JP2002541247A - Method for preparing MKC-442 - Google Patents

Abstract

  (57) [Summary] Disclosed is a method for preparing 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil, also known as MKC-442. Such methods involve the use of any known method, including benzyl cyanide and ethyl-2-bromo-3,3-dimethyl-propionate and activated zinc in the Reformatsky reaction to produce ethyl-2-isopropyl- It involves synthesizing an isopropyl β-ketoester such as 3-oxo-4-phenyl-butyrate. The ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate is then condensed with thiourea in one of two ways. First of all, ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate is condensed with thiourea in a two-base system in the presence of a solvent. A second method for condensing ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate with thiourea is carried out in the presence of potassium t-butoxide to produce benzylisopropyl uracil, which is prepared in a process step ( Desulfurization in B). The benzyl isopropyl uracil resulting from either method is then alkoxyalkylated to produce MKC-442.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is related to US Provisional Application No. 60/128, filed on Apr. 13, 1999.
, No. 925.

This application is in the area of synthetic pharmaceutical chemistry, and in particular, an efficient method for preparing 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil, also known as MKC-442 including.

[0003] In 1983, the etiology of acquired immunodeficiency syndrome (AIDS) was determined to be the human immunodeficiency virus (HIV). It is now believed that the key factor in whether a person infected with HIV develops AIDS is the amount of HIV present in the body at a given time (ie, "viral load"). Researchers have focused on stopping HIV replication and reducing viral load by blocking one or both of the two key enzymes required for viral replication, reverse transcriptase and protease. .

[0004] The first enzyme, reverse transcriptase, is active early in the HIV replication cycle, allowing viruses made from RNA to produce the DNA required for continuous replication. This enzyme can be inhibited by two general classes of drugs defined by its structure as well as its mechanism of action. The first general class, 3'-azide-
3′-deoxythymidine (AZT), 2 ′, 3′-dideoxyinosine (ddI
), 2 ', 3'-dideoxycytidine (ddC) and (-)-cis-2-hydroxymethyl-5- (cytosin-1-yl) -1,3-oxathiolane (3TC
) Reverse transcriptase inhibitors of nucleoside analogues have strong chemical similarity to the natural building blocks of DNA (nucleosides) and interfere with enzyme function by replacing the natural nucleosides used by the enzyme . A second general class of non-nucleoside reverse transcriptase inhibitors, such as MKC-442 and nevirapine, are a wide variety of chemicals that work by binding and modifying the enzyme to reduce functional efficiency. Consists of groups.

[0005] Since 1985, AZT, ddI, ddC, 3TC, 2 ′, 3′-dideoxy-2 ′, 3′-didehydrothymidine (D4T), and cis-2-hydroxymethyl-5- (5-fluoro Cytosine-1-yl) -1,3-oxathiolane (FT
C) has been shown to be effective against HIV. After phosphorylation of the cell to 5'-triphosphate by cellular kinases, these synthetic nucleosides are also incorporated into the growing strand of the viral DNA, and the absence of the 3'-hydroxyl group results in strand termination.

[0006] MKC-442 functions as a non-nucleoside reverse transcriptase inhibitor. MKC
-442 is an allosteric inhibitor because it appears to exert its effect by binding to an "allosteric site", ie, a position other than the binding site of the enzyme. Preclinical studies suggest that MKC-442 may have properties that address some of the therapeutic challenges of HIV. When tested in a cell culture assay against wild-type (drug-sensitive) and several mutant strains of HIV known to be resistant to established non-nucleoside reverse transcriptase inhibitors,
C-442 retained many of its ability to inhibit HIV replication. In these studies, MKC-442 showed higher potency than nevirapine against wild-type and mutant HIV. Preclinical trials of MKC-442 in a two-drug combination with AZT or ddI and a three-drug combination with AZT and Saquina Beer revealed a synergistic inhibition of HIV replication.

[0007] Studies in animals have suggested a good safety and pharmacokinetic profile of MKC-442. Pharmacokinetic analysis in animals indicates good oral bioavailability and excellent penetration into the central nervous system, a key site of HIV replication, with little penetration of many currently marketed anti-HIV drugs showed that. In rats, for example, the concentration of MKC-442 in the brain was 100% of that found in plasma.

[0008] Phase I studies evaluated the pharmacokinetics and tolerance of a single escalating dose of MKC-442 in HIV-infected volunteers. The compounds were generally well tolerated and only a few volunteers experienced mild side effects at high dose levels. In the high dose group, drug concentrations in plasma reached levels much higher than required to suppress 90% of the virus in culture.

[0009] Preliminary data from a phase I / II, double-blind, placebo-controlled study designed to evaluate the safety and efficacy of repeated multiple oral doses of MKC-442 in HIV-infected patients are also currently being evaluated. Have been. MKC- for a total of 49 patients for up to 2 months
442. Doses ranging from 100 mg to 1000 mg twice daily, 6-8
A group of patients was administered at each dose level. The highest dose studied (750
mg and 1000 mg twice daily), after one week all patients had an average 96% reduction in viral load. This decline was largely maintained by the second week, after which the viral load gradually rose to baseline values. In a virus obtained from some patients, a single point mutation at position 13 of reverse transcriptase, which was thought to be related to resistance, was found. A total of 308 patients-MKC-442 at weekly drug exposure
Was well tolerated.

[0010] MKC-442 is described, for example, in US Pat. No. 5,461,060, which is incorporated herein by reference in its entirety.

[0011] Licensed to Ubasawa et al. On February 18, 1997, by Mitsubishi
U.S. Pat. No. 5,60, assigned to Chemical Corporation.
No. 4,209 discloses several 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'-dideoxyribonucleosides including 2', 3'-dideoxycytidine (ddC) are shared with HIV. It discloses a force action.

[0012] Due to the drug activity of MKC-442 alone and in combination with other antivirals,
There is increasing interest in efficient methods for their manufacture.

Since MKC-442 is a 5,6-substituted uracil derivative having an alkoxyalkyl group at the 1-position (see FIG. 1), its synthesis method adds these three 1,5,6-substituents. Steps must be included. A typical synthetic route to MKC-442 involves condensation of an alkoxyalkyl moiety with a 5-substituted thiouracil, followed by desulfurization of the thiouracil, lithiation at position 6, reaction with benzaldehyde, followed by acetylation and optionally MKC-442. Involved the reduction of the hydroxy group using hydrogenolysis to produce See Rosowsky et al. Med. Chem.
Tanaka et al., J. Am. Med. Chem. , 19
92, 35, 4713. Baba et al., Nucleosides and Nucleotides (Nuclee
Osides and Nucleotides), 14 (3-5), 575-
See also 583 (1995). Baba et al. Are well aware that glycosidation of 6-substituted uracils almost always results in the formation of N-3-glycosylated products due to steric hindrance by the substituents, for which reason 6-6 after introduction of the acyclic moiety. -Stated that substituents should be introduced.

[0014] Danel et al. Reported the synthesis of 5,6-disubstituted acyluridine derivatives, which involves inserting the ethoxymethyl part of the molecule at the N-1 position in the last step of the synthesis (J. Med. Chem.). , 1996, 39, 2424-2431; Syn.
thesis, August 1995, 934-936). Danel et al. First react an arylacetonitrile with a 2-bromoester in a Reformatsky reaction to produce ethyl-2-alkyl-4-aryl-3-oxobutyrate (see Scheme 1), a β-ketoester. . The β-ketoester 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 form 6-benzyl-5-ethyluracil. I do. Prior to condensation, silylation of uracil with any alkylating agent is required. Although reasonable on a small scale, Danel's method requires the use of a large excess of Zn and sodium metal to generate the base in situ, a serious safety issue, and the MKC-442 There are substantial weaknesses for preparation on a production scale. The paper also describes a 20-fold excess of ethoxide, which is not reasonable for industrial scale-up. Furthermore, Da
The method of nel requires the use of 15 equivalents of thiourea, which prevents clean crystallization of the thiouracil derivative. In addition, the thiouracil intermediate was not necessarily soluble in the chloroacetic acid solution used, and when the material was heated to reflux, a sticky mass formed and covered the stirrer and flask walls. Finally, when the desulfurization reaction was homogenized with either alcohol or tetrahydrofuran, thiouracil was converted to an unquantified impurity. When the Danel method is scaled up, MKC-4
The yield of 42 was significantly reduced and only 25-50% product with 90% purity was obtained.

[0015]

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In 1996 Orr and Musso reported the synthesis of 5-substituted uracil derivatives (but not 5,6-disubstituted uracils) by the reaction of ethyl phenylpropanoate with thiourea. They note that the prior art literature involves using sodium metal as a base in the preparation of 2-thiouracil from thiourea, which can cause problems in the synthesis and can result in low yields. reported. Syn
thetic Communication, 26 (1), 179-189 (1
996). Orr et al. Disclose ethyl phenylpropanoate and (i) sodium, ethyl formate, diethyl ether, and then thiourea and ethanol with reflux;
ii) lithium diisopropylamide, tetrahydrofuran, ethyl formate at low temperature, then thiourea and ethanol at reflux; (iii) potassium tert-butoxide, ethyl formate, diethyl ether in tetrahydrofuran at room temperature, then thiourea at reflux and 2 -Preparation of 2-thiouracil was compared through reaction with propanol. Orr et al. Reported that the use of lithium diisopropylamide or potassium t-butoxide instead of sodium metal in the preparation of 5-benzyl-2-thiouracil improved the yield of the reaction, and that ethanol was used as the solvent for the condensation of the enolate and thiourea. It was reported that replacement with 2-propanol resulted in improved yield. Since ethyl orthoformate is used, the reaction of Orr cannot be used to provide a 5,6-disubstituted uracil, at best a 5-substituted uracil or 5,5-dihydrouracil (see Scheme 2).

[0017]

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Baba et al. Converted silylated 5-isopropyl-2-thiouracil to the carcinogenic CH. ₃ CH₂OCH₂Cl (KI / CH₂Cl₂) And react with N-1-ethoxyme
A method for producing MKC-442 by obtaining a chill product was reported (Nu
cleosides and Nucleotides, 14 (3-5), 57
5-583 (1995)). Lithium diisopropylamide (LDA)
Tionation resulted in the 6-substituted product. Oxidative hydrolysis of 6-substituted products and it
Subsequent conventional hydrocracking led to MKC-442.

[0019] In view of the fact that MKC-442 is useful in treating HIV-infected patients, a cost-effective and scalable method for producing pure MKC-442 in large quantities, with high purity and high yield There is a need to develop a method. Therefore, 6-
Benzyl-1- (ethoxymethyl) -5-isopropyluracil (MKC-44
It is an object of the present invention to provide an improved synthetic method for preparing 2) with high yield and higher purity.

DISCLOSURE OF THE INVENTION It has been discovered that the reaction of a suitable β-ketoester with thiourea in the presence of potassium t-butoxide can produce a 5,6-disubstituted uracil in good yield.
When the β-ketoester is ethyl-2-isopropyl-3-oxo-4-phenylbutyrate, the resulting product is 6-benzyl-1- (ethoxymethyl) -5-isopropyl, also known as MKC-442. It is uracil.

Therefore, in one embodiment, comprises simultaneously using two bases to efficiently synthesize 5,6-disubstituted uracils from β-ketoesters in high yields,
Methods are provided for producing 5,6-disubstituted uracils. Preferred bases are
Includes potassium carbonate and potassium t-butoxide as coexisting bases in acetonitrile. This embodiment is shown in Scheme 3.

[0022]

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[0023] Non-limiting examples of the synthesis of MKC-442 include any of benzyl cyanide and ethyl-2-bromo-3,3-dimethyl-propionate and activated zinc in a Reformatsky reaction. Ethyl-2- using known methods
Isopropyl β, such as isopropyl-3-oxo-4-phenyl-butyrate
-Ketoesters can be synthesized. The β-ketoester is then reacted in a solvent, such as acetonitrile, with a dibasic system, including but not limited to potassium carbonate and potassium t-butoxide.

Such a method offers several advantages over the prior art. First of all, it is a single-step process in which the reaction proceeds to form the product in yields of over 80%. Further, a two base system yields a higher purity product. For example, the use of potassium carbonate and potassium t-butoxide in acetonitrile results in a product having a purity of 90-95%.

In synthesizing a substituted thiouracil ring, a base with a high pK _b alone requires the starting β
-Destroys ketoester and increases the level of impurities. As a result, use of a base alone of the high pK _b leads to the product of the low yields. Low pK _b of the base tend to have a longer reaction times, it tends to include a residue of the starting material. Therefore, it is beneficial to use a two base system to neutralize these trends.

Bases useful in the two-base system of the present invention include potassium carbonate, potassium t-butoxide, sodium carbonate, diisopropylamine, triethylamine, 2,6-dimethylpyridine, sodium acetate, potassium acetate, ammonia, potassium ethoxide, And potassium cyanide, but are not limited thereto. The base is used in combination with a solvent.

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

In another embodiment of the present invention, preferably in isopropyl alcohol,
Provided is a method for producing a 5,6-disubstituted uracil, comprising reacting a β-ketoester with a thiourea in the presence of potassium t-butoxide. This embodiment of the reaction is shown in Scheme 4.

[0029]

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[0030] Non-limiting examples of the synthesis of MKC-442 include any from benzyl cyanide and ethyl-2-bromo-3,3-dimethyl-propionate and activated zinc in a Reformatsky reaction. Ethyl-2- using known methods
Isopropyl β, such as isopropyl-3-oxo-4-phenyl-butyrate
-Ketoester can then be synthesized and then in the process step (A) according to the invention, in the presence of potassium t-butoxide, ethyl-2-isopropyl-3-oxo-
4-Phenyl-butyrate is condensed with thiourea to produce benzylisopropylthiouracil, which is desulfurized in process step (B), followed by process step (C
)), The benzyl isopropyl uracil is alkoxyalkylated to give MKC-
422 is generated. The improved method of the present invention produces MKC-442 in higher yield and purity, but offers several surprising advantages over the prior art.

[0031] Such a method offers several advantages over the prior art. Replacing NaOEt with t-BuOK in the condensed cyclization reaction of step (A) yields a significantly higher purity product. Although the high yields obtained with t-BuOK are precedent in the literature, the high purity of the resulting products has not been previously known and is unexpected. Further, Na
Replacement of OEt with t-BuOK yields a crude thiouracil that is easy to handle, making the work easier to control and scalable. It is also surprising that the reaction of step (A) occurs for sub-stoichiometric amounts of t-BuOK. Finally, the use of diethoxymethane in the presence of an acid catalyst such as sulfuric acid in step (C), rather than the highly toxic TMS triflate or chloromethyl ether used in the prior art, is a cost advantage. As well as providing safety benefits.

The slow and suppressed generation of hydrogen sulfide in the desulfurization reaction of step (B) is also unexpected. Rapid, uncontrolled gas evolution will hinder scale-up and industrialization of the process.

The alkylation reaction of step (C) has two surprising aspects associated therewith.
First of all, it is surprising that this reaction proceeds very efficiently in the presence of substoichiometric amounts of sulfuric acid. Second, it is very advantageous that HMDS removal is not required. Alkylation proceeds efficiently with substoichiometric amounts of sulfuric acid, even in the presence of HMDS.

DETAILED DESCRIPTION OF THE INVENTION There is provided an improved method for preparing 5,6-disubstituted uracils. This improved method offers several advantages over the prior art. Most importantly, the process of the present invention provides high yields of MKC-442 without loss of product purity.
To produce. In fact, the present method can produce products with a purity of 95% or more.

The present invention comprises at least the following embodiments: An initial method for preparing 5,6-disubstituted uracils, preferably MKC-442, by reacting a β-ketoester with thiourea in a two-base system. Such a method involves the simultaneous use of two bases to efficiently synthesize a 5,6-disubstituted uracil. The method comprises reacting a β-ketoester with thiourea at a temperature in the range of 80-85 ° C in the presence of a dibasic system and a solvent.

[0036] The one-step process of this first method of the present invention involves the conversion of 2,4-disubstituted-3-oxo-butyrate, preferably ethyl-2-isopropyl-3-oxo-4-phenylbutyrate, to a two-base system. And reacting with thiourea in a solvent. Preferred bases for a two base system 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 of 80 ° C 85 ° C. The preferred reaction temperature is about 85 ° C.

As used herein, the term “two-base system” refers to the simultaneous use of two bases in the presence of a solvent. Non-limiting examples of bases included in the two base system include potassium carbonate, potassium t-butoxide, sodium carbonate, diisopropylamine, triethylamine, 2,6-dimethylpyridine, sodium acetate, potassium acetate, ammonia,
Potassium ethoxide, and potassium cyanide. The base is used in combination with a solvent.

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

A second method for preparing a 5,6-disubstituted uracil, preferably MKC-442, comprising: (i) 2,4-disubstituted in the presence of potassium t-butoxide in an organic solvent (A) comprising preparing a 5,6-disubstituted thiouracil by reacting a -3-oxo-butyrate ester with thiourea, wherein the intermediate is a 5,6-disubstituted-2-thiouracil (Ii) the process of step (A), wherein the solvent is an alcohol, preferably isopropyl alcohol; (iii) a molar ratio of thiourea to 2,4-disubstituted-3-keto-butyrate of not more than about 10% excess. (Iv) the method of step (A), wherein the reaction is quenched with water and acetic acid; (v) preferably acetic acid. (B) comprising the method of (A) further comprising desulfurizing the 5,6-disubstituted-2-thiouracil to the 5,6-disubstituted uracil using 10% chloroacetic acid (aqueous) of (Vi) Step (B) further comprising separating thiouracil prior to desulfurization.
(V) the method of step (B), wherein acetic acid is about 25-40%, preferably 35% of the total solvent volume used; (viii) heating the reaction to 85-105 ° C (Ix) producing a solution, the method of step (B); (ix) a step (C) comprising silylating the 5,6-disubstituted uracil of step (v) with a suitable reagent according to known methods; (x) The step wherein the reagent is a silylating agent such as hexamethyldisilizane,
(Xi) the method of step (x), wherein hexamethyldisilizane is reacted in the presence of a catalytic amount of ammonium sulfate; (xii) the alkylation or alkoxyalkylating agent of the product of step C Reacting with ethoxymethane; (xiii) performing the reaction in sulfuric acid or methanesulfonic acid, step (xi)
(xiv) the method of step (xiii) using a substoichiometric amount of sulfuric acid, preferably 0.25 to 0.5 equivalents; (xv) reacting the reaction with refluxing acetonitrile (or other In (alkyl nitrile) of step (xiv), preferably for 2 to 5 hours.

The two-step process of the second method of the present invention includes the steps (A) and (
B). Step (A) comprises cyclizing the isopropyl-β-ketoester with thiourea and potassium t-butoxide in isopropyl alcohol under reflux,
Producing benzyl isopropyl thiouracil. Next, the benzylthiouracil is desulfurized under reflux in step (B) to give a chloroacetic acid solution, 10% Cl2.
AcOH (aqueous), benzyl isopropyl uracil (BIU) using AcOH
Generate Quench the reaction with water and acetic acid, then reduce the crystalline solid to 70-80%
Filtered in yield.

The Stage I method is similar to the method of Danel (J. Med.) In that it eliminates the use of the sodium metal used to generate the base in situ.
Chem. , 1996, 39, 2427-2431). The elimination of sodium has eliminated serious safety concerns at scale-up. Danel also defines a 20-fold excess of ethoxide, but the method of the invention requires only a 5% to 20% excess. In addition, Danel's method produces a thiouracil intermediate which, when heated to reflux, forms a sticky mass covering the agitator and flask walls. In contrast, the use of separately separated thiouracil produced by the method of step (A) of the present invention results in increased conversion and purity. The addition of acetic acid (about 35% of total solvent volume) to the reaction in step (B) achieved two results: it allowed for the formation of a solution when heated to 95 ° C .; Functioned as solvent. The process of the present invention eliminates sticky clumps formed in the Danel process, making the improved process scalable to production sized equipment. In addition, BIU was recovered in approximately 90% yield as> 98% pure material.

In stage II of the process of the present invention, BIU is silylated with 1,1,1,3,3,3-hexamethyldisilizane (HMDS) in the presence of a catalytic amount of ammonium sulfate and then acetonitrile containing sulfuric acid Alkylation with medium diethoxymethane. This method is referred to herein as step (C). Optionally, methanesulfonic acid can be used as an acid catalyst. Conditions were optimized to use a substoichiometric amount (0.25-0.5 equivalent) of concentrated sulfuric acid at reflux in acetonitrile to effect the alkylation. A typical reaction profile is 90 with <3% BIU as an impurity.
A -95% yield of MKC-442 was shown.

The two-step method of the present invention offers several improvements over prior art methods. First, in the condensation cyclization reaction of the step (A), NaOEt is converted to t-BuO.
Substitution with K results in a product of significantly higher purity. The high yields obtained with t-BuOK are precedent in the literature, but in this case the high purity of the products obtained was unexpected. Furthermore, replacement of NaOEt with t-BuOK resulted in a surprisingly easy-to-handle crude thiouracil, which made the operation easier to control and scaleable. Na
OEt resulted in sticky lumps that were difficult to handle, making scale-up difficult. Also,
It is also surprising that this reaction occurs for sub-stoichiometric amounts of t-BuOK.

Another advantage of the method of the present invention is the slow and suppressed generation of hydrogen sulfide in the desulfurization reaction of step (B). The rapid and difficult to control gas evolution would have greatly limited the scalability of the process.

Finally, the alkylation reaction of step (C) has two surprising aspects associated therewith. First, it is surprising that this reaction proceeds very efficiently in the presence of substoichiometric amounts of sulfuric acid. Second, the need for HMDS removal is unexpected and extremely advantageous. Alkylation proceeds efficiently with substoichiometric amounts of sulfuric acid, even in the presence of HMDS. Finally, the use of diethoxymethane as an alkylating agent offers significant advantages over prior art processes using trimethylsilyl triflate or chloromethyl ether. Diethoxymethane is not only cheaper, but also a significantly less toxic reagent, and its use is safer than prior art reagents.

Example 1 Stage I: Synthesis of i-propyl β-ketoester

[0047]

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A 12 liter reaction flask connected to a mechanical stirrer and a pair of condensers was purged with argon and -100 mesh zinc metal (301 g, 4.6 mol) and 500
0 ml tetrahydrofuran (THF) was charged. Catalytic amount of iodine (3.0 g)
Was added and the suspension was stirred until the color disappeared. The activated zinc suspension was then treated with benzyl cyanide (179 g, 1,53 mol) and the mixture was heated to 60 ° C.

Next, the reaction product was treated with ethyl 2-bromoisovalerate (481 g, 2.30 mol).
Processed. About 50 g of the bromoester was added to start the reaction and after 5 minutes the batch temperature was raised to 69 ° C. The remaining ester was then added over 35 minutes to maintain a minimum reflux. After the addition, the reaction was stirred at 65 ° C. for 1 hour.

An in-process sample was obtained and quenched with 1.8 M sulfuric acid. Gas chromatography showed 90% isopropyl β-ketoester and 1% benzyl cyanide. Then the batch was cooled to 35 ° C. and evaporated under reduced pressure to remove 4 L THF. The reaction was then diluted with 3 L EtOAc and carefully quenched with 2.0 L of 1.8 MH ₂ SO ₄ (aq). After 1 hour, the layers were separated. The organics were washed with 1 L of saturated NaHCO ₃ and 500 ml of brine. Separate the aqueous layers with EtOAc
Back-extracted with 250 ml. The organics were combined and concentrated to an oil. 341 g 90% yield, 90% AUC (area under the curve) by gas chromatography. Typically, this material is used as is, but can be distilled at 5 mm Hg, 110-120 ° C. to give 240 g 96% (AUC) by gas chromatography.

Example 2 Synthesis of isopropylbenzyluracil using a two-base system

[0052]

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In a 1000 mL round bottom flask connected to a mechanical stirrer and a condenser, isopropyl β-ketoester (74.0 g, 289 mmol), thiourea (45.6 g,
599 mmol), and acetonitrile (500 mL). Next, potassium t-butoxide (37.0 g, 330 mmol) and potassium carbonate (61.9 g, 448 mmol) were added to the reaction mixture, and the mixture was heated to 82 ° C. After 3 hours, the mixture was cooled to about 40 ° C. and quenched with water (300 mL). The volume of the entire mixture was reduced by half, the pH was adjusted to about 3 (by HCl), and the precipitated solid was collected through filtration. The solid was dried at 45 ° C. for 18 hours to give 62.7 g (81%) of a white solid with 95% purity (AUC).

Synthesis of isopropylbenzyluracil

[0055]

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In a 3000 ml 4-neck round bottom flask connected to a mechanical stirrer and a condenser, isopropyl β-ketoester (259 g, 1.04 mol), thiourea (83 g,
1.1 mol), and 1150 ml of isopropanol. The reaction was then treated with potassium tert-butoxide (140 g, 1.25 mol) in 20 g portions, and the mixture was heated to 85C. After 2 hours, the mixture was cooled to about 60 ° C. and water (6
00 ml). The 2-propanol is removed by evaporation under reduced pressure, the remaining solution is cooled to 30 ° C. and 600 ml of water and 450 ml of acetic acid (pH = 4)
For further dilution. The solid was collected by vacuum filtration and washed with 2 × 250 ml of water. Dry to give 200 g,> 99.5% AUC by HPLC, yield 78
%Met.

166 g of thiouracil were resuspended in a solution containing water (450 ml), glacial acetic acid (200 ml), and chloroacetic acid (250 g). The heterogeneous reaction was heated at 95 ° C. After about 1 hour, a solution had formed, and after an additional hour, a white solid precipitated. The reaction was heated for a further 6 hours, then cooled to room temperature and the product was collected by vacuum filtration. The product, a white crystalline solid, was washed with water (2 × 150 ml) and placed in a vacuum oven (
(60 ° C, 5 mmHg) overnight. The product was separated from the ketoester in an overall yield of 70%, such material having 99.5% AUC purity by HPLC.

Stage 2: Alkylation of isopropylbenzyluracil, synthesis of MKC-442

[0059]

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In a 1 L 3-neck round bottom flask, 6-benzyl-5-i-propyluracil (BIU)
(50 g, 0.20 mol), ammonium sulfate (0.5 g, catalytic) and 1,
1,1,3,3,3-Hexamethyldisilazane (258 g, 1.6 mol) was charged. The slurry was heated to reflux under argon for 4 hours (125-130 ° C)
. The resulting solution was concentrated under reduced pressure (85 ° C., 30 mmHg) to a thick oil (about 125 ml). The oil was resuspended in 350 ml of acetonitrile. Sulfuric acid (4.9 g, 0.0
5 mol) followed by diethoxymethane (36 g, 0.30 ml) at 80 ° C. A precipitate formed after the H ₂ SO ₄ was added, but again a solution formed after about 30 minutes. The solution was stirred for 5 hours. An additional amount of H ₂ SO ₄ was added (2.5 g, 0.0
25 mol) and the mixture was heated for a further 2.5 hours.

After 10 hours, HPLC showed <3% residual starting material and> 90% MKC-442.
showed that. 175 ml of 0.5N KOH at 50-60 ° C (pH = 10-11)
The reaction was quenched with and evaporated under reduced pressure to remove acetonitrile (65
-70 ° C, 30 mmHg). Dilute the remaining slurry with 175 ml of ethyl acetate,
The layers were separated. The aqueous layer was washed with ethyl acetate (50 ml), and the organic fraction was mixed. The organic fraction was concentrated to a volume of about 75 ml, and 75 ml of ethanol was added to add about 75 ml.
Of capacity. The crude MKC-442 was taken up in a further 100 ml of EtOH at 85 ° C., diluted with 60 ml of water, cooled to room temperature and crystallized. After 2 hours, the crude was filtered and washed with 25 ml of 25% EtOH in water. 52g, 94% HPLC
AUC.

This material was then taken up in 115 ml of 85 ° C. EtOH, diluted with 50 ml of water, and the product was cooled to room temperature and crystallized. The product is filtered and 25% EtO in water
H Wash twice with 25 ml, 45 g, 99.8% AUC, 0.10% (AUC)
BIU was obtained.

The present invention has been described with reference to preferred embodiments thereof. Variations and modifications of the invention will be apparent to those skilled in the art from the foregoing detailed description of the invention. All of these variations and modifications are intended to be included within the scope of the present invention.

[Brief description of the drawings]

FIG. 1 shows a structural formula of MKC-442.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Cleary, Daryl G. United States, North Carolina 27514; Chapel Hill, Halcombridge Road 6410 Drive 5703 (72) Inventor Almond, Melitzk Earl United States, North Carolina 27502, Apetux, West Sterlinton Place 10 34 (72) Inventor Omany, Rose United States, North Carolina 27713, Durham, Rolling Woods Drive 4600 (72) Inventor Mangal, Terrance United States, North Carolina 28590, Winterville, Dukes Road・ 120 (72) Inventor Kuzenko, Michael United States, North Carolina ・ 27850, Greenville, Chierry Stone Lane ・ 2034

Claims (24)

[Claims] (1) 2,4-disubstituted-3 in a dibasic system in the presence of a solvent
A 5,6-disubstituted thiouracil is prepared by reacting an oxobutyrate ester with a thiourea; (ii) desulfurizing a chloroacetic acid (aqueous) solution of the 5,6-disubstituted-2-thiouracil in acetic acid (Iii) silylating the 5,6-disubstituted uracil with a suitable reagent according to known methods; (iv) alkylating or alkoxylating the product of step (iii). Reacting with an alkylating agent to form 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil (MKC-442), including 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil ( MKC-442). 2. The method of claim 1, wherein the two-base system comprises potassium t-butoxide and potassium carbonate. 3. The method according to claim 1, wherein the solvent is acetonitrile. 4. The method according to claim 1, wherein the reaction is carried out at a temperature of from 80 to 85 ° C. 5. The method according to claim 1, wherein the dibasic system comprises potassium t-butoxide and potassium carbonate, the solvent is acetonitrile, and the reaction is carried out at a temperature of from 80 to 85 ° C. 6. (i) In the presence of potassium t-butoxide in an organic solvent,
Preparing a 5,6-disubstituted thiouracil by reacting a 4-disubstituted-3-oxobutyrate ester with thiourea to produce a 5,6-disubstituted-2-thiouracil intermediate; (ii) Desulfurization of a chloroacetic acid (aqueous) solution of 5,6-disubstituted-2-thiouracil in acetic acid to produce 5,6-disubstituted-uracil, (iii) 5,6 with suitable reagents according to known methods -Silylating the disubstituted uracil; (iv) reacting the product of step (iii) with an alkylating or alkoxyalkylating agent to give 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil (MKC-442) A method for preparing 6-benzyl-1- (ethoxymethyl) -5-isopropyluracil (MKC-442), comprising producing 7. The method of claim 6, wherein in step (i) the solvent is an alcohol.
The method described in. 8. The method according to claim 7, wherein the alcohol is isopropyl alcohol. 9. The method of claim 6, wherein the molar ratio of thiourea to 2,4-disubstituted-3-keto-butyrate is no more than about a 10% excess. 10. The method of claim 9, wherein the molar ratio of thiourea to 2,4-disubstituted-3-keto-butyrate is no more than about 5% excess. 11. quenching the reaction of step (ii) with water and acetic acid;
The method of claim 6. 12. The reaction of step (ii) is carried out with water and acetic acid of 10% chloroacetic acid (
The method of claim 11, wherein the quenching is with an (aqueous) solution. 13. The method according to claim 6, wherein the thiouracil formed in step (i) is separated before desulfurization. 14. The method of claim 6, wherein in step (ii) acetic acid is about 25-40% of the total solvent volume used. 15. The method of claim 14, wherein in step (ii) the acetic acid is about 35%. 16. The method of claim 6, wherein in step (ii), the reactants are heated to between 85 ° C. and 105 ° C. to form a solution. 17. The method of claim 6, wherein in step (iii) the reagent is a silylating agent. 18. The method according to claim 17, wherein the silylating agent is hexamethyldisilizane.
The method described in. 19. The method according to claim 18, wherein hexamethyldisilizane is reacted in the presence of a catalytic amount of ammonium sulfate. 20. The method according to claim 6, wherein in step (iv) the alkoxyalkylating agent is diethoxymethane. 21. The reaction according to claim 2, wherein the reaction is carried out in sulfuric acid or methanesulfonic acid.
The method according to 0. 22. The method of claim 21 wherein a substoichiometric amount of sulfuric acid is used. 23. The method according to claim 22, wherein the amount of sulfuric acid is 0.25 to 0.5 equivalent. 24. The method according to claim 23, wherein the reaction is carried out in refluxing acetonitrile for 2-5 hours.