AU699789B2 - Colchicine derivatives and the therapeutical use thereof - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT (Original) APPLICATION NO:

LODGED:

COMPLETE SPECIFICATION LODGED:

ACCEPTED:

PUBLISHED:

RELATED ART: NAME OF APPLICANT: INDENA S.p.A.

A A I 0OAC

VA.,

A

*00~ C A CA 40 ACTUAL INVENTOR(S) ADDRESS FOR SERVICE: BOMBARDELLI, Ezio GABETTA, Bruno LORD COMPANY Patent Trade Mark Attorneys 4 Douro Place West Perth, Western Australia, 6005

AUSTRALIA.

"COLCHICINE DERIVATIVES THERAPEUTICAL USE THEREOF' INVENTION TITLE: AND THE The following Statement is a full description of this invention including the best method of performing it known to me/us: WO 96/1118-4 PCT/EP95/03823 COLCHICINE DERIVATIVES AND THE THERAPEUTICAL USE THEREOF SThe present invention relates to novel colchicine derivatives having antiproliferative, antitumoral and antiinflammatory activities, the methods for the preparation thereof and the pharmaceutical formulations containing them. Colchicine is a known pseudo-alkaloid widely used for a very long time in therapy for the treatment of gout; it acts very quickly and specifically, even though it should be used for short i times due to its toxicity. In addition, colchicine is a 10 very potent antiblastic agent, whose action is connected with a mechanism blocking the formation of the mitotic spindle in cell division; this latter aspect :ias been I....investigated thoroughly for any antitumoral activity and a great deal of colchicine derivatives have been 15 prepared for this purpose.

Cilchicine as such and a number of its prepared Sderivatives could not be used due to its high toxicity in terms of risk/benefit ratio. Only one colchicine i derivative, demecolcine, is used in some degree in oncology for the treatment of some leukemia forms. As far as the use in the antiinflammatory field is o concerned, the only marketed colchicine derivative is thiocolchicoside, bearing a thiomethyl moiety at C 10 and a glucose molecule at the hydroxvl in C 3 the therapeutical uses of this derivative related to the muscle relaxant and antiphlogistic effects. The products of the invention differ from those of the prior art in the high therapeutical index. In the antitumoral field, researches have been focused on the research of products WO 96/11184 PCTEP95/03S23 2 having, besides a normal cytotoxicity, a cytotoxicity aimed at cell lint.s resistant to the usual antiblastic medicaments.

The derivatives of the present invention have the general formula 1 S

I

I 10 3

O

X~Me .0.o wherein: X can be an oxygen or sulfur atom; RI and R2, which can be the same or different, are straight or branched alkyl groups or cycloalkyl groups, containing 1 to 6 carbon atoms; or saturated or 0. unsaturated acyl groups, containing 16 to 22 carbon atoms, or a P-D-glucose residue as such or a P-D-glucose 20 residue wherein the hydroxyl in positions 4 and 6 are protected as chetals with aliphatic or aromatic or heteroaromatic aldehydes; Y is a -CH-CH-NH-R 3 group, the methylene group of which being bound to the carbon in position 5 and the methine group, having the same absolute configuration as colchicine, is bound to the tropolone ring, or a -CH-CH20R 4 group, the methine group of which being bound to the carbon in position 5 and to the tropolone ring and having absolute configuration S;

R

3 is an acyl group containing 2 to 6 carbon atoms bearing one to three halogen atoms, preferably fluorine or chlorine, or an acyl group from a natural amino acid, i ~-a31P4~a3p-n Il~aL~ a a De so 0 O a so o ao a.- 6 ae **0 a o~ a *a WO 96/11184 PCTiEP95/03S23 3 wherein the amino group can be free or protected as trifluoroacetamide or benzamide; R4 is an acyl residue of a dicarboxylic acid containing 4 to 6 carbon atoms, or an acyl residue of a natural amino acid, wherein the amino group can be free or protected as trifluoroacetamide or benzamide, or a glycoside residue consisting of D-glucose, D-galactose, L-fucose or L-rhamnose. In this case, the sugars act as chemotactic for melanocytes, hepatocytes, fibroblasts or 10 they give the derivative the characteristic of a prodrug.

Preferred compounds of formula 1 are those wherein X is sulfur.

R1 and R2, which are the same or difFerent, are preferably hydrogen or C 1

-C

6 alkyl.

Y is preferably a group of formula -CH2-CH-NHR 3 as defined above.

For the preparation of the products of the invention, the starting products used are the natural 20 substances colchicine X=O; RI=R 2 =Me; Y=CH 2 -CH-NHAc), 2-O-demethylcolchicine X=O; R1=H; R 2 =Me; Y=CH2-CH- NHAc), 3-0-demethylcolchicine X=O; RI=Me; R2=H; Y=CH2-CH-NHAc), colchicoside X=0; R 1 =Ye; R 2 =0-Dglucose; Y=CH 2 -CH-NHAc) which can be recovered from vegetable materials according to procedures known in literature. These natural substances, 'y treatment with methylmercaptan in alkali solution, according to procedures also known in literature, yield the corresponding thioderivatives which are used as the syntones for the preparation of the derivatives of general formula 1, wherein X is sulfur.

p SWO 9611184 PCTiEP95,03S23 4 For the preparation of the derivatives of general formula 1 wherein R1 and R2 are alkyl or acyl groups, the starting products used are the colchicine or f thiocolchicine syntons demethylated in position 2 or 3.

These syntons are subjected to alkylation or acylation 1 using the well known methods for the phenol derivatization. Analogously, the derivatives of general formula 1 wherein R1 or R 2 are a P-D-glucose residue or a p-D-glucose residue wherein the hydroxyls in 4 and 6 are protected as chetals with aliphatic or aromatic aldehydes, are prepared from colchicine or thiocolchicine syntons demethylated in position 2 or 3.

These syntons are subjected to a reaction with a-bromotetraacetyl-D-glucose or with 2,3-di-O-dichloroacetyl-P- 15 D-glucose containing a chetal group involving the *a hydroxyls in the positions 4 and 6 (cf. Canada Pat. N.

956939). After removing the protective acyl groups by known methods described in literature, the derivatives glucosidated in the position 2 or 3 according to the 20 present invention are obtained.

The derivatives of formula 1 wherein Y is a -CH2-CH-NH-R 3 group are prepared subjecting to Ndesacetylation by acid catalysis the colchicine or thiocolchicine syntons, bearing methoxy or hydroxy groups in the positions 2 and 3, followed by acylation of the primary amino moiety with an acid reactive derivative containing one to three fluorine or chlorine atoms or a natural amino acid, the amino group of which can be free or protected as trifluoroacetamide or benzamide. The derivatives of formula 1 wherein R 3 has the meaning defined above are thereby prepared. The IF~ ~Lq

I

WO 96/11184 PCT/EP95/03S23 derivatives of formula 1 wherein Y is a -CH-CH 2

-OR

4 group are obtained subjecting colchicine or thiocolchicine syntons, bearing methoxy or hydroxy groups in positions 2 and 3, to N-desacetylation followed by treatment with sodium nitrite and acetic acid which contracts of the cycloheptane ring with the formation of the syntons of formula 1 wherein Y=-CH-

CH

2 OH Med. Chem. 36, 544, 1993). The resulting primary alcohol moiety yields, by reaction with suitably 10 activated dicarboxylic acids, or with activated natural .0 amino acids, wherein the amino group can be free or protected as trifluoroacetamide or benzamide, or with o the reactive form of the sugars D-glucose, D-galactose, L-fucose or L-rhamnose, the derivatives of general 15 formula 1 wherein Y=CH-CH 2

OR

4

R

4 having the meaning defined above.

The following table shows the antimitotic activity on tumoral cell lines of some derivatives of the invention. Taxol and colchicine are the comparison 20 substances.

ei

WAM

I

WO 96/11184 PCTIEP95,'03S23 6 TABLE In vitro cytotoxic activity of some novel colchicine derivatives.

Substances IC 50 (n

M

Cell lines Ovary Colon Breast Lung Cell.

resist.

Taxol 6.1 3.5 3.2 1.7 299 1. Colchicine 5.3 3.9 4.4 16 112 Compound IZ 4.2 2.6 6.2 8 26 IIZ 9.4 1.8 5.0 19 11 o IVZ 6.1 2.2 4.1 12 7 15 This table evidences that the novel derivatives have significant advantages on the resistant cell lines, which is the main target for cytotoxic medicaments. The products of the present invention can be incorporated in o pharmaceutical formulations useful for the C 20 admi.. -tration of the medicament. Formulations for the i'"e parenteral, oral, transdermal, epicutaneous administrations can be conveniently prepared.

Among the excipients useful to prepare said formulations, natural and synthetic phospholipids proved to be particularly useful for preparing liposomial forms for the parenteral, transdermal or epicutaneous routes; the latter two formulations being particularly useful in the treatment of arthrosic or peripheral venous inflammatory conditions; said formulations also being useful in the topical treatment of cutaneous epitheliomas and in cutaneous hyperproliferative WO 96111184 PCT/EP95/03S23 7 conditions, such as psoriasis. In the specific antitumoral field, besides the phospholipids which allow for the administration of the medicament in liposomial form, some surfactants such as polyethoxylated castor oil, such as for example Cremoform L50, or polisorbate, such as for example Tween, acting synergistically with the active ingredient, turned out to be particularly useful. In oncology, the products are used at dosages from 1 to 100 mg/m 2 whereas as antiinflammatory the *00> dosages range from 1 to 20 mg per unit dose, one to more times daily. All of the pharmaceutical formulations such as vials, capsules, creams etc. can be prepared with the o main part of said derivatives.

The following examples further illustrate the invention.

SExample I Preparation of N-desacetyl-N-trifluoroacetyl-3-O-demethyl-3-0-cyclopentyl-thiocolchicine, compound IZ X=S; R 1 =Me; R 2

=C

5

H

9

Y=CH

2

CHNH-COCF

3 g of 3-0-demethylthiocolchicine X=S; R 1 =Me; 20 R 2

Y=CH

2 CHNHAc) are dissolved in 300 ml of sulfuric acid and treated at 100'C under nitrogen atmosphere for 36h. The reaction mixture is neutralized, thus separating 12 g of thiocolchicine R 1 =Me; R 2

Y=CH

2 CHNH2). This product is dissolved in acetone and reacted with 3 equivalents of trifluoroacetic anhydride under strong stirring in the presence of anhydrous Na 2

CO

3 After 2h the reaction mixture is filtered and the solution is evaporated to dryness. The residue, consisting of demethyl-N,3-O-bistrifluoroacetylthiocolchicine, is hydrolysed in methanol containing NH 4 C1. The reaction 41111CIIIOPl~rer~pW~-~ ~s*B~aRp lw WO 96/11184 PCT/EP95/03823 8 mixture is evaporated to dryness under vacuum and the residue taken up in acetone. The acetone solution is filtered and refluxed for eight hours with 5 equivalents I of cyclopentyl bromide in the presence of sodium carbonate. Salts are filtered off, the solution is evaporated to dryness and the residue is purified by chromatography on silica gel column using ethyl acetate as eluent. By crystallization from acetone/hexane. 8.6 g of product are obtained, M+a m/z 523.

10 Example II Preparation of N-desacetyl-N-trifluoroacetyl-3-0-demethyl-3-O-isopropyl-thiocolchicine, com- 000 0 o0. pound IIZ X=S; R 1 =Me; R 2 =iPr; Y=CH 2

-CH-NHCOCF

3 o S0 For the preparation of this derivative, the 0 o procedure of example I is repeated, using as reagent 15 isopropyl bromide instead of cyclopentyl bromide. After 0°00: purification of the crude reaction product on silica gel and crystallization, 7.6 g of product are obtained, M+a o m/z 497.

too.

Example III Preparation of N-desacetyl-N-trifluoro- 20 acetylthiocolchicoside, compound IIIZ X=S; R 1 =Me; to**

R

2 =0-D-glucosyl;

Y=CH

2

-CH-NHCOCF

3 g of N-desacetyl-thiocolchicoside X=S;

R

1 =Me; R 2 =p-D-glucose; Y=CH 2

-CH-NH

2 are dissolved in acetone and treated for two hours at 10'C with three equivalents of trifluoroacetic anhydride. The mixture is evaporated to dryness and the residue is crystallized from isopropanol and sJbsequently from ethanol. 8.5 g of product are obtained, M+a m/z 617.

Example IV Preparation of N-(N-trifluoi.acetyl-aphenylglycyl)-desacetylthiocolchicine, compound IVZ (1; X=S; R 1

=R

2 =He; Y=CH 2

-CH-NH-CO-CH(NHCOCF

3 )Ph).

O

a aa o** e 0oo B C 0000° WO 96/11184 PCTIEP95/03823 9 400 mg of N-desacetyl-thiocolchicine X=S; R=

R

2 =Me; Y=CH 2

-CH-NH

2 (1.07 mmol) are dissolved together with 265 mg (1,07 mmol) of L-N-trifluoro-acetyl-aphenyl-glycine in 10 ml of methylene chloride under nitrogen atmosphere. The solution is added 221 mg (1.07 mirol) of N,N-dicyclohexylcarbodiimide, stirring until disappearance of the reagents. The reaction mixture is cooled to -30'C and filtered to remove the precipitated urea. The chloromethylene solution is concentrated and 10 purified by filtration on silica gel eluting with a methylene chloride/methanol 98:2 mixture. Upon crystallization from methylene chloride/ethyl ether, 350 mg of product are obtained, M+a m/z 602.

Example V Preparation of N-(N-trifluoroa:etyl-L-ala- 15 nyl)-desacetylthiocolchicine, compound VZ X=S; R 1

R

2 =Me; Y=CH 2

-CH-NH-CO-CH(NHCOCF

3

)CH

3 400 mg of N-desacetyl-thiocolchicine ,,07 mmol) are treated with one equivalent of N-trifuoroacetyl-Lalanine and one equivalent of N,N-dicyclohexylcarbodiimide in 10 ml of methylene chloride and under nitrogen atmosphere until disappearance of the reagents.

The reaction mixture is cooled to -30'C and filtered to remove the precipitated urea. The chloromethylene solution is concentrated and purified by filtration on silica gel eluting with a methylene chloride/methanol 98:2 mixture. Upon crystallization from methylene chloride/ethyl ether, 94 mg of product are obtained, M+a m/z 540.

Example VI Preparation of N-(N-trifluoroacetylmethionyl)-desacetylthiocolchicine, compound VIZ X=S;

R

1

=R

2 =Me; Y=CH 2

-CH-NHCO-CH(NHCOCF

3

)CH

2

-CH

2 -SMe).

W096h11184 PCT/EP95/0383J The procedure of example IV is repeated, reacting N-trifluoroacetylmethionine. By fractional crystallization, upon chromatographic purificatior, of the reaction residue, from 400 mg of N--des ace tyl thiocolchic ine. 84 mg of product are obtained, W'a m/z 600.

Example VII Preparation of N-(a-phenylglycyl)desacetyithiocoichicine, compound VIIZ X=S; R1 R 2 =Me; Y=CH 2

CH-NHCO-CHNH

2 Ph).

400 mg of the product obtained in example IV are 0010 dissolved in 5 ml of 50% acetone in the nresence of 120 f potassium carbonate and heated at 60'C with 00 stirring for 5 hours. The reaction mixture is cooled, seturated with Nacl and extracted with chloroform. The organic phase is dried over anhydrous sodium sulfate, .0015 then concentrated to dryniess and the residue is chromatographed on silica gel with a methylene chlo~ride /methanol 98:2 mixture. 160 mg 6f product are obtained, Mla m/z 506.

Example VIII Preparation of N-desacetyl-N--trifluoroacet,.'l-3-O-demethyl-3-O-ximeniny.thiocolchicine, com- 0.00pound VIIIZ X=S; R =Me; R =CO(CH 2 7

C=C-CHGCH-

(CH

2 )CH; Y=CH 2

-CH-NH-CO-CF

3 500 mg of thylthiocolchicine X=S; R 1 =Ke; R 2

Y=CH

2

-CH-

NHCOCF

3 are dissolved in 2. 5 ml of' pyridine and added at Q'C with 500 mg of ximeninic acid chloride. The reaction mixture is left to stand overnight at room temperature, then is poured onto ice. The resulting precipitate separated and crystalliz d from da acetone/hexane, M+a m/z 715.

Example IX Preparation of 5,6-dihydro-6(S)-[(O3-D-glu- NW096/11184 PCTBP95/03S23 copyranosyloxy)methYl-l,2,3-trimethoxy-9-(methylthio)- 8H-cycloheptalnaphthalel-8-one, compound IXZ X=S; R =R 2 =Me; Y=Ci--CH 2 -p-D-glucose).

g of N-des ace tylthioco lchicine are treated with sodium nitrite to give 4 g of 5,6-dihydro-6(S)- (hydroxymethyl)-1,2,3-trimethoxy-9-methylthio-8H-cyclohepta~ajnaphthalen-8-one, according to the process described in J. Med. Chem., 36, 544, 1993. The resulting product is treated for 12 hours under ref lux in acetonitrile with 26 g of a-bromotetraacetylglucose in *0the presence of 85 g of mecrccyanide.Sat ar ofiltered off and the solution is evaporated to dryness, .:taken up with 70% acetone and treated for two hours with sodium carbonate. The mixture is neutralized, *015 extracted with etP-Yi1 acetate and chromatographed on silica geal eluting with a methylene chloride-ethanol 9: 1 mixture. 2.1 g of product are obtained, M~a rn/z 536.

Example X Preparation of 5,6-dihydro-6-(S)-(hydroxymethyl)-1,2, 3-trimethoxy--9-(methylthio)-8H-cyclohepta- *eo~ 20 £alnaphthalen-8-one succinyl ester, compound XZ X=S; 0 R 0 R=R 2 =Me; Y=CI-{-CH 2

OCOCH

2 CH 2

CO

2

H).

g of N-desacetyltniocolchicin(6 are treated as in example IX. The resulting naphthalen-8-one is dissolved in pyridine and treated under reflux for 24 hours with a succinic anhydride excess. The rtzaction mixture is cooled, poured into abundant water and extracted with methylerie chloride. The organic phaso is concentrated to small volume and purified on silica gel eluting with a methylene chloride-water-methanol 70:30:5 mixture. After crystallization from methanol, 7 g Of product are obtained, Mla m/z 474.

Claims (9)

1. Compounds of Formula I Y 3 2 R 0 OCH 3 0 XHe o o 0 wherein: on o 0o' 10 X can be an oxygen or sulfur atom; o.0 RI and R 2 which can be the same or different, are straight or branched alkyl groups or cycloalkyl groups, containing 1 to 6 carbon atoms, or saturated or unsaturated acyl a e o" groups, containing 16 to 22 carbon atoms, or a p-D-glucose residue as such or a p-D- glucose residue wherein the hydroxyls in positions 4 and 6 are protected as chetals with 0 15 aliphatic or aromatic or heteroaromn.tic aldehydes; Y is a -CH-CH20R 4 gn'up, the methine group of which being bound to the carbon position 5 and to the tropolone ring 0 and having absolute configuration S; R 4 is an acyl residue of a dicarboxylic acid containing 4 to 6 carbon atoms, or an acyl residue of a natural amino acid, wherein the amino group ca.- be free or protected as trifluoroacetamide or benzamide, or a glycoside -2 20 residue consisting of D-glucose, D-galactose, L-fucose or L-rhrc:-7"ose.

2. Compounds according to claim 1, wherein X is sulphur.

3. Compounds according to claim 1, wherein X is oxygen. 13

4. Compounds according to claim 2 or claim 3, wherein R1 and R 2 which are the same or different, are straight or branched alkyl groups or cycloalkyl groups containing 1 to 6 carbon atoms.

5. A process for the preparation of the compounds of claims 1 to 4, which includes subjecting colchine, 2-0-demethyl colchicine, 3-0-demethylcolchicine, colchicoside to one or more of the following reactions: a) reaction with methylmercaptan in alkali solution; b) alkylation, acylation or glycosylation of the phenol groups; 0 0 c) N-desacetylation follo ed by treatment with sodium nitrite and acetic acid to o a give compounds 1 wherein Y is Y-CH-CH20H and subsequent reaction with derivatives of dicarboxyl;c acids of amino acids or with reactive derivatives of S D-glucose, D-galactose, L-fucose, L-rhamnose. 000

6. Pharmaceutical compositions containing as active ingredient a compound of claims 1 to 4 in admixture with a suitable carrier, particularly natural or synthetic phospholipids.

7. The use of the compounds of claims 1 to 4 for the preparation of medicaments having anti-proliferative, antitumoral and anti-inflammatory activities.

8. Compounds according to Formula I as hereinbefore described in the i. accompanying Examples 9 or C i i~lBC~

9. A process for the preparation of compounds according to Formula I substantially as hereinbefore described in the accompanying Examples 9 or oo 00 0 Do S00 00 0 0 o* oo S0o 000oooo 0 0 ao o o 0000 0 eoe SO S0 S« o o 000 00 0 3 000 0 0 0 Q00 a Pharmaceutical compositions substantially as hereinbefore described in the accompanying Examples 9 or DATED THIS 14 T DAY OF APRIL 1998. INDENA S.p.A. By their Patent Attorneys LORD COMPANY PERTH, WESTERN AUSTRALIA I I I, ABSTRACT The present invention relates to novel colchicine derivatives of Formula I where Y is a -CHCH 2 0R 4 group and the other variables are as defined in claim 1, having anti proliferative, antitumoral and anti-inflammatory activities. The novel compounds have a cytotoxicity on human tumoral cell lines comparable with colchicine, but, in comparison with the latter, they are less toxic and more selective, particularly on cells resistant to the usual medicaments. Some compounds have a marked activity on TNF and inerleukine 2, and therefore are very potent anti-inflammatory agents. They can be included in pharmaceutical formulations useful for the parenteral, oral and topical administrations. R 20 C o 3 O CoMe C