WO1999035140A1

WO1999035140A1 - PYRIDINYLPYRIMIDINE AMINES AS IMMUNOGLOBULINE E (IgE) SYNTHESIS INHIBITORS

Info

Publication number: WO1999035140A1
Application number: PCT/EP1999/000060
Authority: WO
Inventors: Volker Brinkmann; Peter Ettmayer; Christoph Heusser; Max WOISETSCHLÄGER
Original assignee: Novartis Erfindungen Verwaltungs GmbH; Novartis AG
Current assignee: Novartis Pharma GmbH Austria; Novartis AG
Priority date: 1998-01-12
Filing date: 1999-01-08
Publication date: 1999-07-15
Anticipated expiration: 2000-07-12
Also published as: PE20000174A1; CO4940461A1; AR014288A1; AU2516199A

Abstract

Use of the compounds of formula (I) wherein R1 is halogen, phenyl or alkyl and R2 is hydrogen, halogen, alkyl, alkoxy or trifluoromethyl, in free form or salt form, in the treatment of IgE-mediated diseases, including i.a. chronic transplant rejection. The compounds of formula (I) are partly known. A subgroup thereof, namely the compounds of formula (Ib) wherein either R1' is halogen of atomic number 17 or 35 and R2' is hydrogen or alkoxy, or R1' is phenyl or alkyl and R2' is hydrogen, halogen, alkyl, alkoxy or trifluoromethyl, is novel and possesses remarkable cell type specificity. The compounds of formula (Ib) may be prepared e.g. by chlorination or bromination in adjacent position to an N-oxido group or by reaction of the resultant chloro or bromo compound with an organometallic compound.

Description

PYRIDINYLPYRIMIDINE AMINES AS IMMUNOGLOBULINE E (IgE) SYNTHESIS INHIBITORS

The invention relates to pyridinylpyrimidine amines.

It concerns a novel pharmaceutical use of the compounds of formula I

wherein

R, is halogen, phenyl or alkyl and

R₂ is hydrogen, halogen, alkyl, alkoxy or trifluoromethyl, in free form or salt form.

A compound of formula I may be present in free form as base or, where such forms exist, in salt form, particularly acid addition salt form. A compound of formula I in free form may be converted into a salt form in conventional manner and vice- versa.

Halogen preferably is of atomic number 17 or 35, especially chlorine. Phenyl preferably is unsubstituted or substituted by halogen, alkyl or alkoxy. When it is substituted, it preferably is mono- or disubstituted, preferably monosubstituted. Phenyl preferably is unsubstituted. Alkyl and alkoxy preferably independently are of 1 to 4 carbon atoms, they especially are of 1 or 2, even more preferably of 1 carbon atom.

Ri and R₂ preferably are, independently, halogen, preferably chlorine, or alkyl, preferably methyl; more preferably, either Ri and R₂ are both independently halogen, preferably chlorine, or Ri and R₂ are both independently alkyl, preferably methyl.

In a subgroup of compounds of formula I Ri is phenyl and R₂ is as defined above. A preferred subgroup of compounds of formula I (compounds Is) is the compounds of formula I wherein

Ri is chlorine, unsubstituted phenyl or alkyl of 1 or 2 carbon atoms, and R₂ is hydrogen, chlorine, methyl, methoxy or trifluoromethyl, in free form or salt form.

The compounds of formula I are partly known. The compounds of formula la

wherein

Ri ' is halogen, and

R₂' is halogen, lower alkyl or trifluoromethyl, their preparation and their use as protein kinase inhibitors in the treatment of, in particular, tumor diseases and further conditions wherein protein kinases are involved, are described in

Ciba-Geigy WO 95/09851 and/or Ciba-Geigy WO 95/09853.

In a subgroup of compounds of formula la in free form or salt form (compounds lap) Ri' is halogen and R ' is halogen or lower alkyl.

Immunoglobulin E (IgE) is critically involved in the pathogenesis and maintenance of allergic diseases such as atopic dermatitis, allergic asthma, allergic conjunctivitis and allergic rhinitis. To date, patients suffering from atopic dermatitis are mainly treated with local or systemic glucocorticoids, ultraviolet light or, in severe cases, with immunosuppressants such as cyclosporin. Allergic asthma patients are mainly treated with glucocorticoids or theophylline. These compounds suffer from various side effects and are not achieving the goal of reversal of disease progression in addition to alleviation of symptoms. It has been demonstrated recently that interference with IgE production or inactivation of its effector function once it has been synthesized in the body, reduces allergic immune response and, consequently, leads to amelioration of the disease. However, no specific inhibitors of IgE production in human B-lymphocytes are commercially available yet. It has now been found that, surprisingly, the compounds of formula I in free form or salt form act as specific inhibitors of IgE synthesis. Upon systemic or oral administration they strongly suppress immunoglobulin synthesis, in particular the synthesis of immunoglobulin E in B-lymphocytes, i.e. they exhibit isotype specificity. Further, inhibition occurs in a cell-type specific manner.

These activitities can be shown in the following assays. The following abbreviations are used:

ELISA = enzyme-linked immunosorbent assay

FACS = fluorescence-activated cell sorting

HaCat = cell line originating from human adult skin keratinocytes propagated under low calcium conditions and elevated temperature

IgE = immunoglobulin E

IL-4 = interleukin-4

IMDM = Iscove's modified Dulbecco medium

SRBC = sheep red blood cells

TNF-α = tumor necrosis factor - α

TPA = O-tetradecanoylphorbol 13-acetate

1. Isotype specificity: Inhibition of immunoglobulin synthesis induced in primary human B-lymphocytes stimulated by IL-4 with added anti-CD40 antibody:

Normal human B-lymphocytes are purified from tonsils by removing contaminating T-cells with SRBC-rosetting according to M.S. Weiner et al., Blood 42 (1973) 939. The resulting B-cells are more than 95 % pure as judged by CD 19 expression in a FACS analysis. Using 96- well round-bottomed microtiter plates (Costar) 5xl0⁴ B-cells are set up in a final volume of 200 μl/well in IMDM. After pre-incubation with test compound for one hour the cells are cultured to induce IgE production for 9 days at 37°C in air supplied with 5 % CO₂ in the presence of 50 ng/ml of IL-4 and 500 ng/ml of anti-CD40 antibody. The culture cell supernatants are collected and quantitated for IgE, IgG 1 and IgM by standard isotype specific sandwich ELISA.

In this test the compounds of formula I in free form or salt form inhibit IgE production preferentially over IgG (IgGl) and IgM with 50 % inhibitory concentrations (IC₅₀.values) of from about 0.5 nM to about 200 nM.

Similar results are obtained when total splenocytes are used as primary B-cell source. 2. Cell type specificity: Inhibition of proliferation of various cell types: a) HMEC- 1 cells are incubated with increasing amounts of test compound overnight and subsequently stimulated with TNF-α for 16 hours to induce VCAM-1 expression. After fixation, VCAM- 1 positivity is quantitated using an immunohistochemical method. To evaluate anti-proliferative effects of test substances cell numbers are counted by Giemsa dye staining. b) HaCat cells are incubated for 3 days with increasing concentrations of test substance. Cell proliferation is measured using a sulforhodamine B - based colorimetric assay. c) Cytokine production in the T-helper cell clones MoT81 and ChT38 is induced with anti-CD3 monoclonal antibody and TPA for 24 hours in the presence of test compound. IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-γ are quantitated in the supernatants by ELISA. d) Monocyte-derived dendritic cells are co-cultivated with superantigen- or specific allergen- stimulated autologous or allogeneic T-cells for 4 days with increasing concentrations of test compound. The stimulation of T-cell proliferation by antigen-presenting dendritic cells is determined by pulsing with ³H-thymidine for the last 16 hours.

In this test the compounds of formula I in free form or salt form inhibit constitutive proliferation of the above endothelial keratinocyte and T-lymphocyte cell lines with IC₅₀ values of from about 400 nM to more than 5000 nM, well above the concentrations needed to block IgE synthesis.

The compounds of formula I in free form or pharmaceutically acceptable salt form are therefore indicated for use as inhibitors of immunoglobulin synthesis, especially inhibitors of IgE synthesis, in the treatment of IgE-mediated diseases, particularly IgE-mediated allergic diseases, such as atopic dermatitis, particularly in children, urticaria, particularly acute urticaria, allergic asthma, allergic rhinitis, food allergies, allergic conjunctivitis, hayfever, bullous pemphigoid, industrial sensitization and chronic rejection of transplants.

For the above uses the dosage to be used will vary, of course, depending e.g. on the particular compound employed, the mode of administration and the treatment desired. However, in general satisfactory results are obtained when the compounds are administered at a daily dosage of from about 1 mg/kg to about 30 mg/kg animal body weight, suitably given in divided doses two to four times daily. For most larger mammals the total daily dosage is from about 70 mg to about 2000 mg, conveniently administered, for example, in divided doses up to four times a day or in retard form. Unit dosage forms comprise, for example, from about 17.5 mg to about 1000 mg of compound in admixture with at least one solid or liquid pharmaceutically acceptable carrier or diluent.

A compound of formula I in free form or pharmaceutically acceptable salt form may be administered in similar manner to known standards such as glucocorticoids and antihistaminics for use in such indications. It may be admixed with conventional chemotherapeutically acceptable carriers and diluents and, optionally, further excipients, and administered e.g. orally in such forms as tablets and capsules.

Alternatively, it may be administered topically in such conventional forms as aerosols, ointments or creams, parenterally or intravenously. The concentration of active substance will, of course vary depending e.g. on the compound employed, the treatment desired and the nature of the form. In general, however, satisfactory results are obtained in topical application forms at concentrations of from about 0.05 % to about 5 %, particularly from about 0.1 % to about 1 % by weight.

The invention thus comprises the use of a compound of formula I in free form or salt form in the preparation of a medicament for the therapy of IgE-mediated diseases.

Pharmaceutical compositions for use in the therapy of IgE-mediated diseases may be prepared by mixing a compound of formula I in free form or pharmaceutically acceptable salt form together with at least one pharmaceutically acceptable carrier or diluent.

The invention further includes a method of treatment of IgE-mediated diseases which comprises administering a therapeutically effective amount of a compound of formula I in free form or pharmaceutically acceptable salt form to a subject in need of such treatment. A subject in need of such treatment may e.g. be a patient not suffering from, or not treated for, a tumor disease or further condition where protein kinases are involved, or not otherwise undergoing treatment for elevation of depressed immune responses associated with therapy.

The compounds of formula I in free form or pharmaceutically acceptable salt form are well tolerated, as may be determined in conventional manner.

The most preferred compounds of formula I in these indications are: a) N-(3-chlorophenyl)-N-[4-(2-chloropyridin-4-yI)pyrimidin-2-yl]amine (Compound A; of formula la; Example 1 in WO 95/9851); and b) N-(3-methylphenyl)-N-[4-(2-methylpyridin-4-yl)pyrimidin-2-yl]amine (Compound B; of formula lb hereunder; see Example 2). For Compound B the IC₅₀ in the above assay 1. is from about 0.5 nM to about 10 nM. The following activity has for example be determined in the above assay 1.:

In an earlier experiment, an IC₅₀ value of 0.5 nM was obtained

Further compounds of formula I are e.g.: c) N-(3-methylphenyl)-N-[4-(2-chloropyridin-4-yl)pyrimidin-2-yl]amine (Compound C; R, = Cl, R₂ = CH₃; Example 15.2 in WO 95/9853); and d) N-(3-trifluoromethylphenyl)-N-[4-(2-chloropyridin-4-yl)pyrimidin-2-yl]amine (Compound D; R, = Cl, R₂ = CF₃; Example 2 in WO 95/9851).

It has also been found that, although cell type specificity of the compounds of formula I is high, the level of specificity is particularly remarkable for a subgroup of compounds of formula I which is novel and also forms part of the present invention, namely the compounds of formula lb

wherein either RJ is halogen of atomic number 17 or 35 and

R₂" is hydrogen or alkoxy, or Ri " is phenyl or alkyl and

R₂" is hydrogen, halogen, alkyl, alkoxy or trifluoromethyl, in free form or salt form. The invention thus also concerns a compound of formula lb in free form or salt form.

It further concerns a compound of formula lb in free form or pharmaceutically acceptable salt form for use as a pharmaceutical, and a pharmaceutical composition comprising a compound of formula lb in free form or pharmaceutically acceptable salt form together with at least one pharmaceutically acceptable carrier or diluent.

R," preferably is halogen of atomic number 17 or 35 or alkyl, preferably chlorine or methyl, especially methyl. R₂" preferably is halogen or alkyl, preferably halogen of atomic number 17 or 35, especially chlorine, or methyl; it especially is methyl. Even more preferably, R," and R₂" are both methyl.

A preferred subgroup of compounds of formula lb (compounds lbs) is the compounds of formula lb wherein either R," is chlorine and

R " is hydrogen or methoxy, or Ri " is phenyl, methyl or ethyl and

R " is hydrogen, chlorine, methyl, methoxy or trifluoromethyl, in free form or salt form.

In a further subgroup of compounds of formula lb in free form or salt form (compounds Ibp) R₂" is other than hydrogen.

The remarkable cell type specificity of the compounds of formula lb is apparent e.g. from a collection of the IC₅₀ values obtained with the preferred compound of formula la and with the preferred compound of formula lb for inhibition of cell proliferation in various cell types and assays, and their comparison with the IC₅₀ values obtained for inhibition of IgE synthesis in human B-lymphocytes, as appears from the following Table: Comparison of

IC₅₀ values obtained for IgE synthesis inhibition with

IC₅₀ values found to impair cell proliferation (nM)

Assay Compound A Compound B

IgE synthesis 7.2

Cell proliferation:

Primary B-cells (IL-4/anti- CD40 induced) 772 1090

Primary T-cells (dendritic cell 405 1137 induced) HaCat cells (constitutive) 3300 2500

BL2 cells (constitutive) 426 1073

HMEC-2 cells (constitutive) 6350 2450

U266 cells (constitutive) 3869 > 5000

IM9 cells (constitutive) 942 3300

The results show that, compared to inhibition of IgE synthesis, a more than 500fold concentration of Compound B is necessary to impair either induced or constitutive growth of all cell types tested, as compared to an about 60fold concentration for Compound A: thus the window of specificity is approximately 10 times larger for novel Compound B than for known Compound A.

Further, the compounds of formula lb possess beneficial pharmacogalenical properties, such as good solubility in various solvents. Thus the solubility in ethanol is 12J mg/ml for Compound B in free form, as compared with 0.64 mg/ml for Compound A in free form. The invention also provides a process for the preparation of a compound of formula lb in free form or salt form, comprising

a) for the production of a compound of formula Ic

wherein R,^1V is halogen of atomic number 17 or 35 and R₂" is as defined above, reacting a compound of formula II

wherein R₂" is as defined above, with a reagent that introduces chlorine or bromine in the ortho position to the N-oxido group; or

b) for the production of a compound of formula Id

wherein R,'" is phenyl or alkyl and R₂" is as defined above, JO-

reacting a compound of formula Ic with an organometallic compound of formula III

(R _nMe(X)_m III

wherein Me is Al, Zn or Mg; n is 1 to 3; m is O or l; X is halogen; and R,'" is as defined above;

and recovering the resultant compound of formula lb in free form or salt form.

The process of the invention may be effected in conventional manner. Process variant a) can conveniently be performed by reacting a compound of formula II with phosphorous oxychloride or oxybromide, preferably in an inert solvent, e.g. acetonitrile, preferably at elevated temperature. R,^1V preferably is chlorine. Process variant b) can be performed according to known organometallic reactions. It preferably is effected in the presence of a suitable catalyst, such as a Ni- or Pd-catalyst. Conveniently an aprotic solvent such as tetrahydrofuran is used. The reaction preferably is effected at room temperature or at elevated temperature.

The resultant compounds of formula lb can be recovered from the reaction mixture and isolated and purified in known manner.

The starting material of formula II may e.g. be prepared by reacting the compound of formula IV

with a compound of formula V

wherein R₂" is as defined above.

Insofar as its preparation is not specifically described herein, a compound to be used as a starting material is either known, or may be prepared in known manner or analogously to known methods from known compounds.

The following Examples illustrate the invention. All temperatures are in degrees Celsius. m.p. = melting point.

Example 1: N-(3-chlorophenyl)-N-r4-(2-methylpyridin-4-yl)pyrimidin-2-yllamine [process variant b)] Under argon, 952 mg of N-(3-chlorophenyl)-N-[4-(2-chloropyridin-4-yl)- pyrimidin-2-yl]amine (Compound A) and 18 mg of tetrakis-(triphenylρhosphine) palladium are suspended in 10 ml of dry tetrahydrofuran. 2 ml of a 2M solution in heptane of trimethylaluminium are added and the reaction mixture is stirred at 67° for 2.5 hours. The dark solution is poured onto 60 ml of saturated sodium hydrogen carbonate and ice. The aqueous phase is extracted with ethyl acetate (3 x 20 ml) and the combined organic layers are extracted with 300 ml of IN HCl containing 5 % methanol. The aqueous extract is neutralized with solid sodium hydrogencarbonate (pH 8) and the crystals are collected on a sinter funnel, washed 3 times with water, and dried at 60° under reduced pressure. The title compound is obtained (pale yellow crystals; m.p.: 157- 159°). Analogously as described in Example 1 the following compounds of formula lb are obtained:

Example 8: N-r4-(2-chloropyridin-4-yl)pyrimidin-2-vI1-N-(3-methoxyphenyl)amine [Process variant a)] A solution of 2.94 g N-(3-methoxyphenyl)-N-[4-(l-oxidopyridin-4-yl)- pyrimidin-2-yl]amine [formula II; see A) hereunder], 3.31 g of tetraethylammonium chloride and 0.809 ml of pyridine in 18 ml of acetonitrile is heated to reflux and carefully (vigorous boiling at the beginning) treated with 2.80 ml of phosphorous oxychloride. The reaction mixture is heated at reflux for 2 hours, cooled to room temperature and poured onto 7 ml of a stirred solution of 28 % aqueous NH₃ and 60 ml of ice while the quenching temperature is maintained below 30°. After stirring overnight, the product is collected by filtration, rinsed with water containing 30 % acetonitrile and dried in a vacuum oven at 60°. The crude product is purified by passing a hot solution in toluene / methanol (9/1) over silicagel. The title compound is obtained (yellow crystals; m.p.: 154°). Analogously as described in Example 8 the following compound of formula lb is prepared:

The starting material of formula II may be prepared in the following manner:

A) N-(3-methoxyphenyl)-N-[4-(l-oxidopyridin-4-yl)pyrimidin-2-yllamine

a) 4.93 g m-anisidine are dissolved in 8 ml of water and 1 1.8 ml of 37 % aqueous HCl and stirred at 70°. A solution of 3.77 g cyanamide in 3.8 ml of water is added dropwise (the temperature rises to 85°) and the reaction is allowed to proceed at 70-75° for 4 hours. After cooling to room temperature, the solution is poured onto a stirred solution of 5.3 g sodium carbonate in 24 ml of water. After stirring overnight the precipitate is isolated by filtration, rinsed with water and diethyl ether and dried in a vacuum oven at 40°. Bis(3-methoxy- phenyl)guanidine carbonate is obtained (pale white crystals).

b) A mixture of 3.05 g bis(3-methoxyphenylguanidine) carbonate and 3.00 g of 3-dimethylamino-l-(l-oxidopyridin-4-yl)-2-propen-l-one in 30 ml of isopropanol is heated at reflux for 16 hours. After cooling to room temperature, the product is collected by filtration, rinsed with isopropanol and dried in a vacuum oven at 50°. The title compound is obtained (m.p.: 233°).

B) N-phenyl-N-f4-(l-oxidopyridin-4-vI)pyrimidin-2-yl1amine

The title compound (yellow crystals; m.p.: 250°) is prepared analogously as described under A) above, starting from aniline in place of m-anisidine.

Claims

Claims:

1. Use of a compound of formula I

wherein

R, is halogen, phenyl or alkyl and

R₂ is hydrogen, halogen, alkyl, alkoxy or trifluoromethyl, in free form or salt form, in the preparation of a medicament for the therapy of IgE-mediated diseases.

2. Use according to claim 1 of a compound of formula la

wherein

Ri' is halogen and

R₂' is halogen, lower alkyl or trifluoromethyl, in free form or salt form.

3. Use according to claim 2 of a compound of formula la in free form or salt form wherein Ri' is halogen and R₂' is halogen or lower alkyl (a compound lap).

4. Use according to claim 1 , whereby the compound of formula I is a) N-(3-chlorophenyl)-N-[4-(2-chloropyridin-4-yl)pyrimidin-2-yl]amine (Compound A) or b) N-(3-methylphenyl)-N-[4-(2-methylpyridin-4-yl)pyrimidin-2-yl]amine (Compound B), in free form or salt form.

5. A method of treatment of IgE-mediated diseases which comprises administering a therapeutically effective amount of a compound of formula I as defined in claim 1 in free form or pharmaceutically acceptable salt form to a subject in need of such treatment.

6. A compound of formula I as defined in claim 1 , which is of formula lb

wherein either R," is halogen of atomic number 17 or 35 and

R₂" is hydrogen or alkoxy, or R," is phenyl or alkyl and

R₂" is hydrogen, halogen, alkyl, alkoxy or trifluoromethyl, in free form or salt form.

7. A compound according to claim 6 in free form or salt form wherein R₂" is other than hydrogen (a compound Ibp).

8. The compound according to claim 6 which is N-(3-methylphenyl)-N-[4-(2-methylpyridin- 4-yl)pyrimidin-2-yl]amine (Compound B) in free form or salt form.

9. A compound according to claim 6 in free form or pharmaceutically acceptable salt form for use as a pharmaceutical, or a pharmaceutical composition comprising a compound according to claim 6 in free form or pharmaceutically acceptable salt form together with at least one pharmaceutically acceptable carrier or diluent.

10. A process for the preparation of a compound according to claim 6 comprising

a) for the production of a compound of formula Ic

wherein R,^1V is halogen of atomic number 17 or 35 and R₂" is as defined in claim 6, reacting a compound of formula II

wherein R₂" is as defined in claim 6, with a reagent that introduces chlorine or bromine in the ortho position to the N-oxido group; or b) for the production of a compound of formula Id

wherein R,'" is phenyl or alkyl and R₂" is as defined in claim 6, reacting a compound of formula Ic with an organometallic compound of formula III

(R₁ "')_nMe(X)_m III

wherein Me is Al, Zn or Mg; n is 1 to 3; m is 0 or 1; X is halogen; and R,'" is as defined in this claim;

and recovering the resultant compound of formula lb in free form or salt form.