NO150242B - ANALOGUE PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE 2-BETA-D-RIBOFURANOSYLTIAZOL-4-CARBOXAMIDE ESTERS - Google Patents

Description

Combating malignant tumors in humans and animals

is still a goal that has not been reached. During the last decades, the understanding of malignant tumors has made significant progress, but it has so far been unsuccessful in combating the malignant disease state.

Common treatment of both humans and other valuable animal species affected by malignant tumors currently includes surgical removal of the tumor, local radiation treatment of the affected animal, and chemotherapy by administering a chemotherapeutic agent to the animal. The fact that a significant number of patients who are attacked by malignant tumors die is not due to the primary tumor, but instead to metastases of the primary tumor to secondary sites in the body. If a primary tumor is detected early, it can usually be removed

by surgery, radiotherapy or chemotherapy or combinations of these. However, the metastatic colonies of these primary tumors are much more difficult to detect and remove,

and a less successful treatment of them remains a serious medical problem.

Tumors are normally classified as either benign or malignant. A malignant tumor differs from the benign one by its ability to penetrate both surrounding tissue and colonize distant sites via metastasis. Certain organs are more prone to metastasis than others. This group includes the lungs, brain, liver, ovaries and adrenal glands. It has further been suggested that both surgery and irradiation of a primary tumor actually promote metastasis.

Because the current cancer treatment is not able to

to provide any successful treatment of malignant tumors and metastasis thereof, it is clear that there is a need for additional chemotherapeutic agents.

In an article entitled Synthesis and Antiviral

Activity of Certain Thiazole C-Nucleosides, J. Med. Chem. 1977, vol. 20, No. 2, 256, the inventor and his co-workers have described the synthesis and some preliminary in vitro antiviral activity of the compounds 2-O-D-ribofuranosylthiazole-4-carboxamide and 2-(2,3,5-tri-O-acetyl-Ø -D-ribofuranosyl)-thiazole-4-carboxamide in an in vitro test system using three types of virus, type 1 herpes simplex virus, type 3 parainfluenza virus and type 3 rhinovirus. The in vitro activity of the compound 2-0-D-ribofuranosylthiazole-4-carboxamide against these three viruses was only moderate. With the compound 2-(2,3,5-tri-O-acetyl-Ø-D-ribofuranosyl)thiazole-4-carboxamide only moderate activity was obtained with type 1 herpes simplex virus, while the activity was negative with type 3 parainfluenza virus and type 13 rhinovirus. While some marginal in vitro antiviral activity was found as seen from the above, on the other hand in vivo antiviral activity for both 2-0-D-ribof uranosylthiazole-4-carboxamide and 2-(2 , 3 ,5-tri- O-acetyl-[beta]-D-ribofuranosyl)thiazole-4-carboxamide, as judged

by the number of dead experimental animals, negative. In the in vivo studies, the number of deaths for the test animals for both 2-0-D-ribofuranosylthiazole-4-carboxamide and 2-(2,3,5-tri-O-acetyl-Ø-D-ribofuranosyl)thiazole-4-carboxamide was equal to or exceeded the number of deaths with placebo control animals, showing that both compounds 2-0-D-ribofuranosylthiazole-4-carboxamide and 2-(2,3,5-tri-O-acetyl-Ø-D-ribofuranosyl)thiazole-4- carboxamide does not exhibit any useful in vivo antiviral activity.

With respect to the above in vitro antiviral study of both 2-O-D-ribofuranosylthiazole-4-carboxamide and 2-(2,3,5-tri-O-acetyl-Ø-D-ribofuranosyl)thiazole-4- carboxamide, these compounds were examined against viruses against which the known antiviral compound RIBAVIRIN has positive antiviral activity. On the basis of the preliminary marginal in vitro activity of 2-O-D-ribofuranosylthiazole-4-carboxamide against these experimental viruses, it was assumed that the activity spectrum of 2-O-D-ribofuranosylthiazole-4-carboxamide would be similar to the activity spectrum of the compound RIBAVIRIN< ®>. RIBAVIRIN<®> is known to be an active in vitro antiviral and also an in vivo antiviral, and is also known not to exhibit any significant antitumor activity. Moreover, certain derivatives of RIBAVIRIN such as its 5<1->monophosphate are also known to be inactive as antitumor compounds. It was reasonable to assume, by comparing the preliminary in vitro antiviral activity of 2-D-ribo.furanosylthiazole-4-carboxamide with that of RIBAVIRIN", that 2-0-D-ribofuranosylthiazol-4-carboxamide would show positive in vivo antiviral activity and negative antitumor activity similar to RIBAVIRIN ® In contrast, the compound 2-0-D-ribofuranosylthiazole-4-carboxamide had no useful in vivo antiviral activity and is unexpectedly found to have positive antitumor activity.

It has been found that certain esters of the compound 2-3-D-ribofuranosylthiazole-4-carboxamide including 2-(5-0-phosphoryl-Ø-D-ribofuranosyl)thiazole-4-carboxamide exhibit antitumor activities of such importance that they are useful as antitumor agents in vivo.

Certain esters of 2-3-D-ribofuranosylthiazole-4-carboxamide

has been shown to exhibit significant antitumor activity in vivo. The present invention relates to the production of these compounds which can be used for the treatment of malignant tumors in warm-blooded animals. The antitumor properties of 2-/3-D-ribofuranosylthiazole-4-carboxamide esters are exploited by administering to a warm-blooded animal an effective amount of a pharmaceutical preparation containing the active compound in an amount of at least approx. 0.1% by weight, based on the total weight of the preparation.

The new compounds produced according to the invention have the formula: where R., is lower alkanoyl or

and physiologically acceptable salts thereof.

Particularly preferred alkanoyl groups as meaning for are acetyl, propionyl, butyryl and isobutyryl.

Particularly important as acceptable salts are alkali metal and ammonium and substituted ammonium salts, such as sodium, potassium and ammonium salts.

For use in pharmaceutical preparations, a pharmaceutical carrier is used of such a nature that it allows the administration of a suitable concentration of the active compounds produced according to the invention as solutions or suspensions by injection to a warm-blooded animal to be treated. Depending on the animal having the malignant tumor, the type of tumor and the location of the tumor, administration by injection will be intravenous, intramuscular, intracerebral, subcutaneous or intraperitoneal.

Alternatively, the preparation can be suitably prepared

in suitable pharmaceutical carriers which allow administration by other routes such as oral administration, ophthalmic administration, local administration or administration by suppositories.

The starting compound used in the method according to the invention, compound 1, 2-0-D-ribofuranosylthioazole-4-carboxamide, is preferably prepared as described in example 1. An alternative synthesis of this compound is described in J. Org. Chem., vol. 41, No. 26, 1976, 4074.

Certain esters of 2-O-D-ribofuranosylthiazole-4-carboxamide, compound 1, such as 2-(5-0-phosphoryl-Ø-D-ribofuranosyl)-thiazole-4-carboxamide, compound 2, are prepared as described in Example 2. Other esters, such as the monoester 2-(5-0-acetyl-Ø-D-ribofuranosyl)thiazole-4-carboxamide, compound 3, can be prepared by the synthesis described in example 3. For other preferred carboxylic acid esters, acetic anhydride can be replaced by a suitable anhydride such as propionic anhydride or butyric anhydride.

Alternatively, the appropriate acid chloride can be used instead of the acid anhydride.

The alkanoyl groups can be selected from acids such as acetic acid, propionic acid, n-butyric acid, isobutyric acid, valeric acid, caproic acid, enanthic acid and caprylic acid. If a phosphoryl group is chosen, the phosphoryl ester can be in the form of a free acid or as a salt form. Acceptable salts of the phosphate portion of the phosphoryl ester may be selected from the group consisting of alkali and alkaline earth salts, e.g. of sodium, potassium, calcium, magnesium, lithium, ammonium and substituted ammonium, trialkylammonium, dialkylammonium, alkylammonium, e.g. triethylammonium, trimethylammonium, diethylammonium, octylammonium, cetyltrimethylammonium and cetylpyridinium.

As preferred esters prepared according to the invention, compounds 2 and 3 can be mentioned. When phosphoryl esters are prepared, the phosphate groups are preferably formed as a salt, in particular as a sodium salt or another alkali metal salt or ammonium salt.

Ester forms of compound 1, as shown in the examples, are useful for delivering the compound to the affected site in a patient. Although we shall not be bound by any theory here, it is possible that if the phosphoryl ester of compound 1, the 5'-phosphate is used, it is possible that enzymes present in vivo cleave the phosphate enzymatically to supply in situ compound 1 Compound 2, the phosphoryl ester of compound 1, as shown in the examples, has been found to be an effective anti-tumor agent when injected into a patient. At the present time, it is not clear whether the activity is in the form of the 5'-phosphate or whether this is split enzymatically to compound 1. Furthermore, it is possible that compound 1 can be formed in situ by other enzymatic reactions to compound 2.

In preparing a suitable preparation, a desired ester of formula I is mixed with a suitable pharmaceutical carrier which may be as simple as sterilized water, or it may be a compound carrier containing suitable agents to suitably mimic certain biological environments, i.e.

pH- and salt-adjusted solution suitable for intravenous, intramuscular or other injections.

When choosing a suitable pharmaceutical carrier, consideration must be given to the type of tumour, the location of the tumor and the state of health and age of the patient. In addition, one must also take into account

the chemical reactivity of the ester. Thus, a carboxylic acid ester will preferably be suspended or dissolved in a suitable non-acidic medium. A phosphoryl ester can suitably be used in the presence of a suitable buffer or as a salt as mentioned above.

The compounds produced according to the invention are preferably mixed with a suitable pharmaceutical carrier so that the compound preferably dissolves in the carrier. Alternatively, suspensions, emulsions or other forms of the compounds produced according to the invention can be used. The pharmaceutical carrier can, in addition to containing a solubilizing or suspending agent, also contain suitable diluents, buffers, surfactants and other similar agents that are typically used in pharmaceutical carriers. The total composition of the pharmaceutical carrier will, however, be chosen so that it fits the place where the effect is to be exerted, the concentration of the active ingredient and other parameters in accordance with standard pharmaceutical practice.

The compounds produced according to the invention are appropriately mixed with the pharmaceutical carrier so that they are present in a concentration of at least 0.1% by weight of the total preparation. Preferably, they are present in the pharmaceutical carrier in a concentration of approx. 10 to approx. 90% by weight of the total preparation.

Effective amounts of the compounds produced according to the invention typically vary from approx. 2.5 mg per kg total body weight of the warm-blooded animal to be treated, to approx.

200 mg/kg per day. The preferred range is from 12.5 mg/kg to approx. 100 mg/kg. An even more preferred area is from approx. 15 mg/kg to approx. 50 mg/kg. As is the case with the others

factors mentioned above, the amount of compound used for treating an affected animal is dependent on such parameters as tumor type, tumor site, the form in which the compound is administered, and the patient's physical size and condition. In any case, the amount used must be sufficient to provide a therapeutically effective amount of the agent to the patient

•in an appropriate volume, which can be easily determined by those skilled in the art at

basis of what is stated here.

In Example 4 which is included as an illustration below, an animal with a tumor was treated once daily with the indicated test compound. Depending on the clinical situation, the daily dose of the other compounds prepared according to the invention can be administered in a similar way, but the daily doses can also be broken up into smaller doses which are equal to the total daily dose. Thus, e.g. a patient to be treated with 50 mg/kg is appropriately treated 4 times daily with doses of 12.5 mg/kg.

A preparation used to inhibit malignant tumors in warm-blooded animals can conveniently be prepared by adding the compounds prepared according to the invention to a pharmacologically compatible solvent followed by sterilization and introduction into suitable ampoules that can be sealed, in a known concentration. Appropriate doses of the compound are then taken from the ampoule and administered by injection into the patient.

Example 1

2- 0- D- ribofuranosylthiazole- 4- carboxamide, compound 1

(source material)

Ethyl 2-(2,3,5-tri-O-benzoyl-O-D-ribofuranosyl)-thiazole-4-carboxamide was used as prepared in Srivastova et al, J. Med. Chem. 1977, vol. 20, No. 2, 256. A concentrated solution

of ethyl 2-(2,3,5-tri-O-benzyl-Ø-D-ribofuranosyl)thiazole-4-carboxamide (5.0 g, 8.31 mmol) in methanol (15 mL) was stirred with methanolic ammonia (saturated at 0°C, 100 ml) in a pressure vessel at room temperature for 2 days. The solvent was evaporated, and the residue was chromatographed through a column (2.4 x 35 cm) of silica gel (100 g) packed in ethyl acetate. Elution of the column with a solvent system (ethyl acetate-1-propanol-water, 4:1:2, v/v; top layer) removed the fast migrating methyl benzoate and benzamide. The slow-migrating, UV- and sugar-positive major fractions were collected, and the solvent was evaporated in vacuo. The thus obtained residue (syrup)

was easily recrystallized from ethanol-ethyl acetate to give 1.6 g (74%) of pure product, compound 1: m.p. 144-145°C,

[α]<25>p- 14.3° (c, 1, DMF); UV Amax PH 1,237 nm (8640);

UV ^max<PH><11> 238 nm ( 8100);

<1>H NMR (Me2SO-d6) 6 7.5-7.8 [S (br), 2, CONH2],

"4 NMR (Me2SO-d6-D2O) 6 4.99 (d, 1, J = 5 Hz, E±), 8.25 (s, 1, H5). Analysis (C<gH>12N2<0>5 <S>) C, H, N, S.

Example 2

2-(5-O-phosphoryl-ø-D-ribofuranosyl)thiazole-4-carboxamide (2-g-D-ribofuranosylthiazole-4-carboxamide-5'-phosphate),

connection 2

Water (151 mg, 8.4 mmol) was carefully added to a solution (which was kept at 0°C with stirring) of freshly distilled phosphoryl chloride (2.0 g, 13.2 mmol), pyridine (1.21 g , 14.4 mmol)

and acetonitrile (2.3 g, 56.7 mmol). 2-6-D-ribofuranosylthiazole-4-car box amide, compound 1 (dried over P-jO^- and powdered,

800 mg, 3.0 mmol), was added to the solution, and the reaction mixture was stirred continuously for 4 hours at 0°C. The reaction mixture was poured into ice water (ca. 50 ml) and the pH was adjusted to 2.0 with 2N sodium hydroxide. The solution was added to a column of activated carbon (20 g) and the column was washed thoroughly with water until the eluate was salt-free. The column was eluted with a solution of ethanol-water-concentrated ammonium hydroxide (10:10:1) and the fractions (25 ml each) were collected.

The fractions containing pure (TLC, silica gel, acetonitrile-

0.1N ammonium chloride (7:3)) nucleotide, compound 2, was collected and evaporated to dryness under vacuum. The anhydrous residue was dissolved in water and passed through a column of Dowex 50W-X8 (20-50 mesh, H+<->form, 15 ml). The column was

washed with water, and the fraction containing the nucleotide was collected. The solution was concentrated to a small volume (5 ml) and passed through a column of Dowex 50W-X8 (20-50 mesh, Na<+> form, 15 ml). The column was washed with water. The fraction containing the nucleotide was lyophilized. The residue was triturated with ethanol, collected by filtration and dried (P2°5^ to give 560 mg (47%) of compound 2 as monosodium dihydrate in crystalline form.

Analysis calculated for CgH^1^OgPSNa-2H20:

C 27.13 H 4.04 N 7.04 P 7.78 S 8.05

Found:

C 27.42 H 3.87 N 7.07 P 8.03 S 8.41.

Example 3

2-( 4- O- acetyl- ff- D- ribofuranosyl) thiazol- 4- carboxamide compound 3

A solution of 2-(2,3-O-isopropylidene-Ø-D-ribofuranosyl)-thiazole-4-carboxamide (1.5 g, 5 mmol) (prepared according to Fuertes et al, J. Org. Chem., vol .41, No. 26, 1976, 4074) in anhydrous pyridine (20 mL) was cooled in an ice-water bath and acetic anhydride (2.5 mL) was added slowly with stirring. The reaction solution was allowed to warm to room temperature, and stirring was continued for 15 hours. The solvent was evaporated in vacuo and the residue was dissolved in ethyl acetate and washed with water. The ethyl acetate portion was evaporated in vacuo, and the residue was dissolved in 80% acetic acid (25 ml). The solution was heated on a steam bath for 30 minutes, and the solvent was removed in vacuo. The residue was dissolved in ethyl acetate, washed once with water and dried over MgSO 4 . The ethyl acetate portion was evaporated, and the crude product was passed through a column of silica gel (100 g, packed in chloroform) and eluted with 20% (v/v) ethyl acetate in chloroform. The nucleoside-containing fractions were collected and evaporated to give 1.05 g (70%) of compound <3> (Cn,Hn .N_O,S).

11 14 l. o

As an illustrative example of the use of the compounds produced according to the invention, example 4 is given below. This example illustrates the effectiveness of the compounds using standard samples against certain malignant tumors. The samples used in this example were performed by the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute. The trials were monitored by this institution using their standard protocols and procedures. All trials were in accordance with these protocols, and all trials were judged according to the criteria defined in these protocols. The following representative example illustrates the activity of the compound prepared according to the invention against sample tumor systems from the National Cancer Institute.

In the following example, the abbreviation IP means intra-peritoneal. Mean and median survival times are calculated according to Instruction 14 (revised 6/78) of the Screening Data Summary, Developmental Therapeutics Program, Division of

Cancer Treatment, National Cancer Institute.

In the example below, the carrier that was

used for the active ingredient injected (without active ingredient) in the control animals in the same amount as the amount of vehicle injected into the animals that also received the active ingredient, in order to eliminate any effect of the vehicle on the experiments.

Example 4

Compound 2, 2-(5-O-phosphoryl-3-D-ribofuranosyl)-thiazole-4-carboxamide is shown to be active against L-1210 lymphoid leukemia in Examples 4a and 4b. The dosage amounts of the test compound are as indicated in Tables Ia and Ib below for Examples 4a and 4b respectively. In these examples, 36 control animals were used, and 6 experimental animals for each of the dose quantities indicated in tables Ia and Ib. Salt water was used as a vehicle for the test compound. No toxicity of the test compound was found in the test animals of Examples 4a and 4b. No experimental animal survived beyond day 10 in example 4a, with a mean day of death of day 8.3, and beyond day 11 in example 4b with a mean day of death of day 10.1.

For both the control groups and the test compound groups,

tumors were induced by IP inoculation of tumor cells on day 0, followed by initiation of test compound treatment on day 1, where compound 2 was given once daily for 5 days. The trial results are expressed as T/C (treated/control).

Claims (2)

<1-> Analogous method of preparation for the preparation of a therapeutically active compound with the formula where R-. is lower alkanoyl or characterized in that the compound 2-3-D-ribofuranosylthiazole-4-carboxamide is reacted in the presence of a base with an acyl anhydride or acyl chloride corresponding to R^, preferably a carboxylic acid anhydride or phosphoric anhydride or a carboxylic acid chloride or phosphoric acid chloride. 2. Method according to claim 1 for the production of 2-(5-0-phosphoryl-Ø-D-ribofuranosyl)-thiazole-4-carboxamide, characterized in that 2-3-D-ribofuranosylthiazole-4-carboxamide in the presence of a base is reacted with phosphoryl chloride in a polar solvent, the reaction mixture is treated with water, and the product is isolated, preferably by desalting the reaction mixture with activated charcoal and elution of the product from the charcoal.