KR20020040657A - Process for preparing MKC-442 - Google Patents

Abstract

A process for preparing 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil, also known as MKC-442, is disclosed. This method comprises ethyl-2-isopropyl-3, including from zinc and benzyl cyanide and ethyl-2-bromo-3,3-dimethyl-propiotate activated in the Reformatsky reaction using any known method. Methods for synthesizing isopropyl β-ketoesters, such as -oxo-4-phenyl-butyrate. The ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate is then condensed to thiourea in one of two ways. First, ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate is condensed to thiourea in a two-base system in the presence of a solvent. The second method of condensing ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate with thiourea is carried out in the presence of potassium-t-butoxide to form benzyl isopropyl thiourea, which is step (B) Desulfurization at MKC-442 is prepared by alkoxyalkylation of the benzyl isopropyl uracil produced from either method.

Description

Manufacturing method of MBC-442 {Process for preparing MBC-442}

This application claims the benefit of US Provisional Patent Application No. 60 / 128,925, filed April 13, 1999.

This application includes, in the field of synthetic pharmaceutical chemistry, in particular, an effective method for preparing 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil, also known as MKC-442.

The pathogenesis of AIDS was identified in 1983 as human immunodeficiency virus (HIV). It is now believed that an important factor in whether HIV infected persons express AIDS or not is the amount of HIV in the body at a given time (ie, "viral load"). The researchers focus on stopping HIV replication and reducing viral load by blocking one or both of the two important enzymes, reverse transcriptase and protease, necessary for viral replication.

The first enzyme, reverse transcriptase, is initially active in the HIV replication cycle and allows viruses composed of RNA to produce the DNA needed for continuous replication. This enzyme can be inhibited by two general classes of drugs defined by their structure and their mechanism of action. The first general class is 3'-azido-3'-deoxythymine (AZT), 2 ', 3'-dideoxyinosine (ddI), 2', 3'-dideoxycytidine (ddC), and ( Nucleoside analog reverse transcriptase inhibitors, such as-)-cis-2-hydroxymethyl-5- (cytocin-1-yl) -1,3-oxathioran (3TC), are natural DNA building blocks. It is chemically very similar to (nucleoside) and displaces the natural nucleoside used by the enzyme, thereby interfering with the action of the enzyme. The second general class consists of a wide variety of chemicals that act non-nucleoside reverse transcriptase inhibitors such as MKC-442 and nevirapine to act less efficiently by binding to and modifying enzymes.

Since 1985, AZT, ddI, ddC, 3TC, 2 ', 3'-dideoxy-2', 3'-didehydrothymine (D4T), and cis-2-hydroxymethyl-5- (5-fluoro A number of Neuleoside reverse transcriptase inhibitors, including cytosine-1-yl) -1,3-oxathiolan (FTC), have been shown to be effective against HIV. After cellular phosphorylation to 5'-triphosphate with cellular kinases, these synthetic nucleosides were also inserted into the renal strand of viral DNA, terminating the chain due to the absence of the 3'-hydroxy group.

MKC-442 acts as a non-nucleoside reverse transcriptase inhibitor. MKC-442 is considered an allosteric inhibitor because it appears to show its activity by binding to the enzyme's "allosteric position", that is, elsewhere outside the binding site. Preclinical studies suggest that MKC-442 may have the characteristics to undertake some of the therapeutic challenges of HIV. When tested in cell culture assays for mutant strains of HIV known to be resistant to wild-type (drug sensitivity) and conventional nonnucleoside reverse transcriptase inhibitors, MKC-442 retained its ability to inhibit HIV replication. In this study, MKC-442 showed better potential than nevirapine for wild type and HIV mutant strains. Preclinical studies of two drug mixtures of MKC-442 and AZT or ddI and three drug mixtures of MKC-442 and AZT and squinavir demonstrated that HIV replication was synergistically inhibited.

In animal studies, an appropriate safety and pharmacokinetic profile for MKC-442 has been proposed. Pharmacokinetic analysis of animals showed good oral bioavailability and good penetration into the central nervous system, an important site of HIV replication, which is insufficiently penetrated by many anti-HIV drugs currently on the market. For example, the concentration of MKC-442 in the rat brain was 100% of that in plasma.

Phase I studies evaluated the pharmacokinetics and resistance of a single increase in MKC-442 in HIV-infected volunteers. The compounds were generally resistant and only some participants experienced a small amount of adverse effects at higher dose levels. In the group receiving higher doses, the concentration of drug in the plasma reached a level higher than required to inhibit 90% of the virus in the culture.

Preliminary data have been evaluated from a phase I / II double-blind, placebo-controlled trial designed to assess the safety and efficacy of repeated multiple oral administrations of MKC-442 in HIV-infected patients. A total of 49 patients were treated with MKC-442 for up to two months. Doses ranging from 100 mg to 1000 mg twice daily were given to groups of 6 to 8 patients at each dosage level. At the highest dose tested (750 mg and 1000 mg twice daily), viral load decreased to an average of 95% in all patients after one week. After a gradual increase towards the baseline level in the viral load, this decrease usually lasts two weeks. A single point mutation at position 13 of the reverse transcriptase, which may be associated with resistance, has been found in viruses obtained from some patients. In over 308 patients exposed to the drug, MKC-442 was resistant.

MKC-442 is described, for example, in US Pat. No. 5,461,060, which is incorporated herein by reference in its entirety.

U.S. Pat. No. 5,604,209, assigned to Uvasawa et al. On February 18, 1997, and then assigned to Mitsubishi Chemical Corporation, discloses certain 6-benzyl-1-ethoxymethyl-5-substituted uracil derivatives including MKC-442 and 2 ', 3'-dideoxyinosine (ddI), 3'-azido-3'-deoxythymidine (AZT), AZT triphosphate, and 2', 3'-dideoxycytidine (ddC) It is published that certain 2 ', 3'-dideoxyribonucleosides have a synergistic effect on HIV.

Since MKC-442 alone and in combination with other antiviral agents exhibits pharmacokinetic activity, there is a growing interest in how to prepare them effectively.

Since MKC-442 is a 5,6-substituted uracil derivative having an alkoxyalkyl group at position 1 (see Figure 1), its synthesis method should include the addition of these three 1,5,6-substituents. do. A typical method of synthesis of MKC-442 is to condense the alkoxyalkyl moiety with a 5-substituted thiouracil, subsequently desulfurize the thiouracil, lithiate at position 6, react with benzaldehyde, and subsequently acetyl And finally reduce the hydroxy group using hydrocracking to yield MKC-442 [Rosowsky, et al., J. Med Chem., 1981, 24, 1177; Tanaka, et al., J. Med Chem., 1992, 35, 4713. and references. Baba et al., Nucleosides and Nucleotides, 14 (3-5), 575-583 (1995). Because Baba et al. Are steric hindrance by substituents, the glycosylation of 6-substituted uracil almost without exception forms an N-3-glycosylated product, and for this reason inserts an acyclic site After that, it was specifically mentioned that 6-substituents should be inserted.

Danel et al. Reported the synthesis of 5,6-disubstituted acyluridine derivatives, including the insertion of an ethoxymethyl moiety of the molecule at the N-1 position in the final step of the synthesis ( J. Med Chem., 1996, 39 , 2427-2431; Synthesis , Auguest 1995, 934-936). Danel et al. React arylacetonitrile with 2-bromoester in a Reformatsky reaction to form ethyl-2-alkyl-4-aryl-3-oxobutyrate, i.e., β-keto ester (Scheme 1). Reference). The β-keto ester is then condensed with thiourea in the presence of sodium ethoxide to form 2-thiouracil, which is refluxed overnight with chloroacetic acid in aqueous acetic acid to give 6-benzyl-5-ethyluracil. The uracil must be silylated before condensation with a specially selected alkylating agent. The Danel method has a substantial disadvantage in that small quantities are adequate, while for mass production of MKC-442, metal sodium must be used to produce bases in excess of Zn and in situ in relation to severe stability. In addition, more than 20-fold ethoxide is described herein, which is inappropriate for industrial scale-up. The Danel method also requires the use of 15 equivalents of thiourea to prevent pure crystallization of the thiouracil derivatives. In addition, thiouracyl intermediates are not soluble in chloroacetic acid solution at all, and when the material is heated to reflux, a viscous mass is formed and coats the agitator and flask walls. Finally, in the case of homogenizing the desulfurization reaction with alcohol or tetrahydrofuran, thiouracil is converted into undefined impurities. In the case of scale-up of the Danel method, the yield of MKC-442 will be drastically reduced while providing 25-50% product with only 90% purity.

Scheme 1

In 1996 Orr and Nusso reported the reaction of ethyl phenylpropanoate with thiourea to synthesize 5-substituted uracil derivatives (but not 5,6-disubstituted uracil). The use of metal sodium as a base in the preparation of 2-thiouracil from thiourea in the conventional prior art has reported that it can cause problems in the synthesis and lead to low yields. In Synthetic Communications, 26 (1), 179-189 (1996), Orr et al. Include ethyl phenyl propanoate and (i) sodium, ethylformate, diethyl ether, and then thiourea and ethanol at reflux; (ii) lithium diisopropylamide, tetrahydrofuran, ethyl formate at low temperature, followed by thiourea and ethanol at reflux; (iii) Preparation with potassium t-butoxide in tetrahydrofuran, ethyl formate, diethyl ether at ambient temperature, followed by thiourea and 2-propanol at reflux. Orr et al. Found that the use of lithium diisopropylamide or potassium t-butoxide in place of metal sodium in the preparation of 5-benzyl-2-thiouracil improves the yield of the reaction and condenses enolate using thiourea. Substitution with 2-propanol instead of ethanol as the solvent has been reported to improve yield. The Orr reaction cannot be used to give 5,6-disubstituted uracil while using ethyl orthoformate, but can be used to give 5-substituted uracil or 5,5-dihydrouracil (Scheme) 2).

Scheme 2

^a reagent: (i) EtOH, Et ₂ O, HCl, reflux; (ii) EtOH, H ₂ , PtO ₂ ; (iii) Me ₂ CO, R ₃ Br, K ₂ CO ₃ ; (iv) Na, HCO ₂ Et, Et ₂ O followed by thiouracil, EtOH, reflux; (v) LDA, THF, HCO ₂ Et, -60 ° C-78 ° C, followed by thiourea, EtOH, reflux; (vi) t-BuOK, HCO ₂ Et, Et ₂ O, ambient temperature in THF; Followed by thiourea, 2-PrOH, reflux.

Baba et al. Reacted silylated 5-isopropyl-2-thiouracil with carcinogenic CH ₃ CH ₂ OCH ₂ (KI / CH ₂ Cl ₂ ) to obtain an N-1-ethoxymethyl product. Reported methods for preparing the (Nucleoside and Nucleotides, 14 (3-5), 575-583 (1995)). Lithiation with lithium diisopropylamide (LDA) affords a 6-substituted product. The 6-substituted product is oxidatively hydrolyzed and usually hydrogenated to yield MKC-442.

In connection with the fact that MKC-442 is useful for the treatment of HIV-infected patients, there is a need to develop low-cost, measurable methods for producing pure MKC-442 in large quantities, with high purity and high yield. It is therefore an object of the present invention to provide a synthetic process for preparing 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil (MKC-442) with improved high yields and higher purity.

Summary of the Invention

It has been found that 5,6-disubstituted uracil can be produced in good yields by appropriately reacting β-keto esters with thiourea in the presence of potassium t-butoxide. If the β-keto ester is ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate, the product is 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil, also known as MKC-442. to be.

Therefore, in one aspect, there is provided a method for producing 5,6-disubstituted uracil, comprising simultaneously using two bases to efficiently synthesize 5,6-disubstituted uracil from a β-keto ester with high yield. . Preferred bases include potassium carbonate and potassium t-butoxide as co-bases in acetonitrile. This surface is shown in Scheme 3.

Scheme 3

Examples of MKC-442 synthesis include, but are not limited to, isopropyl β-keto esters, such as ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate, with zinc activated in any known method, the Reformatsky reaction. Benzyl cyanide and ethyl-2-bromo-3,3-dimethylpropionate. The β-keto ester is then reacted with a two-base system in a solvent, such as acetonitrile, but not limited to potassium carbonate and potassium t-butoxide.

The method provides several advantages over the prior art. First, the reaction is a single-step method of forming the product at a yield of greater than 80%. In addition, the products are obtained in higher purity by a two-base system. For example, the use of potassium carbonate and potassium t-butoxide in acetonitrile gives a product having a purity of 90-95%.

In the synthesis of substituted thiouracil rings, the high pK _b base alone destroys the starting material β-keto ester and has a higher level of impurity. As a result, the use of a high pK _b base alone lowers the yield of the product. Lower pK _b bases tend to react for longer periods of time and contain residual starting material. Therefore, it is advantageous to use a two-base system to counteract this trend.

Bases useful in the two-base system of the present invention include, but are not limited to, potassium carbonate, potassium t-butoxide, sodium carbonate, di-isopropyl amine, triethyl amine, 2,6-dimethyl pyridine, sodium acetate, potassium acetate , Ammonia, potassium ethoxide, and potassium cyanide. Bases are used in admixture with a solvent.

Solvents useful in the two-base system of the present invention include, but are not limited to, acetonitrile, butyronitrile, isobutyronitrile, chloroacetonitrile and other alkyl nitrile solvents, isopropyl alcohol, methanol, ethanol, propanol, butanol, And other alcoholic solvents.

In another aspect of the invention, there is provided a process for preparing 5,6-disubstituted uracil comprising reacting β-keto ester with thiourea, preferably in the presence of potassium t-butoxide in iso-propyl alcohol. do. This aspect of this reaction is shown in FIG. 4.

Scheme 4

Examples of MKC-442 synthesis include, but are not limited to, from zinc and benzyl cyanide and ethyl-2-bromo-3,3-dimethyl-propionate activated in the Reformatsky reaction using any known method. Condensation of ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate with thiourea in the presence of potassium t-butoxide in step (A) of the invention to form benzyl isopropyl thiuracil, After desulfurization in process B), the benzyl isopropyl uracil is alkoxyalkylated in step (C) to form MKC-442, such as ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate. Isopropyl-β ketoester can be synthesized. The improved process of the present invention provides some unexpected advantages over the prior art while at the same time producing MKC-442 with higher yields and purity.

The method provides several advantages over the prior art. In the ring condensation reaction of step (A), NaOEt is replaced with BuOK to obtain a higher purity product. It has been reported in the prior art that the yield was increased by using t-BuOK, but the increased purity of the present products obtained to date is unknown and unexpected. In addition, NaOEt was substituted with t-BuOK to obtain a crude thiouracil that is easy to pore, so that post-treatment was easy and measured by a balance. Surprisingly, the reaction of step (A) is carried out with t-BuOK of sub-stoichiometric equyvakebt amount. Finally, the use of diethoxy methane in the presence of a catalyst such as sulfuric acid in step (C) rather than the highly toxic TMS triflate or chloromethyl used in the prior art offers the advantage of cost savings and stability.

The slowing and controlled release of hydrogen sulfide in the desulfurization reaction of step (B) is also unexpected. The fast pace and uncontrollable release of gases would have hampered the scale up and commercialization of the process.

The alkylation reaction of step (C) has two unexpected aspects in this regard. First, this reaction is carried out very efficiently in the presence of less than stoichiometric equivalents of sulfuric acid. Second, it is easy in that there is no need to remove HMDS. Alkylation is carried out in an effective fashion in the presence of less than stoichiometric equivalents of sulfuric acid, even HMDS.

Provided is an improved process for preparing 5,6-disubstituted uracil. This improved method offers several advantages over the prior art. Most notably, the process of the present invention produces MKC-442 at higher yields without compromising the purity of the product. In practice, the process can yield products with a purity of at least 95%.

The present invention includes at least one of the following aspects:

The first method for producing 5,6-disubstituted uracil, preferably MKC-442, by reacting β-keto ester with thiourea in a two-base system. The method involves the simultaneous use of 2 bases to effectively synthesize 5,6-disubstituted uracil. The method comprises reacting the β-keto ester with thiourea in the presence of a solvent and a 2-base system at a temperature in the range of 80-85 ° C.

One step of the first process of the present invention is the reaction of 2,4-disubstituted-3-oxo-butyrate, preferably ethyl-2-isopropyl-3-oxo-phenylbutyrate with thiourea in a two-base system and a solvent It involves making. Preferred bases for two-base systems include, but are not limited to, potassium t-butoxide and potassium carbonate. Preferred solvents for the reaction include, but are not limited to, acetonitrile. The reaction is carried out at a temperature in the range from 80 ° C to 85 ° C. Preferred reaction temperatures are about 85 ° C.

As used herein, the term "2-base system" means the use of two bases simultaneously in the presence of a solvent. Examples of bases included in two base systems include, but are not limited to, potassium carbonate, potassium t-butoxide, sodium carbonate, di-isopropyl amine, triethyl amine, 2,6-dimethylpyridine, sodium acetate, potassium acetate , Ammonia, potassium ethoxide, potassium cyanide. The base is used in admixture with a solvent.

Solvents useful in the two-base system of the present invention include, but are not limited to, acetonitrile, butylonitrile, isobutylonitrile, chloroacetonitrile and other alkyl nitrile solvents, isopropyl alcohol, methanol, ethanol, propanol, butanol, and Other alcoholic solvents.

The second method of preparing 5,6-disubstituted thiouracil, preferably MKC-442, comprises the following methods:

(i) reacting the 2,4-disubstituted-3-oxo-butyrate ester with thiourea in the presence of potassium t-butoxide in an organic solvent to produce a 5,6-disubstituted thiouracil (A Where the intermediate is 5,6-disubstituted-2-thio-uracil;

(ii) the process of step (A) wherein the solvent is an alcohol, preferably isopropyl alcohol;

(iii) the method of step (A) in which the molar ratio of thiourea to 2,4-disubstituted-3-keto-butyrate does not exceed about 10% excess, more preferably 5% excess;

(iv) The method of step (A) quenching the reaction with water and acetic acid:

(v) preferably further desulfurizing 5,6-disubstituted-2-thiouracil with 5,6-disubstituted-uracil using 10% chloroacetic acid (aqueous solution) in acetic acid (A) (B) comprising the method of;

(vi) the method of step (B) further comprising isolating thiouracil prior to desulfurization;

(vii) the process of step (B) wherein acetic acid is about 25 to 40%, preferably about 35% of the total solvent volume used;

(viii) the method of step (B) of heating the reactants to 85 ° C. to 105 ° C. to form a solution;

(ix) silicifying the 5,6-disubstituted uracil of step (v) with any suitable reagent according to known methods;

(x) the method of step (C) wherein the reagent is a silylating agent such as hexamethyldisilizane;

(xi) the process of step (x) of reacting hexamethyldisilizane in the presence of a catalytic amount of ammonium sulfate;

(xii) reacting the product of step (C) with an alkylating agent or an alkoxyalkylating agent such as diethoxymethane;

(xiii) the process of step (xii) in which the reaction is carried out in sulfuric acid or methanesulfonic acid;

(xiv) the process of step (xiii) using less than stoichiometric equivalents of sulfuric acid, preferably from 0.25 to 0.5 equivalents; And

(xv) The method of step (xiv), when refluxing acetonitrile (or other alkyl nitrile), is preferably carried out for 2-5 hours.

The two step method of the second method of the invention comprises steps (A) and (B) in step I. Step (A) involves cyclization of the isopropyl-β-ketoester to thiourea and potassium t-butoxide in isopropyl alcohol at reflux to produce benzylisopropyl thiouracil. Benzyl thiouracil in step (B) is then desulfurized with chloroacetic acid solution, 10% ClAcOH (aqueous solution), AcOH under reflux to produce isopropyluracil (BIU). Reaction workup includes termination with water and acetic acid followed by filtration of the crystalline solid at 70-80% yield.

The step I method differs from Danel's method (J. Med. Chem. 1996, 39,2427-2431) in that no metal sodium is used to generate the isotopic base used by Danel. Eliminating sodium solves the serious stability concerns of mass production. Only 5% -20% excess is required by the method of the present invention, while Danel also specified a 20-fold excess of ethoxide. The Danel method also produces thiouracil intermediates that, upon reflux heating, form viscous masses that coat the stirrer and flask walls. In contrast, the separation and thiuracil produced by the method of step (A) of the present invention were used to increase the conversion and purity. Two tasks were performed by adding acetic acid (about 35% of the total solvent volume) to the reaction in step (B): forming a solution when heated to 95 ° C .; If the reactants are large, they act as recrystallization cosolvents. The method of the present invention removes the viscous mass formed in the Danel method while making an improved method for measurable device size. In addition, BIU was recovered at about 90% yield of more than 98% pure material.

In step II of the process of the invention, the BIU is silylated with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) in the presence of a catalytic amount of ammonium sulfate, and in acetonitrile containing sulfuric acid Alkylated with diethoxymethane. The method is referred to herein as step (C). Optionally, methanesulfonic acid can also be used as acid catalyst. To effectively effect alkylation, less than stoichiometric equivalents (0.25-0.5 equivalents) of concentrated sulfuric acid in acetonitrile were used at reflux for 2-5 hours to optimize the conditions. Typical reaction profiles showed 90-95% yield MKC-442 with less than 3% BIU as impurities.

The two step method of the present invention provides several improvements over the prior art method. First, the purity of the product increases more significantly by replacing NaOEt with t-BuOK in the cyclocondesation reaction of step (A). The prior literature also increased the yield by using t-BuOK, but in this case the increased purity of the product obtained was unexpected. Subsequently, NaOEt was substituted with t-BuOK to obtain crude thiouracil, which was unexpectedly good in pore, thereby making handling and measurement possible. NaOEt was used to obtain a viscous mass that was difficult to pore, which made scale-up difficult. Surprisingly, this reaction also occurs with less than stoichiometric equivalents of t-BuOK.

Another advantage with the process of the present invention is that the release of hydrogen sulfide in the desulfurization reaction of step (B) is slowed down and controlled. Rapid and difficult to control gas release severely limits the scalability of the method's scale measurement.

Finally, the alkylation reaction of step (C) has some unexpected aspects in this regard. First, surprisingly this reaction is very effective in the presence of less than stoichiometric equivalents of sulfuric acid. Second, the removal of HMCS is unexpectedly very easy. In an efficient fashion, alkylation is carried out with less than stoichiometric equivalents of sulfuric acid in the presence of HMDS. Finally, there is a significant advantage over the preceding process using trimethylsilyl triflate or chloromethyl ether using diethoxymethane as alkylating agent. Because diethoxymethane is a lower cost and significantly less toxic reagent, it is used more safely than the preceding reagents.

Step I: Synthesis of i-propyl β-ketoester

The 12 L reaction flask, equipped with a mechanical stirrer and twin condenser, was clarified with argon and filled with -100 mesh zinc metal (301 g, 4.6 mol) and 5000 ml of tetrahydrofuran (THF). A catalytic amount of iodine (3.0 g) was added and the suspension was stirred until the color faded. The activated zinc suspension was then treated with benzyl cyanide (179 g, 1.53 mol) and the mixture was heated to 60 ° C.

The reaction was then treated with ethyl 2-bromoisovalerate (481 g, 2.30 mol). About 50 g of bromo ester was added and after 5 minutes the reaction was initiated while raising the batch temperature to 69 ° C. The remaining ester was then added over 35 minutes to maintain minimal reflux. After addition, the reaction was left to stir at 65 ° C. for one hour.

In-process samples were obtained and terminated with 1.8 M sulfuric acid. Gas chromatography showed 90% isopropyl β-ketoester, and 1% benzyl cyanide. The batch was then cooled to 35 ° C. and 4 L of THF was removed by evaporation under reduced pressure. The reaction was then diluted with 3 L EtOAc and carefully terminated with 2.0 L 1.8 M H ₂ SO ₄ (aq). After 1 hour, the layers were separated. The organics were then washed with 1 L of saturated NaHCO ₃ and 500 mL brine. The aqueous layers were back extracted with 250 mL each of EtOAc. The organics were mixed and concentrated to an oil. 341g of 90% by gas chromatography yield, exhibited a 90% AUC (area under the curve). The material is generally used but can be distilled at 5 mmg Hg, 110-120 ° C. to give 240 g of 96% (AUC) by gas chromatography.

Example 2

Isopropyl benzyl uracil synthesis using two-base system

A 1000 ml round bottom flask equipped with a mechanical stirrer and twin condenser was filled with isopropyl-β-ketoester (74.0 g, 289 mmol), thiourea (45.6 g, 599 mmol), and acetonitrile (500 ml) I was. The reaction mixture was then charged with potassium t-butoxide (37.0 g, 330 mmol), potassium carbonate (61.9 g, 448 mmol) and heated to 82 ° C. After 3 hours, it was cooled to 40 ° C. and terminated with water (300 mL). The total volume of the mixture was halved, the pH was adjusted to about 3 (using HCl) and the precipitated solid was collected by filtration. The solid was dried at 45 ° C. for 18 hours to give 62.7 g (81%) of a white solid with a purity of 95% (AUC).

Isopropyl benzyl uracil synthesis

A 3000 ml four necked round bottom flask equipped with a mechanical stirrer and twin condenser was charged with isopropyl-β-ketoester (259 g, 1.04 mol), thiourea (83 g, 1.1 mol), and 1150 ml of isopropanol I was. The reaction was treated with potassium t-butoxide (140 g, 1.25 mol) added in 20 g portions and the mixture was heated to 85 ° C. After 2 hours, the mixture was cooled to 60 ° C. and terminated with water (600 mL). 2-propanol was removed by evaporation under reduced pressure and the remaining solution was cooled to 30 ° C. and diluted with 600 mL of water and 450 mL of acetic acid (pH 4). The solid was collected by vacuum filtration and washed with 2 x 250 mL water. Dry to 200 g and show greater than 99.5%, 78% yield by HPLC.

166 g of thiouracil was resuspended in a solution containing water (450 mL), glacial acetic acid (200 mL), and chloracetic acid (250 g). Heterogenous reactions were heated at 95 ° C. After about 1 hour a solution formed and after a further 1 hour a white solid precipitated out. The reaction was heated for an additional 6 hours, then cooled to room temperature and the product collected by vacuum filtration. The white crystalline solid product was washed with water (2 × 150 mL) and dried overnight in a vacuum oven (60 ° C., 5 mm Hg). The product was separated from the ketoesters with a total yield of 70% and had an AUC purity of 99.5% by HPLC.

Step 2: alkylation of isopropyl benzyl uracil, synthesis of MKC-442

A 1 L three neck round bottom flask was charged with 6-benzyl-5-i-propyl uracil (BIU) (50 g, 0.20 mol), ammonium sulfate (0.5 g, catalyst) and 1,1,1,3,3,3-hexa Charged with methyldisilazane (258 g, 1.6 mol). The slurry was heated to reflux (125-130 ° C.) under argon for 4 hours. The resulting solution was concentrated to concentrated oil (125 mL) under reduced pressure (85 ° C., 30 mm Hg). The oil was resuspended in 350 ml of acetonitrile. Sulfuric acid (4.9 g, 0.05 mol) followed by diethoxymethane (36 g, 0.30 mol) was added at 80 ° C. After H ₂ SO ₄ addition, a precipitate formed, but after about 30 minutes the solution reformed. The solution was stirred for 5 hours. An additional amount of H ₂ SO ₄ (2.5 g, 0.025 mol) was added and the mixture was heated for an additional 2.5 hours.

After 10 hours, HPLC showed less than 3% residual starting material and more than 90% MKC-442. The reaction was terminated with 175 mL of 0.5N KOH (pH 10-11) at 50-60 ° C. and acetonitrile was removed by evaporation under reduced pressure (65-70 ° C., 30 mHg). The remaining slurry was diluted with 175 mL ethyl acetate and the layers partitioned. The aqueous layer was washed with ethyl acetate (50 mL) and the organic fractions were mixed. The organic fraction was concentrated to a volume of about 75 mL, then 75 mL was added to a volume of about 75 mL. The crude MKC-442 was then dissolved in additional 100 ml EtOH at 85 ° C., diluted with 60 ml water, cooled to ambient temperature and crystallized. After 2 hours, the crude was filtered off and washed with 25 ml of 25% EtOH in water. 52 g, 94% HPLC AUC.

At 85 ° C. the material was dissolved in 115 mL of EtOH and diluted with 50 mL of water and the product was left to cool to ambient temperature and crystallized. The product was filtered and washed twice with 25 ml 25% EtOH in water to give 45 g, 99.8% AUC with 0.10% (AUC) BIU.

The invention has been described in connection with its preferred embodiment. Modifications or variations of the present invention will be apparent to those skilled in the art from the foregoing detailed description of the invention. All such changes or modifications are intended to fall within the scope of the present invention.

Claims (24)

(i) reacting the 2,4-disubstituted-3-oxo-butyrate ester with thiourea in a 2-base system in the presence of a solvent to prepare a 5,6-disubstituted thiouracil; (ii) desulfurizing 5,6-disubstituted-2-thiouracil in chloroacetic acid (aqueous solution) in acetic acid to form 5,6-disubstituted-uracil; (iii) silylating 5,6-disubstituted uracil according to known methods with an appropriate reagent; (iv) 6-benzyl-1 comprising reacting the product of step (iii) with an alkylating agent or an alkoxyalkylating agent to form 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil (MKC-442) -(Ethoxymethyl) -5-isopropyluracil (MKC-442). The method of claim 1, wherein the two-base system comprises potassium t-butoxide and potassium carbonate. The method of claim 1 wherein the solvent is acetonitrile. The process of claim 1 wherein the reaction is carried out at a temperature between 80-85 ° C. The process of claim 1 wherein the two-base system comprises potassium t-butoxide and potassium carbonate, the solvent is acetonitrile and the reaction is carried out at a temperature between 80-85 ° C. (i) reacting 2,4-disubstituted-3-oxo-butyrate ester with thiourea in the presence of potassium t-butoxide in an organic solvent to prepare a 5,6-disubstituted thiouracil, 5,6-disubstituted To form a 2-thio-uracil intermediate; (ii) desulfurizing 5,6-disubstituted-2-thiouracil in chloroacetic acid (aqueous solution) in acetic acid to form 5,6-disubstituted-uracil; (iii) silylating 5,6-disubstituted uracil according to known methods with an appropriate reagent; (iv) 6-benzyl-1 comprising reacting the product of step (iii) with an alkylating agent or an alkoxyalkylating agent to form 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil (MKC-442) -(Ethoxymethyl) -5-isopropyluracil (MKC-442). The process of claim 6 wherein the solvent in step (i) is an alcohol. 8. The method of claim 7, wherein the alcohol is isopropyl alcohol. The method of claim 6, wherein the molar ratio of thiourea to 2,4-disubstituted-3-keto-butyrate does not exceed about 10% excess. 10. The method of claim 9, wherein the molar ratio of thiourea to 2,4-disubstituted-3-keto-butyrate does not exceed about 5% excess. The process of claim 6, wherein the reaction of step (ii) is terminated with water and acetic acid. 12. The process of claim 11, wherein the reaction of step (ii) is terminated with 10% chloroacetic acid (aqueous solution) in water and acetic acid. The process of claim 6, wherein the thiouracil formed in step (i) is separated prior to desulfurization. 7. The process of claim 6 wherein acetic acid in step (ii) is about 25-40% of the total solvent volume used. 15. The process of claim 14 wherein the acetic acid in step (ii) is about 35%. The method of claim 6, wherein in step (ii) the reactants are heated to between 85 ° C. and 105 ° C. to form a solution. The method of claim 6, wherein the reagent in step (iii) is a silylating agent. 18. The method of claim 17, wherein the silylating agent is hexamethyldisilizane. 19. The process of claim 18, wherein hexamethyldisilizane is reacted in the presence of a catalytic amount of ammonium sulfate. 7. The process of claim 6 wherein the alkoxyalkylating agent in step (iv) is diethoxymethane. The process of claim 20, wherein the reaction is carried out in sulfuric acid or methanesulfonic acid. The method of claim 21 wherein less than stoichiometric equivalents of sulfuric acid are used. The method of claim 22 wherein the amount of sulfuric acid is 0.25-0.5 equivalents. The method of claim 23, wherein the reaction is carried out with reflux of acetonitrile for 2-5 hours.