Patent Description:
<NUM>-Ureido-<NUM>,<NUM>-dihalo-quinoline-<NUM>-carboxylate compounds, particularly diphenylureido-dichlorokynurenic acid (DCUKA) compounds and esters thereof, reportedly have analgesic activity and are useful in treating chronic pain and alcohol dependence, as well as preventing relapse in alcohol addicted subjects. One difficulty in the commercial development of the <NUM>-ureido-<NUM>,<NUM>-dihalo-quinoline-<NUM>-carboxylate compounds has been the harsh conditions needed for preparing <NUM>,<NUM>-dihalo-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylates, which are intermediates in the synthesis of the <NUM>-ureido-<NUM>,<NUM>-dihalo-quinoline-<NUM>-carboxylate compounds. A published approach for preparing DCUKA ester compounds (e.g., the methyl or ethyl ester), is illustrated in Scheme <NUM> for the ethyl (Et) ester. Reaction A in Scheme <NUM> below comprises heating a mixture of <NUM>,<NUM>-dichloroaniline <NUM> and diethyl acetylenedicarboxylate (e.g., <NUM> in tetrahydrofuran (THF) at about <NUM> to afford Michael adduct <NUM>. Reaction B involves heating Michael adduct <NUM> at about <NUM> in diphenyl ether solvent to afford ethyl <NUM>,<NUM>-dichloro-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester <NUM>. Ester <NUM> is then reacted with chlorosulfonyl isocyanate in acetonitrile, followed by quenching with hydrochloric acid (collectively Reaction C) to afford ethyl <NUM>-amino-<NUM>,<NUM>-dichloro-quinoline-<NUM>-carboxylate ester <NUM>, which is then reacted with diphenylcarbamoyl chloride in dimethylformamide (DMF) in the presence of a NaH to afford the DCUKA ethyl ester (Reaction D) or if desired, the DCUKA acid can be isolated by in situ hydrolysis of the ester group. Reaction B in Scheme <NUM> is a potential impediment to commercial scale production of DCUKA compounds due to the high temperature of the reaction, as well as the expense of the solvent, and the difficulties of purifying the product and recovering the solvent. The processes described herein address this problem. <NPL>) describes a synthesis of <NUM>-hydroxycoumarin and <NUM>-hydroxy-<NUM>-quinolone derivatives. <CIT> discloses aminoquinoline compounds useful for treating chronic pain, addiction, and other conditions.

A method for preparing an alkyl <NUM>,<NUM>-dihalo-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester of Formula IV, below, is described herein. The method comprises cyclizing a dialkyl <NUM>-(<NUM>,<NUM>-dihalophenylamino)ethylene-<NUM>,<NUM>-dicarboxylate compound of Formula III with P<NUM>O<NUM> in methane sulfonic acid (often referred to as Eaton's reagent) in a heated continuous flow reactor to form the alkyl l,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester of Formula IV. Optionally, a cosolvent such as dichloromethane can be included with the Eaton's reagent. A method of preparing diphenylureido-dihalokynurenic acid esters of Formula VI from the alkyl <NUM>,<NUM>-dihalo-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester of Formula IV is also described. Diphenylureido-dihalokynurenic acid alkyl esters (e.g., such as DCUKA ethyl ester) are analgesic agents useful in treating chronic pain and alcohol dependence, as well as preventing relapse in alcohol addicted subjects. Scheme <NUM> summarizes the synthesis of diphenylureido-dihalokynurenic acid alkyl esters of Formula VI according to the methods described herein.

In the compounds of Formulas I, II, III, IV, V and VI of Scheme <NUM>, each X independently is a halogen, e.g., F, Cl, Br or I, and each R is a C<NUM> to C<NUM> alkyl group, e.g., methyl (Me), ethyl (Et), propyl (Pr), isopropyl (iPr) , butyl (Bu), and the like. In one preferred embodiment, each X is Cl. In another preferred embodiment, each R is ethyl. In yet another preferred embodiment, each X is Cl, and each R is ethyl.

In Scheme <NUM>, Reaction (a) is a Michael addition reaction which typically involves adding the dialkyl acetylenedicarboxylate of Formula II to a heated solution of the <NUM>,<NUM>-dihaloaniline of Formula I. The dialkyl <NUM>-arylamino-ethylene-<NUM>,<NUM>-dicarboxylate Michael adduct of Formula III can be isolated and purified, or can be used in Reaction (b) in crude form.

The previously reported cycloacylation of Compound <NUM> (Formula III where each X is Cl and each R is ethyl) by Tabakoff et al. is entirely thermally driven by heating a solution of Compound <NUM> at about <NUM> in a high boiling point solvent such as diphenyl ether (illustrated as Reaction A in Scheme <NUM>). The reaction typically is performed in a bulk reactor, and can be difficult to perform in a flow reactor due to the high temperatures and the relatively low solubility of the product. The high temperature required for the reaction also poses a safety issue, and requires that Compound <NUM> must be relatively free of unreacted <NUM>,<NUM>-dichloroaniline (Compound <NUM>), since any aniline present in the reaction will react with the ester group to form an amide under the high temperature conditions of the reaction.

In the methods described herein, cycloacylation of the Michael adduct of Formula III to form the <NUM>,<NUM>-dihydro-<NUM>-oxoquinoline compound of Formula IV, Reaction (b) of Scheme <NUM>, is performed at much lower temperature utilizing P<NUM>O<NUM> in methanesulfonic acid (e.g., about <NUM> to about <NUM> % by weight (wt%) P<NUM>O<NUM> in methanesulfonic acid; also known as Eaton's reagent) than the prior known thermal cycloacylation. As described herein, Reaction (b) is performed in a flow reactor in neat Eaton's reagent as the sole solvent, or with Eaton's reagent and a hydrocarbon or halogenated hydrocarbon cosolvent.

While the use of Eaton's reagent has been reported by <NPL>) for cycloacylation of aniline derivatives having other substitution patterns on the phenyl ring, the cycloacylation reaction with a <NUM>,<NUM>-disubstituted-aniline Michael adduct, such as compound <NUM> of Scheme <NUM>, has not been reported or suggested. In fact, Zewge et al. reported (footnote <NUM>) that the cycloacylation of aniline derivatives with Eaton's reagent did not proceed effectively in the presence of cosolvents, and that aniline derivatives with meta substituents provided messy reaction profiles, with a yield of only <NUM>% desired product in the case of a meta-methoxy aniline compound (footnote <NUM>). Thus, the effective cycloacylation of the <NUM>,<NUM>-dihaloaniline Michael adducts of Formula III with Eaton's reagent is unexpected. Even more surprising is that a cosolvent can be included with the Eaton's reagent in a flow reaction, given the very negative results reported by Zewge et al. with cosolvents.

Another unexpected advantage of using Eaton's reagent for cycloacylation Reaction (b) is that relatively high levels (up to <NUM> % or more) of unreacted dihaloaniline of Formula I (e.g., Compound <NUM>) can be present during the cycloacylation without significantly interfering with the reaction. Thus, in one preferred embodiment, the isolated crude product of Formula III obtained from the Michael addition, Reaction (a), which contains significant quantities of the unreacted aniline of Formula I, is used in the cycloacylation without further purification, other than isolating the crude product from the solvent used in the Michael addition reaction.

The crude product of Formula IV obtained from the cycloacylation reaction can be isolated by trituration of the product-containing portion of effluent from the flow reactor (e.g., by adding the effluent to chilled water) to obtain the crude <NUM>,<NUM>-dihydro-<NUM>-oxoquinoline of Formula IV as a granular solid precipitate. The granular solid can then be purified by slurrying the precipitate in a solvent (e.g., acetonitrile, methanol, or isopropanol; preferably isopropanol or acetonitrile) with mild heating (e.g., about <NUM>) to remove impurities into the liquid phase, and then recovering the remaining solids from the slurry (e.g., by filtration) to obtain a substantially purified (<NUM> % or greater by HPLC) compound of Formula IV.

Reaction (c) in Scheme <NUM> is conversion the <NUM>,<NUM>-dihydro-<NUM>-oxoquiniline of Formula IV to a <NUM>-aminoquinoline compound of Formula V by reaction of the <NUM>,<NUM>-dihydro-<NUM>-oxoquiniline compound with chlorosulfonyl isocyanate in an aprotic solvent (e.g., acetonitrile). The crude <NUM>-aminoquinoline product can be purified by slurrying the product in a solvent (e.g., ethyl acetate, ethanol, or isopropanol) with mild heating (e.g., about <NUM>) to remove impurities into the liquid phase, and recovering the remaining solids from the slurry to obtain a substantially purified (> <NUM> % by HPLC) compound of Formula V.

Reaction (d) in Scheme <NUM> is conversion of the <NUM>-aminoquinoline compound of Formula V to the diphenylureido-dihalokynurenic acid ester of Formula VI by reaction with diphenylcarbamoyl chloride in the presence of a base (e.g., sodium hydride) in a polar aprotic solvent (e.g., dimethylacetamide; "DMAc").

The following non-limiting embodiments are provided to illustrate the methods described herein.

A first embodiment is a method for preparing an alkyl <NUM>,<NUM>-dihalo-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester. The method comprises the sequential steps of:.

wherein in Formula III and Formula IV, each X independently is a halogen atom (e.g., Cl); and each R independently is C<NUM> to C<NUM> alkyl (e.g., Et). Optionally, step (i) can be performed in the presence of a hydrocarbon or halogenated hydrocarbon cosolvent (e.g., dichloromethane).

The method also can include purifying the ester of Formula IV, if desired. The purification is performed by:.

A second embodiment is a method for preparing an alkyl <NUM>,<NUM>-dihalo-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate comprising the sequential steps of:.

wherein in Formula I, Formula II, Formula III, and Formula IV each X independently is a halogen atom (e.g., Cl); and each R independently is C<NUM> to C<NUM> alkyl (e.g., Et). Optionally, step (iii) can be performed in the presence of a hydrocarbon or halogenated hydrocarbon cosolvent (e.g., dichloromethane).

The method of the second embodiment also can include purifying the ester of Formula IV, if desired. The purification is performed by:.

A third embodiment is a method for preparing a diphenylureido-dihalokynurenic acid alkyl ester comprising performing steps (i), (ii), and (iii) of the first embodiment; and then:.

The ester of Formula V can be isolated in step (viii) by any desired method. In some embodiments, the ester of Formula V is isolated in step (viii) by adjusting the pH of the acidic mixture of step (vii) to about <NUM> to about <NUM> to form a second precipitate comprising the ester of Formula V, which is then recovered, e.g., by filtration or centrifugation.

The method of the third embodiment also can include purifying the ester of Formula V, if desired. The purification is performed by:.

The diphenylureido-dihalokynurenic acid ester of Formula VI can be isolated in step (x) by any desired method. In some embodiments, the ester of Formula VI is isolated in step (x) by adding the solution from step (ix) to about <NUM> to about <NUM> volumes of an aqueous acid (e.g., <NUM> wt% acetic acid) with stirring to form a precipitate comprising the ester of Formula VI, which is then recovered, e.g., by filtration or centrifugation.

If desired, the method of the third embodiment can include purifying the diphenylureido-dihalokynurenic acid ester of Formula VI, as well. The purification is performed by:.

A fourth embodiment is a method for preparing a diphenylureido-dihalokynurenic acid alkyl ester comprising performing steps (i), (ii), (iii), (iv), and (v) of the second embodiment; and then:.

The ester of Formula V can be isolated in step (x) by any desired method. In some embodiments, the ester of Formula V is isolated in step (x) by adjusting the pH of the acidic mixture of step (ix) to about <NUM> to <NUM> to form a second precipitate comprising the ester of Formula V, which is then recovered, e.g., by filtration or centrifugation.

The method of the fourth embodiment also can include purifying the ester of Formula V, if desired. The purification is performed by:.

The diphenylureido-dihalokynurenic acid ester of F Formula VI can be isolated in step (xii) by any desired method. In some embodiments, the ester of Formula VI is isolated in step (xii) by adding the solution from step (xi) to about <NUM> to about <NUM> volumes of an aqueous acid (e.g., <NUM> wt% acetic acid) with stirring to form a precipitate comprising the ester of Formula VI, which is then recovered, e.g., by filtration or centrifugation.

If desired, the purified ester of Formula VI can be converted to the corresponding carboxylic acid (e.g., to DCUKA) or to a salt thereof by hydrolysis. In addition, the carboxylic acid or the carboxylate salt can be converted to an addition salt with a strong acid, such as p-toluenesulfonic acid, such that the heterocyclic nitrogen of the diphenylureido-dihalokynurenic acid is protonated.

The drawing schematically illustrates a flow reactor for use in the methods described herein.

As noted above, the previously reported procedures for the synthesis of DCUKA ethyl and methyl esters, illustrated in Scheme <NUM> for the ethyl ester, involves four stages, beginning with the treatment of <NUM>,<NUM>-dichloroaniline <NUM> with a diethyl acetylenedicarboxylate <NUM> in THF under reflux conditions to obtain Michael adduct <NUM>, which then is thermally cyclized at <NUM> in diphenyl ether (Ph<NUM>O) to afford ethyl <NUM>,<NUM>-dichloro-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester <NUM>. Ester <NUM> is then treated with chlorosulfonyl isocyanate under reflux conditions to provide <NUM>-aminoquinoline <NUM>. The amino group of Compound <NUM> is then reacted with diphenylcarbamoyl chloride in the presence of NaH in DMF to afford the target DCUKA ethyl ester <NUM>. Ester <NUM> and the corresponding acid (where the ethyl group, Et, is replaced by H) are analgesics, which are useful for treating chronic pain and alcohol dependence, as well as other related conditions.

A significant advance in the synthesis of DCUKA esters is described herein, which comprises the use of a solution of diphosphorus pentoxide in methane sulfonic acid (also known as Eaton's reagent) for the cycloacylation reaction, which eliminates the requirement of high temperature in the cycloacylation reaction and is amenable to use in flow reactor conditions.

As illustrated by the preparation of Compound <NUM> (Formula III in which each X is Cl and each R is Et), the aza-Michael reaction is carried out by dropwise addition of a THF solution of about <NUM> equivalents (eq) of diethyl acetylenedicarboxylate <NUM> to a THF solution of about <NUM> eq of <NUM>,<NUM>-dichloroaniline <NUM>, and then heating the resulting mixture at about <NUM> for several hours (h), e.g., about <NUM> to <NUM> hours, to produce a crude Michael addition product <NUM> as a mixture of cis and trans isomers. Compound <NUM> has been obtained at a conversion of about <NUM> % (based on consumption of <NUM>,<NUM>-dichloroaniline <NUM>) under these conditions, which did not improve upon heating the reaction for <NUM>. As described elsewhere herein, conversions were determined by HPLC area under curve (AUC) measurements, unless otherwise specified. The conversion to Compound <NUM> can be increased to about <NUM>% by addition of another <NUM> eq of the diethyl acetylenedicarboxylate and additional heating at <NUM> for about <NUM>. However, diethyl acetylenedicarboxylate is not thermally stable and decomposes with a significant energy release, so it is preferable to limit the diethyl acetylenedicarboxylate charge to less than one equivalent (e.g., about <NUM> eq) for safety purposes. Upon consumption of about <NUM>% of the <NUM>,<NUM>-dichloroanilne (as determined by HPLC), THF was removed by distillation and the crude Michael adduct mixture can be directly used in the subsequent cycloacylation reaction to produce Compound <NUM>, as described below. This procedure was found to be suitable for the production of the Michael adduct material in a multi-kilo scale.

The cycloacylation step to form a compound of Formula IV, such as Compound <NUM> (Formula IV where x is Cl and R is Et) was originally carried out at <NUM> in diphenyl ether. Attempts at performing this reaction in a continuous flow reactor under such conditions, to provide a more practical large-scale synthesis, led to partial precipitation of the cyclized product from the diphenyl ether into the reactor lines causing hazardous conditions due to high back pressure. To circumvent this problem, alternative cycloacylation methods were considered.

The cycloacylation and compatibility of compounds of Formula III (e.g., Compound <NUM>) to highly acidic Eaton's reagent was investigated at a small scale by bulk reaction of Compound <NUM> with neat Eaton's reagent at a concentration of about <NUM>-<NUM> of Eaton's reagent per gram of Compound <NUM> (also referred to <NUM>-<NUM> volumes of Eaton's reagent, for convenience). The reaction was performed at <NUM>, while following the reaction by high-performance liquid chromatography (HPLC). The HPLC results showed the consumption of Compound <NUM> with the formation of a new peak without significant byproducts.

With success in a small scale bulk cycloacylation, the procedure was examined under flow conditions and optimized further. The cycloacylation of Compound <NUM> (which has two meta-chloro substituents) with neat Eaton's reagent proceeded surprisingly well under flow conditions, even with crude Compound <NUM> containing <NUM>-<NUM>% unreacted dichloroaniline (Compound <NUM>). The flow reaction provided significantly higher conversions of (e.g., <NUM>-<NUM> % based on the initial amount of Compound <NUM>) in comparison to a bulk cycloacylation reaction of Compound <NUM>, which provided only about <NUM>% conversion after heating for about <NUM>, with no further increase in conversion thereafter. Unexpectedly, cosolvents (e.g., dichloromethane) did not significantly interfere with the cycloacylation reaction under flow conditions, in contrast to the results reported by Zewge et al. (footnote <NUM>), which indicated that cosolvents all but shut down the reaction, at least at the <NUM>-hour reaction timeframe examined.

Advantageously, crude Compound <NUM>, comprising about <NUM> mol % of <NUM> and about <NUM> to <NUM>% of unreacted <NUM>,<NUM>-dichloroaniline <NUM>, can be utilized in the cycloacylation with Eaton's reagent without significant interference from the unreacted aniline compound. For example, in a reaction utilizing about <NUM> grams of crude Compound <NUM> dissolved in about <NUM> (<NUM> volumes) of Eaton's reagent at <NUM>, with a <NUM> residence time and a backpressure of about <NUM> kPa (<NUM> pounds per square inch (psi)), afforded crude product comprising about <NUM> % of <NUM> and about <NUM> % <NUM>,<NUM>-dichloroaniline based on AUC measurements from HPLC data, indicating a very high conversion of Compound <NUM> in the crude starting material to Compound <NUM> (e.g., greater than <NUM> %).

As noted above, under flow reactor conditions, the cycloacylation reaction also proceeds surprisingly well with cosolvents added to Eaton's reagent. This is in stark contrast to reported attempts at similar cycloacylations using Eaton's reagent with a cosolvent (toluene, xylene and sulfolane) in bulk reactions at <NUM>:<NUM> and <NUM>:<NUM> ratios of solvent to Eaton's reagent, which were reported Zewge et al. to be very sluggish, with best conversion being only about <NUM> % after <NUM> at <NUM> with <NUM>:<NUM> toluene/Easton's reagent (Zewge et al. footnote <NUM>).

Isolation of Compounds of Formula IV. As illustrated by the reaction of crude Compound <NUM> with Eaton's reagent to produce Compound <NUM>, the crude product from the cycloacylation reaction (Reaction (b) of Scheme <NUM>) can be readily isolated as a granular solid by trituration, e.g., by adding the product-containing effluent to cold (e.g., <NUM> to <NUM>) water (typically about <NUM> to <NUM> volumes of water per volume of effluent) with vigorous stirring (typically over a period of several minutes to several hours) to precipitate the product, followed by filtering to recover the precipitate, and drying the recovered precipitate under vacuum. Optionally, the water can include a base to neutralize or partially neutralize the methane sulfonic acid and any phosphoric acid that may form upon adding the effluent to the water. The trituration procedure can be used even when a cosolvent such as dichloromethane is present in the effluent.

The crude precipitate, which may still contain some unreacted Compound <NUM>, significant amounts of unreacted <NUM>,<NUM>-disubstituted aniline <NUM>, as well as some side products, can be further purified by slurring crude Compound <NUM> in an aqueous solution of a basic salt such as sodium acetate (e.g., about <NUM> to <NUM> of aqueous salt solution per gram of crude Compound <NUM>). Alternatively, or in addition, the crude Compound <NUM> can be purified by slurrying the solid in an organic solvent in which the product has moderate to low solubility, such as a C<NUM>-C<NUM> alcohol (e.g., methanol or isopropanol) or acetonitrile. With isopropanol and acetonitrile, purities of up to <NUM> % can be achieved, with recoveries of up to <NUM> % of the theoretical amount of desired product in the crude solid product.

With high quality Compound <NUM> in hand, evaluation of the amination reaction was initiated. Chlorosulfonyl isocyanate was added to a suspension of Compound <NUM> in acetonitrile at <NUM>. Although chlorosulfonyl isocyanate is one of the most reactive isocyanates according to literature, there was no exotherm observed during the addition. After completion of addition, the mixture was heated to reflux (about <NUM>-<NUM>), for about <NUM> during which the reaction was followed by HPLC. Upon reaching a temperature of about <NUM>-<NUM>, the reaction mixture became a clear brown solution and a continuous evolution of CO<NUM> was observed, which ceased at a temperature of about <NUM>-<NUM>. A new broad peak appeared in the HPLC profile, with concomitant consumption of Compound <NUM>. At this point, the mixture was cooled to about <NUM>-<NUM> and <NUM> HCl in methanol was added, followed by heating to reflux at about <NUM> for an additional <NUM> to quench the intermediate product formed in the initial reaction and to release the <NUM>-aminoquinoline product, Compound <NUM>. The appearance of a new peak in the HPLC profile, with the disappearance of the wide peak confirmed the progress of the reaction to release the product.

Isolation of Compounds of Formula V. As part of initial optimization, <NUM> N NaOH was added to the reaction mixture directly at <NUM>-<NUM> to adjust the pH of the mixture to about <NUM>-<NUM>, as measured by a pH meter. The resulting thick slurry was filtered and washed with water to collect the tan colored filter cake, which was conditioned at <NUM>-<NUM>. The HPLC purity of the filter cake was assessed as about <NUM>% with no loss of product in filtrate and washes.

Some ester exchange from ethyl to methyl was observed with HCl in methanol, so the procedure was modified by using HCl-ethanol, instead. Thus, it is preferable that the alcohol used for the quenching solution have the same alkyl group as the ester in the reaction product. To enhance process safety, chlorosulfonyl isocyanate preferably is added to a solution of Compound <NUM> at about <NUM> instead of <NUM>, which results in controlled evolution of CO<NUM> during the entire addition. Following this procedure, crude Compound <NUM> is isolated in up to <NUM>% yield at a purity of up to <NUM>%.

Purification of Crude Compounds of Formula V. Crude Compound <NUM> was readily purified by slurrying in a variety of solvents, including ethanol, isopropanol and ethyl acetate. For examples, slurrying in <NUM> to <NUM> volumes of isopropanol (i.e., <NUM> to <NUM> per gram of crude product) resulted in a recovery of about <NUM> % of Compound <NUM> at purities of <NUM> to <NUM> % as determined by HPLC. Similar results were obtained with <NUM> to <NUM> volumes of ethanol, albeit with slightly lower recovery (<NUM>-<NUM> %) and slightly higher purity (<NUM> to <NUM> %). Slurry purification with about <NUM> to <NUM> volumes ethyl acetate afforded the highest recoveries (<NUM> to <NUM> %) at HPLC purities of <NUM> to <NUM> %.

The carbamylation of Compounds of Formula V to form the diphenylureido-dihalokynurenic acid esters of Formula VI is illustrated by the conversion of Compound <NUM> to form DCUKA ethyl ester <NUM>. The carbamylation is carried out in a polar aprotic solvent such as dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), propylene carbonate (PC), and the like, by addition of diphenylcarbamoyl chloride to a solution of the compound of Formula V, e.g., Compound <NUM>, in the presence of a an alkali metal hydroxide such as NaOH, or an alkali metal hydride such as NaH. DMAc is a preferred solvent for the reaction, and <NUM>% sodium hydride is the preferred base.

A number of bases were evaluated for the reaction. Lithium and sodium hexamethyldisilazine (LiHMDS and NaHMDS) in THF solvent both led to loss of Compound <NUM> as determined by HPLC, but without formation of the desired DCUKA ethyl ester Compound <NUM>, instead the acid form of Compound <NUM> (where Et is replaced by H) was formed. Similar results were observed with potassium tert-butoxide and sodium ethoxide. Weaker bases such as pyridine, diisopropylethylamine in toluene, and cesium carbonate in DMF also failed to afford the desired product, as did neat pyridine.

The use of NaH in THF also was unsuccessful, while reactions were effective in DMF, DMAc, NMP and propylene carbonate (PC). Use of NaOH and LiOH in DMAc did result in formation of Compound <NUM>, with NaOH providing <NUM> to <NUM> % conversion, although a longer reaction time (<NUM>) was required. LiOH provided only <NUM>% conversion in DMAc. Overall, the use of <NUM>% NaH in DMAc solvent provided a reduced reaction time, the highest conversion, and the best safety for large scale batches, since DMAc is essentially inert in presence of NaH. In summary, <NUM>% NaH with DMAc provided the desired product in over <NUM> % conversion within <NUM> after the complete addition of the base.

Isolation and Purification of Compounds of Formula VI. Upon completion of the carbamylation reaction, as confirmed by HPLC, quench methods were evaluated. Initially the reaction mixture was slowly charged into cold water (<NUM> to <NUM>) for the precipitation of product. While this procedure does work, some hydrolysis of the ester group can occur, so it is desirable to quench the reaction in an acidic solution, such as <NUM> wt% acetic acid (AcOH) to avoid hydrolysis of ethyl ester in Compound <NUM>. The crude yield of the product, after filtration to recover the filter cake consistently was about <NUM> to <NUM> % with about <NUM> to <NUM>% purity by HPLC. To remove the byproduct DCUKA acid that is formed during the reaction, acid/base workup followed by crystallization or a slurry wash purification was developed. For example, the crude filter cake can be dissolved in about <NUM> volumes of dichloromethane (DCM) and washed with about <NUM> volumes of <NUM> NaOH solution followed by about <NUM> volumes of <NUM> citric acid solution, which removes some of the impurities observed in the HPLC. Alternatively, <NUM> volumes of <NUM>%/<NUM>% v/v EtOAc/MEK can be used to dissolve the crude DCUKA acid, in place of DCM.

As alternatives, a crystallization method and a slurry purification method were developed to purge two unknown impurities observed in the crude product. Slurry purification with isopropanol (IPA) and ethanol (EtOH) was effective at removing the impurities with good overall recovery. Upon removal of DCM to dryness, the crude Compound <NUM> was suspended in about <NUM> volumes IPA or EtOH and slurried at <NUM> overnight, followed by cooling to <NUM>. Filtration led to highly pure (><NUM>%) Compound <NUM> from both IPA and EtOH, and the <NUM>H-NMR spectra exhibited no additional peaks except solvents.

Crystallization initiated by a solvent swap from DCM to EtOH also provided high purity Compound <NUM>, since this material is relatively insoluble in EtOH. Alternatively, a mixture of EtOAc and MEK can be used to dissolve the crude DCUKA acid, and this solvent combination can be removed by solvent swapping with EtOH as in the case of DCM. The organic phase was subjected to distillation at <NUM> to reduce the DCM volume level about <NUM>- to <NUM>-fold from initial volume, and then EtOH was added dropwise continuously to remove DCM further during the distillation. After about <NUM> volumes of EtOH was added, the mixture was maintained at about <NUM> for about <NUM> to afford a DCM to EtOH ratio of about <NUM>:<NUM> as determined by <NUM>H-NMR analysis. The resulting slurry was slowly cooled to <NUM> and maintained at <NUM> overnight. Filtration followed by HPLC analysis of filter cake confirmed the complete removal of both impurities. Overall the isolation and purification methods afforded Compound <NUM> in in about <NUM> % recovery with ><NUM>% purity.

As yet another alternative, DCM can be eliminated from the crystallization process for the compounds of Formula VI (e.g., Compound <NUM>). Several solvents were screened to replace DCM, including EtOAc/EtOH, EtOAc/THF, EtOAc/MEK. A solvent ratio of <NUM>/<NUM> to <NUM>/<NUM> was screened. From all the screened solvents only EtOAc/MEK was successful to dissolve Compound <NUM> at <NUM> vol, with a <NUM>/<NUM> ratio of EtOAc/MEK.

Hydrolysis of Compounds of Formula VI to Form Acids of Formula VII.

Compounds of Formula VI can be hydrolyzed to the corresponding salt of Formula VII or the acid of Formula VIII:
<CHM>
where M is a cation (e.g., Na+). The salt of Formula VII is formed by base hydrolysis in an aqueous solvent (e.g., a mixture of water and an alcohol) followed by adjusting the pH to about <NUM> to precipitate the salt. For example, addition of a base such as sodium hydroxide (e.g., <NUM> eq) to an aqueous solution (e.g., a water-alcohol mixture) of Compound <NUM> with mild heating (e.g., <NUM>) resulted in complete hydrolysis to the salt Compound <NUM> (Formula VII where each X is Cl and M is Na). Adjusting the pH to about <NUM> with a mild acid (e.g., acetic acid) results in precipitation of the salt from the solution. Compound <NUM> can then be isolated by filtration, and purified by slurrying the filtrate with an alcohol (e.g., methanol) and with water. The carboxylic acid of Formula VIII (e.g., DCUKA) can too be formed from the salt of Formula VII by neutralization with a strong acid.

If desired, addition salts of diphenylureido-dihalokynurenic acids can be formed by treating the salt of Formula VII with an excess of a strong acid, such as p-toluenesulfonic acid (TSOH), to form the addition salt of Formula IX:
<CHM>
wherein x is a halogen. For example, addition of <NUM> equivalents of TsOH to a solution of Compound <NUM> resulted in formation of the TsOH addition salt, Compound <NUM>, at very high conversions (up to about <NUM>%).

The attached drawing schematically illustrates a reactor <NUM> for performing the cycloacylation reaction. Reactor <NUM> comprises a flow pipe <NUM> having influx section <NUM>, conditioning coil section <NUM>, reactor coil section <NUM>, and efflux section <NUM>. Influx section <NUM> is in fluid flow connection with pump <NUM>, which is in fluid flow connection with conditioning coil section <NUM>. Conditioning coil section <NUM> is in fluid flow connection with reactor coil section <NUM>. Conditioning coil section <NUM> and reactor coil section <NUM> are housed within heated chamber <NUM>. Reactor coil section <NUM> is in fluid flow connection with efflux section <NUM>. In operation, a reaction mixture is charged into reactant reservoir <NUM> and is drawn into pump <NUM> via influx section <NUM> of pipe <NUM>, and expelled from pump <NUM> into conditioning coil section <NUM>, where the reaction mixture them flows into reactor coil section <NUM>, and then out of heated chamber <NUM> via efflux section <NUM> of pipe <NUM> into effluent reservoir <NUM>.

In use, flow of the reaction mixture is in the direction of broad arrows A. Heated chamber <NUM> is heated to a temperature sufficient to heat the contents of reactor coil section <NUM> to a desired reaction temperature. The total internal volume of conditioning coil section <NUM> and reactor coil section <NUM>, in combination with the pumping rate of pump <NUM>, are selected to afford a desired residence time for the reaction mixture within heated chamber <NUM>. Typically, pipe <NUM> is initially filled with a reaction solvent, which is displaced by the reaction mixture as pumping proceeds. Effluent that does not contain any of the product from the cycloacylation reaction typically is discarded before collecting effluent containing the product. In order to maintain pumping efficiency, reactant reservoir <NUM> is refilled as needed, or alternatively, additional reactant reservoirs can be connected to pump <NUM> as one reservoir is depleted. In the final stage of operation, solvent can be pumped through pipe <NUM> to displace the last remaining reaction mixture through the reactor.

The following non-limiting examples are provided to illustrate certain aspects and features of the methods described herein.

All the reagents and solvents were purchased from commercial sources and used as received. <NUM>H-NMR spectra were obtained using <NUM> and <NUM> BRUKER AVANCE <NUM> or AVANCE <NUM> spectrometers. Tetramethylsilane from NMR solvents was used as internal reference. HPLC analyses were performed using VARIAN PROSTAR instrument.

The elution gradient is found in Table <NUM>.

<NUM>,<NUM>-Dichloroaniline <NUM> (<NUM>, <NUM> mol, <NUM> eq) was charged into a one liter, three-neck, jacketed reactor equipped with mechanical stirrer, reflux condenser and temperature probe, followed by THF (<NUM>, <NUM> vol). Diethyl acetylenedicarboxylate <NUM> (<NUM>, <NUM> mol, <NUM> eq) was then added into this reactor neat. The addition of diethyl acetylenedicarboxylate was carefully monitored for any exotherm. After the completion of addition, the resulting mixture was stirred at about <NUM>-<NUM> during which the reaction was followed by HPLC (about <NUM>µL of reaction mixture was dissolved in <NUM> of MeCN, <NUM>µL of which was then injected onto the HPLC column). The end point of for the reaction was selected to be at least <NUM>% conversion of <NUM>,<NUM>-dichloroanilne (retention time: RT=<NUM>) into Compound <NUM> (RT=<NUM>), based on HPLC area under curve (AUC) measurements. Upon achieving this conversion, reflux condenser was replaced with distillation head to remove THF under reduced pressure. The end point of THF distillation was determined by <NUM>H-NMR. The <NUM>H-NMR of the resulting product was consistent with the structure of Compound <NUM>.

In a <NUM>-neck, <NUM> jacketed-reactor equipped with mechanical stirrer, temperature probe and reflux condenser, was charged Compound <NUM> (<NUM>, <NUM> eq, <NUM> mol) followed by acetonitrile (MeCN; <NUM>, <NUM> volumes) at <NUM>. The slurry was heated to <NUM> and chlorosulfonyl isocyanate was added slowly (<NUM>, <NUM> eq, <NUM> mol,) over <NUM> using syringe pump (<NUM>/mL). A controlled evolution of CO<NUM> was observed during the addition of the isocyanate. After complete addition, the mixture was stirred at <NUM> for additional <NUM>. Samples were taken before and after chlorosulfonyl isocyanate addition to monitor the consumption of Compound <NUM> (RT: <NUM>) to the initial intermediate product (RT: <NUM>-<NUM>) by HPLC. Upon complete conversion, <NUM> HCl-EtOH (<NUM>, <NUM> eq, <NUM> volumes) was added over <NUM> at <NUM> using syringe pump (<NUM>/min). The addition was followed by HPLC as the disappearance intermediate <NUM>-<NUM> and appearance of new peak at <NUM>. The batch was then subjected to vacuum distillation at <NUM>-<NUM> (internal temperature) to reduce the batch volume by about one half. The resulting slurry was then cooled to <NUM> and the pH (<NUM>-<NUM>) was adjusted using <NUM> N NaOH (<NUM>, <NUM> eq. <NUM> volumes) to pH about <NUM> to <NUM>. The resulting thick slurry was warmed to <NUM> and stirred for addition <NUM> and filtered to collect the filter cake. Both filter cake and filtrate were analyzed by HPLC. The filter cake, Compound <NUM>, was conditioned in vacuum oven at <NUM>-<NUM> overnight. The dry weight of the isolated crude was <NUM> (crude yield=<NUM>%, purity: <NUM> %).

The crude Compound <NUM> (<NUM>) was charged into a <NUM>-neck <NUM> jacketed reactor equipped with mechanical stirrer, temperature probe and reflux condenser. EtOAc (<NUM>, <NUM> volumes) was charged into the reactor and the slurry was stirred at <NUM> for <NUM> then heated to <NUM> and stirred for <NUM>. After <NUM>, the mixture was slowly cooled to <NUM> with stirring. The slurry was filtered to collect the filter cake and both filter cake and filtrate were analyzed by HPLC. The filter cake was further conditioned at <NUM>-<NUM> for <NUM> that provided <NUM> Compound <NUM> in <NUM>% yield with <NUM>% purity by HPLC.

Compound <NUM> (<NUM>, <NUM> mol, <NUM> eq) was added into a <NUM>-neck, <NUM> jacketed reactor equipped with mechanical stirrer and temperature probe, followed by DMAc (<NUM>, <NUM> vol) and the resulting solution was cooled to <NUM> then diphenylcarbamoyl chloride (<NUM>, <NUM> mol, <NUM> eq) was added as solid in single portion. The solution was stirred for <NUM> at <NUM> then NaH (<NUM>%, <NUM>, <NUM> mol, <NUM> eq) was added in portions over <NUM> at <NUM>. Upon completion of addition, the deep red mixture was stirred for additional <NUM>. The progress of reaction was monitored by HPLC. A <NUM> wt% AcOH solution (<NUM>, <NUM> volumes based on the DMAc volume) was added to another <NUM>-neck <NUM> jacketed reactor equipped with mechanical stirrer and temperature probe, and cooled to about <NUM>. The DMAc solution was then slowly pumped into the <NUM> wt% AcOH solution to quench the intermediate product and precipitate out crude Compound <NUM> by keeping the internal temperature around <NUM>. Upon complete quench, the slurry (pH=<NUM>) was stirred at <NUM> for an additional <NUM>, and then was filtered to collect the filter cake. The filter cake was washed with water (2x140 mL, 2x2 vol) and the filter cake, filtrate, and washes were analyzed by HPLC. The collected filter cake was further conditioned at <NUM>-<NUM> for overnight in a vacuum oven to yield <NUM> (<NUM>%) with <NUM>% purity by HPLC.

The crude Compound <NUM> (<NUM>) was charged into a <NUM>-neck <NUM> jacketed-reactor and DCM (<NUM>, <NUM> volumes) was added to dissolve the material. Upon complete dissolution, <NUM> N NaOH (<NUM>, <NUM> volumes) was added into the reactor and the mixture was vigorously stirred for about <NUM>-<NUM> and then stirring was stopped to provide a phase separation. Organic phase was collected and charged back into the reactor and the <NUM> N NaOH wash was repeated for <NUM> times. Upon completion of <NUM> N NaOH washes (3x3 volumes), the organic layer was washed with <NUM> citric acid (<NUM>, 1x3 volume). Both organic and aqueous phases were analyzed after every wash by HPLC, which showed that a <NUM> impurity was partially purged in base washes, but still remained in organic phase in addition to a <NUM> impurity.

The organic phase (<NUM>) was transferred into a <NUM> reactor equipped with mechanical stirrer, thermocouple, addition funnel and Dean-Stark condenser, and was slowly heated to <NUM> to remove DCM. Upon removal DCM to about <NUM> volumes EtOH was continuously charged dropwise to maintain <NUM> volumes of solvent in the still pot, while DCM was distilled away. The temperature of the mixture was slowly raised to <NUM> with constant dropwise addition of EtOH during which crystallization/precipitation was observed. In total, about <NUM> of EtOH (<NUM> volumes) was added, and the mixture was maintained at <NUM> for about <NUM>. <NUM>H-NMR confirmed a <NUM>:<NUM> ratio of EtOH:DCM and a successful swapping of most of DCM to EtOH.

The slurry was slowly cooled over <NUM> to <NUM> and maintained at <NUM> overnight with stirring. The slurry was then filtered to collect the solid, and the both filter cake and filtrate were analyzed by HPLC. HPLC data of filter cake confirmed the purging of both <NUM> and <NUM> RT impurities in EtOH. The filter cake was further conditioned at <NUM> in a vacuum oven for <NUM> to provide <NUM> of purified Compound <NUM> in <NUM>% yield with over <NUM>% purity by HPLC.

In another preparation, Compound <NUM> (<NUM>, <NUM> mol, <NUM> equiv) was dissolved in DMAc (<NUM>, <NUM> vol), and cooled to <NUM> in a four-neck, <NUM>-L, jacketed reactor equipped with mechanical stirrer and temperature probe. Diphenylcarbamoyl chloride (<NUM>, <NUM> mol, <NUM> eq) was added to the solution of Compound <NUM> as solid in single portion. The resulting reaction solution was stirred for <NUM> at <NUM> then NaH (<NUM>%, <NUM>, <NUM> mol, <NUM> equiv) was added in portions over <NUM> at <NUM>. Upon completion of the addition, the obtained brown mixture was stirred for additional <NUM>. The progress of reaction was monitored by HPLC, and was complete after <NUM>. In another three-neck, <NUM>-L, jacketed reactor equipped with mechanical stirrer and temperature probe was charged, <NUM> wt % AcOH solution and cooled to <NUM>. Main reaction mixture was slowly added into <NUM> wt % AcOH solution (<NUM>, <NUM> vol based on DMAc) at <NUM> to precipitate out crude Compound <NUM> by keeping the internal temperature around <NUM>. Upon complete quenching of the reaction mixture, the resulting slurry (pH = <NUM>) was stirred at <NUM> for additional <NUM> then filtered to collect the filter cake. The filter cake was washed with water (<NUM> × <NUM>, <NUM> × <NUM> vol, based on DMAc) and the filter cake, filtrate, and washes were analyzed by HPLC. The filter cake collected was further conditioned at <NUM>-<NUM> for overnight (<NUM>) in a vacuum oven to yield <NUM> (<NUM> %) of Compound <NUM> with <NUM>% purity.

The crude Compound <NUM> was split into two parts; part <NUM> (<NUM>) was charged into a three-neck, <NUM>-L jacketed-reactor and dissolved in ethyl acetate: methyl ethyl ketone (<NUM>, <NUM>:<NUM>, <NUM> vol) at <NUM>. Upon complete dissolution, the solution was cooled to about <NUM>, and <NUM> N NaOH (<NUM>, <NUM> vol) added into the mixture with vigorous stirring for about <NUM>-<NUM>, after which the stirring was stopped to allow for a phase cut. The organic phase was collected and charged back into the reactor and was washed <NUM> times with <NUM> N NaOH. The organic layer was then washed with <NUM> citric acid (<NUM>, <NUM> × <NUM> vol). Both organic and aqueous phases were analyzed after every wash by HPLC.

The washed organic phase (<NUM>) was transferred into a <NUM> reactor jacketed reactor and was slowly heated to <NUM> under vacuum to reduce the amount of ethyl acetate and methyl ethyl ketone to about <NUM> vol. Ethanol (<NUM>, <NUM> vol) then was added to reactor and mixture was distilled down to <NUM> vol again. Final mixture was cooled to about <NUM>. The resulting slurry was filtered and washed with ethanol <NUM> (<NUM> vol) and then dried in vacuum oven at <NUM> for <NUM> days to afford Compound <NUM> was a white solid. (<NUM>, <NUM>% yield, <NUM>% purity).

Compound <NUM> was hydrolyzed with sodium hydroxide to form the sodium salt, Compound <NUM>. The reaction conditions were demonstrated using GMP lot (run <NUM>, Table <NUM>) on <NUM> scale. The reaction proceeded using <NUM> eq of NaOH and was complete within <NUM> hours at <NUM> on a <NUM> scale (see the <NUM> scale run from Table <NUM>. After the hours, the reaction mixture was cooled to room temperature and filtered. The solid was slurried in methanol (<NUM> vol) and water (<NUM> vol). The pH of the slurry was adjusted to <NUM> using glacial acetic acid and the mixture was stirred overnight, after which the pH was adjusted again to <NUM> and the slurry was filtered, washed with methanol (<NUM> vol), and dried in a vacuum oven overnight at <NUM> to afford the desired product in <NUM>% yield (<NUM>). Different conditions were screened as well, as set forth in Table <NUM>, all of which resulted in high conversions to Compound <NUM>.

Formation of Compound <NUM> was achieved by addition of about <NUM> equivalents ofp-toluenesulfonic acid to Compound <NUM>. Various reaction conditions were examined, as shown in Table <NUM>. A <NUM> scale run (Table <NUM>) is provided as an illustration of the process. The dried Compound <NUM> material was slurried in ethyl acetate (<NUM>, <NUM> vol) and warmed to about <NUM>. A <NUM> aqueous solution of p-toluenesulfonic acid (prepared from <NUM> ofp-toluenesulfonic acid monohydrate dissolved in <NUM> of water) was added to the ethyl acetate slurry, which was then stirred at <NUM> for <NUM> hours and then was cooled to <NUM> over <NUM> hour. Proton NMR at this point showed about <NUM>% conversion to the desired salt. The slurry was then rewarmed to <NUM> and was treated with an additional portion of <NUM> p-TsOH solution (<NUM>, <NUM> of p-toluenesulfonic acid in <NUM> of water) and was stirred at <NUM> for <NUM> hours, after which proton NMR showed complete conversion to the tosylate addition salt. The slurry then was cooled to <NUM> over <NUM> hour, aged for <NUM> hour at <NUM>, and was filtered. The filter cake was washed with ethyl acetate (<NUM>×<NUM>, <NUM>×<NUM> vol) and dried at <NUM> to afford <NUM> of the desired product in <NUM>% yield.

Claim 1:
A method for preparing an alkyl <NUM>,<NUM>-dihalo-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester of Formula IV, comprising the sequential steps of:
(i) heating a first solution comprising a compound of Formula III dissolved in a reagent comprising <NUM> to <NUM> wt% P<NUM>O<NUM> in methanesulfonic acid, at a concentration of <NUM> to <NUM> grams of the compound of Formula III per mL of the reagent:
<CHM>
at a selected temperature in the range of <NUM> to <NUM> by pumping the first solution through a heated continuous flow reactor coil at a pumping flow rate sufficient to provide a residence time of <NUM> to <NUM> minutes in the heated coil, and collecting an effluent flowing out of the heated coil comprising an alkyl <NUM>,<NUM>-dihalo-<NUM>,<NUM>-dihydro-<NUM>-oxoquinoline-<NUM>-carboxylate ester of Formula IV:
<CHM>
(ii) adding the effluent collected from step (i) to at least <NUM> volumes of water per volume of the effluent, with stirring, while maintaining a water temperature of <NUM> or less, to form a first precipitate comprising the ester of Formula IV; and
(iii) recovering the first precipitate;
wherein in Formula III and Formula IV, each X independently is a halogen atom; and each R independently is C<NUM> to C<NUM> alkyl.