing to their characteristic immunomodulatory activity (inhibition of IL-12, increase of IL-10), the substances according to the invention are suitable for the treatment and/or prevention of inflammation, in particular of the skin and mucous membranes and of blood vessels, and for the treatment and/or prevention of autoimmune diseases.

These diseases include, in particular, inflammation of the skin (e.g., atopic dermatitis, psoriasis, eczema), inflammation of the respiratory tract (e.g., bronchitis, pneumonia, bronchial asthma, ARDS (adult respiratory distress syndrome), sarcoidosis, silicosis/fibrosis), inflammation of the gastrointestinal tract (e.g., gastroduodenal ulcer, crohn's disease, ulcerative colitis), and also diseases such as hepatitis, pancreatitis, cecronitis, peritonitis, nephritis, aphthous ulcer, conjunctivitis, keratitis, uveitis, rhinitis.

Autoimmune diseases include, for example, arthritis-type diseases (e.g., rheumatoid arthritis, HLA-B27, and related diseases), as well as multiple sclerosis, juvenile diabetes, or lupus erythematosus.

Other indications are sepsis, bacterial meningitis, cachexia, graft rejection, graft-versus-host reactions, as well as reperfusion syndrome and atherosclerosis.

The medicament of the invention comprises at least one compound of the general formula I and further carriers, fillers, solvents, diluents, dyes and/or binders. The choice of auxiliary substances and the amounts used depend on whether the medicament is to be administered orally, intravenously, intraperitoneally, intradermally, intramuscularly, intranasally, buccally or topically. Suitable formulations for oral administration are tablets, chewable tablets, dragees, capsules, granules, drops, juices or syrups. Solutions, suspensions, instantly reconstitutable anhydrous formulations and sprays are suitable for parenteral and topical administration and inhalation. A prolonged-action preparation of a compound of the invention in dissolved form, optionally with the addition of a skin-penetration-promoting agent, on a support film or on a plaster, is an example of a suitable transdermal administration form. It is also possible to delay the release of the compounds of the invention from oral or transdermal formulations.

The dosage of the active ingredient to be administered to a patient varies depending on the weight of the patient, the method of administration, the indication and the severity of the disease. In general, at least one compound of the invention of formula I is administered in an amount of 1 to 150. mu.g/kg.

Examples

TABLE 1

Example numbering	The structure of the compound of formula I, wherein	Name (R)
			1 (comparative example)	R1＝COOHR2＝HR3＝CH2-NR5R6Wherein R is5And R6Together with the N atom representing a morpholine ring	N- (1-Morpholino-4-ylmethyl-2, 6-dioxopiperidin-3-yl) o-carbamoylbenzoic acid
2 (comparative example)	R1＝HR2＝HR3＝CH2-NR5R6Wherein R is5And R6Is represented by a morpholine ring together with the N atom	N- (1-Morpholino-4-ylmethyl-2, 6-dioxopiperidin-3-yl) benzamide
			3 (according to the invention)	R1＝CONR5R6Wherein R is5And R6Together with the N atom representing a morpholine ring R2＝HR3＝CH2-NR5R6Wherein R is5And R6Together with N atomShown as a morpholine ring	2- (morpholino-4-carbonyl) -N- (1-morpholino-4-ylmethyl-2, 6-dioxopiperidin-3-yl) benzamide
4 (according to the invention)	R1＝COOCH3R2＝HR3＝OH	N- (1-hydroxy-2, 6-dioxopiperidin-3-yl) o-carbamoylbenzoic acid-methyl ester
			5 (according to the invention)	R1＝CONR5R6Wherein R is5And R6Together with the N atom representing a morpholine ring R2＝HR3＝CH2-NR5R6Wherein R is5And R6Together with the N atom representing a piperidine ring	2- (morpholino-4-carbonyl) -N- (1-piperidin-4-ylmethyl-2, 6-dioxopiperidin-3-yl) benzamide
6 (according to the invention)	R1＝COOC2H5，R2＝HR3＝CH2-NR5R6Wherein R is5And R6Together with the N atom representing a piperidine ring	N- (2, 6-dioxo-1-piperidin-1-yl-methyl-piperidin-3-yl) o-carbamoylbenzoic acid-ethyl ester

The substances of Table 1 were analyzed by NMR hydrogen spectroscopy (instrument: DPX 300 Avance from Bruken, Inc.; 300 MHz; solvent: DMSO-d)₆(ii) a Chemical shift value data in ppm).Example 11.97-2.16(m，2H，CH₂)；2.52-2.86(m，2H，CH₂)；3.20-3.75(m，8H，CH₂)；4.50-4.65(m，2H，NCH₂N); 4.64-4.82(m, 1H, CH); 7.49-7.66(m, 3H, aromatic); 7.79-7.85(d, 1H, aromatic); 8.60-8.72(d, 1H, CONH).Example 21.94-2.20(m，2H，CH₂)；2.68-2.95(m，2H，CH₂)；3.28-3.70(m，8H，CH₂)；4.52-4.65(m，2H，NCH₂N); 4.83-4.96(m, 1H, CH); 7.42-7.58(m, 3H, aromatic); 7.85-7.92(m, 2H, aromatic); 8.85-8.89(d, 1H, CONH).Example 31.92-2.24(m，2H，CH₂)；2.69-3.02(m，2H，CH₂)；3.12(s，2H，CH₂)；3.35-3.77(m，12H，CH₂)；4.52-4.68(m，2H，NCH₂N); 4.74-4.98(m, 1H, CH); 7.28-7.32(d, 1H, aromatic); 7.45-7.59(m, 2H, aromatic); 7.74-7.80(d, 1H, aromatic); 8.78-8.88(d, 1H, CONH).Example 42.01-2.18(m，2H，CH₂)；2.58-2.88(m，2H，CH₂)；3.79(s，3H，COOCH₃) (ii) a 4.70-4.80(m, 1H, CH); 7.54-7.72(m, 3H, aromatic); 7.77-7.81(d, 1H, aromatic); 8.77-8.82(d, 1H, CONH); 10.21(s, 1H, NOH).Example 51.38-1.46(m，6H，CH₂)；1.96-2.16(m，2H，CH₂)；2.20-2.32(m，4H，CH₂)；2.60-2.92(m，2H，CH₂)；3.26-3.75(m，8H，CH₂)；4.57-4.70(m，2H，NCH₂N); 4.72-4.88(m, 1H, CH); 7.28-7.30(d, 1H, aromatic); 7.43-7.57(m, 2H, aromatic); 7.70-7.74(m, 1H, aromatic); 8.75-8.78(d, 1H, CONH).Example 61.18-1.26(t，3H，CH₃)；1.36-1.48(m，6H，CH₂)；1.95-2.16(m，2H，CH₂)；2.22-2.34(m，4H，CH₂)；2.60-2.90(m，2H，CH₂)；4.08-4.18(q，2H，OCH₂)；4.55-4.68(m，2H，NCH₂N); 4.70-4.80(m, 1H, CH); 7.54-7.72(m, 3H, aromatic); 7.78-7.83(d, 1H, aromatic); 8.78-8.82(d, 1H, CONH).Study of the Activity of immunomodulators

Human monocytes were isolated from Peripheral Blood Mononuclear Cells (PBMCs) obtained from heparinized whole blood by Ficoll density gradient centrifugation. After isolation, PBMCs were incubated with monoclonal antibodies against the monocyte-specific surface molecule CD14 and coupled to superparamagnetic microbeads (microbead) (Miltenyi Biotech, Bergisch Gladbach). To perform a positive (positive) selection of labelled monocytes from a cell mixture of PBMCs, the whole cell suspension is applied to a column of a strongly magnetic carrier matrix and the column is placed in a magnetic field, by which method microbeads-loaded cells are attached to the carrier matrix, while unlabelled cells are passed through the column and discarded, after the matrix is removed from the magnetic field, the demagnetized column is washed with a buffer to elute the antibody-loaded cells. The purity of the CD 14-positive monocyte population obtained was about 95-98%. These monocytes (at 10)⁶Cell/ml medium (RPMI, supplemented with 10% fetal calf serum) density) was combined with test substance dissolved in DMSO at 37 ℃ and 5% CO₂And incubated for 1 hour. Then 20. mu.g/ml LPS (obtained from E.coli) was added. After 24 hours, cell-free culture medium supernatants were removed and analyzed to determine the levels of cytokines IL-12 and IL-10.

IL-12 and IL-10 concentrations in cell culture supernatants were determined by sandwich-ELISA using two anti-IL-12 and anti-IL-10 monoclonal antibodies (Biosource Europe, Fluurus, Belgium). Standard control curves for human IL-12 and IL-10, respectively, are also included. The limit of detection by IL-12 ELISA was 10pg/ml, whereas by IL-10 ELISA was 15 pg/ml.

TABLE 2 Effect of test Compounds on IL-12 and IL-10 production by LPS-activated monocytes

Substance(s)	Concentration of	% IL-12 production compared to control (100%)	% IL-10 production compared to control (100%)
				Example 1 compound example 2 compound example 3 compound (second experiment) example 4 compound example 5 compound	10μg/ml2μg/ml0.4μg/ml6.0μg/ml2.0μg/ml0.66μg/ml0.22μg/ml10μg/ml2μg/ml0.4μg/ml6.0μg/ml2.0μg/ml0.66μg/ml0.22μg/ml10μg/ml2μg/ml0.4μg/ml6.0μg/ml2.0μg/ml0.66μg/ml0.22μg/ml	767890105104102993046874953596423406647576788	10984876999109114127121108131133123117112189122134134122115

EXAMPLE 6 dexamethasone pentoxifylline cyclopentapyrazine

6.0μg/ml2.0μg/ml0.66μg/ml0.22μg/ml1μM0.1μM0.01μM0.001μM50μg/ml5μg/ml50μM0.5μM0.005μM

384761796620837472323058

1301241321023435641057481287992

The results in Table 2 show that known immunomodulators such as dexamethasone, pentoxifylline, and cyclopentapyrazole, both inhibit IL-12 and IL-10 production in the case of LPS-activated monocytes. In contrast, the structurally similar benzamides substituted with carboxyl groups (example 1) showed only a little effect at high doses.

Surprisingly, the esters (examples 4 and 6) and amides (examples 3 and 5) of the substituted benzamides of the present invention exhibit immunomodulatory activity in a studied model. These compounds each potently inhibited the synthesis of IL-12 monocyte activated by LPS at concentrations of 10, 6 and 2. mu.g/ml. But unlike known immunomodulators, they increase IL-10 synthesis. This type of signature (significant inhibition of IL-12, increase of IL-10 in the same concentration range of the substance) suggests that they are a new class of immunomodulators.

Claims (4)

1. Substituted benzamides of the formula I,

wherein R is¹Represents a radical corresponding to the formula COOR⁴Where R is⁴Represents a compound having 1 to 6C atoms

A linear or branched alkyl group; or correspond to the formula CONR⁵R⁶Where R is⁵And R⁶Is composed of

Identical or different and denotes straight-chain or branched alkyl having 1 to 6C atoms,

or together with the N atom, represents a morpholine ring; r²Represents hydrogen, and R³Represents hydroxy, or CH₂-NR⁵R⁶Where R is⁵And R⁶Together with the N atom, represents a morpholine ring or a piperidine ring.

2. Substituted benzamides of the formula I according to claim 1, characterized in that R¹The radicals representing radicals corresponding to the formula COOR⁴Where R is⁴Represents a linear or branched alkyl group having 1 to 6C atoms; or correspond to the formula CONR⁵R⁶Where R is⁵And R⁶Together with the N atom represents a morpholine ring; r²Is H and R³Represented by hydroxy or a group CH₂-NR⁵R⁶Where R is⁵And R⁶Together with the N atom, is represented by a morpholine ring.

3. An immunomodulator comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.

4. Use of a compound according to claim 1 for the preparation of a medicament for the treatment and/or prevention of inflammatory or autoimmune diseases.