Title of Invention	SYNTHESIS OF HISTAMINE DIHYDROCHLORIDE
Abstract	The invention disclosed herein relates to (fee preptration of pharmaceutical grades of histamine dihydrochloride using a two step non-enzymatic synthetic method. The invention disclosed herein describes the synthesis of histamine dihydrochloride by the non-enzymatic decarboxylation of bistidine and the step-wise conversion of tha decarboxylated product to the dihydrochloride salt form. The invention disclosed herein considers a final product of histamine dihydrochloride containing less than each of the following: 0.8 % L-histidine HCl monohydrate, 0.1 % individual ethomatographic Impurities,to be acceptable for pharmaceutical use.

Title of Invention

SYNTHESIS OF HISTAMINE DIHYDROCHLORIDE

Abstract

The invention disclosed herein relates to (fee preptration of pharmaceutical grades of histamine dihydrochloride using a two step non-enzymatic synthetic method. The invention disclosed herein describes the synthesis of histamine dihydrochloride by the non-enzymatic decarboxylation of bistidine and the step-wise conversion of tha decarboxylated product to the dihydrochloride salt form. The invention disclosed herein considers a final product of histamine dihydrochloride containing less than each of the following: 0.8 % L-histidine HCl monohydrate, 0.1 % individual ethomatographic Impurities,to be acceptable for pharmaceutical use.

Full Text	SYNTHESIS OF HISTAMINE DIHYDROCHLORIDE Background of the Invention Histamine is a campound possessing significant biological activity mediated by pharmacological receptors. Histamine has lone been contemplated as a molecule having primarily negative biological effects. Recently, however, new uses for histamine as a powerful pharmaceutical agent have come to light. For example, histamine has been used in conjunction with interferen-alpha to activate NK cells in the presence of monocytes. See U.S. Patent No. 5.728,378. To take full advantage of the therapeutic properties of histamine, it is necessary to obtain large quantities of the compound in a pharmaceutical grade. Histamine occurs widely in nature as a result of putrefactive processes and a derivative, histamine dihydrochloride, is sold commercially for use as a standard in assays and as e component in certain allergy diagnostic kits. The source of this histemine is often a natural one end as such contains a variety of contaminants that render it unsuitable for pharmaceutical use. There are also synthetic protocols for the synthesis of histemine dihydrochlorida known in the art. Histamine dihydrochloride can be conveniently synthesized by exploiting the decarboxylation of histidine. Using this synthesis pathway, histidine is decarboxylated and subsequently treated to form the dihydrochloride salt form of the molecule. For example, Hashimoto et al., discussed the preparation of histamine using cycloherenone as a catalyst for the decarboxylation of histidine. (Hashimoto, M.. et al., Chemistry Letters, 883-896 (1986)). The Hashimoto, et al., paper reported the isolation of histamine dihydrochloride at a 95% yield, using 2-cyclohexen-1-one as the catalyst, from the reaction involving histidine and 1 % v/v of 2-cyclohexen-1-one in 10 parts of refluxing cydohexanol (26 hours). The Hashimoto method also teaches the use of toluene and HCI gas bubbled through the resulting decarboxylated solution to precipitate out and harvest the final histamine dihydrochloride product. Attempts to reproduce the Hashimoto procedure to generate pharmaceutically pure amounts of histamine failed. Additional amounts of the catalyst were required to make the procedure operative and a substantial number of impurities were present In the final product. Moreover, those impurities were difficult to remove. In view of these results, it was found that the Hashimoto procedure is an unsuitable method for generating large quantities of pharmaceutically acceptable histamme. The use of acetophenone as a catalyst for the decarboxylation of histamine has also been reported. Sea DD 56 793 A. We recreated the method described in the Japanese patent to Akimasa, et al., patent, Japanese Patent No. 05,255,204 (1983), and used 0.26 equivalents of acetophenone and ID parts of diethylene glycol as the solvent for the derarboxylation reaction. Although the Akimasa et al. method was far more efficient in convertine- histidina to histamine. it failed to consistently yield a pharmaceutical grade product. Like the final product using the Hashimoto method, impurities were observed in the final product made using the Akimasa method during the HPLC analysis. Although the conditions with acetophenone and diethylene glycol looked promising, there existed a problem related to the work-up. Both histamine toe base and the dihydrochloride salt are readily soluble in water, therefore, ft was difficult to utilize any extraction technique to separate the product from the diethylene glycol solvent, which was -1- usually removed by a water enraction. Futhermore, the histamine dihydrochloride was also readily soluble in -dithylene-glycol thus-the-directions b -filiratiDn.ww-also-imposslbier The reaction conditions of TaVano et al.. involving pentan-3'One were also recreated. {Heterocycks, 6:1167 {1977}}. See also G8 1008594 A, which used 2,4-jfihydiQxybenzophenDns in the synthesis process. The results from these experiments showed no imprev&ment over the acetophenone conditions described above. A consistent source of pharmaceutical orade histamme is required. especiaDy in view of the new-found pharmaceutics! applications for hist a mine. The standard methods used by the art wherein histamfna is purified from natural sources, fail to yield hist ami ns of a sufficiently high grade for pharmaceutical uses. Moreover, the synthetic methods practiced in the art also fail to yield histam'me of a sufficiently hfth grade. Accordinpjy. there is a need in tha art for an improvBd method by which to produce pharmaceutical grade histamine dihydrochioride. Summary of the Invention" The Invention disclosed herein relates to ihe preparation of pharmaceutical Grades of histamine clhydrechloride using a two step non-enzymatic synthetic method. Ona embodiment of the invention is a method for the synthesis of histamine dihydrochloride comprising: decarbonating a L-histldme containing sorutiao, whereby a histamine containing solution is formed in the absence of a decarboiylating enzyme: forming 8 hist ami ne monohydrochloride containinfl solution from the histamire containing solution; and forming a histamrna dthydrochlorrde-comaining solution from the histamine monohydroehtoride containing solution. Accordingly, the present invention provides a method for trie synthesis of histamine dihydrochloride comprising: forming a rtistamine monohydrochloride containing solution from a decartxwyiated Uiistidine-containing solution by addition of hydrogen chloride; and forming a histamine dihydrochloride containing solution from the histamine monohydrochloride containing solution by addition of hydrogen chloride, wherein the resulting histamine dihydrochloride comprises less than 2% total impurities as measured by HPLC analysis. One aspect of This embodiment further comprises triturating the histamine containing solution, for eiampla, tha histamine containing solution can be triturated with a msthylene chloride solution. In another aspect of this embodiment, tha rnstamihe rnonohydrochtoride containing solution is formed by addition of en effective amount of hydrochloric acid in an isopropanol solution. For example, the effective amount of hydrochloric acid u about 0.1 to Q.9 molar equivalents of hydrochloric acid to histamine free base. In another example, the effective amount of hydrochloric acid is about 0.6 molar equivalents of hydrochloric acid to ftstamine free base. Stilt another aspect of this embodiment further comprises rtia step of isolating a pharmaceutical grade of histamine dihydrocnlsride from the histsmino dihydrochloride comeininrj solution. Another embodiment of the invention disclosed herein is a method for synthesizing a pharmaceutical grade of histamine dihydrochloride comprising: dBcarbciyiating a i-hisiidine containing solution, whereby a histamine containing solution is formed in the absence of a decarbonating enryme; forming a hi stamina monohydrochloride containing solution from the histamine containing solution; forming a histamine (Sihydrochloride containing solution from the htstamine moneh'ydrochloiide containing solution; and isolating the histamine dihydro chloride from the histamine dihydrochlorfde containing solution. In one aspect of this embodiment, the histamine dihydrochloride contains equal t" of less than each of the following: 0.8% l-histidine HCI monohydrate, 0.1 % individual chrorr.atographic impurities, and 2% total impurities. -2- Briff Description of the Accompanying Drawings Figure 1 shows a reaction method taught in the an. Figure 2 shows the method of the intention disclosed herein discussed in Examples 5 and 6. Darailed Description of the Invention The invention disclosed herein relates to the preparation of pharmaceutical grades of histamine dihydrochloride using a two step non-ewynwtic synthetic method. The invention disclosed herein describes the synthesis of hisiammc dihydrochloride try the non-enzymatic dacarboiylation of htstidine and the step-wise conversion of the decarboiytated product to the dihydrocbloride salt form. The invention disclosed herein considers a final product of histamrne dihydrochloride containing, less than each of the following: 0.6% i-histidine HCI monohyrJrete. 0.1 V" ehrotnatographic impurities {defined below), and 2% total impurities, to be acceptable for pharmaceutical use Synthetic "methods of synthesizing histamifte dlhydrnchloride known in the art fail to yield product of a sufficient purity to be used as a pharmaceutical compound. Figure 1 shows a deearboiylation method taughi in the art. The method steps of the invention disclosed herein are shown in Figure 2. Prior art methods were used to generate histamine dihydrochloride from i-histidine starting material in an attempt to generate pharmaceutical grades of synthetic histamma dihydraehloride. The starting material was reacted with the prior art catalyst a-letialone and cyclohexane. Afier completion Df the reaction, the sample was cooled and hydrochloric acid was bubbled into the solution to convert the histamine free base inio tha dihydtochlorid* salt forni. 71w precipitata that formed ws filmed, washed, and driai The final product produced by the prior an method was found to contain an tmacccptafah/ Nh number of contaminants. The crude material produced using the prior an method had a purity of 92-94% with one major impurity at 3-5 and five to eight other impuritiBS at > 0.1 ft. Additional purification steps or recrystafcations were performed and o were substantially effective at removing most of these contaminants. Nevertheless, two unidentified irnpurhies remained "t lewis above 0.1%. These impurities ehrted after the histamir* dihydrochloride product and were refened w as chromatooraphic impurities or contaminants. Thus, product made by this method was enaccept abi"f>r pharmaceutical use. In view of ihese results, a new procedure was designed to synthesije histamine dihydrochloride ot-the desired purity. This new procedure involved the decartwxylation of Uiistidine \|o-ammo4lor 5Hmidazolepropionic acid (CtHgNA) to yield histawine. Fonowing dKarboiylation, ihe solution containing the histamine free base was tmuraied with mcihvlene chloride to precipitate the product. The product was then filtered and washed. Ths fittered product was subsequently treated with hydrochloric add in isopropanol to precipitate a crude histalhine moflohydfochloride salt. This product was filtered and isolated. The crude salt can be subsequently purified by recrysta&ation techniques or it can proceed to the fwil modification step of the present method. Neil, the monohydrochloride salt was treated again with a hydrochloric acrd/rsopropanol solution to generate the histamine dihildrocWoride form of the molecule. The final form of the produti was then decolorized and washed. These steps, known as recrystafeation, can be repwtedly extensively to yield histamine dihydrochloride of pharmaceutical purity. -3- All steps ware performed under a nitrogen gas atmosphere The purity of the final product was analyzed through a number of analytical methods including HPLC analysis. The invention disclosed herein contemplates the use of 3 number of catalysts or radical initiators 10 facilitate the decarboxylation reaction. An appropriate catalyst is one thai will efficiently catalyze the decarboxylation of histidine when that precursor compound is in a neutral solvent and heated for a number of hours to yield an acceptably pure final product. Electron-enriched ketones are preferred as they tend to reduce the number of impurities present in the final product. For example, a group of suitable catalysts comprises: benzoyl peroxide, 2,2'-azobisisobutyronitrile (AIBN1, 2-cyclohexen-l-one, acetophenone, 4'-bromoacetophenone, benzophenone, p-nitroacetophenone, p-methylacerophenorre. p-meihoiyactgphenone. p-methylaceiophenone/1 -meirry Wpiperidone, and p-meihyUeeiephenonefAcOH, The decwboxylation reaction eonditioas promote the deetrfcoiyiation of the starling materials while minimrzirty ihfl formation of.unwamed.contaminants. The reaction conditions include conducting several method steps in the presence of an inert gastjor example, nitrogen. The reaction conditions further include conducting the decarboiytation step at a range of temperatures between about 145 to 170"°C. Preferably, the reaction is carried out ai a range of temperatures from about 150 to 165C. or ate range Df temperatures from about 160 to 165"C. A number of solvents are contemplated for USQ in the invention disclosed herein. The solvents in which certain steps of the reaction are conducted may effect the reaction time which rs required to catalyze the dacarcoxytattort of htsttdme. Solvents us cable in the invention disclosed herein include: cyclohexanot n-methrpyrrondinone (NMPI. difethylenegJvcol). d((ethyleneg,lycol]methyl ether, 2methylaxy0ihlether, Iburwwl, metfioxyemartol, cydohexanol/nlMP (in a 3:1 ratio), (frmethyif ormimide, and tetramethylenesulfone. Another parameter of the reaction disclosed herein is the method of creating the salt form of hiitamirw by Treating the reaction mixture with hydrogen chloride. The impurity profile oi the final product was found to be effected by the molar equivalency of acid added durinp, the precipitation of the monohydrochloride crude salt. It is possible to control the eiiem e impurity formation by preparing a solution of hydrogen chloride of a known concentration in isopropancl end treating the reaction mtiture therewith. A range Df molar equivalents of hydrogen chloride (KCt) in isopropanol (ISA) may be used to practice the method of the invention disclosed herein. A range of about 0.01 to 2 rnolar equivalents may be used 10 create the salt form of hiitamine. Alternatively, a range of about 0.05 to 1.4 molar equivalents may be used. In another alternative, a range of about 0.1 to 0.S molar equivalents may be used. In yet another alternative, about 0.5 molar equivalents may DB used. Tha ratio selected to practice the invention disclosed herein should result in the ultimate generation of a final product with an acceptable (evet of impurities so that the final product may be used as a pharmaceutical composition. The concentration of the acidic solution used to create the tail form was not critical. For example, the concentration of HCI in ISA may range from about 6 to & N. However, The number of moles of acid introduced is ciucial to isolating a pharmaceuticsIry acceptable grade of the final product. The addition of too much acid causes 4. impurities to precipitate with the fnonohydfochlDride salt that are eatrenrely difficult to eliminate during the subsequent formation of the hisramine dihydrochloride raft. The relationship between (he method of sail formation and the generation of contaminants w" not appreciated in the an. Various co-solvents may be used during the addition of HCI in isopropanol to effect precipitation of the monotiydroriiloride salt form of the molecule (salt precipitation). Co-solvents useahle m the invention disclosed herein include: meihylene chloride, cydoheianol tDhiene and tett-butyl methyl ether (TBME). The ultimate purity of the final product is of particular concern. Additional method steps to purify the final product we afse contemplated. For example, recrysTai!iifition is a process ot repeated crystallization in order 10 purify a wbstanee. A number of solvents w contemplated for use in this purification process. These solvents include: methyl chloride, 2-prcpanol, methanol, cthanol (ETOH!, me chariot/acetone, water, methanol/eihyl acetats, water/aceiDne. methanol/ethanol, witc7methanol, methanol/hezane, water/methanolfaceione, methanol/methylene chloride, 2-propanolfethanDl, mathanoli2-propano!, acetone/2-propanol, acitane/ethanol. From a toiicological viewpoint, a non-toiic solvent such as ETOH is preferred. The presence of color in the various solutions obtained during the synthesis pathway was observed. Activated canon may be added to remove some of the color before or as a step of the recrystaliizanon process. The invention disclosed herein further contemplates the use of derivitizmg chemical reactions to assist in the purification of htstsmirte dihydrachloride. Accordingly, it is contemplated that chemical derrvatives of various impurities would bfl made during trie hisiamine dftydrochioride ptocess of the invention disclosed herein to facilitate the removal of ttnse impurities. The creation of one such derivative involves the addition of a lert-butoiycarbonyl group to a molecule of interest. Other modifying groups such as behzyloxycarbonyt groups (CBZ) ait also contempts tsd. The following examples discuss methods addressing the decarboiyfaii&n of histidtne as well as the isolation of the hisiamine product AJso discussed are methods of purification of the crude histamine dihydrocMoride product using multiple recrystaUizstion steps. Charcoal mediated decoloration is also discussed. The efficiency of various method steps as welt at the purity of the final product may be analyzed usinp the methods desired beJow. One or more monitoring steps may be used to assay the efficiency of the decarboiyletion step. AliBinativeiy, various assay methods well known in the art may be used to anafyw trie purity of the final product. An example of such a monitoring step is the performance of thin layer chromatography (TLC), a procedure weB known in the art, on various reaction products. Fur esamsle, reactions could be monitored using TIC (mobile phase: CHaCffcHjQiNHiOH: 7.5:2.0:0.5; and ninhydrin spray). This monitoring siep may be performed anytime after the decaitoiytation step. Particular embodiments of the invention are discussed in detail below. The following examples are for illustrative purposes only and should not be interpreted as limitations of ihe claimed invention. TherB are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention. 5- EXAMPLES Preparation of KFsTumine Dihydrochlpridg Th" fallowing txamples discos* the synthesis of histarnini dihvdrochloride from the precursor compound L-histidine Eiisting histamme synthash protocols, while capable of yielding hi sr am me dihydfocWoride. suffer from the Hmnnion of producing an impure final product The Eiamples below discuss various improvemenii in histamirw (tmydrocrilaride synthesis and teach the preparation of a phi rmtceutie ally acceptable grade of his!imine dihydrochloride. Example \| Preparation of 500 6rams of Crude Hisramme pihydrocfiloride A method for the synthesis of a 500 gram cample of hisiamine dihydrocMoride is described below. A twelve liter (12-U. 4 necked, round bouom flask equipped with a thermometer, mechanical stirrtr, condtnsir and nitrogen bubbler wax dialed with 7.5 I of cycMeianol (the solvent), 750 grams of i-histidint (the itAsuaiti and U3 ml ef aceiophenoni (the utatysU. The suspmsioo was agitated In a nitiooen atmosphere that was maintained throughout the reaction. The suspension was heated to reflui and maintained at that temperature (150 1650 far a minimum of 40 hours. A small sample was withdrawn for an inprocess assay to determine the eiterrt of histidine decarborylation. The suspension was cooftd to below 8OC and 1875 ml of toluene 919$ charged. This minure wai further cooled to room lemptraiurf. The minure was filtered through a Buchner funneJ into a fresh 12 L, 4 necked round-bottom flask. "Die fresh flask containing the filtrate was equipped with a theimomgtcr. mechanical stirrer, hydrogen tWoriflr trap and vacuum trap, and prrpvtti for gaseous hydrogen chloride addition. With agitation, the solution was cooled to btlow 10°C. Maintaming thi batch temperature below 20°C. a minimum of 441 grams 12.5 iquivalems) of gaseous hydrogen chloride was charged. Upon completion of the hydrogen chloride addition, the resulting thick yellowish suspension was agitated "t room Temperature for ena how. The suspension was again filrtnd through a Buchner funnel. The filter cake was rimed with a mixture of 375 ml of cycloheianri and 375 mf of toluene, followed by two 750 ml wishes of tolutne and twa 750 ml rinses of hezanes. The cake was dried on the filter with suction for 1 minimum of 30 minutu. Tna filter cake contained a substantial amount of cyelonexarwl which was removed through triTuratiwi The wet firm c^ke was charged to a I2-L 4-necked round bottom flask equipped with 1 mechanical stirrer and nitrogen bubbler. Ethanel (ETOHJ in a volume of 7.5 I was also charged. The suspension was agitated at room temperature for 4 hours. Tfte suspension was filtered through a Buchner funnel and the fitter cake rinsed with 400 ml of hoanes. The filter eaki was dried in a vacuum own at SO B5CC ovemrght. The product of this method produced 504 of crude hisiamine dihydroUilonde grams (a 56.6% yield) at 94.4% a7a purity determined using high performance liquid chromatography (HPLC). The product was racrysiatiied to improve the purity of the final product. A 12-1. 4-netked. round-bottomed flask equipped with a thermometer, mechanical stirref, condenser, addition funnel and nitrogen bubbler was charged wrth the 503 grams of crude hisramine dihydrochloride product synthesized o6- above. Additionally, 4.5 L of ETOH and 200 ml of water wete added to the reaction flask to dissolve the fitter cake. The suspension was agitated under a nitrogen atmosphere. Ths saspeasisrr was famed ta reflux, Maimaming the suspension tinder reflux, water was charged drop-wise to ths suspension ismi most of the Sofia's were dissolved. Hie solution was cooled to below 7S°c. The solutton was charged with a mixture of 50 grams of NUCHA8 SA {Westvaea, New Ysrk, NY) arts' 50 grams of CEUTE {XT. Baker, Haywsrt, CA). This suspension was heated ihen Itsaied ta refSu* and maintained at that temperature lor D.5 hours. The suspension was cooled to 65"7SC and then filtered through a CELITE bed into a clean, dry 12-1.4-necked, round-bottam flask. The filter aks was rinsed with a mitture of 450 mt of ETOH and 5Q mi of water. The filtered solution was slowly coaled to room temperature wiih stirring overnight. The solution was further caoied to 0-S°C ht 2 hours. At O-S^C, the saspensien was filtered through a Buchner funnel. The filter cake was washed three times with 200 mi cf ETOH chiiistl to S-5°C. The filter cake was dried m a vacuum oven at 60-S5°C After recrystalBzaitoit, the ffeal product "ss 2SS grams, {a SdA% yields ai %B,V& afg HPLC purity. The HPLC protocol is discussed in Example 7 below. Additional rounds of recrysiallizatiDrt were performed to increase tha parity of the final pfoducu However, twa unknown impurities, {RRt 1,3, 1.5} were stii! present above irte 0,1% threshold level after recrysiallizaliDn. Typfcaliy, the ffrsi mipurity !RRi 1.31 was at 8.2-0.4$i aia snd the wtani impurity {Rftt 1.5) at D.5-0.S%. A second recrysta1!izati(in of the sampie discussed above reduced the impurity levels w 0.10.2% and 0.4-0.5%, respectively. Tht impunties sppeared te grow vahm the samples were reanalyzed after a rwrnaer of days, indicating stability ceitcems fer the fins! product. InstabiBly of the product n^flRt stphm why the -mi filter "cake discasssd above showed 99,9% a/a HPLC purity but only 99.1% was obtained after the batch was dried. Subsequent treatments with dichloromethane or charwa! treatments were unable to remove the impurities. Eampte2 Catalysts for tha ttecartmiylation of t-His'tidine In view of the results discussed above, a number Bf rriaoificatisRs tg the symhssis method were undenakBtt These modificatians sei^ht to reta the levels of the unknown impurities ta an acceptable level, 8m variable examined cortcemed tlte nature af the catalyst used is the decafbetylatisn reaction. A variety of sther catalysts were Biammed. including acettphenone, to determrne what role, if any. they play in the formation of the chrornatographic Bnpurities. Table 1 shows the catalysts used in this siutiy. The catalysts were used at 0.3 equivaSsnis. -7, TABLE 1 The results m Table 1 indicate that p-mathylacetophonone was superior to acecophenons at diminising the level of impurities found in the final product . In conduct using p- methylacetophenone in conjunction with a base (1-metyyl-4-piperidodel showed no improvment in the level of impurity generation, while introducting an acid (acetic acid) considerably elevated the impurities found in the final product. Further p-methoxyacetophenone offered an advantage over acetophenone with respect to containing generation but did not produce a significant enhancement vesus p-methylacstophenone upon solaring the imtftyfecatophtiHMie upon isafaimg ihe imisahyrfrscWiiHiJe salt. The data suggest that eaidysts with an electron-defttlsit kstfint esMblt sn torresss kt lbs ^Katton Bf te^mtks 1mm$ in the fisal f^EHtoct, wtesas eteetraiwiricM teteuis s^ws# 3 dp crease \|R mpurftY geswatan, Bastd K tfesse m^t$( acetspt"meBS was replaced with p-metltyiatetophenone ss tffl cai^st used In the teffioiYteticra reactian of the mwmtia& isciose^ her^n. Method of production Histamine Salt Forms Another parameter explored, whldt concerned the generation of acceptably pure histamtne dihydrocMoridB, itmhtti the msiar equivahmey of acid atMssi duilng the prscipstgtion st tbs cmils salt, it is ens of ihs surprafaa iscovetffis af ihe isvtftttei ^scte^ii Nmis that a ndvctisn in tfis af^oafii of csni^^"s presem in tfce fmal pre&tef is related te tto mmm of add ystd 10 OHM ite s# fwn of ths m^scuie, k ^fer an pmcedafes 3 tjasniity ef 2.5 melsr eqwvatenis t# hyaregen cWufiite JHCil gas was tntioduced into a station cenuinmg ths ^ecarboiySaigfl UctMim fliisiamine free feass! to geserate a cruds ifihydrachisrMe salt The presssst Exsmpls eiamsfies rtss effect of a^mg a vamty of molar eimtv^ms sf h¥ocWofic acW te the hist^wne free (me sofetion by btratod^ tiw Kid dsstMwd in isopropanoi ftSAt A nfiscy of H0 concsfttratiofis were 4ru"tv"" m SSA and tested far their eifscts m the pfataisfi sf impiHims. Tte synthesis pretocsl was fcfiawsd as terlssd sfeave except that 0.3 e^waltRis of P-"!hyi^8toptoJ8n8 iwitfc tehiese ss ths ca-sshfeni for (he tdllon of the HC1 were tissd. The HPtC preiscrf of ¦a- Example 7 below was used ID determine the presence of impurities. TN results of this, range of acid concentrations are fisted in Table 2 below. TABLE 2 ¦. Equivalents of HCI and Their Effect of Impurity Generation The results shown in Table 2 illustrate how the amount of acid charged to the solution containing the hislamine free base dramatically altered the revel of the TWO impurities present in the product The observed decrease was likely attributable to the impurities possessing less of a basic character than that of the histamine free base. As a consequence, the histamine free base likely undergoes prolonation first followed by the impurities. The use of 0.5 molar eaurvalents of HCI provided the most favorable results with regards to limiting the levels of impurities found in the product. Under these conditions, the crude product isolated was the monohydrochloride salt as determined by titration for chloride content. Accordingly, to synthesize a djhydrochloride form of histamine of an acceptably high purity, an intermediate purification step involving the intentional generation of monohydrochloride salt was adopted. Using this method, however, it would be necessary to add an additional equivalent of HCI in a later synthesis step so as to produce the dihydrochloride form of the molecule. Additional experiments were performed to examine the effect of small changes in acid concentration on product purity and yield. The results of these experiments are shown in Table 3. These results were taken from products formed from a 100 nil reaction mixture with 0.3 equivalents of p-methylacetopbenone and CH2Cl2 as the co-solvent. The selection of CK2CI2is discussed in detail in Example 4. TABLE 3 Small Variations of Acid Equivalents and Their Effect on Product Formation The equivalency window was narrowed to determine the effect that relatively small variation in the amount of acid had on the impurity profile in the crude salt, The data shown in Table 3 support the previous observation that a decrease in the quantity of acid charged results in a decrease in the amount of impurities found in the final product, as -9- well as a decrease in the yield. In future experiments 0.6 molar equivalents of HCl versus the starting material was used. For larger amounts of product using larger amounts of starling material, the amount of acid required is 0-85 molar equivalents of HCl per mole of free base, as determined by assay- This amount of HCl calculated represents approximately 0.6 molar equivalents versus the starting material of L-fiistidine. Example 4 Co-solyents.for Use Purina Salt Formation The next variable examined to improve the synthesis of histamine dihydrochloride concerned co-solvent used during the acid addition step of the procedure. Previously, toluene was used as the co-solvent. To explore The passible effect of the co-solvenr on the purity of the final product, a variety of eo-sclvems were used in the precipitation step. As above, the purity of the resulting samples was assayed using the HPLC method described in Example 7. The results are shown in Table 4. TABLE 4 The Effect of Methvlsne Chloride and Other Go-sofvents on Knal Product Purity The reaction conditions for the results produced in Table 4 were p-methvlacetoohenone present in 0,3 equivalents. 0.6 equivalents of HCt/lPA and 5 pans of co-solvent for precipitation. The results in Table 4 show that melhylene chloride provides superior results with respect to impurity formation as compared to other co-solvents. Example 6 Preparation of Crude Histamina Monohydrochloride The procedure described below Teaches the preparation of histamine'monohydrochloride, A two liter (2-L), 3-necked, round-hottomed flask (the reactor) was equipped with a thermometer, mechanical stirrer, condenser and nitrogen purge system was charged with 1 L of r.ycloheianol, 100 gm of i-histidine and 25.9 m! of p-methylaceiDphenone. Cyetohexanol has a melting point of 22-22"C and may require heating to generate a liquid that can be Transferred to the reactor. The suspension had 8 white coloration, with a temperature of between 20-25°C and 3 volume of 1050 ml. The suspension was agitated in The presence of a nitrogen atmosphere that was maintained throughout the reaction. The suspension was heated to reflux U60-165°C} and maintained under reflui for 30 hours. A small sample was withdrawn to determine what percentage of the starting material had been decarboxylated. The suspension should contain _<_ a l-histidine. in the event of an incomplete reaction continue heating suspension at reflux> o10- for an additional 3-5 hours and then resample. The formation of a dear, homflfl"mjus solution indicates the consumption of ttie starting material and the completion of the decarboiylation reaction. Oncfl the reaction was complete, the suspension was cooled to about 20-25°C. Then the reactor was charged with 300 ml of roettiylene chloride. This mature was further cooled to room temperature. The mixture was filtered through a Buctiner funnel into another 2-L 3 necked round-bottomed flask. The first reactor was then washed twice wnh 100 ml mBthytene chloride that was then used ta rinse the filter. This fittrsti&n step removed any residual i-h IST i dine. The second reactor containing Die filtrate was equipped with a thermometer, mechanical stirrer, addition funnel and nitrogen purge system. After the wasting step and the re-establishment of the nitrogen atmosphere m the reactor, the fill/ate was heated to 30-35°C. An aliquot of the solution was withdrawn and assayed for the content of histamme free base. The results from the assay were used to calculate the amount of acid required IO generate the monohydrodiloride salt. The amount of Mid required was 0.85 molar equivalents of HCI per mote of histamine free base. With vigorous agitation. 50.5 ml of a 7.65M HCI jsoprnpanol (HCI/ISA) solution was added dropwise at a rate where trie temperature of the solution did not exceed 4Q°C. Given the "0thermic nature of this method step, addition of the HCI/1SA solution occurred over the time of an hour. The resulting light beige suspension was allowed to cool to 20-25°C over 1 hour and agitated for a minimum of 2 hours. The 7.65 M HCI in isopropanol solution was prepared by babbling 27.9 g of HCI g" into 100 m! of isopropanol chilled to 5-10°C. The cooled suspension W3S f jttered through a Buchner funnel under a stream of nitrogen and the filter cake rinsed three.rimes with 100 ml of a 1:1 meAytene chloridefcycloheiartol solution. The filer cake was then washed three times with 100 ml Df metfiylene chloride. Since the monohydrochlonde salt was readily soluble in water, The humidity of the laboratory may Nave an effect on the yield of the product. Therefore, exposure af the filter cake to moisture during the filtration step was minimized by performing the operation under a stream of nitrogen. The wet filter cake was then charged to a It 3-necked round bottom flasV equipped with a thermometer, mechanical stirrex and nitrogen purge system for mcthylene tmuiation. The solid was suspended in 500 ml of methylens chloride and agitated for 1 hour under nitrogen. The methylene irituration assisted in the removal of residual cyclohexanol and enabled the product to be dried more effectively, as was seen in the subsequent steps described below. The suspension, under a stream of nitrogen, was filtered and the solid material was washed twice with 75 ml of methylene chloride. The fitter taks was dried in a vacuum even at 5S-60°C fo; 16 hours. Table 5 below shows the results of the method described m this £iample. This method was practiced three times and the product yields from each were compared. oIV TABLE 5 Crude Yields of Histamfne Manohydrochloride t Weight of solids and corresponding percentage yiBMs were corrected for solvent content. t In Experiments 2 and 3. the filter cake was dried for 6 rather than 16hours. Example 6 Preparation of Histamine DJhvdrochloride by Oecarbotvlatipn of l-Histidine Example 6 shows a procedure for tht synthesis of histamine dihydrochloride from the monohydnichloride precursor product produced with the method of Example 5. A one bter (1 I) thfewiecked, round bottom flask Ithe reactor) equipped with i mechanical siir bar, an addition funnel. 3 condenser, a nitrogen purge system, and thermometer was placed in a heating mantle. The reactor was charged with 40 grams of hisrsmrne monohydrocMoride, 32 ml K20 (distilled}, and 260 ml of a IX ETON solution consisting of 99.5% ETCH and 0.5fc toluene. A nitrogen atmosphere was maintained throughout the reaction as the histimme monohydrochtoride salt was very hygroscopic. The next step of the mtihod entailed the addition of a HCl/ISA solution to convert the histamme moflohydrochloride salt to the dihydrochloride form. To the reactor was added 41.5 ml of 6.65M HC1/1SA solution (1X5 equivalents). As discussed above, the addition of tha acid solution was exothermic, therefore, the acid was added over a 15 minute time frame. During the initial stages of the acid addition, a dear solution was generated, however this quickly returned to a thick off-white suspension after approximately 75% of the acid was introduced. After addition of the acid was complete, the resulting thick, off-white suspension was heated to reflux (78 80°C) in an oil baih. The soM matter in the suspension gradually dissolved to form an ember solution. One the solid matter was completely dissolved, the reactor was removed from the oil bath. The reactor was then charged with NUCHAR SA charcoal \|2 grams! and CELITE \|2 grams). This suspension was heated to reflux for 25 minuies. Maintenance of temperature was important as the product would precipitate at about 60°C. The hot, black suspension was filtered through a bed of CELITE into a fresh 1L 3-necked, round bottom flask equipped with a mechanical stifrer and thermometer. The CELITE bed served as a barrim to prevent the flow of lh" charcoal through the filtering unit. Tna fresh reactor had been preheated in an oil bath and the charging of the reactor also occurred in thrs oil bath. The first reactor containing the reaction mtiture was rinsad twice with 40 mi of ETOH 1X solution at a temperature of 60-65°C. This solution was filtered and added to thfl filtrate produced above. The addition of the rinse volume produced some precipitate in the filtrate. The total volume of solution was then agitated by stirring ai 60-6 5°C for 30 minutes. o12- The suspension (hisumins dihydrochlflride) was than slowly cooled to 25°C aver 1 hour, and agitated at 20-25°C for 2 hours and then cooled to 0-5°C for 2 more hours. The suspension was thmi filtered undar a stream of nitrogen and the filter cake washed three times with 40 ml of cold ETON tX. The filter cake was then weighed and dried in a vacuum oven at 55-60°C for 16 hours. The results of three different eiperimenu convening histamine monohydrochloride to th" dihydroehloride sail form are shown m Table 6. TABLES Yields flf Histamine DitivdiocMoride " The percent yield figure was based on the hist a mine me no hydra chloride corrected for solvent content. Erample7 An HPtC Method ID Atsav. Identify and Dgiermine Purhv of Bisiamine Dirty dfpehloride This example discusses the use of HPLC to quantitat* and identify hist ami ne dihydrochloride and to Qgamitaie related substances and degrtdantt in the final product The method employed a complete HPtC system with gradient and UV detection capabJroes. For chrortwtoaraphic purity determinations, a system containing, a computerized data acquisition system w" utolzBd. Other equipment used included: a Waters Symmetry C-16, 5 Jim, 4.6 i 350 mm column; an anarytittl balance with 0.01 mg or 0.01 g resolution; volumetric glassware; and a column heater. Reagents and standard! used included: a USP htstamme dihydrochloride reference standard or equivalent; mtlhanol, HPLC grade: "cetonitrite, HPIC gtade; 1-heptane sutfonk acid, sodium salt. Ushw Scientific fPittsbureh, PA) HPLC grade or equivalent; sodium phosphate, monobasic, monohydrate, ACS reagent grade; D-, i-histidine monohydftchioride. monohydrate, iSigmi. St Louis, MO); 1 H sodium hydroxide solution: 1 N hydrochloric acid solution; purified water; and benzv) alcohol, ACS reagent grade or equivalent. Two mobile phase buffers were' prepared. Mobile Phasfi A (MPA) contained 0.02 M sodium phosphate monobasic and 0.005 M heptanesulfonic acid, pH adjusted to 3.0. Mobile- phase B (MPB) contained acatonitriifl (ACNJ/methanol (M"0H): 20/15 IvM. Standards and samplw wne prepared for the assay and epromatographic pumy determifiKiorts. The assay standards involved the preparation of histamine dirtyCrochlortde standard solutions at three concentrations. 0.08 mgfml. 0.80 mgfni and 0.72 mg/ml oi-htstidine monohydrothlorrde, moiwhydrate standards were prepared at 0.008 mg/ml. Similartv, assay samples were prepared m duplicate to contain 0.8 mg/ml of synthetically produced hisiamine dmydrochlorida while limit of quantrration (LOQ) solution was prepared at 0.0006 me/ml of histamine dihydrochloride. The sensitivity of the method for Histamine has been determined to be 0.07% for ihe limit of quamiration and 0.03% for the limit of detection. Photodiode arra* peak purity studies have demonstrated the specificity for histamine. o13- Following preparation of the various standards and samples, the HPLC system was equilibreted. Once equilibrated, the flow rate from the waste line was checked at the initial condition setting [i.e., 10% MPB at 1.5 mlJmmuie). T>e flow raw was 1.5 ml/mmrte ± 0.15 ml/mmure. A water bland injection was made after the system equilibrated to condition the column prior to tha start of the assay. Once these preparations w"e complex, the resolution solution of 0.7 mg/mi t 0,1 ragftnl fcittamine dihytfrochloride was injected. The fesoMrtimt TT betwwn a 1 mglmi benzyl alcohol solutipn peak and histamiM peaks was calculated. Further, this process was repeated five (5) limes and a standard deviation was calculated. For the assay, a standard curve was Generated. The standard check was performed averv four to s" sample injections and fell wilhin the following parameters: the tailing factor was not > 2.0; the resolution was > 1.5. the relative standard deviation of the histamine peak responses was not > 2.0%; and the correlation coefficient of the standard curve was not less than 0.995. To calibrate the chromatognphic purity, a single injection of the resolution solution was made. The resolution "R" between benzyl alcohol and the histamine peaks was calculated and so was the tailing factor of the fristamine peak. Since the resolution and tailing factors met tha specifications, three consecutive injections of the LQQ sample were performed. The relative standard deviation for ttiB three histamine peak responses were tabulated. In general, th" tailing factor was not > 2.0-. the resototton was p.reatfir than 1.5. and the relative standard deviation of the histamine peak responses was not greater than 10%. Since tha above parameters Were met, the final hisTamiAe dihydrochhride samples were tested Tht operating parameters for the HPLC are listed in Table 7. The gradient parameters are listed in Table 8. .14. TABU 7 Operating Parameters TABLE 8 Gradient Parameters Retention times: histidrne - approximately 3 minutes histamine - approximately 12 minutes Use of this analytical system provided the method required to determine itie purity of the histamine Ahydrachloride sample produced in the aforementioned examples. Example 8 HPLC Analysts of Histamme DihvdrDchloridef rodoct The histamine dihydrochloriife pro (bets from Example 6 were subjected to the HPLC analysis described in Example 7 to determine the purity of the samples and to establish whether the final products met the criteria of purity set for the method of the invention disclosed herein. For use as a pharmaceutical agent, the nistirnine dlKyrfrochlorkJe must possess minimal chromatograpWc impurities. Individual impurities found at levefs above 0.1% a/a .generally require toiicological qualification. Three lots of hist ami tie dihydrochloride were generated using the methods of Examples 5 and 6. Their purity is described in Table 9. 15- TABLE 9 HPIC Analysis Remits The results described in Table 9 show that the final histammt dihydrochloride pro dud tails within acceptable standards set for the invention disclosed herein. First the levd of Impurity #1 was found ro be below the limit if quamhation for the assay. Second. Impurity 12 was found it livits slightly above the 0.1% threshold and will therefore be qualified through toxic oteojcil testing. The sptdficaTion level for impurity has been estabtrshed as The invention disclosed hwtin describes a novel, non-eniynuttc method for producing pharmaceutical grade histamine dihvdrochioride. One sign'rfictnt advantage of the method described herein is that it yields histamine AhydrochJonde at a purity level higher then is otherwise presently available. Finally, the forgoing examples are not intended to timit the scope of the present invention, which is sac forth In the following claims, in particular, various equivalents and substitutions will be recognized by those of ordinary skill in the art in v>ew of the fcregomg disclosure, and these are contemplated to he within the scope of the disclosed invention. 16 WE CLAIM : 1. A method for the synthesis of histamine dihydrochloride comprising: forming a histamine monohydrochloridc containing solution from a decarboxylated L-histidine-containing solution by addition of hydrogen chloride; and forming a histamine dihydrochloride containing solution from the histamine monohydrochloride containing solution by addition of hydrogen chloride, wherein the resulting histamine dihydrochloride comprises less than 2% total impurities as measured by HPLC analysis. 2. The method as claimed in claim 1, comprising triturating the histamine containing solution. 3. The method as claimed in claim 2, wherein the histamine containing solution is triturated with a methylene chloride solution. 4. The method as claimed in claim 1, wherein the histamine monohydrochloridc containing solution is formed by addition of hydrochloric acid in an isopropanol solution. 5. The method as claimed in claim 4, wherein the hydrochloric acid is about 0.1 to 0.9 molar equivalents of hydrochloric acid to histamine free base. 6. The method as claimed in claim 4, wherein the hydrochloric acid is about 0.6 molar equivalents of hydrochloric acid to histamine free base. 7. The method as claimed in claim 1, comprises the step of isolating a pharmaceutical grade of histamine dihydrochloride from the histamine dihydrochloride containing solution. 8. A method for synthesizing a pharmaceutical grade of histamine dihydrochloride comprising: forming a histamine monohydrochloride containing solution from a decarboxylated L-histidine-containing solution by addition of hydrogen chloride; and forming a histamine dihydrochloride containing solution from the histamine monohydrochloride containing solution by addition of hydrogen chloride; isolating the histamine dihydrochloride from the histamine dihydrochloride containing solution, wherein the resulting histamine dihydrochloride comprises less than 2% total impurities as measured by HPLC analysis. 9. The method as claimed in claim 8, wherein the histamine dihydrochloride contains equal to or less than each of the following: 0.8% L-histidinc HC1 monohydrate, and 0.1% individual chromatographic impurities. 17 The invention disclosed herein relates to (fee preptration of pharmaceutical grades of histamine dihydrochloride using a two step non-enzymatic synthetic method. The invention disclosed herein describes the synthesis of histamine dihydrochloride by the non-enzymatic decarboxylation of bistidine and the step-wise conversion of tha decarboxylated product to the dihydrochloride salt form. The invention disclosed herein considers a final product of histamine dihydrochloride containing less than each of the following: 0.8 % L-histidine HCl monohydrate, 0.1 % individual ethomatographic Impurities,to be acceptable for pharmaceutical use.

Full Text

SYNTHESIS OF HISTAMINE DIHYDROCHLORIDE
Background of the Invention
Histamine is a campound possessing significant biological activity mediated by pharmacological receptors. Histamine has lone been contemplated as a molecule having primarily negative biological effects. Recently, however, new uses for histamine as a powerful pharmaceutical agent have come to light. For example, histamine has been used in conjunction with interferen-alpha to activate NK cells in the presence of monocytes. See U.S. Patent No. 5.728,378. To take full advantage of the therapeutic properties of histamine, it is necessary to obtain large quantities of the compound in a pharmaceutical grade.
Histamine occurs widely in nature as a result of putrefactive processes and a derivative, histamine dihydrochloride, is sold commercially for use as a standard in assays and as e component in certain allergy diagnostic kits. The source of this histemine is often a natural one end as such contains a variety of contaminants that render it unsuitable for pharmaceutical use. There are also synthetic protocols for the synthesis of histemine dihydrochlorida known in the art.
Histamine dihydrochloride can be conveniently synthesized by exploiting the decarboxylation of histidine. Using this synthesis pathway, histidine is decarboxylated and subsequently treated to form the dihydrochloride salt form of the molecule. For example, Hashimoto et al., discussed the preparation of histamine using cycloherenone as a catalyst for the decarboxylation of histidine. (Hashimoto, M.. et al., Chemistry Letters, 883-896 (1986)). The Hashimoto, et al., paper reported the isolation of histamine dihydrochloride at a 95% yield, using 2-cyclohexen-1-one as the catalyst, from the reaction involving histidine and 1 % v/v of 2-cyclohexen-1-one in 10 parts of refluxing cydohexanol (26 hours). The Hashimoto method also teaches the use of toluene and HCI gas bubbled through the resulting decarboxylated solution to precipitate out and harvest the final histamine dihydrochloride product.
Attempts to reproduce the Hashimoto procedure to generate pharmaceutically pure amounts of histamine failed. Additional amounts of the catalyst were required to make the procedure operative and a substantial number of impurities were present In the final product. Moreover, those impurities were difficult to remove. In view of these results, it was found that the Hashimoto procedure is an unsuitable method for generating large quantities of pharmaceutically acceptable histamme.
The use of acetophenone as a catalyst for the decarboxylation of histamine has also been reported. Sea DD 56 793 A. We recreated the method described in the Japanese patent to Akimasa, et al., patent, Japanese Patent No. 05,255,204 (1983), and used 0.26 equivalents of acetophenone and ID parts of diethylene glycol as the solvent for the derarboxylation reaction. Although the Akimasa et al. method was far more efficient in convertine- histidina to histamine. it failed to consistently yield a pharmaceutical grade product. Like the final product using the Hashimoto method, impurities were observed in the final product made using the Akimasa method during the HPLC analysis.
Although the conditions with acetophenone and diethylene glycol looked promising, there existed a problem related to the work-up. Both histamine toe base and the dihydrochloride salt are readily soluble in water, therefore, ft was difficult to utilize any extraction technique to separate the product from the diethylene glycol solvent, which was

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usually removed by a water enraction. Futhermore, the histamine dihydrochloride was also readily soluble in -dithylene-glycol thus-the-directions b -filiratiDn.ww-also-imposslbier
The reaction conditions of TaVano et al.. involving pentan-3'One were also recreated. {Heterocycks, 6:1167 {1977}}. See also G8 1008594 A, which used 2,4-jfihydiQxybenzophenDns in the synthesis process. The results from these experiments showed no imprev&ment over the acetophenone conditions described above.
A consistent source of pharmaceutical orade histamme is required. especiaDy in view of the new-found pharmaceutics! applications for hist a mine. The standard methods used by the art wherein histamfna is purified from natural sources, fail to yield hist ami ns of a sufficiently high grade for pharmaceutical uses. Moreover, the synthetic methods practiced in the art also fail to yield histam'me of a sufficiently hfth grade. Accordinpjy. there is a need in tha art for an improvBd method by which to produce pharmaceutical grade histamine dihydrochioride.
Summary of the Invention"
The Invention disclosed herein relates to ihe preparation of pharmaceutical Grades of histamine clhydrechloride using a two step non-enzymatic synthetic method. Ona embodiment of the invention is a method for the synthesis of histamine dihydrochloride comprising: decarbonating a L-histldme containing sorutiao, whereby a histamine containing solution is formed in the absence of a decarboiylating enzyme: forming 8 hist ami ne monohydrochloride containinfl solution from the histamire containing solution; and forming a histamrna dthydrochlorrde-comaining solution from the histamine monohydroehtoride containing solution.
Accordingly, the present invention provides a method for trie synthesis of histamine dihydrochloride comprising:
forming a rtistamine monohydrochloride containing solution from a decartxwyiated Uiistidine-containing solution by addition of hydrogen chloride; and
forming a histamine dihydrochloride containing solution from the histamine monohydrochloride containing solution by addition of hydrogen chloride, wherein the resulting histamine dihydrochloride comprises less than 2% total impurities as measured by HPLC analysis.
One aspect of This embodiment further comprises triturating the histamine containing solution, for eiampla, tha histamine containing solution can be triturated with a msthylene chloride solution. In another aspect of this embodiment, tha rnstamihe rnonohydrochtoride containing solution is formed by addition of en effective amount of hydrochloric acid in an isopropanol solution. For example, the effective amount of hydrochloric acid u about 0.1 to Q.9 molar equivalents of hydrochloric acid to histamine free base. In another example, the effective amount of hydrochloric acid is about 0.6 molar equivalents of hydrochloric acid to ftstamine free base. Stilt another aspect of this embodiment further comprises rtia step of isolating a pharmaceutical grade of histamine dihydrocnlsride from the histsmino dihydrochloride comeininrj solution.
Another embodiment of the invention disclosed herein is a method for synthesizing a pharmaceutical grade of histamine dihydrochloride comprising: dBcarbciyiating a i-hisiidine containing solution, whereby a histamine containing solution is formed in the absence of a decarbonating enryme; forming a hi stamina monohydrochloride containing solution from the histamine containing solution; forming a histamine (Sihydrochloride containing solution from the htstamine moneh'ydrochloiide containing solution; and isolating the histamine dihydro chloride from the histamine dihydrochlorfde containing solution.
In one aspect of this embodiment, the histamine dihydrochloride contains equal t" of less than each of the following: 0.8% l-histidine HCI monohydrate, 0.1 % individual chrorr.atographic impurities, and 2% total impurities.
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Briff Description of the Accompanying Drawings
Figure 1 shows a reaction method taught in the an.
Figure 2 shows the method of the intention disclosed herein discussed in Examples 5 and 6.
Darailed Description of the Invention
The invention disclosed herein relates to the preparation of pharmaceutical grades of histamine dihydrochloride using a two step non-ewynwtic synthetic method. The invention disclosed herein describes the synthesis of hisiammc dihydrochloride try the non-enzymatic dacarboiylation of htstidine and the step-wise conversion of the decarboiytated product to the dihydrocbloride salt form. The invention disclosed herein considers a final product of histamrne dihydrochloride containing, less than each of the following: 0.6% i-histidine HCI monohyrJrete. 0.1 V" ehrotnatographic impurities {defined below), and 2% total impurities, to be acceptable for pharmaceutical use
Synthetic "methods of synthesizing histamifte dlhydrnchloride known in the art fail to yield product of a sufficient purity to be used as a pharmaceutical compound. Figure 1 shows a deearboiylation method taughi in the art. The method steps of the invention disclosed herein are shown in Figure 2.
Prior art methods were used to generate histamine dihydrochloride from i-histidine starting material in an attempt to generate pharmaceutical grades of synthetic histamma dihydraehloride. The starting material was reacted with the prior art catalyst a-letialone and cyclohexane. Afier completion Df the reaction, the sample was cooled and hydrochloric acid was bubbled into the solution to convert the histamine free base inio tha dihydtochlorid* salt forni. 71w precipitata that formed w*s filmed, washed, and driai The final product produced by the prior an method was found to contain an tmacccptafah/ Nh number of contaminants.
The crude material produced using the prior an method had a purity of 92-94% with one major impurity at 3-5* and five to eight other impuritiBS at > 0.1 ft. Additional purification steps or recrystafcations were performed and o were substantially effective at removing most of these contaminants. Nevertheless, two unidentified irnpurhies remained "t lewis above 0.1%. These impurities ehrted after the histamir* dihydrochloride product and were refened w as chromatooraphic impurities or contaminants. Thus, product made by this method was enaccept abi"*f>r
pharmaceutical use.
In view of ihese results, a new procedure was designed to synthesije histamine dihydrochloride ot-the desired purity. This new procedure involved the decartwxylation of Uiistidine |o-ammo4lor 5Hmidazolepropionic acid (CtHgNA) to yield histawine. Fonowing dKarboiylation, ihe solution containing the histamine free base was tmuraied with mcihvlene chloride to precipitate the product. The product was then filtered and washed. Ths fittered product was subsequently treated with hydrochloric add in isopropanol to precipitate a crude histalhine moflohydfochloride salt. This product was filtered and isolated. The crude salt can be subsequently purified by recrysta&ation techniques or it can proceed to the fwil modification step of the present method. Neil, the monohydrochloride salt was treated again with a hydrochloric acrd/rsopropanol solution to generate the histamine dihildrocWoride form of the molecule. The final form of the produti was then decolorized and washed. These steps, known as recrystafeation, can be repwtedly extensively to yield histamine dihydrochloride of pharmaceutical purity.
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All steps ware performed under a nitrogen gas atmosphere The purity of the final product was analyzed through a number of analytical methods including HPLC analysis.
The invention disclosed herein contemplates the use of 3 number of catalysts or radical initiators 10 facilitate the decarboxylation reaction. An appropriate catalyst is one thai will efficiently catalyze the decarboxylation of histidine when that precursor compound is in a neutral solvent and heated for a number of hours to yield an acceptably pure final product. Electron-enriched ketones are preferred as they tend to reduce the number of impurities present in the final product. For example, a group of suitable catalysts comprises: benzoyl peroxide, 2,2'-azobisisobutyronitrile (AIBN1, 2-cyclohexen-l-one, acetophenone, 4'-bromoacetophenone, benzophenone, p-nitroacetophenone, p-methylacerophenorre. p-meihoiyac*tgphenone. p-methylaceiophenone/1 -meirry Wpiperidone, and p-meihyUeeiephenonefAcOH,
The decwboxylation reaction eonditioas promote the deetrfcoiyiation of the starling materials while minimrzirty ihfl formation of.unwamed.contaminants. The reaction conditions include conducting several method steps in the presence of an inert gastjor example, nitrogen. The reaction conditions further include conducting the decarboiytation step at a range of temperatures between about 145 to 170"°C. Preferably, the reaction is carried out ai a range of temperatures from about 150 to 165*C. or ate range Df temperatures from about 160 to 165"C.
A number of solvents are contemplated for USQ in the invention disclosed herein. The solvents in which certain steps of the reaction are conducted may effect the reaction time which rs required to catalyze the dacarcoxytattort of htsttdme. Solvents us cable in the invention disclosed herein include: cyclohexanot n-methrpyrrondinone (NMPI. difethylenegJvcol). d((ethyleneg,lycol]methyl ether, 2methylaxy0ihlether, Iburwwl, metfioxyemartol, cydohexanol/nlMP (in a 3:1 ratio), (frmethyif ormimide, and tetramethylenesulfone.
Another parameter of the reaction disclosed herein is the method of creating the salt form of hiitamirw by Treating the reaction mixture with hydrogen chloride. The impurity profile oi the final product was found to be effected by the molar equivalency of acid added durinp, the precipitation of the monohydrochloride crude salt. It is possible to control the eiiem e* impurity formation by preparing a solution of hydrogen chloride of a known concentration in isopropancl end treating the reaction mtiture therewith.
A range Df molar equivalents of hydrogen chloride (KCt) in isopropanol (ISA) may be used to practice the method of the invention disclosed herein. A range of about 0.01 to 2 rnolar equivalents may be used 10 create the salt form of hiitamine. Alternatively, a range of about 0.05 to 1.4 molar equivalents may be used. In another alternative, a range of about 0.1 to 0.S molar equivalents may be used. In yet another alternative, about 0.5 molar equivalents may DB used. Tha ratio selected to practice the invention disclosed herein should result in the ultimate generation of a final product with an acceptable (evet of impurities so that the final product may be used as a pharmaceutical composition.
The concentration of the acidic solution used to create the tail form was not critical. For example, the concentration of HCI in ISA may range from about 6 to & N. However, The number of moles of acid introduced is ciucial to isolating a pharmaceuticsIry acceptable grade of the final product. The addition of too much acid causes
4.

impurities to precipitate with the fnonohydfochlDride salt that are eatrenrely difficult to eliminate during the subsequent formation of the hisramine dihydrochloride raft. The relationship between (he method of sail formation and the generation of contaminants w" not appreciated in the an.
Various co-solvents may be used during the addition of HCI in isopropanol to effect precipitation of the monotiydroriiloride salt form of the molecule (salt precipitation). Co-solvents useahle m the invention disclosed herein include: meihylene chloride, cydoheianol tDhiene and tett-butyl methyl ether (TBME).
The ultimate purity of the final product is of particular concern. Additional method steps to purify the final product we afse contemplated. For example, recrysTai!iifition is a process ot repeated crystallization in order 10 purify a wbstanee. A number of solvents w contemplated for use in this purification process. These solvents include: methyl chloride, 2-prcpanol, methanol, cthanol (ETOH!, me chariot/acetone, water, methanol/eihyl acetats, water/aceiDne. methanol/ethanol, witc7methanol, methanol/hezane, water/methanolfaceione, methanol/methylene chloride, 2-propanolfethanDl, mathanoli2-propano!, acetone/2-propanol, acitane/ethanol. From a toiicological viewpoint, a non-toiic solvent such as ETOH is preferred.
The presence of color in the various solutions obtained during the synthesis pathway was observed. Activated canon may be added to remove some of the color before or as a step of the recrystaliizanon process.
The invention disclosed herein further contemplates the use of derivitizmg chemical reactions to assist in the purification of htstsmirte dihydrachloride. Accordingly, it is contemplated that chemical derrvatives of various impurities would bfl made during trie hisiamine dftydrochioride ptocess of the invention disclosed herein to facilitate the removal of ttnse impurities. The creation of one such derivative involves the addition of a lert-butoiycarbonyl group to a molecule of interest. Other modifying groups such as behzyloxycarbonyt groups (CBZ) ait also contempts tsd.
The following examples discuss methods addressing the decarboiyfaii&n of histidtne as well as the isolation of the hisiamine product AJso discussed are methods of purification of the crude histamine dihydrocMoride product using multiple recrystaUizstion steps. Charcoal mediated decoloration is also discussed.
The efficiency of various method steps as welt at the purity of the final product may be analyzed usinp the methods desired beJow. One or more monitoring steps may be used to assay the efficiency of the decarboiyletion step. AliBinativeiy, various assay methods well known in the art may be used to anafyw trie purity of the final product. An example of such a monitoring step is the performance of thin layer chromatography (TLC), a procedure weB known in the art, on various reaction products. Fur esamsle, reactions could be monitored using TIC (mobile phase: CHaCffcHjQiNHiOH: 7.5:2.0:0.5; and ninhydrin spray). This monitoring siep may be performed anytime after the decaitoiytation step.
Particular embodiments of the invention are discussed in detail below. The following examples are for illustrative purposes only and should not be interpreted as limitations of ihe claimed invention. TherB are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention.
5-

EXAMPLES Preparation of KFsTumine Dihydrochlpridg
Th" fallowing txamples discos* the synthesis of histarnini dihvdrochloride from the precursor compound L-histidine Eiisting histamme synthash protocols, while capable of yielding hi sr am me dihydfocWoride. suffer from the Hmnnion of producing an impure final product The Eiamples below discuss various improvemenii in histamirw (tmydrocrilaride synthesis and teach the preparation of a phi rmtceutie ally acceptable grade of his!imine dihydrochloride.
Example | Preparation of 500 6rams of Crude Hisramme pihydrocfiloride
A method for the synthesis of a 500 gram cample of hisiamine dihydrocMoride is described below.
A twelve liter (12-U. 4 necked, round bouom flask equipped with a thermometer, mechanical stirrtr, condtnsir and nitrogen bubbler wax dialed with 7.5 I of cycMeianol (the solvent), 750 grams of i-histidint (the itAsuaiti and U3 ml ef aceiophenoni (the utatysU. The suspmsioo was agitated In a nitiooen atmosphere that was maintained throughout the reaction.
The suspension was heated to reflui and maintained at that temperature (150 165*0 far a minimum of 40 hours. A small sample was withdrawn for an inprocess assay to determine the eiterrt of histidine decarborylation. The suspension was cooftd to below 8O*C and 1875 ml of toluene 919$ charged. This minure wai further cooled to room lemptraiurf. The minure was filtered through a Buchner funneJ into a fresh 12 L, 4 necked round-bottom flask.
"Die fresh flask containing the filtrate was equipped with a theimomgtcr. mechanical stirrer, hydrogen tWoriflr trap and vacuum trap, and prrpvtti for gaseous hydrogen chloride addition. With agitation, the solution was cooled to btlow 10°C. Maintaming thi batch temperature below 20°C. a minimum of 441 grams 12.5 iquivalems) of gaseous hydrogen chloride was charged. Upon completion of the hydrogen chloride addition, the resulting thick yellowish suspension was agitated "t room Temperature for ena how.
The suspension was again filrtnd through a Buchner funnel. The filter cake was rimed with a mixture of 375 ml of cycloheianri and 375 mf of toluene, followed by two 750 ml wishes of tolutne and twa 750 ml rinses of hezanes. The cake was dried on the filter with suction for 1 minimum of 30 minutu. Tna filter cake contained a substantial amount of cyelonexarwl which was removed through triTuratiwi
The wet firm c^ke was charged to a I2-L 4-necked round bottom flask equipped with 1 mechanical stirrer and nitrogen bubbler. Ethanel (ETOHJ in a volume of 7.5 I was also charged. The suspension was agitated at room temperature for 4 hours. Tfte suspension was filtered through a Buchner funnel and the fitter cake rinsed with 400 ml of hoanes. The filter eaki was dried in a vacuum own at SO B5CC ovemrght. The product of this method produced 504 of crude hisiamine dihydroUilonde grams (a 56.6% yield) at 94.4% a7a purity determined using high performance liquid chromatography (HPLC). The product was racrysiatiied to improve the purity of the final product.
A 12-1. 4-netked. round-bottomed flask equipped with a thermometer, mechanical stirref, condenser, addition funnel and nitrogen bubbler was charged wrth the 503 grams of crude hisramine dihydrochloride product synthesized
o6-

above. Additionally, 4.5 L of ETOH and 200 ml of water wete added to the reaction flask to dissolve the fitter cake. The suspension was agitated under a nitrogen atmosphere.
Ths saspeasisrr was famed ta reflux, Maimaming the suspension tinder reflux, water was charged drop-wise to ths suspension ismi most of the Sofia's were dissolved. Hie solution was cooled to below 7S°c. The solutton was charged with a mixture of 50 grams of NUCHA8 SA {Westvaea, New Ysrk, NY) arts' 50 grams of CEUTE {XT. Baker, Haywsrt, CA). This suspension was heated ihen Itsaied ta refSu* and maintained at that temperature lor D.5 hours. The suspension was cooled to 65"7S*C and then filtered through a CELITE bed into a clean, dry 12-1.4-necked, round-bottam flask. The filter aks was rinsed with a mitture of 450 mt of ETOH and 5Q mi of water.
The filtered solution was slowly coaled to room temperature wiih stirring overnight. The solution was further caoied to 0-S°C ht 2 hours. At O-S^C, the saspensien was filtered through a Buchner funnel. The filter cake was washed three times with 200 mi cf ETOH chiiistl to S-5°C. The filter cake was dried m a vacuum oven at 60-S5°C
After recrystalBzaitoit, the ffeal product "ss 2SS grams, {a SdA% yields ai %B,V& afg HPLC purity. The HPLC protocol is discussed in Example 7 below. Additional rounds of recrysiallizatiDrt were performed to increase tha parity of the final pfoducu However, twa unknown impurities, {RRt 1,3, 1.5} were stii! present above irte 0,1% threshold level after recrysiallizaliDn.
Typfcaliy, the ffrsi mipurity !RRi 1.31 was at 8.2-0.4$i aia snd the wtani impurity {Rftt 1.5) at D.5-0.S%. A second recrysta1!izati(in of the sampie discussed above reduced the impurity levels w 0.1*0.2% and 0.4-0.5%, respectively. Tht impunties sppeared te grow vahm the samples were reanalyzed after a rwrnaer of days, indicating stability ceitcems fer the fins! product. InstabiBly of the product n^flRt stphm why the -mi filter "cake discasssd above showed 99,9% a/a HPLC purity but only 99.1% was obtained after the batch was dried. Subsequent treatments with dichloromethane or charwa! treatments were unable to remove the impurities.
E*ampte2 Catalysts for tha ttecartmiylation of t-His'tidine
In view of the results discussed above, a number Bf rriaoificatisRs tg the symhssis method were undenakBtt These modificatians sei^ht to reta the levels of the unknown impurities ta an acceptable level, 8m variable examined cortcemed tlte nature af the catalyst used is the decafbetylatisn reaction. A variety of sther catalysts were Biammed. including acettphenone, to determrne what role, if any. they play in the formation of the chrornatographic Bnpurities. Table 1 shows the catalysts used in this siutiy. The catalysts were used at 0.3 equivaSsnis.
-7,

TABLE 1

The results m Table 1 indicate that p-mathylacetophonone was superior to acecophenons at diminising the level of impurities found in the final product . In conduct using p- methylacetophenone in conjunction with a base (1-metyyl-4-piperidodel showed no improvment in the level of impurity generation, while introducting an acid (acetic acid) considerably elevated the impurities found in the final product. Further p-methoxyacetophenone offered an advantage over acetophenone with respect to containing generation but did not produce a significant enhancement vesus p-methylacstophenone upon solaring the imtftyfecatophtiHMie upon isafaimg ihe imisahyrfrscWiiHiJe salt. The data suggest that eaidysts with an electron-defttlsit kstfint esMblt sn torresss kt lbs ^Katton Bf te^mtks 1mm$ in the fisal f^EHtoct, wtesas eteetraiwiricM teteuis s^ws# 3 dp crease |R mpurftY geswatan, Bastd K* tfesse m^t$( acetspt"meBS was replaced with p-metltyiatetophenone ss t*ffl cai^st used In the teffioiYteticra reactian of the mwmtia& isciose^ her^n.
Method of production Histamine Salt Forms
Another parameter explored, whldt concerned the generation of acceptably pure histamtne dihydrocMoridB, itmhtti the msiar equivahmey of acid atMssi duilng the prscipstgtion st tbs cmils salt, it is ens of ihs surprafaa iscovetffis af ihe isvtftttei ^scte^ii Nmis that a ndvctisn in tfis af^oafii of csni^*^"s presem in tfce fmal pre&tef is related te tto mmm of add ystd 10 OHM ite s# fwn of ths m^scuie, k ^fer an pmcedafes 3 tjasniity ef 2.5 melsr eqwvatenis t# hyaregen cWufiite JHCil gas was tntioduced into a station cenuinmg ths ^ecarboiySaigfl UctMim fliisiamine free feass! to geserate a cruds ifihydrachisrMe salt The presssst Exsmpls eiamsfies rtss effect of a^mg a vamty of molar eimtv^ms sf h¥*ocWofic acW te the hist^wne free (me sofetion by btratod^ tiw Kid dsstMwd in isopropanoi ftSAt
A nfiscy of H0 concsfttratiofis were 4ru"tv"" m SSA and tested far their eifscts m the pfataisfi sf impiHims. Tte synthesis pretocsl was fcfiawsd as terlssd sfeave except that 0.3 e^waltRis of P-"!hyi^8toptoJ8n8 iwitfc tehiese ss ths ca-sshfeni for (he td**llon of the HC1 were tissd. The HPtC preiscrf of
¦a-

Example 7 below was used ID determine the presence of impurities. TN results of this, range of acid concentrations are fisted in Table 2 below.
TABLE 2 ¦. Equivalents of HCI and Their Effect of Impurity Generation

The results shown in Table 2 illustrate how the amount of acid charged to the solution containing the hislamine free base dramatically altered the revel of the TWO impurities present in the product The observed decrease was likely attributable to the impurities possessing less of a basic character than that of the histamine free base. As a consequence, the histamine free base likely undergoes prolonation first followed by the impurities.
The use of 0.5 molar eaurvalents of HCI provided the most favorable results with regards to limiting the levels of impurities found in the product. Under these conditions, the crude product isolated was the monohydrochloride salt as determined by titration for chloride content. Accordingly, to synthesize a djhydrochloride form of histamine of an acceptably high purity, an intermediate purification step involving the intentional generation of monohydrochloride salt was adopted. Using this method, however, it would be necessary to add an additional equivalent of HCI in a later synthesis step so as to produce the dihydrochloride form of the molecule.
Additional experiments were performed to examine the effect of small changes in acid concentration on product purity and yield. The results of these experiments are shown in Table 3. These results were taken from products formed from a 100 nil reaction mixture with 0.3 equivalents of p-methylacetopbenone and CH2Cl2 as the co-solvent. The selection of CK2CI2is discussed in detail in Example 4.
TABLE 3 Small Variations of Acid Equivalents and Their Effect on Product Formation

The equivalency window was narrowed to determine the effect that relatively small variation in the amount of acid had on the impurity profile in the crude salt, The data shown in Table 3 support the previous observation that a decrease in the quantity of acid charged results in a decrease in the amount of impurities found in the final product, as
-9-

well as a decrease in the yield. In future experiments 0.6 molar equivalents of HCl versus the starting material was used. For larger amounts of product using larger amounts of starling material, the amount of acid required is 0-85 molar equivalents of HCl per mole of free base, as determined by assay- This amount of HCl calculated represents approximately 0.6 molar equivalents versus the starting material of L-fiistidine.
Example 4
Co-solyents.for Use Purina Salt Formation
The next variable examined to improve the synthesis of histamine dihydrochloride concerned co-solvent used during the acid addition step of the procedure. Previously, toluene was used as the co-solvent. To explore The passible effect of the co-solvenr on the purity of the final product, a variety of eo-sclvems were used in the precipitation step. As above, the purity of the resulting samples was assayed using the HPLC method described in Example 7. The results are shown in Table 4.
TABLE 4 The Effect of Methvlsne Chloride and Other Go-sofvents on Knal Product Purity

The reaction conditions for the results produced in Table 4 were p-methvlacetoohenone present in 0,3 equivalents. 0.6 equivalents of HCt/lPA and 5 pans of co-solvent for precipitation. The results in Table 4 show that melhylene chloride provides superior results with respect to impurity formation as compared to other co-solvents.
Example 6 Preparation of Crude Histamina Monohydrochloride
The procedure described below Teaches the preparation of histamine'monohydrochloride, A two liter (2-L), 3-necked, round-hottomed flask (the reactor) was equipped with a thermometer, mechanical stirrer, condenser and nitrogen purge system was charged with 1 L of r.ycloheianol, 100 gm of i-histidine and 25.9 m! of p-methylaceiDphenone. Cyetohexanol has a melting point of 22-22"C and may require heating to generate a liquid that can be Transferred to the reactor. The suspension had 8 white coloration, with a temperature of between 20-25°C and 3 volume of 1050 ml. The suspension was agitated in The presence of a nitrogen atmosphere that was maintained throughout the reaction.
The suspension was heated to reflux U60-165°C} and maintained under reflui for 30 hours. A small sample was withdrawn to determine what percentage of the starting material had been decarboxylated. The suspension should contain _<_ a l-histidine. in the event of an incomplete reaction continue heating suspension at reflux> o10-

for an additional 3-5 hours and then resample. The formation of a dear, homflfl"mjus solution indicates the consumption of ttie starting material and the completion of the decarboiylation reaction.
Oncfl the reaction was complete, the suspension was cooled to about 20-25°C. Then the reactor was charged with 300 ml of roettiylene chloride. This mature was further cooled to room temperature. The mixture was filtered through a Buctiner funnel into another 2-L 3 necked round-bottomed flask. The first reactor was then washed twice wnh 100 ml mBthytene chloride that was then used ta rinse the filter. This fittrsti&n step removed any residual i-h IST i dine.
The second reactor containing Die filtrate was equipped with a thermometer, mechanical stirrer, addition funnel and nitrogen purge system. After the wasting step and the re-establishment of the nitrogen atmosphere m the reactor, the fill/ate was heated to 30-35°C. An aliquot of the solution was withdrawn and assayed for the content of histamme free base. The results from the assay were used to calculate the amount of acid required IO generate the monohydrodiloride salt. The amount of Mid required was 0.85 molar equivalents of HCI per mote of histamine free base.
With vigorous agitation. 50.5 ml of a 7.65M HCI jsoprnpanol (HCI/ISA) solution was added dropwise at a rate where trie temperature of the solution did not exceed 4Q°C. Given the "0thermic nature of this method step, addition of the HCI/1SA solution occurred over the time of an hour. The resulting light beige suspension was allowed to cool to 20-25°C over 1 hour and agitated for a minimum of 2 hours. The 7.65 M HCI in isopropanol solution was prepared by babbling 27.9 g of HCI g" into 100 m! of isopropanol chilled to 5-10°C.
The cooled suspension W3S f jttered through a Buchner funnel under a stream of nitrogen and the filter cake rinsed three.rimes with 100 ml of a 1:1 meAytene chloridefcycloheiartol solution. The filer cake was then washed three times with 100 ml Df metfiylene chloride. Since the monohydrochlonde salt was readily soluble in water, The humidity of the laboratory may Nave an effect on the yield of the product. Therefore, exposure af the filter cake to moisture during the filtration step was minimized by performing the operation under a stream of nitrogen.
The wet filter cake was then charged to a It 3-necked round bottom flasV equipped with a thermometer, mechanical stirrex and nitrogen purge system for mcthylene tmuiation. The solid was suspended in 500 ml of methylens chloride and agitated for 1 hour under nitrogen. The methylene irituration assisted in the removal of residual cyclohexanol and enabled the product to be dried more effectively, as was seen in the subsequent steps described below.
The suspension, under a stream of nitrogen, was filtered and the solid material was washed twice with 75 ml of methylene chloride. The fitter taks was dried in a vacuum even at 5S-60°C fo; 16 hours.
Table 5 below shows the results of the method described m this £iample. This method was practiced three times and the product yields from each were compared.
oIV

TABLE 5 Crude Yields of Histamfne Manohydrochloride

t Weight of solids and corresponding percentage yiBMs were corrected for solvent content. t In Experiments 2 and 3. the filter cake was dried for 6 rather than 16hours.
Example 6 Preparation of Histamine DJhvdrochloride by Oecarbotvlatipn of l-Histidine
Example 6 shows a procedure for tht synthesis of histamine dihydrochloride from the monohydnichloride precursor product produced with the method of Example 5.
A one bter (1 I) thfewiecked, round bottom flask Ithe reactor) equipped with i mechanical siir bar, an addition funnel. 3 condenser, a nitrogen purge system, and thermometer was placed in a heating mantle. The reactor was charged with 40 grams of hisrsmrne monohydrocMoride, 32 ml K20 (distilled}, and 260 ml of a IX ETON solution consisting of 99.5% ETCH and 0.5fc toluene. A nitrogen atmosphere was maintained throughout the reaction as the histimme monohydrochtoride salt was very hygroscopic.
The next step of the mtihod entailed the addition of a HCl/ISA solution to convert the histamme moflohydrochloride salt to the dihydrochloride form. To the reactor was added 41.5 ml of 6.65M HC1/1SA solution (1X5 equivalents). As discussed above, the addition of tha acid solution was exothermic, therefore, the acid was added over a 15 minute time frame. During the initial stages of the acid addition, a dear solution was generated, however this quickly returned to a thick off-white suspension after approximately 75% of the acid was introduced.
After addition of the acid was complete, the resulting thick, off-white suspension was heated to reflux (78 80°C) in an oil baih. The soM matter in the suspension gradually dissolved to form an ember solution. One* the solid matter was completely dissolved, the reactor was removed from the oil bath. The reactor was then charged with NUCHAR SA charcoal |2 grams! and CELITE |2 grams). This suspension was heated to reflux for 25 minuies. Maintenance of temperature was important as the product would precipitate at about 60°C.
The hot, black suspension was filtered through a bed of CELITE into a fresh 1L 3-necked, round bottom flask equipped with a mechanical stifrer and thermometer. The CELITE bed served as a barrim to prevent the flow of lh" charcoal through the filtering unit. Tna fresh reactor had been preheated in an oil bath and the charging of the reactor also occurred in thrs oil bath.
The first reactor containing the reaction mtiture was rinsad twice with 40 mi of ETOH 1X solution at a temperature of 60-65°C. This solution was filtered and added to thfl filtrate produced above. The addition of the rinse volume produced some precipitate in the filtrate. The total volume of solution was then agitated by stirring ai 60-6 5°C for 30 minutes.
o12-

The suspension (hisumins dihydrochlflride) was than slowly cooled to 25°C aver 1 hour, and agitated at 20-25°C for 2 hours and then cooled to 0-5°C for 2 more hours. The suspension was thmi filtered undar a stream of nitrogen and the filter cake washed three times with 40 ml of cold ETON tX. The filter cake was then weighed and dried in a vacuum oven at 55-60°C for 16 hours. The results of three different eiperimenu convening histamine monohydrochloride to th" dihydroehloride sail form are shown m Table 6.
TABLES Yields flf Histamine DitivdiocMoride

" The percent yield figure was based on the hist a mine me no hydra chloride corrected for solvent content.
Erample7 An HPtC Method ID Atsav. Identify and Dgiermine Purhv of Bisiamine Dirty dfpehloride
This example discusses the use of HPLC to quantitat* and identify hist ami ne dihydrochloride and to Qgamitaie related substances and degrtdantt in the final product The method employed a complete HPtC system with gradient and UV detection capabJroes. For chrortwtoaraphic purity determinations, a system containing, a computerized data acquisition system w" utolzBd. Other equipment used included: a Waters Symmetry C-16, 5 Jim, 4.6 i 350 mm column; an anarytittl balance with 0.01 mg or 0.01 g resolution; volumetric glassware; and a column heater. Reagents and standard! used included: a USP htstamme dihydrochloride reference standard or equivalent; mtlhanol, HPLC grade: "cetonitrite, HPIC gtade; 1-heptane sutfonk acid, sodium salt. Ushw Scientific fPittsbureh, PA) HPLC grade or equivalent; sodium phosphate, monobasic, monohydrate, ACS reagent grade; D-, i-histidine monohydftchioride. monohydrate, iSigmi. St Louis, MO); 1 H sodium hydroxide solution: 1 N hydrochloric acid solution; purified water; and benzv) alcohol, ACS reagent grade or equivalent.
Two mobile phase buffers were' prepared. Mobile Phasfi A (MPA) contained 0.02 M sodium phosphate monobasic and 0.005 M heptanesulfonic acid, pH adjusted to 3.0. Mobile- phase B (MPB) contained acatonitriifl (ACNJ/methanol (M"0H): 20/15 IvM.
Standards and samplw wne prepared for the assay and epromatographic pumy determifiKiorts. The assay standards involved the preparation of histamine dirtyCrochlortde standard solutions at three concentrations. 0.08 mgfml. 0.80 mgfni and 0.72 mg/ml oi-htstidine monohydrothlorrde, moiwhydrate standards were prepared at 0.008 mg/ml. Similartv, assay samples were prepared m duplicate to contain 0.8 mg/ml of synthetically produced hisiamine dmydrochlorida while limit of quantrration (LOQ) solution was prepared at 0.0006 me/ml of histamine dihydrochloride. The sensitivity of the method for Histamine has been determined to be 0.07% for ihe limit of quamiration and 0.03% for the limit of detection. Photodiode arra* peak purity studies have demonstrated the specificity for histamine.
o13-

Following preparation of the various standards and samples, the HPLC system was equilibreted. Once equilibrated, the flow rate from the waste line was checked at the initial condition setting [i.e., 10% MPB at 1.5 mlJmmuie). T>e flow raw was 1.5 ml/mmrte ± 0.15 ml/mmure. A water bland injection was made after the system equilibrated to condition the column prior to tha start of the assay.
Once these preparations w"e complex, the resolution solution of 0.7 mg/mi t 0,1 ragftnl fcittamine dihytfrochloride was injected. The fesoMrtimt TT betwwn a 1 mglmi benzyl alcohol solutipn peak and histamiM peaks was calculated. Further, this process was repeated five (5) limes and a standard deviation was calculated.
For the assay, a standard curve was Generated. The standard check was performed averv four to s" sample injections and fell wilhin the following parameters: the tailing factor was not > 2.0; the resolution was > 1.5. the relative standard deviation of the histamine peak responses was not > 2.0%; and the correlation coefficient of the standard curve was not less than 0.995.
To calibrate the chromatognphic purity, a single injection of the resolution solution was made. The resolution "R" between benzyl alcohol and the histamine peaks was calculated and so was the tailing factor of the fristamine peak. Since the resolution and tailing factors met tha specifications, three consecutive injections of the LQQ sample were performed.
The relative standard deviation for ttiB three histamine peak responses were tabulated. In general, th" tailing factor was not > 2.0-. the resototton was p.reatfir than 1.5. and the relative standard deviation of the histamine peak responses was not greater than 10%.
Since tha above parameters Were met, the final hisTamiAe dihydrochhride samples were tested Tht operating parameters for the HPLC are listed in Table 7. The gradient parameters are listed in Table 8.
.14.
TABU 7 Operating Parameters

TABLE 8 Gradient Parameters
Retention times: histidrne - approximately 3 minutes
histamine - approximately 12 minutes
Use of this analytical system provided the method required to determine itie purity of the histamine Ahydrachloride sample produced in the aforementioned examples.
Example 8 HPLC Analysts of Histamme DihvdrDchloridef rodoct
The histamine dihydrochloriife pro (bets from Example 6 were subjected to the HPLC analysis described in Example 7 to determine the purity of the samples and to establish whether the final products met the criteria of purity set for the method of the invention disclosed herein. For use as a pharmaceutical agent, the nistirnine dlKyrfrochlorkJe must possess minimal chromatograpWc impurities. Individual impurities found at levefs above 0.1% a/a .generally require toiicological qualification. Three lots of hist ami tie dihydrochloride were generated using the methods of Examples 5 and 6. Their purity is described in Table 9.
15-

TABLE 9 HPIC Analysis Remits
The results described in Table 9 show that the final histammt dihydrochloride pro dud tails within acceptable standards set for the invention disclosed herein. First the levd of Impurity #1 was found ro be below the limit if quamhation for the assay. Second. Impurity 12 was found it livits slightly above the 0.1% threshold and will therefore be qualified through toxic oteojcil testing. The sptdficaTion level for impurity has been estabtrshed as The invention disclosed hwtin describes a novel, non-eniynuttc method for producing pharmaceutical grade histamine dihvdrochioride. One sign'rfictnt advantage of the method described herein is that it yields histamine AhydrochJonde at a purity level higher then is otherwise presently available.
Finally, the forgoing examples are not intended to timit the scope of the present invention, which is sac forth In the following claims, in particular, various equivalents and substitutions will be recognized by those of ordinary skill in the art in v>ew of the fcregomg disclosure, and these are contemplated to he within the scope of the disclosed invention.
16

WE CLAIM :
1. A method for the synthesis of histamine dihydrochloride comprising:
forming a histamine monohydrochloridc containing solution from a decarboxylated L-histidine-containing solution by addition of hydrogen chloride; and
forming a histamine dihydrochloride containing solution from the histamine monohydrochloride containing solution by addition of hydrogen chloride, wherein the resulting histamine dihydrochloride comprises less than 2% total impurities as measured by HPLC analysis.
2. The method as claimed in claim 1, comprising triturating the histamine containing
solution.
3. The method as claimed in claim 2, wherein the histamine containing solution is
triturated with a methylene chloride solution.
4. The method as claimed in claim 1, wherein the histamine monohydrochloridc
containing solution is formed by addition of hydrochloric acid in an isopropanol solution.
5. The method as claimed in claim 4, wherein the hydrochloric acid is about 0.1 to 0.9
molar equivalents of hydrochloric acid to histamine free base.
6. The method as claimed in claim 4, wherein the hydrochloric acid is about 0.6 molar
equivalents of hydrochloric acid to histamine free base.
7. The method as claimed in claim 1, comprises the step of isolating a pharmaceutical
grade of histamine dihydrochloride from the histamine dihydrochloride containing solution.
8. A method for synthesizing a pharmaceutical grade of histamine dihydrochloride
comprising:
forming a histamine monohydrochloride containing solution from a decarboxylated L-histidine-containing solution by addition of hydrogen chloride; and
forming a histamine dihydrochloride containing solution from the histamine monohydrochloride containing solution by addition of hydrogen chloride;
isolating the histamine dihydrochloride from the histamine dihydrochloride containing solution, wherein the resulting histamine dihydrochloride comprises less than 2% total impurities as measured by HPLC analysis.
9. The method as claimed in claim 8, wherein the histamine dihydrochloride contains
equal to or less than each of the following: 0.8% L-histidinc HC1 monohydrate, and 0.1%
individual chromatographic impurities.
17
The invention disclosed herein relates to (fee preptration of pharmaceutical grades of histamine dihydrochloride using a two step non-enzymatic synthetic method. The invention disclosed herein describes the synthesis of histamine dihydrochloride by the non-enzymatic decarboxylation of bistidine and the step-wise conversion of tha decarboxylated product to the dihydrochloride salt form. The invention disclosed herein considers a final product of histamine dihydrochloride containing less than each of the following: 0.8 % L-histidine HCl monohydrate, 0.1 % individual ethomatographic Impurities,to be acceptable for pharmaceutical use.

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Patent Number

205849

Indian Patent Application Number

IN/PCT/2001/00601/KOL

PG Journal Number

15/2007

Publication Date

13-Apr-2007

Grant Date

13-Apr-2007

Date of Filing

07-Jun-2001

Name of Patentee

MAXIM PHARMACEUTICALS INC .

Applicant Address

SUITE 400, 8899 UNIVERSITY CENTER LANE, SAN DIEGO,

Inventors:

#	Inventor's Name	Inventor's Address
1	YEH WEN-LUNG	120 CHELWOOD DRIVE THORNHILL, ONTARIO L4J 7H6,
2	ANTCZAK CASIMIR	103 TIMPSON DRIVE, AURORA ONTARIO L4G 5W5,
3	MCGOLRICK JEFFRY DAVID	540 PICKERING CRESCENT NEWMARKET ONTARIO L3Y 8G9
4	ROTH MICHAEL JOSEPH	44 SCHSEFER PLACE, BOLTON ONTARIO L7E 1W3
5	WRONA MARK	282 DEWITT ROAD STONEY CREEK ONTARIO L8E 241,

PCT International Classification Number

C07 D 233/54

PCT International Application Number

PCT/US99/30379

PCT International Filing date

1999-12-20

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/113,933	1998-12-23	U.S.A.