KR100333749B1 - O6-substituted guanine derivatives, their preparation and their use in tumor cell therapy - Google Patents

Abstract

The formula (I) and the O ⁶ -heterylalkyl- or naphthylalkylguanine derivative

Wherein X is O or S, R " and R "'are alkyl or substituted derivatives thereof, and R ' is H Or alkylhydroxyalkyl, R is (i) a cyclic group having at least one 5- or 6-membered heterocyclic ring or a substituted derivative thereof, optionally fused to it by a carbon ring or heterocyclic ring , Each heterocyclic ring has at least one heteroatom selected from O, N or S, or R is (ii) naphthyl or a substituted derivative thereof, and pharmaceutically acceptable salts thereof are O ⁶ - And exhibit the ability to reduce alkylguanine-DNA alkyltransferase (ATase) activity. A method for preparing this compound is also described. This compound is used in combination with an alkylating agent in a chemotherapeutic agent for tumor cells.

Description

O6-substituted guanine derivatives, their preparation and their use in tumor cell therapy

Figure 1 is a graph of the residual activity percent of purified recombinant human ATase after incubation with different concentrations of various deactivators. Each point represents the average of three measurements. The 50% residual activity line is used to calculate the concentration of the inactivating agent required to cause a 50% reduction in I ₅₀ value, ATase activity.

Second turn O ⁶ of two different levels for the sensitization of Raji cells to BCNU (0.1 and 1.0 / μM) - benzyl guanine (BeG) and O ⁶ -, alkylating agent concentration indicating the effect of the sensitizer ethenyl guanine (B.4205) ([mu] g / ml). 90% growth lines D ₉₀ value, that is used to calculate the dose of BCNU was 90% growth, i.e. 10% growth inhibition as compared to untreated control.

FIG. 3 shows the percentage of four cell growths for the alkylating agent concentration ([mu] M), which represents the effect of BeG and B.4205 on four different concentrations (0.1, 0.5, 1.0 and 5.0 [mu] M) on sensitization of Raji cells to temozolomide Graph.

FIG. 4 is a graph plotted from FIG. 3 (ie, the dose of temozolomide that was 50% greater than the untreated control), which is the D ₅₀ value for the inactivating agent concentration (μM).

FIG. 5 shows the effect of two cell growths on the inactivation agent concentration, showing the growth inhibitory effect of O ⁶ -furfuryl guanine (B.4203) and B.4205 on Chinese hamster V79 cell (RJKO) and its parent (+120) It is a percentage graph.

FIG. 6 is a graph of two cell growth percentages versus inactivation agent concentration, showing growth inhibitory effects of B.4203 and B.4205 on two Xeroderma pigmentosum subclones.

FIG. 7 shows the inactivating agents B.4203, B.4205, B.4212 (O ⁶ -piperonyl guanine) and B.4226 (O ⁶ - [2-benzo (b) thienylmethyl] guanine 0.0 > (uM) < / RTI > indicating the effect of the degradation products.

FIG. 8 shows the ATase activity for time (h), which shows a decrease in the ATase activity of the A375 xenografts in untreated mice against untreated control, corn oil treated control group and BeG (30 mg / kg) and B.4205 Activity (fm / mg) plot.

9 to 13 are graphs showing the results of the xenograft study. The upper graph in each figure shows the percentage of tumor volume over time (days) of A375 tumor xenografts in uncoated mice treated with BCNU alone, in combination with BCNU, and in combination with BCNU and B.4205. In the angle view, the lower graph shows the number of surviving mice in each treatment group for the time (days) after treatment as illustrated in the upper graph.

Details of each drawing are as follows.

Figure 14 is a graph of three survival percentages for the temozolomide concentration ([mu] M), indicating the survival of the bone marrow cells after treatment in combination with an inactivating agent (10 [mu] M) or DMSO (control) while increasing the dose of temozolomide .

DETAILED DESCRIPTION OF THE INVENTION

Technical field

The present invention relates to O ⁶ -substituted guanine derivatives, a process for their preparation and their use in tumor cell therapy. In particular, the present invention relates to guanine derivatives having a heteroalkyl or naphthylalkyl substituent at the O ⁶ position, and their ability to reduce O ⁶ -alkylguanine-DNA alkyltransferase (ATase) activity in tumor cells .

Background technology

Alkyl -DNA alkyl guanine O ⁶ with a transferase activity decreases - - O ⁶ in order to improve the efficiency of the chemotherapeutic alkylating agent which is used to kill the tumor cells to use an alkyl guanine derivatives have been proposed. There is increasing evidence that the toxicity and mutagenic effects of alkylating agents in mammalian cells are mostly the result of alkylation at the O ⁶ -position of guanine in DNA.

The restoration of O ⁶ - alkyl guanine is mediated by ATase, which is a restoration protein. This protein acts on O ⁶ - alkyl guanine residues by stoichiometrically transferring the alkyl group to its cystine residue in the self - inactivating process.

The importance of ATase in cell protection against the biological effects of alkylating agents is clearly demonstrated by the transfer and expression of cloned ATase genes or cDNAs into ATase deficient cells. This is evidenced by several drugs, mainly methylated or chloroethylated DNA Provide tolerance for.

Although the mechanism of O ⁶ - methyl guanine - induced apoptosis in ATase - deficient cells is not yet clear, the death of O ⁶ - chloroethyl guanine is mediated by the cyclic ethanoguanine intermediate to the cytosine residue on the opposite strand and the DNA strand Cross-linking occurs and this process is prevented by ATase-mediated chloroethyl group removal or complex formation.

The use of O ⁶ -methylguanine and O ⁶ -n-butylguanine to reduce ATase activity has been studied (Dolan et al., Cancer Res., (1986) 46, pp. 4500; Dolan et al., Cancer Chemother Pharmacol., (1989) 25, pp. 103). O ⁶ -benzylguanine derivatives have been proposed to reduce ATase activity in order to make ATase-expressing cells more sensitive to the cytotoxicity of chloroethylating agents (Moschel et al., J. Med. Chem., 1992, 35, 4486 ). U.S. Patent No. 5.09143 million and International Patent Application No. WO 91/13898 (. Moschel et al) O ⁶ is of the formula -, the host cells in the tumor, which comprises administering an effective amount of a benzylated guanine derivative to a host O ⁶ - Discloses a method for reducing alkylguanine-DNA alkyl-transferase levels:

Wherein Z is hydrogen, or

And R < ^a > is a benzyl group or a substituted benzyl group. The benzyl group may be optionally substituted with one or more substituents selected from the group consisting of halogen, nitro, phenyl or substituted phenyl, aryl, 1-4C alkyl, 1-4C alkoxy, up to 4C alkenyl, up to 4C alkynyl, , monoalkylamino, dialkylamino, trifluoromethyl, hydroxy, hydroxymethyl, and SO _n R ^b (wherein n is 0, 1, 2 or 3 and R ^b is hydrogen, 1-4 carbon atoms alkyl or Aryl, and the like). J. Med. Chem., 1994, 37, 342-347 (published after the priority date of the present application) describes a test on an O ⁶ -benzylguanine analog having a larger substituent on the benzylic ring or at position 9.

The compound No. < / RTI > 6 is O ⁶ - (2-pyridylmethyl) guanine, which is referred to herein as O ⁶ - (2-picolyl) guanine. However, in the results and review on pages 342-343 of the paper by Chae Mi Young et al. 6 is not emphasized as a subject of interest, but is categorized as " the remaining compounds exhibiting intermediate activity " (literature 342 pages 12-15 line). The authors have confirmed their initial observation that the allyl or benzyl substituent at the O ⁶ position of guanine efficiently inactivated ATase (line 343, pp. 21-23).

O ⁶ - benzylguanine has limited utility as an ATase inactivating agent. This results in a longer survival time in the administered animal, which is more stable than desirable. This has a potent toxicity level in combination with the chloroethylating agent, which may be associated with survival time alone and also undesirable.

The compounds show ATase inactivation properties different from O ⁶ - benzylguanine and in some cases their activity is up to 8 times greater than O ⁶ - benzylguanine. Different half-life and toxicity characteristics were also observed. It is therefore an object of the present invention to provide novel compounds useful for decreasing ATase activity in order to improve the efficacy of chemotherapeutic agents such as chloroethylated or methylated anti-tumor agents.

Another object of the present invention is to provide a pharmaceutical composition containing a compound useful for decreasing ATase activity. It is yet another object of the present invention to provide a method for reducing ATase activity in tumor cells. It is yet another object of the present invention to provide a method for treating tumor cells in a host.

DISCLOSURE OF INVENTION

Accordingly, the present invention provides an O ⁶ -substituted guanine derivative of formula I and a pharmaceutically acceptable salt thereof:

In this formula,

Y is H, ribosyl, deoxyribosyl, or Wherein X is O or S, R " and R "" are alkyl or substituted derivatives thereof;

R ' is H, alkyl or hydroxyalkyl;

R is (i) at least one 5- or 6-membered heterocyclic ring optionally fused with a carbon ring or a heterocyclic ring, each of which is a heterocyclic ring having at least one heteroatom selected from O, N, Or a substituted derivative thereof; or

(ii) naphthyl or a substituted derivative thereof.

R may suitably be a 5- or 6-membered heterocyclic ring or a benzo derivative thereof, and in the latter case an O ⁶ -alkyl guanine moiety may be attached to R in a heterocyclic ring or a benzene ring.

In a preferred embodiment, R is a 5-membered ring containing S or 0, with or without a second ring fused thereto.

Preferably R is a heterocyclic ring having at least one S atom; More preferably R is a 5-membered heterocyclic ring having at least one S atom; Most preferably R is a thiophene ring or a substituted derivative thereof.

Alternatively, R may be a heterocyclic ring having at least one O atom, specifically a 5-membered heterocyclic ring having at least one O atom, and more particularly R is a furan ring or a substituted derivative thereof .

Alternatively, R may be a heterocyclic ring having at least one N atom, specifically a 6-membered heterocyclic ring having at least one N atom, and in particular, R may be a pyridine ring. In the definition of Y, the term "substituted derivative" includes the substitution by one or more of the following groups: hydroxy, alkoxy, amino, alkylamino, amido or ureido.

In the definition of R, the term " substituted derivatives " refers to the groups: alkyl, alkenyl, alkynyl, halo, haloalkyl, nitro, cyano, hydroxyalkyl, SO _n R " (n) is 0, 1 or 2, or substitution of a heterocyclic ring and / or carbon ring ring (s) by at least one of the carboxyl or ester groups of the formula -COOR ⁵ wherein R ⁵ is H or alkyl. .

Halo, haloalkyl, cyano, SO _n R "" (as defined above) and -COOR ⁵ wherein R ⁵ is alkyl are preferred substituents.

The alkyl, alkenyl or alkynyl group preferably contains 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms, and most preferably 1 to 5 carbon atoms. Halo includes iodo, bromo, chloro or fluoro.

Examples of compounds of the present invention (with compounds B.4214 and B.4218 not included herein) are shown in Table 1.

Table 1

Among the compounds of Table 1, compounds B.4214 and B.4218 are not compounds of the present invention. Compound B.4210 (i.e., a compound of formula I wherein R is 2-pyridyl and R ' is H and Y is H) is not a preferred compound of the present invention.

Particularly preferred compounds of the present invention are:

B.4205 O ⁶ -enylguanine

B.4206 O ⁶ - (3-thienylmethyl) guanine

B.4212 O ⁶ -piperonyl guanine

B.4226 O ⁶ - (2-Benzo [b] thienylmethyl) guanine

B.4266 O ⁶ - (2-benzofuranylmethyl) guanine

B.4275 O ⁶ - (5-thiazolylmethyl) guanine

And substituted by a halo, cyano or ester group in the heterocyclic ring of R,

B.4229 O ⁶ - (5-methoxycarbonylpurfuryl) guanine

B.4269 O ⁶ - (5-Bromothenyl) guanine

B.4273 O ⁶ - (5-cyanofurfuryl) guanine

≪ / RTI >

Other preferred compounds include

B.4209 O ⁶ methyl guanine -3- furyl

B.4276 O ⁶ - (2-Benzo [b] thienylmethyl) guanosine

B.4277 O ⁶ - (4-picolyl) guanine

.

The most preferred compounds of the present invention are those that more effectively inactivate ATase in vitro and / or mammalian cells and / or tumor xenografts than O ⁶ -benzylguanine (BeG) and more effectively inhibit ATase in mammalian cells and / or tumor xenografts than nitroglycerin Sensitizing < / RTI > for the killing or growth inhibitory effect of the urea and / or methylating agent.

Also preferred compounds should reduce toxicity to normal tissues and / or whole organisms when used in combination with such agents as compared to BeG. Preferred compounds should not be toxic by themselves or exhibit minimal toxicities in the amount required to inactivate ATase, and any hydrolysis products of chemically unstable preferred compounds should not be toxic. Although the present invention is not limited by any theory, it may be necessary for a preferred compound to be less stable than BeG to perform spontaneous chemical degradation immediately after achieving maximum activation of ATase: Any action of the metabolic process which may act to occur will be minimized. Preferred compounds should be such that the human bone marrow or other normal cells are less susceptible to the toxic effects of the alkylating agents, so as not to exacerbate the known toxicity of these agents or to induce new toxicity in normal human tissues.

Preferred compounds of the invention are those having a relatively low I ₅₀ value (eg, less than or equal to 1.0 μM, and more specifically less than or equal to 0.04 μM) in Table 4 and / or buffers I of Table 4 ) And / or phosphate buffered saline (PBS) (representing conditions in physiological media) (e.g., less than 20 hours in buffer 1 or 16 hours or less in PBS).

A relatively short half-life can be regarded as an indicator that the compounds of the present invention are less stable than O ⁶ -benzylguanine due to the reactivity of RR'CH- and tend to disintegrate by hydrolysis in physiological media.

The effect of the RR'CH- group in a compound of formula I that allows it to function as an ATase inhibitor is determined by electron, stereosuggestion, and physicochemical factors. The steric factor may be related to the environmental properties of the cysteine receptor site in ATase. Preferably, R 'is H. The secondary carbon atom bonded to O ⁶ (as in DL Compounds B.4214 or B.4217) was found to significantly reduce the inactivation activity due to the large volume of substitutional substituents.

Preferably, the cyclic group of R has no methyl groups at adjacent positions even though the effect of the adjacent substitution is apparently less when R is a heterocyclic group than in the naphthyl isomers B.4213 and B.4265 (as in B.4222). ).

Physico-chemical factors such as stability, solubility and water-lipid distribution are involved in the selection of compounds for in vivo use, for example affecting the formulation, absorption and delivery. The choice of compound can also be influenced by its different distribution to different tissues.

One embodiment of the present invention provides a pharmaceutical composition, wherein Y, R and R 'contain a compound of formula 1 as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient . Optionally, the composition may comprise an alkylating agent such as a chloroethylating agent or a methylating agent.

In another embodiment, the invention provides a pharmaceutical composition comprising a compound of formula 1, wherein Y, R and R 'are as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, In particular, there is provided a method of reducing ATase activity in a host, comprising administering to the host an effective amount of a pharmaceutical composition as defined above.

Alternatively, this method can be defined as a method of reducing the ATase-mediated DNA restoration activity in a host.

The present invention relates to a pharmaceutical composition comprising a compound of formula 1 wherein Y, R and R 'are as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, The method comprising administering to the host an effective amount of a pharmaceutical composition as hereinbefore described and administering to the host an effective amount of a composition containing an alkylating agent. The present methods are well known to be sensitive to the action of alkylating agents, such as melanomas and gliomas, and those resistant to treatment with an alkylating agent alone, which can be overcome by the use of an inactivating agent according to the present invention, Can be used for treatment.

The invention also relates to a process for the preparation of a compound of formula (I), comprising: reacting a solution of sodium hydride and RR'CHOH (wherein R and R 'are as defined above) in an organic solvent, preferably at or below room temperature; Amino-N, N, N-trimethyl-1H-purin-6-aminium chloride or 2-amino-6-chloropurine riboside; Treating the reaction mixture with weak acid and ether; And extracting the desired product. ≪ Desc / Clms Page number 3 >

The present invention will be described in more detail with reference to the accompanying drawings.

Mode for carrying out the invention

O ⁶ -heteryl alkylguanine derivatives having ATase inactivation properties can be synthesized by modifying the standard preparation method given below.

2-Amino-N, N, N-trimethyl-1H-purin-6-aminium chloride was prepared according to Kiburis et al. Chem. Soc. (C), 1971, 3942. Details of the reaction conditions of this quaternary salt and sodium benzyloxide (to obtain O ⁶ -benzylguanine) not disclosed in MacCoss, Chen and Tolman, Tetrahedron Lett., 1985, 26, 1815 are described in MacCoss, Tolman, Wagner and Hannah, European Patent Application No. 184,473, which are not suitable for the preparation of comparatively sensitive analogues, the present inventors have devised the following standard production process.

Standard Preparation of O ⁶ -heterylalkylguanine (Formula I, Y = H)

Sodium hydride (60% in oil, 0.8 g, 20 mmol) is added to a solution of RR'CHOH (56 mmol, ca 5 ml) in DMSO (5 ml) and the mixture is stirred at room temperature for 1 h. For solid or high molecular weight alcohols, up to 10 ml of DMSO may be used instead of 5 ml. 2-Amino-N, N, N-trimethyl-1H-purin-6-aminium chloride (2.29 g, 10 mmol) was added and stirring was continued for an additional hour.

Then, after the change of the UV spectrum (λmax 312 → 284 nm), the almost transparent solution is treated with acetic acid (1.7 ml). Cool, dilute with ether (300 ml), place the mixture aside (2 h) and collect the solid (A). Triturate with water to obtain the product. The second fraction can be obtained by evaporation of the ether-DMSO filtrate and subsequent grinding of the residue by ether and water. Alternatively, the product may be extracted from the solid (A) by warm acetonitrile. The recrystallized compound _{TLC (C 6 H 6 -MeOH,} 4: 1) showed a single point in the characterized by analysis and their NMR spectra. Often they contain a crystallization solvent. Melting point and analysis data are shown in Table 2, and UV and < 1 > H NMR data are shown in Table 3.

NMR spectra were measured on a Bruker WP80 or MSL 300 instrument.

This standard method of preparation (variations indicated by symbols in Table 2) was used to prepare the compounds listed in Tables 2a and 3a. In compounds B.4217 and B.4219, R 'is methyl and in the remaining compounds, R' is H. The O ⁶ -benzylguanine compounds listed in Table 4 below and the compounds B4214, B4218 and B4231 were also prepared by this standard method for comparative test purposes.

The changes indicated by the symbols in Table 2 are as follows:

a For the preparation of this compound, 5 mmol of sodium hydride per mmol of quaternary salt is used. Using the standard amount (2mmol), 40% of the quaternary salt can be recovered in the reaction experiment.

b This compound was treated with 2M-NaOH (2.5ml) at room temperature for 4h to give the title compound as a white solid, after hydrolysis of the methyl ester B.4229 (145mg, 0.5mmol) in 2-methoxyethanol (2.5ml) . A solid is obtained which is neutralized with acetic acid (0.32 ml, 5.5 mmol), gentle evaporation, trituration with water (3 ml) and filtration to give acid B.4234 upon extraction with hot methanol. c The requirement is based on the monohydrate of a mixture of 4 parts of the sodium salt of the acid and 3 parts of the acid.

Requirement: Na, 4.3%, found: Na, 4.44%.

d 3 mmol of alcohol RCH ₂ OH per mmol of quaternary salt is used instead of the standard 5.6 mmol.

e RCH ₂ ONa is prepared in DMF (2.5 ml at -10 ° C, ₃ mmol of RCH ₂ OH, ₂ mmol of sodium hydride) for alcohols too sensitive to sodium hydride in DMSO at room temperature.

After 15-20 minutes, 1 mmol of quaternary salt is added and stirring is continued for 2-3 hours at room temperature.

The product is extracted with acetonitrile.

Preparation of riboside (formula I, Y = ribosyl)

The alkoxide solution prepared according to the standard method as described above from sodium hydride (60% in oil; 120 mg, 3 mmol) and RR 'CHOH (4.6 mmol) in anhydrous DMSO (2 ml) was treated with 2-amino-6-chloropurine riboside 302mg, 1mmol) and stirred at room temperature for 5 minutes, then at 60-65 < 0 > C for 15 minutes. The reaction is then terminated as indicated by the change in UV spectrum ([lambda] max 311 - > 284 nm). After cooling, it is thoroughly triturated with ether (100 + 15 ml) and filtered to give a solid which is treated with water (10 ml). The pH is reduced from 11 to 8 by passing CO ₂ over a short period of time. The inorganic material is removed by filtration and the dried residue is extracted with methanol (4x10ml each containing a drop of pyridine) at room temperature. Almost all of the methanol is evaporated and a small amount of ether is added to obtain a nearly pure product with TLC (C ₆ H ₆ -MeOH 4: 1).

It is dissolved and concentrated at 40 占 폚 or lower and recrystallized from trace amounts of methanol and pyridine, sometimes with a small amount of ether being added occasionally.

This method was used to prepare the compounds listed in Tables 2b and 3b. It is difficult to remove traces of impurities, but the purity of the nucleoside is about 90% in the NMR spectrum. The yield is about 30-40%.

The starting alcohol RR'CHOH is generally prepared by reduction of the corresponding aldehyde, commonly available as sodium borohydride. A different approach was used for the precursors of ether B.4229 and nitrile B.4273. Sucrose is was converted via a 5-chloromethyl-puru furfural 5-hydroxymethyl-furfural ^1. The oxidation to the aldehyde of ² to obtain a carboxylic acid methylamide This required for B.4229 by ^three methods Bochy (Bocchi) 5- (hydroxymethyl) was esterified with ^4-furoate. The oxime ³ of the aldehyde was dehydrated by the method of Carotti et al ⁶ ;

The crude reaction product was treated with concentrated aqueous ammonia in methanol before extraction with dichloromethane. The distillation provided the 5-cyanofurfuryl alcohol ⁷ required for B.4273.

For B.4266, a 2-aldehyde was obtained in the Vilsmeier reaction ⁸ of benzofuran to obtain the desired alcohol ⁹ , while for B.4226, 2-aldehyde was added by treatment with lithiating and dimethylformamide followed by addition of an alcohol ¹⁰ .

For B.4274, dimethyl tartrate was oxidized to methyl glyoxylate and ¹¹ it was reacted with tosylmethyl isocyanide to give ¹² -methyloxazole-5-carboxylate ¹⁸ .

This was reduced with aluminum hydride to alcohol ¹⁵ by the method of Fallab ¹⁴ .

For B.4275, bromomalonaldehyde ¹⁶ and 2-aminothiazole-5-carboxaldehyde ¹⁷ with thiourea were obtained. Deamidation with amyl nitrite ¹⁷ followed by reduction with sodium borohydride afforded 5-hydroxymethyl thiazole ¹⁴ .

For B.4271 carbonyl alcohol 5-aza piperazine (melting point 82-84 ℃; Found, C, 54.60; H, 4.58 ; N, 9.09; C 7 H 7 NO 3 has C, 54.90; H, 4.61; N, 9.15%) was prepared from the corresponding aldehyde ¹⁸ .

For B.4278 Chemistry nitro 1-methyl-pyrrole-2-carboxaldehyde (carboxaldehyde), and was reduced to the alcohol ²⁰ by sodium borohydride ^19.

The preparation of O ⁶ -enyl guanine (B.4205) is now described by certain embodiments.

Preparation of O ⁶ -tenylguanine

A solution of the behenyl alcohol (3.18 ml, 33.6 mmol) in DMSO (3 ml) was initially treated with sodium hydride (60% in oil; 0.48 g, 12 mmol) with care and stirring. After 1 hour, 2-amino-N, N, N-trimethyl-1H-purin-6-aminium chloride (1.37 g, 6 mmol) was added and stirring was continued for an additional hour. Acetic acid (1.0 mml) was added with brief cooling and the mixture was diluted with ether (180 ml). After 1-2 hours, a solid (2.09 g) was collected. The ether was evaporated from the filtrate and the residue remained after distillation of DMSO and excess thienyl alcohol (boiling point 48-57 DEG C / 0.2 mm), which was triturated with ether to give a second solid fraction (0.36 g). The combined solids were rubbed with water (6 ml) (triturated) to give the product (1.335 g, 90%) which showed a strong point in TLC (C ₆ H ₆ -MeOH, 4: 1) with only minor impurities. The residue was dissolved in hot ethanol (30 ml) and the filtrate was purified through celite and concentrated using a rotary evaporator (up to 10 ml) to give B.4205 (1.125 g, 71% of material containing 1/3 EtOH per mole of solvent) .

Compounds of formula I wherein Y is R " XCHR ", (secco-nucleoside) can be prepared by reacting O ⁶ -benzylguanine with a -chloroether (MacCoss et al., Tetrahedron Lett., European Patent Application No. 184,473, 1986, 27, 877; Moschel, McDougall, Dolan, Stine, and Pegg, J. Med. Chem., 1992, 35, 4486), or alkyl bromides (e.g., Kjellberg, Liljenberh and Johansson, Tetrahedron Lett. ≪ / RTI >

Representative " sugar " components corresponding to R " XCHR ", which derive seco-nucleoside, are described, for example, in McCormick and McElhinney, J. Chem. Soc., Perkin Trans. 1, 1985, 93; Lucey, McCormick and McElhinney, J. Chem. Soc., Perkin Trans. 1, 1990, 795.

Compounds of formula I (nucleosides) wherein Y is ribosyl or deoxyribosyl are synthesized by the method described in Synthesis of O ⁶ -benzylguanine riboside and 2-deoxyribose (Moschel et al., 1992, ef. Gao, Fathi, Gaffney et al., J , 1981, 103 5489) (see Preparation of the riboside). The reaction can be carried out in the same manner as in the above-mentioned method.

Industrial availability

The amount of the compound of the present invention to be used depends on the effective amount necessary for treating the tumor cells. A suitable dosage to be the concentration of the compound of the invention in the tumor cells to be treated which results in a decrease in ATase activity is, for example, about 1-2000 mg / kg, preferably 1-800 mg / kg, especially 1- 120 mg / kg.

The pharmaceutical compositions of the present invention can be prepared in conventional forms using conventional excipients as described, for example, in WO 91/13898, the contents of which are incorporated herein by reference.

This composition may contain an inactivating agent according to the invention together with an alkylating agent.

Alternatively, the composition may comprise two parts, one containing an inactivating agent and the other containing an alkylating agent. Methods of administering a compound of the present invention to a host may also be a conventional method as described, for example, in WO 91/13898. To administer the inactivating agent according to the present invention to a patient, the pharmaceutical composition may be administered orally in saline or 3% ethanol (in saline) for intravenous injection, or in a suitable vehicle such as 40% polyethylene glycol 400 in aqueous salt solution, May contain an inactivation agent in powder form in suitable capsules.

The alkylating agent may be administered in conventional dosage forms, for example as described in WO 91/13898, as known in the art, or preferably as a single dose, preferably immediately after administration of the ATase inactivating agent or up to 24 hours, preferably about 2 hours, May be administered at a lower dosage than that used in standard therapies. Decreases in dosage may be necessary since inactivating agents can generally be involved in increasing the properties of the alkylating agent. Examples of the chloroethylating agent include 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU), 1- (2-chloroethyl) -3-cyclohexyl-1-nitroso urea (CCNU) Potemustine, mitozolomide and clomethone, and McCormick, McElhinney, McMurry and Maxwell J. Chem. Soc., Perkin Trans. I. 1991, 877 and Bibby, Double, McCormick, McElhinney, Radacic, Pratesi and Dumont Anti-Cancer Drug Design, 1993, 8, Examples of methylating agents include temozolomide (GB 2 104 522) and dacarbazine, proccarbazine and streptozocin.

Way

Purification of recombinant ATase

From cDNA cloning and overexpression of the ATase have been already reported ² 3. Purification of recombinant proteins is affinity chromatography or ion DEAE- cellulose with a DNA- cellulose column described by ^{25, 26,} such as Wilkinson - by exchange chromatography It was accomplished.

In the latter case, the ATase protein was partially purified by ammonium sulfate precipitation (30-60%) and dialyzed against 10 mM Tris-HCl pH 7.5, 1 mM DTT, 2 mM EDTA, 10% glycerol before being loaded onto DEAE-cellulose column. The ATase was then eluted by a 0-0.1 M NaCl gradient. The purified human ATase protein remained active for more than one year when stored at high concentration at -20 ° C in buffer I [50 mM Tris / HCl (pH 8.3) / 3 mM dithiothreitol / 1 mM EDTA] It could be thawed and re-frozen several times without substantially reducing it.

Culture with ATase and ATase analysis

The compound to be tested was dissolved in DMSO to a final concentration of 10 mM and diluted in buffer I (IBSA) containing 1 mg / ml bovine serum albumin immediately prior to use. Recombinant ATase was diluted in IBSA and titrated to ensure that the reaction was carried out under ATase (not substrate) restriction conditions.

In each assay, a fixed amount of ATase (60-75 fmol) was incubated with varying amounts of O ⁶ -benzylguanine or test compound in a total volume of 200 μl IBSA containing 10 μg of calf thymus DNA for 1 hour at 37 ° C Lt; / RTI > After addition of [ ³ H] -methylated-DNA substrate (100 μl containing 6.7 μg of DNA and 100 fmol of O ⁶ -methyl guanine), the culture was continued at 37 ° C for 1 hour until the reaction was complete. ²¹ [ ³ H] -methylated protein was recovered by acid hydrolysis of the previously described DNA and quantitated by liquid scintillation counting.

Samples were typically analyzed in duplicate, and the experiments were repeated several times. I ₅₀ is the concentration of inactivating agent required to produce a 50% reduction in ATase activity.

Cell culture and preparation of extract

The mammalian cells were cultured under standard conditions. For example, Raji (human lymphocyte lineage cells from bucket's lymphoma) cells were grown in suspension culture in RPMI medium supplemented with 10% horse serum. A375M cells are human melanoma cells from which the following xenografts have been colonized by subcutaneous injection into hairless mice: WiDr cells are human colon tumor cell lines; Hamster +120 cells are mitosolomide-selected secondary cell lines of Chinese hamster lung fibroblast V79 cell line called RJKO; Yoshida cell is a rat carcinosarcoma cell line and YR _bus is its subpubule-resistant subcellular line; XP cells are SV40-transfected fibroblast cell lines derived from the skin of Xeroderma pigmentosum patients, pHGhAT26 cells are clones of these cells infected with mammalian cell expression vectors containing human ATase cDNA, and pHMGla cells are simply expression vectors Which does not contain human ATase cDNA). Cell pellets were resuspended in cold (4 캜) buffer containing 2 ug / ml of rupeptin and sonicated for a period of 10 seconds at a peak distance of 12 mu peak.

After cooling in ice, the cells were further sonicated for 10 seconds at 18 l.

Immediately after ultrasonic digestion, 0.01 volume of 8.7 mg / ml phenylmethanesulfonyl fluoride in ethanol was added and the sonicate was centrifuged at 15000 g for 10 minutes at 4 DEG C to become a pellet cell debris. The supernatant was stored for measurement of ATase activity (see below).

Stability of the inactivating agent at 37 캜

The inactivating agent (10 mM in DMSO) was diluted to 0.1 mM in pre-warmed degassing buffer I (1 mM EDTA, 50 mM Tris pH 8.3) or PBS (pH 7-7.2). PBS (phosphate buffered saline) is 0.8% NaCl, 0.02% KCl, 0.15% Na 2 H 2 PO 4, 0.02% KH 2 PO 4, pH 7.2. Samples were immediately transferred to a CARY13 spectrophotometer (cuvette block maintained at 37 DEG C) and scanned at appropriate wavelengths (according to the optical properties of the compound) at intervals of 3-10 minutes up to 80 hours. The results are expressed as the percentage of absorbance change versus time and the extrapolated T1 / 2 value (half-life).

Inactivation of ATase activity in mammalian cells

Cells were diluted to 10 < ⁶ > / ml in a culture medium containing an appropriate concentration of deactivation agent or equivalent volume of vehicle (DMSO). After incubation for 2 hours at 37 ° C, cells were harvested by centrifugation, washed twice with PBS, and the resulting cell pellets (between 1 and 2 x 10 ⁷ per pellet) were stored at -20 ° C. ATase activity was determined as described above in two volumes of sonicated degraded cell extract and expressed as a percentage of the remaining activity based on that present in the untreated control group (e.g., 350-450 fm / mg in Raji cells) .

The value of I ₅₀ (i.e. the concentration of inactivating agent required to reduce ATase activity to 50%) was extrapolated to this data.

Sensitization of Raji and A375M cells to BCNU and temozolomide

The sensitization of A375M cells to the cytotoxic effect of BCNU and temozolomide after pretreatment with a 2 hour inactivating agent was analyzed using XTT assay ²² . Briefly, cells were seeded in 96 well plates at 1000 cells / well prior to addition of medium containing the appropriate concentration of inactivating agent or equivalent volume of vehicle and incubated at 37 [deg.] C for 30 minutes. After incubation for 2 hours at 37 ° C, medium containing increasing doses of BCNU, temozolomide or equivalent vehicle was added and cells were allowed to grow for 6 days. At this point the XTT solution was added and the cells were further incubated at 37 ° C for 4 hours. The resulting red / orange formazan reaction composition was quantitated by measuring the absorbance at 450 nm with a microtiter plate reader.

The sensitization of A375M cells to the cytotoxic effect of BCNU was analyzed by MTT assay ²⁴ , which is different from the XTT assay described above. A375M cells (1000 / well) were grown for 24 hours, then treated with an inactivating agent and treated with BCNU for 2 hours. After 6 days, MTT solution (4 mg / ml) was added and the cells were incubated at 37 ° C for 3 hours. The medium was aspirated, and the resulting purple formazan crystal was dissolved in DMSO (120 μl), and cell viability was determined by measuring the absorbance at 540 nm using a microtiter plate reader.

From this data, the percent growth of cells relative to the growth of control wells was determined for a range of BCNU or temozolomide doses in the presence and absence of inactivating agents.

^{_{BCNU (D 90. C / D}} 90. I) Raji sensitization is (dose with a 90% growth, i.e. 10% growth inhibition compared to that is the non-treated control group), the D ₉₀ calculated for BCNU alone using data for Was determined by dividing by BCNU and the use of an inactivating agent (D _90. ^I ).

Thus, the value of one (1) indicates no sensitization by the inactivating agent. Raji sensitization to temozolomide and A375M sensitization to BCNU were measured using corresponding D ₅₀ values (i.e., doses with 50% growth inhibition).

Sensitization of mammalian cells to inactivating agents or their hydrolyzate

To assess the effect on cell growth only, Xeroderma pigmentosum cells and Chinese hamster V79 cells were exposed to selected deactivators at increasing concentrations (up to 600 [mu] M) for 2 hours at 37 [deg.] C. In some cases, the inactivating agent was hydrolyzed at 37 ° C for 20 hours and then added to Raji cells to evaluate to what extent the decomposition product of the inactivating agent could inhibit cell growth.

After 6 days, the degree of cell growth was determined as described above.

Sensitization of bone marrow cells to temozolomide (GM-CFC analysis)

Primary human bone marrow samples were collected from patients undergoing cardiothoracic surgery for granulocyte / macrophage colony-forming cells (GM-CFC) analysis. After removing red blood cells from the sample, the cells were resuspended in 10 μM deactivator or equivalent volume of DMSO, 20% fetal bovine serum, 10% 5637 regulated medium and 0.3% fetal bovine serum as the growth factor source in a 1 ml Petri dish containing the appropriate dose of temozolomide To a concentration of 1-2 x 10 < ⁵ > / ml in a 300 mOsM Iscove's medium containing high-grade agar, and cultured at 37 [deg.] C in an environment of 5% CO ₂ and 95% air. After 9 days, the number of colonies containing more than 50 cells was counted. Colonies expressed precursor cells of the granulocyte / macrophage lineage (huGM-CFC). Survival rates were expressed as% of colonies in a zero dose of temozolomide.

Xenograft study

animal

BALB-C induced athymic male mice (nu / nu athymic) weighing 25-35 g were obtained from the indoor breeding colonies of the Paderborn Cancer Institute (England, Manchester M20 9BX, Wilmslow Road, Christie Hospital NHS Trust) ). The animals were housed in a sterile environment until needed for the experiment. In one experiment, mice were obtained from Harlan-Olak (England, Oxon. OX60PT, Bicester, Blackthorn, Shaw's Farm, Harlan UK Limited) (OLAC mouse). These mice, which weigh between 17 and 23 g, were then found to be more sensitive to the toxic effects of the combination (inactivating agent and BCNU) treatment. For this reason, small doses were administered.

cell

A375M (human melanoma) cells were grown in DMEM containing 10% fetal bovine serum. Cells were prepared by incubation with 0.01% trypsin for in vivo inoculation.

tumor

Cells (10 ⁶ ) in 100 μl of PBS were injected subcutaneously into the right flank of 8-10 weekly nu / nu athymic mice. To make them experimental animals, these cells were allowed to develop into tumors for 3-4 weeks. A 2 mm x 2 mm x 2 mm sized tumor mass was subcutaneously implanted in the right flank. These animals were prepared for use in inactivating agent experiments after 7-10 days.

Chemical treatment

Nu / Nu mice were treated with either 30 or 60 mg / kg O ⁶ -benzylguanine or B.4205 and appropriate vehicle control (ip) 60-90 min before treatment with 10-25 mg / kg BCNU (ip). O ⁶ -benzylguanine and B.4205 were prepared as 5 mg / kg solution in corn oil and BCNU (2 mg / ml) in PBS + 3% ethanol.

Tumor measurement

Xenograft tumor measurements were performed every 1-2 days by a technician using a digital caliper. Tumor volume was calculated using the equation (hxw x1) π / 6. Laboratory animals were also weighed every 1-2 days. Measurements were continued until tumors within the control animals reached the maximum allowed volume (i.e., 1 cm x 1 cm x 1 cm).

result

Tables 4, 5 and 6 show the physical, biochemical and in vivo data for the respective deactivators. The enumerated experiments were described in the Methods section.

Table 5

Table 6

Figure 1 shows the in vitro ATase inactivation test results using four compounds. B.4206 was somewhat more effective than BeG, but B.4212 and B.4205 were significantly better under the test conditions used. B.4203 was not as effective as BeG.

In FIG. 2, in a 0.1 μM inactivation agent, B.4205 was more effective than BeG in sensitizing Raji cells to the growth inhibitory effect of BCNU; With 1.0 μM deactivators, B.4205 and BeG were shown to be equally effective.

In FIG. 3, B.4205 was more effective than BeG for sensitization of Raji cells to the growth inhibitory effect of temozolomide in 0.1 μM deactivation agent, but as dose of inactivating agent increased, sensitization by BeG remained the same It became more effective than by B.4205.

Thus, the clear dose response of BeG and the absence of dose response of B.4205 are more evident in FIG.

5 shows that while some inhibition of B79 cells is produced by B.4203 with an excess of 100 [mu] M (i.e., at least 100 times greater than the I ₅₀ dose for this compound), B.4205 is used up to maximum concentration , This shows that there is no such effect.

FIG. 6 shows that XP cells are sensitive to the growth inhibitory effect of B.4203, but these cells are more sensitive than V79 cells to the effect of B.4205. However, the dosage required for growth inhibition was at least 100 times that required for ATase inactivation.

It can be concluded that the inhibitory effect of these inactivating agents on the innate growth does not significantly contribute to sensitization of the cells to the growth inhibitory effect of the alkylating agent.

FIG. 7 shows that no substantial growth inhibitory effect is produced in Raji cells when the given compound is hydrolyzed (see Methods) before it is added to cells without the addition of an alkylating agent. Therefore, under these test conditions, decomposition of reagents can not be expected to contribute to growth inhibition using a combination of these reagents and alkylating agents.

Figures 8 through 13 show the results of a more detailed xenograft study.

Following exposure to O ⁶ -benzylguanine or B.4205, the consumption and recovery of ATase activity was measured in the A375 tumor xenograft (Figure 8) extract prepared from the tissue of the animal sacrificed 2 hours or 24 hours after the inactivation agent Respectively. FIG. 9 to 13 with the O ⁶ in combination with BCNU as described in the methods section turning-benzyl guanine or or present a hairless mouse in sensitization of A375 tumor xenografts for B.4205. The top graph in each figure shows the percentage of tumor volume increase over time of the test. The lower graph shows the number of animals in each treatment group and the number of survivors after treatment. The graph shows that B.4205 and O ⁶ -benzylguanine (BeG) can be compared with a decrease in tumor volume, and the combination of BCNU and B.4205 is substantially less toxic than the combination of BCNU and BeG. In malignant melanomas, malignant melanomas have been reported in the United States (Balch, CM, Houghton, A. & Peters, L. (1989), Cutaneous Melanoma in Cancer: Principles and Practice of Oncology, De Vita, S. & Rosenberg, SA (eds), pp1499-1542, Lipincott; Philadelphia) BCNU, a human melanoma xenograft that was grown in uninjured mice was a clinically highly relevant animal model system.

FIG. 14 shows the test results of sensitization of human bone marrow cells to temozolomide as described in the method section above. Bone marrow cells were obtained from three patients, cognized as C, D and E, respectively. Survival curves were associated with the cytotoxic effect of inactivating agent / temozolomide combination therapy. This effect is preferably reduced.

Results for Patient C and E are B.4205 is O ⁶ - benzyl glue rather indicates that with a small sensitization effect than the (O ⁶ BeG); Although there is no difference in patient D, the results are regarded as reasonable deviations from the scientific test on the patient.

Table 7 shows the toxic effects of inactivating agents only in bone marrow cells from five patients A through E, including the same Patients C through E in FIG. 17. This result for B.4205 is comparable to that for O ⁶ -benzylguanine. It is noted that patient B cells are almost twice as sensitive to BeG and B.4205 as compared to other patients.

Table 7

Summary of the Invention

1) All compounds were tested for efficacy to inactivate recombinant human ATase under standard conditions in laboratory experiments. Under these conditions, the two compounds (B.4214 and B.4217) failed to inactivate ATase to the highest concentration used, which was a compound in which R 'is a methyl group. The rest of compounds were inactivated ATase with 0.0045 to about I ₅₀ value of 95μ mol range. The eight compounds (B.4205, B.4206, B.4212, B.4226, B.4266, B.4269, B.4273, B.4275) had lower I ₅₀ values than the BeG values ).

2) Inactivators were hydrolyzed in aqueous solution at different rates (half-life 0.17 to less than 80 hours), but this is not related to their potency, such as ATase inactivation (Table 4).

3) the active compound in vitro inactivation of ATase (I ₅₀ <1.0μM) also generally vitro analysis approximately 1.5 times the only slightly higher (mean value than those found using recombinant protein in) I ₅₀ value ATase in human cells Lt; / RTI &gt;

4) B.4205 inactivated ATase with the same effect (I ₅₀ value 0.02-0.03) in human, rat and Chinese hamster cells. For B.4203, the range was 0.04 to 0.12.

In the cell lines used, B.4205 and B.4203 were up to seven times more effective than BeG (Table 5).

5) B.4203 and B.4205 were toxic to the studied XP cells and B.4203 was toxic to the studied V79 cells, but the sensitization to BCNU was at least 100 times higher than the observed concentration (FIGS. 5 and 6).

6) Raji and A375M cells were sensitized for inhibition of BCNU growth when compounds that are effective against ATase inactivation in vitro (I ₅₀ <1.0 μM) and Raji cells (I ₅₀ <1.0 μM) were also tested ).

7) At an inactivation agent concentration of 0.1 μM, B.4205 was more effective than BeG in sensitizing Raji cells to the growth inhibitory effect of temozolomide (FIG. 3).

8) In vitro assays can be used to predict compounds that are likely to be the most effective sensitizing agents of mammalian cells against the growth inhibitory effects of BCNU and related cytotoxic agents.

9) B.4205 was about the same or slightly more effective than BeG on sensitization of human melanoma xenografts grown in hairless mice to BCNU growth inhibitory effect (FIGS. 9-13).

10) B.4205 was as effective as BeG on the inactivation of ATase in human melanoma xenografts grown in hairless mice (FIG. 8).

11) In vitro assays and / or xenograft ATase reduction studies may be used to predict compounds that are likely to be the most effective sensitizers of melanoma species implants for the suppressive effects of BCNU and related cytotoxic agents.

12) In a BeG comparison that resulted in death of animals treated to 70%, B.4205 had very little effect on sensitization of reared mice with human melanoma xenografts for acute toxic effects under the conditions used 13 to 13).

13) BeG sensitizing GM-CFCs in three bone marrow samples were tested for the toxic effects of temozolomide, but two of them were scarcely sensitized by B.4205.

Therefore, this test may be used to predict possible bone marrow suppression effects of ATase inactivators when used in combination with BCNU and related drugs (FIG. 14).

Claims (14)

An O ⁶ -alkyl guanine derivative of formula I and a pharmaceutically acceptable salt thereof. In this formula, Y is H, ribosyl, or deoxyribosyl; R 'is H or C ₁ to C ₅ alkyl; R is (i) a 5-membered heterocyclic ring having at least one heteroatom selected from O, N or S, optionally fused with a benzene or pyridine ring, or a 6-membered heterocyclic ring having at least one N atom Wherein said cyclic group is optionally substituted on the heterocyclic ring or carbocyclic ring or both rings with one or more C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, halo, C ₁ -C ₅ haloalkyl, nitro, cyano, C ₁ -C ₅ hydroxyalkyl, SO _n R "" ( _wherein R "" is C ₁ -C ₅ alkyl and n = 0, 1 or 2) , Or a carboxyl or ester group of formula -COOR ⁵ (wherein R ⁵ is H or C ₁ -C ₅ alkyl); or (ii) one or more C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, halo, C ₁ -C ₅ haloalkyl, nitro, cyano, C ₁ -C ₅ hydroxy Alkyl, SO _n R "" _wherein R "" is C ₁ -C ₅ alkyl and n = 0, 1 or 2, or a group of formula -COOR ^{5 wherein} R ⁵ is H or C ₁ -C ₅ alkyl Naphthyl which is optionally substituted by a carboxyl group or an ester group. The compound of claim 1, wherein R is a cyclic group having a 5-membered heterocyclic ring having at least one heteroatom selected from O, N or S fused to a benzene ring, wherein the O ⁶ -alkyl guanine moiety is a heterocycle or benzene Lt; RTI ID = 0.0 &gt; R &lt; / RTI &gt; in the ring. The method of claim 1, wherein, R is, optionally, one or more C ₁ -C ₅ alkyl in the heterocyclic ring, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, halo, C ₁ -C ₅ haloalkyl, nitro, cyano, C ₁ -C ₅ hydroxyalkyl, SO _n R "" (wherein R "" is C ₁ -C ₅ alkyl and n = 0, 1 or 2), or a group represented by the formula -COOR ⁵ (formula R ⁵ of the compound is characterized in that the H or C ₁ -C ₅ groups may be substituted by carboxyl group or an ester of an alkyl), a 5-membered heterocyclic ring having one S atom, which. The method of claim 1, wherein, R is a thiophene ring, a furan ring, and its C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, halo, C ₁ -C ₅ haloalkyl, , Nitro, cyano, C ₁ -C ₅ hydroxyalkyl, SO _n R "" _wherein R "" is C ₁ -C ₅ alkyl and n = 0, 1 or 2. Or a substituted derivative having at least one substituent selected from a carboxyl group or an ester group of formula -COOR ^{5 wherein} R ⁵ is H or C ₁ -C ₅ alkyl. The compound of claim 1 or 2 wherein R is halo, C ₁ -C ₅ haloalkyl, cyano, SO _n R "" wherein R "" is C ₁ -C ₅ alkyl and n = 0, a) or -COOR ⁵ (wherein R ⁵ is a compound comprising both C ₁ -C ₅ ring heterocycle substituted by alkyl), or a carbocyclic ring or two rings. 5. A compound according to claim 1 or 4, wherein R is selected from thiophene ring, furan ring and its substituted derivatives selected from halo- and cyano-substituted derivatives thereof. 7. A compound according to claim 6, characterized in that RR'CH- is cyano or a bromine-substituted derivative thereof. The method according to claim 1, O ⁶ -thenyl guanine; O ⁶ - (3-thienylmethyl) guanine; O ⁶ -piperonyl guanine; O ⁶ -furfurylguanine; O ⁶ - (3-furylmethyl) guanine; O ⁶ - (2-benzo [b] thienylmethyl) guanine; O ⁶ - (2-benzofuranylmethyl) guanine; O ⁶ - (5-thiazolylmethyl) guanine; O ⁶ - (5-methoxycarbonylpurfuryl) guanine; O ⁶ - (5-bromothenyl) guanine; O ⁶ - (5-cyanopurfuryl) guanine; O ⁶ - (2-benzo [b] thienylmethyl) guanosine; O ⁶ - (4-picolyl) guanine; O ⁶ - (2-naphthylmethyl) guanine; &Lt; / RTI &gt; 9. A pharmaceutical composition for the treatment of tumor cells, characterized by comprising a compound of claims 1, 4 or 8 and a pharmaceutically acceptable excipient. 10. The composition of claim 9, further comprising an alkylating agent, wherein the composition is present in one portion containing an inactivating agent together with the alkylating agent, or in two portions in which one portion contains an inactivating agent and the other portion contains an alkylating agent &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof. 11. The composition of claim 10, wherein the alkylating agent is selected from 1,3-bis (2-chloroethyl) -1-nitroso urea (BCNU) and temozolomide. A medicament for treating tumor cells, which comprises reducing the activity of O ⁶ -alkylguanine-DNA alkyltransferase in a tumor cell comprising the compound of any one of claims 1, 4 or 8 and a pharmaceutically acceptable excipient. Reacting a solution of sodium hydride and RR'CHOH (wherein R and R 'are as defined in claim 1) in an organic solvent with a solution of 2-amino-N, N, N-trimethyl- Lt; RTI ID = 0.0 &gt; I &lt; / RTI &gt; as defined in claim 1, characterized in that it comprises the steps of: &Lt; / RTI &gt; The method according to claim 1, R is (i) a 5-membered heterocyclic ring having at least one heteroatom selected from O, N or S, optionally fused with a benzene or pyridine ring, or a 6-membered heterocyclic ring having at least one N atom Wherein said cyclic group is optionally substituted on the heterocyclic ring or carbocyclic ring or both rings with one or more C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, halo, C ₁ -C ₅ haloalkyl, nitro, cyano, C ₁ -C ₅ hydroxyalkyl, or SO _n R "" _wherein R "" is C ₁ -C ₅ alkyl and n = 0, 1 or 2 ); or (ii) one or more C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, halo, C ₁ -C ₅ haloalkyl, nitro, cyano, C ₁ -C ₅ hydroxy Alkyl, or naphthyl optionally substituted by SO _n R "" _wherein R "" is C ₁ -C ₅ alkyl and n = 0, 1 or 2.