WO2001074778A1

WO2001074778A1 - (1-adamantylamino)pyridines and method of their preparation

Info

Publication number: WO2001074778A1
Application number: PCT/PL2001/000029
Authority: WO
Inventors: Zygmunt Kazimierczuk; Witold Lasek; Agata Sikorska
Original assignee: FUNDACJA ROZWOJU DIAGNOSTYKI I TERAPII
Current assignee: FUNDACJA ROZWOJU DIAGNOSTYKI I TERAPII
Priority date: 2000-04-04
Filing date: 2001-04-03
Publication date: 2001-10-11
Anticipated expiration: 2002-10-04
Also published as: AU2001248926A1; PL339418A1; PL207853B1

Abstract

New (1-adamantylamino)pyridines of formula (2) wherein substituents R1, R2, R3, R4 or R5 are adamantylamino group, hydrogen atom, methyl group or halogen atom, the molecule of formula (2) containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an-ortho position to the amino group, and the method of preparation of the new compounds. Studies have shown that these compounds stimulate secretion of TNF-α in some neoplastic cell lines.

Description

(l-ADAMANTYLAMINO) PYRIDINES AND METHOD OF THEIR PREPARATION

An object of the invention are new (1-adamantylamino)~pyridines, salts thereof with inorganic and organic acids and method of their preparation.

New (1 -adamantylamino )-pyridines of formula 2 are substituted at heterocydic ring by alkyl groups or by halogen atoms. Many derivatives of adamantane exhibit biological activity.

The most known compounds of this group are derivatives of 1 -adamantane (amantidine, rimantidine and tromantidine) used as antiviral drugs or in therapy of Parkinson's disease [W.L. Davies et al., Science, 144, 862, (1964), R. S. Schwab et al., J. Am. Med. Assoc, 208, 1168 (1969)]. Selected adamantyl esters of phthalimide substituted by amino acids inhibit growth of various bacteria strains to an extent that is comparable to activity of some antibiotics [A.Orzeszko, J. Stefaήska, B. Starosciak, Z. Kazimierczuk, Acta Biochim. Polon., paper in press]. Our studies on biological activity of (l-adamantylamino)-pyridine derivatives have shown, that the derivatives stimulate secretion of TNF-α. Tumour necrosis factor - TNF-α is a cytokine exhibiting antineoplastic and immunomodulating properties. From a structural point of view it is identical with cachectin secreted by macrophages in the course of neoplastic processes and infection [B. Beutler et al., Nature, 320, 584, 1986]. Factors that stimulate secretion of TNF-α by macrophages are bacterial endotoxins of lipopolysaccharides (LPS) type. Recently, it has been found that also chemical compounds, such as e.g. 5,6- dimethylxanthenone-4-acetic acid, can stimulate secretion of TNF-α within a tumour [W. R. Joseph et al., Cancer Res. 59, 633, (1999] and this can possibly constitute a new targeted method of treatment of some tumours, e.g. solid tumours.

(I-Adamantylamino)pyridine derivatives and salts thereof constituting an object of the present invention exhibit considerable activity as substances that cause an increase in production of tumour necrosis factor of alpha-type (TNF-α) in some cell lines.

Our studies on an effect of stimulation of production of TNF-α by (l-adamantylamino)pyridine derivatives were conducted on genetically modified B78 murine melanoma cells, subjected to transduction by a gene for human TNF- α (the line was referred to as B78/TNF) [W. Lasek, A. Mackiewicz, A. Czajka, T. Switaj, J. Gotab, paper in press]. These cells constitutively secreted human TNF-α in the culture. Exponentially growing B78/TNF cells (2 x 10⁵ cells per 1 ml_) were incubated for 24 hours at 37°C in Dulbecco's medium comprising 10% of bovine foetal serum and the respective concentration of tested compounds (100, 10, 1 and 0.1 μM). TNF-α concentrations were measured with a use of ELISA test. More intense secretion of TNF-α, depending on a position and a number of methyl groups in tested compounds were observed, which is shown on Fig. 1.

In a Table below selected results of TNF-α secretion for that line with an addition of compounds of formula 2 of the same concentration (10 μM) are given.

Table

New compounds stimulating TNF-α production that we have synthesized can constitute a promising group of drugs to be used for topical treatment of neoplastic solid tumours.

The most important aspect of the invention are new (1 -adamantylamino) pyridines of formula 2:

wherein substituents Ri , R₂, R₃, R₄ or R5 denote adamantylamino group, hydrogen atom, methyl group or halogen atom, the compound of formula 2 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group.

According to the invention, the new compounds are: 2-(1 -adamantylamino)pyridine, 3-(1 -adamantylamino)pyridine,

2-(1-adamantylamino)^-methylpyridine,

2-(1-adamantylamino)-5-methylpyridine,

2-(1-adamantylamino)-6-methylpyridine,

2-(1-adamantylamino)-4,6-dimethylpyridine,

4-(1 -adarnantylamino)-pyridine,

3-(1-adamantylamino)-6-chloropyridine,

2-(1 -adamantylamino)-6-chloropyridine hydrochloride.

Another important aspect of the invention is a method of preparation of new (l-adamantylamino)pyridines of formula 2,

wherein substituents R-i, R₂, R3, R4 or R5, denote adamantylamino group, hydrogen atom, methyl group or halogen atom, the compound of formula 2 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, consisting in that, a pyridine derivative of formula 1 ,

wherein R^ R₂, R3, R4 or R5, denote an amino group, a hydrogen atom, a methyl group or a halogen atom, the molecule of formula 1 containing one amino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, is subjected to reaction with 1 -adamantanol at elevated temperature in an acid solution, to yield a product of formula 2, wherein substituents R^ R₂ ,R3, R4 or R₅ are as defined above, the compound of formula 2 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, and the product of formula 2 is isolated and converted by a known method into a salt, preferably hydrochloride.

In the method according to the invention, trifluoracetic acid or ortophosphoric acid is used to provide an acid reaction solution, the ortophosphoric acid being used at concentration of 85-100%, preferably 90%.

In the method of the invention, the reaction is carried out at elevated temperature, within the range of 90-120°C, preferably at boiling temperature of trifluoroacetic acid.

The final new (l-adamantylamino)pyridines are isolated from a reaction mixture by diluting reaction mixture with water and adjusting it to pH 7.0-14.0.

The resulting precipitate is isolated by crystallisation or adsorption chromatography on silica gel. In the process of the invention, adamantylation of an exocyclic amino group of pyrimidine derivatives results from formation of an adamantyl carbocation from 1 -adamantanol in a trifluoroacetic acid solution at elevated temperature.

It should be stressed that the reaction does not take place e.g. in case of aniline, where the amino group in an acid solution is protonated. A successful course of the reaction of aminopyridines with the adamantyl carbocation is conditioned by the fact that it is a nitrogen atom of the heterocydic ring which is subjected to protonation and not the amino group, which captures carbocations, to yield (l-adamantylamino)pyridine derivatives as final products.

As already mentioned, the new compounds exhibit activity stimulating production of tumour necrosis factor - TNF-α and can constitute a promising group of drugs for topical treatment of neoplastic solid tumours. Accordingly, the present invention covers pharmaceutical or veterinary compositions comprising at least one compound defined by formula 2 as a single active substance used or in combination with a pharmaceutically acceptable and compatible carrier and an excipient. Hereinafter and in the patents claims the term "pharmaceutical composition" is meant as a mixture of an active substance combined with a pharmaceutically acceptable and compatible carrier and an excipient, to be used for treatment of humans and animals.

For the purpose of topical treatment, the compounds of formula 2 can be formulated as an ointment or a cream or in another form comprising the compound of formula 2 suspended in a liquid or solid carrier, used for preparation of such pharmaceutical forms. An addition of particular carriers to provide protection of an active substance from its decomposition or oxidation is also possible.

For the purpose of systemic treatment, compounds of formula 2 can be formulated as injections or infusion solutions into a tumour. In all these cases the pharmaceutical form has to be sterile and a liquid should enable a use of syringe. Another possible form is a dry pharmaceutical composition to be diluted or suspended prior to administration in an injection liquid containing e.g. liquid polyethylene glycol, propylene glycol, glycerol or the like. Preferably, prolonged absorption of an injection composition can be achieved through a use of known compounds delaying absorption and/or sustaining release, such as aluminium monostearate and gelatine.

A unit dose can contain e.g. a main active ingredient in an amount from about 0.1 to about 500 mg, preferably from about 0.1 to about 250 mg per day.

In the embodiment examples of the invention chemical reagents were from Aldrich (apart from solvents). Column chromatography was performed on silica gel 60H (Merck). Analytical thin-layer chromatography was performed on aluminium plates with silica gel 60F254 (Merck). Melting points were determined with a use of Gallenkamp-5 apparatus in glass capillaries. UV spectra were measured on Kontron-Uvikon-940 spectrophotometer. NMR spectra (ppm) were measured on Varian-Gemini-200 MHz spectrometer.

The following examples illustrate the invention, while not limiting its scope: Example 1

2-(1 -Adamantylamino)pyridine.

To a solution of 2-aminopyridine (0.94g, 10 mmol) in 8 cm³ of trifluoroacetic acid,

1 -adamantanol (1.52g) was added while stirring. The resulting reaction mixture was heated at reflux for 5 hrs while stirring. The reaction mixture was left to reach a room temperature, poured into 100 cm³ of mixture water-methanol (8:2) and adjusted to pH 7 with an aqueous ammonia solution. The resulting precipitate was filtered off, washed with water and crystallized from an ethanol-water mixture (1 :1 ) v/v to yield 810 mg of colourless crystals, mp 163-166°C. Yield 44%. TLC

(CH₂CI₂/MeOH 9:1 , silica gel): R_f 0.69. UV (H₂O/MeOH 4:1 ): λ_max: 241 nm, 308nm.

¹H-NMR (CDC ): 1.70 (bs, 6H, H-Ada), 2.04 (bs, 6H, H-Ada) 2.12 (bs; 3H, H-Ada),

6.05 (m, 2H, H-arom.), 7.35 (m, 1 H, H-arom.), 8.01 (m, 1 H, H-arom.). MS:

228(100), 227(36), 213(11 ), 185(24), 172(14), 171 (75), 132(48).

Elemental analysis: Calcd for C15H₂0N2 (228.34): C 78.90, H 8.83, N 12.27; Found:

C 78.80, H 8.91 , N 12,15.

Example 2.

3-(1 -Adamantanylamino)pyridine

To a solution of 3-aminopyridine (0.94g) in 25 cm³ of 90% orthophosphoric acid,

1 -adamantanol (1.21g) was added while stirring in an oil bath (105°C). Stirring at that temperature was continued for 30 minutes. The mixture was poured into 100 cm³ of ice water and neutralised with 20% KOH solution. The resulting precipitate was filtered off, washed with water and crystallised from ethanol-water mixture

(1 :1 , v/v) to yield 0.99g of colourless crystals, mp 227-229°C. Yield 55%. TLC

(CHCb/MeOH 9:1 , silica gel): R_f .0.6, UV (H₂O/MeOH 4:1 ): λ_max: 225 nm, 267 nm,

350 nm. ¹H-NMR (CDCI₃): 1.61 (bs, 6H, H-Ada), 1.71 (bs, 6H, H-Ada), 2.14 (bs,

3H, H-Ada), 7.10 (m, 2H, H-arom.), 8.00 (m, 1H, H-arom.), 8.12 (m, 1 H, H-arom).

MS: 228(55), 172(11 ), 171 (77), 136(11), 135(100), 107(13).

Elemental analysis: Calcd for C₁₅H₂oN₂ (228.34): C 78.90, H 8.83, N 12.27; Found

C 78.78, H 8.94, N 12.18 Example 3.

2-(1-Adamantanylamino)-4-methylpyridine

To a solution of 2-amino-4-methylpyridine (1.08g) in 10 cm³ of trifluoroacetic acid, 1 -adamantanol (1.21 g) was added while stirring. The resulting reaction mixture was heated at reflux for 5 hrs while stirring. The reaction mixture was left to reach a room temperature, poured into 100 cm³ of water-methanol mixture (9:1) and adjusted to pH 7 with an aqueous ammonia solution. The resulting precipitate was filtered off and crystallised from mixture hexane-ethyl acetate (1:1 , v/v) to yield 0.91 g of colourless crystals, mp 123-125°C. Yield 47%. TLC (CH₂CI₂/MeOH 9:1 , silica gel): R_f 0.44 UV (H₂O/MeOH 4:1): λ_max: 243 nm, 309nm. ¹H-NMR (CDCI₃): 1.62 (bs, 6H, H-Ada), 1.70 (bs, 6H, H-Ada) 2.14 (bs, 3H, H-Ada), 2.39 (s, 3H, H- Me), 6.45 (m, 1 H, H-arom.), 6.76 (s, 1 H, H-arom.), 7.59 (d, 1 H, H-arom.). Elemental analysis: Calcd for de-Hz_.^ (242.37): C 79.29, H 9.15, N 11.56; Found: C 79.40, H 9.11 , N 11.45.

Example 4.

2-(1-Adamantylamino)-5-methylpyridine

To a solution of 2-amino-5-methylpyridine (1.08g) in 10 cm³ of trifluoroacetic acid,

1 -adamantanol (1.22g) was added while stirring. The resulting reaction mixture was heated at reflux for 5 hrs while stirring. The reaction mixture was left to reach a room temperature, poured into 120 cm³ of water-methanol mixture (9:1 ) and adjusted to pH 7 with an aqueous ammonia solution. The resulting precipitate was filtered off and crystallised from mixture hexane-diethyl ether (1 :1 , v/v) to yield 0.81 g of colourless crystals, mp 235-238°C. Yield 42%. TLC (CH₂CI₂/MeOH 95:5, silica gel): R_f 0.70, UV (H₂O/MeOH, 4:1 ) λ_max: 243 nm, 321 nm. ¹H-NMR (CDCI₃): 1.58

(bs, 6H, H-Ada), 1.68 (bs, 6H, H-Ada), 2.15 (bs, 3H, H-Ada), 2.33 (s, 3H, H-Me),

6.78 (d, 1 H, H-arom.), 7.43 (d, 1 H, H-arom.), 7.72 (s, 1 H, H-arom.).

Elemental analysis: Calcd for C₁₆H22N2 (242.37): C 79.29, H 9.15, N 11.56 ;

Found: C 79.41 , H 9.08, N 11 ,41. Example 5.

2-(1-Adamantylamino)-6-methylpyridine

To a solution of 2-amino-6-methylpyridine (1.08g) in 10 cm³ of trifluoroacetic acid, 1 -adamantanol (1.22g) was added while stirring. The resulting reaction mixture was heated at reflux for 6 hrs while stirring. The reaction mixture was left to reach a room temperature, poured into 100 cm³ of water-ethanol mixture (9:1 ) and adjusted to pH 7 with an aqueous ammonia solution. The resulting precipitate was filtered off and absorbed on silica gel, which was then passed through a column with silica gel (3 x 15 cm). Chromatography was performed with chloroform (150 cm³), then with chloroform-methanol mixture (95:5) as an eluent. Fractions containing the product were evaporated to dryness, and the residue was crystallised from hexane to yield 0.42 g of colourless crystals, mp 123-127°C. Yield 22%. TLC (CH₂CI₂/MeOH 95:5, silica gel): R_f 0.55. UV (H₂O/MeOH, 4:1 ): λ_max: 241 nm, 324nm. ¹H-NMR (CDCl₃): 1.61 (bs, 6H, H-Ada), 1.72 (bs, 6H, H- Ada), 2.14 (bs, 3H, H-Ada), 2.49 (s, 3H, H-Me), 6.41 (m, 1 H, H-arom.), 6.74 (m, 1 H, H-arom.), 7.52 (s, 1 H, H-arom.).

Elemental analysis: Calcd for Cι₆H₂₂N2 (242.37): C 79.29, H 9.15, N 11.56; Found: C 79.40, H 9.07, N 11 ,43.

Example 6.

2-(1-Adamantylamino)-4,6-dimethylpyridine

To a solution of 2-amino-4,6-dimethylpyridine (1.21g) in 10 cm³ of trifluoroacetic acid, 1 -adamantanol (1.22g) was added while stirring. The resulting reaction mixture was heated at reflux for 8 hrs while stirring. The reaction mixture was left to reach a room temperature, poured into 100 cm³ of water -methanol mixture (9:1) and adjusted to pH 7 with an aqueous ammonia solution. The resulting precipitate was filtered off and absorbed on silica gel, which was then passed through a column with silica gel (3 X 15 cm). Chromatography was performed with chloroform (150 cm³) and then with chloroform-methanol mixture (95:5) as eluents. Fractions containing the product were evaporated to dryness, and the residue was crystallized from hexane to yield 0.76 g of colourless crystals, mp 128-130°C. Yield 37%. TLC (CH₂CI₂/MeOH 95:5, silica gel): R_f 65. UV (H₂O/MeOH, 4:1 ): λ_max: 244 nm, 320nm. ¹H-NMR (CDCI₃): 1.61 (bs, 6H, H-Ada), 1.72 (bs, 6H, H-Ada), 2.11 (bs, 3H, H-Ada), 2.45 and 2.49 (2s, 6H, H-Me), 6.63 and 6.95 (2s, 2H, H- arom.), 6.74 (m, 1 H, H-arom.), 7.52 (s, 1 H, H-arom.).

Elemental analysis: Calcd for C₁₇H₂₄N₂ (256.39): C 79.64, H 9.44, N 10.93 ; Found: C 79.49, H 9.37, N 10,84.

Example 7.

3-(1-Adamantylamino)-6-chloropyridine

To a solution of 3-amino-6-chloropyridine (1.29 g) in 20 cm³ of 90% orthophosphoric acid, 1-adamantanol (1.21g) was added while stirring in an oil bath (105°C). Stirring at that temperature was continued for 2 hrs. The mixture was poured into 100 cm³ of ice water and neutralised with 20% KOH solution. The resulting precipitate was filtered off, washed with water and absorbed on silica gel, which was then passed through a chromatographic column with silica gel (3 x 15 cm), chloroform being used as an eluent. Fractions containing the product were evaporated to dryness, and the residue was crystallized from hexane to yield 0.26 g of colourless crystals, mp 124-126°C. Yield 12%. TLC (CHCI3, silica gel): R_f 0.23. UV (H₂0/MeOH, 4:1 ): λ_max: 258 nm, 310nm. ¹H-NMR (CDCb): 1.68 (bs, 6H, H-Ada), 1.85 (bs, 6H, H-Ada), 2.13 (bs, 3H, H-Ada), 7.07 (d, 2H, H-arom.), 7.88 (t, 1 H, H-arom.)

Elemental analysis: Calcd for C₁₅H₁₉N₂CI (262.79): C 68.56, H 7.29, N 10.66; Found: C 68.49, H 7.37, N 10,74.

Example 8.

2-(1 -Adamantylamino)-6-chloropyridine hydrochloride

To a solution of 2-(1-adamantylamino)-6-rnethylpyridine (2.42 g) in 25 cm³ of anhydrous ethanol, 5 cm³ of anhydrous ethanol saturated with hydrogen chloride was added. The resulting solution was evaporated to dryness and the residue was treated with 20 cm³ of acetone-diethyl ether mixture (1 :1 , v/v). The precipitate was filtered off and dried under reduced pressure over KOH to yield 2.51 g of yellowish crystals, mp 127-130 °C. Yield 90%. TNF Production byB78/TNF9 clone (10 uM)

Control (No pyridine derivative added)

1

Claims

Patent claims

1. New (1 -adamanty!amino)pyridines of formula 2,

wherein substituents Ri, R₂, R3, R₄ θr Rsare adamantylamino group, hydrogen atom, methyl group or halogen atom, the molecule of formula 2 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, and salts thereof.

2. A new compound according to claim 1 , which is 2-(1- admantyl- amino)pyridine.

3. A new compound according to claim 1 , which is 3-(1- adamantyl-amino)pyridine.

4. A new compound according to claim 1 , which is 2-(1- adamantyl- amino) -4- methylpyridine.

5. A new compound according to claim 1 , which is 2-(1- adamantyl- amino)-5- methylpyridine.

6. A new compound according to claim 1 , which is 2-(1- adamantyl- amino)-4,6- dimethylpyridine.

7. A new compound according to claim 1 , which is 2-(1- adamantyl- amino)-6- methylpyridine.

8. A new compound according to claim 1 , which is 4-(1 -adamantyl- amino)pyridine.

9. Method of preparation of new (l-adamantylamino)pyridines of formula 2,

wherein substituents Ri, R₂, R3, R₄ or Rsare adamantylamino group, hydrogen atom, methyl group or halogen atom, the compound of formula 2 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, by reacting 1 -adamantanol with aminopyridine derivatives, wherein a pyridine derivative of formula 1 ,

wherein Ri, R₂, R3, R₄ θr R5 are amino group, hydrogen atom, methyl group or halogen atom, the molecule of formula 1 containing one amino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, is reacted with 1-adamantol at elevated temperature in an acid solution, to yield a compound of formula 2,

wherein substituents R<_\ , R₂, R₃, ₄ or R5 are as defined above, the compound of formula 2 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, and the product of formula 2 is isolated or optionally converted by a known method into a salt.

10. A method according to claim 9, wherein trifluoroacetic acid is used to provide an acid reaction solution.

11. A method according to claim 9, wherein orthophosphoric acid is used to provide an acid reaction solution.

12. A method according to claim 11 , wherein orthophosphoric acid at concentration of 85-100%), preferably 90%, is used.

13. A method according to claim 9, wherein the reaction is carried out at elevated temperature, preferably 90-120°C, or preferably at boiling temperature of trifluoroacetic acid.

14. A method of isolation of a final product of formula 2, according to claim 9, wherein the product is isolated from a reaction mixture of pH 7.0-14.0.

15. A method of isolation of a final product of formula 2, according to claim 14, wherein the product is isolated from a reaction mixture through crystallization.

16. A method of isolation of a final product of formula 2, according to claim 14, wherein the product is isolated from a reaction mixture by adsorption chromatography on silica gel.

17. Use of new compounds of formula 2 stimulating secretion of TNF-α in neoplasfically altered cells, according to claims 1 -8, as active substances in pharmaceutical compositions.

18. Pharmaceutical composition stimulating secretion of TNF-α in neoplasfically altered cells, comprising an effective, stimulating TNF-α secretion in neoplastic cells, amount of the compound of formula 2 according to claims 1-8 together with a pharmaceutically acceptable carrier.

19. A method of treatment of neoplastic solid tumours comprising administration of an effective, stimulating TNF-α secretion in neoplastic cells, amount of the compound of formula 1 according to claims 1-7 together with a pharmaceutically acceptable carrier.