Prodrugs of 6-mercaptopurine and 6-thioguanine

Abstract: Prodrugs for the treatment of kidney tumors are disclosed. The prodrugs are conjugates that can require the cooperation of multiple enzymes in the kidney to release 6-­ mercaptopurine or 6-thioguanine, or can selectively release 6-thioguanine. The prodrugs have the formula: wherein R₁ is H or NH₂, x is 1 or 2 and R₂ represents various carboxylic acid or ester groupings, generally containing an animo and/or amido group.

Description: This invention relates to compounds capable of targeting anti-tumor drugs to the kidney. More specifically, it relates to prodrug conjugates of 6-mercaptopurine and 6-thioguanine that are designed to require exposure to multiple enzymes present in the kidney prior to releasing an anti-tumor agent. The chemical modification of a biologically active drug to give a new chemical from which the active drug can be generated by enzymatic action is an important strategy to target drug action to specific cells and tissues and thereby decrease toxicity or side effects on non-target cells. N. Bodor et al. , 22 Ann. Rep. Med. Chem. 303-313 (1987); T. Krenitsky et al. , 81 P.N.A.S. USA 3209-3213 (1984); J. Hjelle et al. , 229 J. Pharmacol. Exp. Ther. 372-380 (1984); S. Magnan et al. , 25 J. Med. Chem. 1018-1021 (1982); M. Orlowski et al. , 212 J. Pharmacol. Exp. Ther. 167-172 (1980). The disclosure of these articles and of all other articles listed herein are incorporated by reference as if fully set forth herein. Such compounds are known as "prodrugs". In A. Elfarra et al. FASEB Journal, Abstract 4922 (March 20, 1988) it was reported that S-(6-purinyl)-L-cysteine, the cysteine derivative of the anti-tumor and immunosuppressant drug 6-mercaptopurine, could be a potential prodrug. In this regard, 6-mercaptopurine is known to be effective in the treatment of various types of tumors. However, it is not actively transported from the G.I. tract, and is readily metabolized by xanthine oxidase to generate biologically inactive metabolites. Thus, it must be given in large doses for a long time to be effective. The concept is that the β-lyase needed to release the 6-mercaptopurine is predominantly found in the kidney. Further, the kidney transport system tends to concentrate amino acid derivatives. This development is of great importance since currently there is no satisfactory chemotherapy technique for treating kidney tumors. While this S-(6-purinyl)-L-cysteine approach had some success, even greater specificity is desired. Further, 6-mercaptopurine may have certain disadvantages for some patients which renders use of 6-thioguanine alternatives desirable. In separate work, our laboratory has reported that gamma-glutamyl transpeptidase, cysteinyl glycine dipeptidase, and cysteine conjugate β-lyase are all present in the kidney and may cooperatively act to release an S-(1,2-dichlorovinyl) thio moiety from S-(1,2-dichlorovinyl glutathione). A. Elfarra et al. , 35 Biochem. Pharm. 283-288 (1986). Also, it has been reported that the renal acylase and the renal esterase are prevalent in the kidney. It has also been reported in A. Elfarra et al. , 83 P.N.A.S. USA 2667, 2670 (1986) that a homocysteine S-conjugate of dichlorovinyl could undergo transamination in the kidney to yield the corresponding 2-keto acid, and that non-enzymatic elimination under the conditions present in the kidney would then release the dichlorovinyl group. The S-conjugate of the hydroxy analogue is also toxic as it is metabolized to the keto-acid in the kidney by amino acid oxidases. See L. Lash et al . 276 Arch. Biochem. Biophys. 322 (1990). Thus, it can be seen that a need exists for the development of more specific kidney prodrugs. One embodiment of the present invention provides a compound having the formula: where R₁ is H or NH₂; and where R₂ is: or (where R₃ is alkyl, preferably -CH₃ or -CH₂CH₃). Another embodiment of the present invention provides a compound having the formula: where R₁ is NH₂; and where R₂ is: or (where R₃ is H or alkyl). Preferably R₂ is -CH(NH₂)-COOH. In another aspect, the invention provides a compound having the formula: where R₁ is H or NH₂; and where R₂ is: or Preferably R₂ is -CH(NH₂)-COOH, -CH(OH)-COOH or -CO-COOH. The compounds of the present invention are useful for inhibiting tumor development in a mammalian kidney. Most of the above prodrugs require multiple enzyme activity prior to releasing 6-mercaptopurine or thioguanine. In this regard, the glutathione variants require action by gamma-glutamyl transpeptidase (which releases a glutamate moiety), cysteinyl glycine dipeptidase (which releases a glycine residue), and cysteine conjugate β-lyase. The cysteinyl glycine variants require use of the latter two enzymes to release the anti-tumor agent. The acetyl variants require use of renal acylase and then β-lyase. The ester variants require use of renal esterase and renal β-lyase. Because these five enzymes are predominantly found in the kidney, because of the kidney's ability to concentrate amino acid derivatives, and because significant concentrations of the combination of these five enzymes are not found in many other parts of the body, specificity can be achieved through use of these compounds. Similarly, S-conjugates of homocysteine (and of hydroxy analogues thereof) require enzymatic modification to the keto-acid form, followed by further action in the kidney to release the drug. The objects of the invention therefore include: (a) providing compounds of the above kind; and (b) providing methods for synthesizing such compounds. A detailed synthesis for S-(6-purinyl)-L-cysteine is described below Synthesis of the claimed compounds will then be described with reference to this synthesis. Basically, 6-chloroguanine or 6-chloropurine are reacted with the amino acid precursor (glutathione, cysteinyl glycine, N-acetyl-L-cysteine or cysteine alkyl ester, e.g. cysteine methyl ester or cysteine ethyl ester) under the specified conditions to yield the compound of interest. The compounds are then tested on rat kidney. Materials 6-Chloropurine, 6-chloroguanine, glutathione, cysteinylglycine, N-acetyl-L-cysteine, 6-mercaptopurine, and 2-chlorobenzothiazole can be obtained from Aldrich Chemical Co. (Milwaukee, WI). Sephadex LH-20 was supplied by Pharmacia Inc. (Piscataway, NJ). L-cysteine, aminooxy-acetic acid, cysteine methyl ester, cysteine ethyl ester, and bovine serum albumin can be purchased from Sigma Chemical Co. (St. Louis, MO). As standard for enzyme activity, S-[2-benzylthiazolyl]-L-cysteine ("BTC") was prepared as previously described D. Dohn et al. , 120 Anal. Biochem. 379-386 (1982). Supplies for proton NMR were obtained from Wilmad Glass Co. (Buena, NJ). Other cysteinyl alkyl ester starting compounds can be prepared using standard ester formulation reactions. All other reagents were of the highest grade commercially available. Synthesis Of S-6-Purinyl-L-Cysteine 6-Chloropurine (152 mg; 1.0 mmol) and L-cysteine (182 mg; 1.5 mmol) were dissolved in a mixture of 2.5 mL of 1 N NaOH solution and 2.5 mL of methanol. This lead to the displacement of the chlorine by the disodium salt of cysteine. The reaction mixture was kept under nitrogen and heated in a water bath for 45 minutes at 50°C with continuous stirring. The reaction mixture was kept in an ice bath until S-6-purinyl-L-cysteine ("PC") was isolated by using either semi-preparative reverse phase HPLC or by low pressure Sephadex LH-20 chromatography. For the Sephadex 1H-20 chromatography, the reaction mixture (pH 10.2-10.5) was applied to a 2.2 x 76 cm Sephadex 1H-20 column which was kept at 4°C because of the apparent instability of PC when the Sephadex 1H-20 chromatography was carried out at room temperature. Product was separated from the reaction mixture using water, adjusted to pH 10.5 with 1 N NaOH, as eluent. The flow rate was 0.68 mL/minute and 5 minute fractions were collected using a fraction collector. Fractions 55-62 which contained the product were pooled, lyophilized and stored in a desiccator at -20°C. The yield of the compound was approximately 15%. For the isolation of the compound by semi-preparative HPLC, samples of the reaction mixture (adjusted to pH 5-6 with acetic acid) were applied into a Whatman Partisil 10 ODS 3 magnum-9 HPLC column (9.4 mm x 250 mm) attached to a Beckman 114M HPLC pump, a Rheodyne injection valve, a 3 cm long C-18 guard column, a Spectroflow 757 variable wavelength detector (Kratos Analytical Inc., NJ) and a 4270 SP Integrator (Spectra Physics Inc., NJ). The eluent was acetonitrile:water (10:90 % v/v); flow rate was 2.0 mL/minute; detection wavelength was 266 nm. The fractions that contained the product (retention time 11-12 minutes) were collected and kept on ice until it was lyophilized. Analytical HPLC analyses were done as described for the semi-preparative HPLC except that a 4.6 x 250 mm Whatman ODS 10 um column was used, and the flow rate was 1 mL/minute. TLC were developed using 250 um Silicagel GF plates (Analtech Inc., DE) with isopropanol:water:ammonium hydroxide (9:2:1 5 v/v/v). Product was visualized with a UV light (PC had a maximal UV absorption at 284 nm; Fig. 3) or a ninhydrin spray. Rf values of the product, 6-chloropurine, L-cysteine and L-cystine were 0.42, 0.67, 0.05 and 0.2 respectively with the indicated solvent system. Tests On Kidneys Male Sprague-Dawley rats (250-350 g) (Charles River Laboratories, Wilmington, MA), were killed by decapication, and the kidneys were removed. Subcellular fractionation by centrifugation was performed as described by D. Dohn et al. , 120 Anal. Biochem. 379-386 (1982). These subcellular fractions were used as the enzyme source without further purification. The protein concentrations of the different fractions were determined according to the procedure of O. Lowry et al. , 193 J. Biol. Chem. 265-275 (1951) with bovine serum albumin as the standard. In a final volume 1.2 mL, each incubation tube contained sodium borate buffer (pH 8.6, 0.1 M); the prodrug (3 mM); and the kidney homogenate (4 mg/mL). Some tubes contained 1 mM aminooxyacetic acid, an inhibitor of β-lyase. Control incubations were carried without the addition of the enzyme. Enzymatic reactions were conducted for 20 minutes at 37°C in a Dubnoff metabolic incubator with continuous shaking. Reactions were terminated by placing the tubes on ice followed by filteration with 0.2 um Acro LC 13 membrane filters (VWR Scientific Inc., Chicago). HPLC analyses were carried as described earlier with the additional use of an authentic sample of 6-mercaptopurine as a reference. Renal β-lyase activity was determined with the prodrug as the substrate and the amount of 6-mercaptopurine formed was determined by HPLC. The assay mixture contained in a final volume of 2 mL: prodrug (2-16 mM), sodium borate buffer (pH 8.6; 0.1 M), and enzyme solution (0.6-1.2 mg/mL). The reaction mixture was incubated at 37°C for 20 minutes and the reaction was terminated as described above. A portion (20 uL) of the filtrate was injected into the HPLC system. The standard curve obtained by plotting the peak area of the 6-mercaptopurine peak was linear over the range of 1 to 350 uM of 6-mercaptopurine in the incubation mixture. To determine the effectiveness of the prodrug as a substrate for renal β-lyase, the rates of 6-mercaptopurine production were compared with the rates of metabolism of the prototype β-lyase substrate, BTC (0.5-6 mM in .1 M sodium borate buffer, pH 8.6) to form 2-mercaptobenzothiazole (D. Dohn et al. , 120 Anal. Biochem. 379-386 (1982)). The research showed that 6-mercaptopurine is generated by the β-lyase-dependent metabolism of the prodrug. Additional studies showed that 6-mercaptopurine formation was dependent upon incubation time, and substrate- and protein concentrations. The results of the fractionation of various kidneys showed β-lyase activity in different subcellular fractions with the prodrug as the substrate, and show that most of the prodrug dependent β-lyase activity was present in the cytosolic and mitochondrial fractions. These results are in agreement with the subcellular localization of renal β-lyase. The ability of the kidneys to concentrate amino acid derivatives will result in the accumulation of prodrugs in renal proximal tubular cells where it would be metabolized by β-lyase (and the other required enzymes) to form 6-mercaptopurine or 6-thioguanine. Thus, the kidneys will be exposed to high concentrations of 6-mercaptopurine without exposing other portions of the body to adverse concentrations of such compounds. This has been verified by experiments in vivo on rats. To synthesize the claimed compounds, one reacts 6-chloropurine or 6-chloroguanine with the precursor thio compound (L-cysteine, glutathione, cysteinyl-glycine, cysteine methyl ester, cysteine ethyl ester, other cysteine alkyl ester, or N-acetyl-L-cysteine) under similar conditions to yield S-(6-purinyl)-N-acetyl-L-cysteine; S-(6-purinyl)-cysteinyl-glycine; S-(6-purinyl)-glutathione; S-(6-purinyl)-cyteine methyl ester, S-(6-purinyl)-cysteine ethyl ester, other S-(6-purinyl)-cysteine alkyl esters; S-(6-guanyl)-N-acetyl-L-cysteine; S-(6-guanyl)-cysteinylglycine; S-(6-guanyl)-cysteine methyl ester, S-(6-guanyl)-cysteine ethyl ester; other S-(6-guanyl)-cysteine alkyl esters; and S-(6-guanyl)-glutathione. S-(6-guanyl)-L-cysteine can also be formed. To create the homocysteine variants, one begins by making S-(6-purinyl)-L-homocysteine (Compound H1). To do this, sodium metal is gradually added to a stirred solution of L-homocysteine (Sigma Chemical Co.; 2 mmol) in liquid ammonia (150 ml) until a blue color persisted for ten minutes. The white solid obtained after the evaporation of the ammonia was redissolved in 15 ml water containing EDTA (ethylene diamine tetraacetic acid; Aldrich Chemical Co.; 5 mg). 6-Chloropurine (2 mmol; Aldrich Chemical Co.) was added and the pH of the solution was adjusted to 10.8 using a 10% solution of sodium hydroxide (Aldrich Chemical Co.). After refluxing for 2.7 hours, the reaction mixture was cooled to room temperature and the pH was adjusted to 2.7 using a 20% solution of trifluoroacetic acid (Aldrich Chemical Co.). Portions of the reaction mixture (2 ml each) were applied to a Pharmacia KX26 column, and the product was separated using water (pH 2.5) as eluent. The flow rate was 2.5 ml/min; UV-detection was at 254 nm; Ve was 458 ml. The collects were pooled, lyophilized, and then stored in a desicator. Synthesis of the thioguanine variant (Compound H2) can be achieved using 6-chloroguanine (Aldrich Chemical Co.) instead of 6-chloropurine. Synthesis of the homocysteine compounds where R₂ is (Compounds H3 and H4 respectively) will be achieved by a modification of the method of M. Winitz et al ., 78 J. Amer. Chem. Soc. 2423-2430 (1956). One reacts sodium nitrite (Aldrich Chemical Co.) with Compounds H1 and H2, respectively. The synthesis of related compounds where R₂ is can be achieved by the reaction of Fremy's salt with Compounds H3 and H4, respectively. See generally A. Garcia-Rafo et al . 51 J. Org. Chem. 4285 (1986) (Fremy's salt). Further Testing At 30 minutes after treatments of rats ( in vivo) with S-purinyl-L-glutathione (0.8 mmol/kg, i.p.), 6-mercaptopurine, and its further metabolites 6-thiouric acid and 6-methylmercaptopurine, were detected in the kidneys at concentrations of nearly 20 nmol metabolites/g wet tissue. These compounds were not detected at all in plasma or liver in this experiment (albeit with some dosages, kill times, and other prodrugs small amounts of 6-mercaptopurine can be detected in the plasma or liver). At 24 hours after treatments of rats with S-(purinyl)-L-glutathione, S-(purinyl)-N-acetyl-L-cysteine, or S-(purinyl)-L-cysteine ethyl ester (0.4 mmol/kg, i.p.), 6-mercaptopurine and its metabolites were present in urine at levels corresponding to 4, 2, and 1% of the theoretically possible administered dose, respectively. When rats were given S-(6-purinyl)-homocysteine (0.4 mmol/kg), the amounts of 6-mercaptopurine and the products of its further metabolism which were detected in urine at 24 hour post treatment were at levels corresponding to four times that amount found for S-(6-purinyl)-L-cysteine. When rats were given the homocysteine variant (0.8 mmol/kg, i.p.) the concentration of metabolites (6-mercaptopurine, 6-methylmercaptopurine, and 6-thiouric acid) present in liver and kidneys at 30 minute post treatment were nearly 10 and 90 nmol/g wet tissue, respectively. No metabolites were detected in plasma. These results show that this compound is a selective kidney precursor of 6-mercaptopurine. This concept of providing a multiplicity of enzymes to control release 6-mercaptopurine or 6-thioguanine provides a highly advantageous system, and yields prodrugs with improved specificity. Also, thioguanine prodrugs are highly desirable. It will be appreciated that the above describes only the preferred embodiments of the invention. A number of other modifications and changes are within the scope of the invention. For example, DL (as distinguished from L stereochemistry) is included. Also, equivalents such as radiolabelled variants are included.

Claims: A compound having the formula: wherein R₁ is H or NH₂; and R₂ is or (where R₃ is alkyl). A compound according to claim 1, wherein R₃ is -CH₃ or -CH₂CH₃. A compound according to claim 1 or 2 wherein R₁ is hydrogen. A compound according to claim 1 or 2 wherein R₁ is NH₂. A compound having the formula: where R₁ is NH₂; and R₂ is or (where R₃ is alkyl). A compound according to claim 5 wherein R₂ is A compound having the formula: where R₁ is H or NH₂; and R₂ is or A compound according to claim 7 wherein R₂ is or A compound according to claim 7 or 8 wherein R₁ is H. A compound according to claim 7 or 8 wherein R₁ is NH₂. A compound as claimed in any one of the preceding claims for use in increasing mercaptopurine or 6-thioguanine in a mammalian kidney.

Inventors: ELFARRA ADNAN, ELFARRA, ADNAN
Applicants: WISCONSIN ALUMNI RES FOUND, WISCONSIN ALUMNI RESEARCH FOUNDATION
Application year: 1990
Main classification: C07D473
IPC codes: A61P35, C07D473, A61K38, A61K31, A61P43, C07K5, A61P13, A61P15