Carbobenzoxy Derivatives of Histidine, Imidazole and Benzimidazole1

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6416

ABRAHAM PATCHORNIK, ARIEH BERGERAND EPHRAIM KATCHALSKI [CONTRIBUTION FROM

THE

DEPARTMENT OF BIOPHYSICS, WEIZMANN

INSTITUTE OF

V O l . 79

SCIENCE]

Carbobenzoxy Derivatives of Histidine, Imidazole and Benzimidazole1 BY ABRAHAM PATCHORNIK, ARIEHBERGERAND EPHRAIMKATCHALSKI RECEIVEDJUSE 5, 1957

1,N-Dicarbobenzoxyhistidine(L and DL) ( I ) were obtained from benzyl chloroformate and histidine (L and DL, respectively). Compound I was decarbobenzoxylated by amines or alcohols yielding the corresponding IC-carbobenzoxy derivatives 11. 1,N-Dicarbobenzoxy-L-histidine methyl ester hydrochloride (111) was obtained from I either by direct esterification or via the chloride hydrochloride IV. 1-Carbobenzoxyimidazole hydrochloride (V) and 1-carbobenzoxybenzimidazole hydrochloride ( V I ) were prepared. The 1-carbobenzoxy groups of V and V I were found t o be similar in reactivity t o those of I and 111. 1,3-Dicarbobenzoxy-2-hydroxybenzimidazoline( V I I ) and the corresponding 2-methoxy and 2-benzyloxy derivatives (VI11 and I X ) were synthesized, and their reactivity toward sodium methoxide and hydrogen chloride was investigated. Non-aqueous titrations were applied t o the investigation of the structure and the reactivity of these compounds (see Table I ) .

The synthesis of various X-acyl derivatives of histidine has been described. The N-benzoylj3e4 N-p-nitrobenzoy16 and N-carbobenzoxy6 derivatives were prepared from the amino acid and the respective acyl chlorides in dilute alkali; N-acetylhistidine7 was synthesized from histidine and acetic anhydride in glacial acetic acid. Although the imino group of imidazole can be acylated, 1,N-di(2-naphthylsulfonyl) -histidinea is the only 1-acyl substituted histidine known. The failure to prepare histidines substituted in the 1-position by acetyl or benzoyl is probably due to the susceptibility of the imidazole acyl bond to alkaline hydrolysis. 1,N-Diacylhistidine ester derivatives, however, are readily obtained from histidine ester and an acyl chloride under anhydrous conditions.4,9,10 Action of excess acyl chloride on histidine ester in aqueous sodium carbonate causes opening of the imidazole ring which undergoes the well known Bamberger reaction.'l Because of the importance of the carbobenzoxy derivatives of amino acids in peptide synthesis,12 and since little was known concerning the synthesis and properties of carbobenzoxy derivatives of histidine, an investigation of the reaction between L- and DL-histidine and benzyl chloroformate was undertaken. Dicarbobenzoxy-L-histidine (I) was obtained by coupling L-histidine with two moles of benzyl chloroformate in aqueous solution a t O " , a t pH 0 to 10, followed by acidification to pH 2.4 whereupon i t precipitates. Excess mineral acid must be avoided since the product is amphoteric. The crude dicarbobenzoxy derivative is unstable, decomposing a t room temperature within several hours to give N-carbobenzoxy-L-histidine(11). Two considerably more stable forms of I, I a and Ib, were obtained on crystallization from methanol and (1) A preliminary report on this work was presented a t t h e 2 I s t hIeeting of t h e lsrael Chemical Society; A . Patchurnik, Bull. Res. C O I L I IoCf l. r r a e i , 6 8 , 10 (19.57), ( 2 ) See K. Hofmann, "Imidazole and I t s Derivatives," Interscience Publishers, Inc., New York, N.Y . , 1933. (3) H . Pauly, Ber., 43, 2243 (1910). ( 4 ) 0. Gerngross, Z . physioi. Chem., 108, 50 (1919). ( 5 ) H. Pauly, ibid., 64, 75 (1910). (0) M. Brrgmann and L. Zervas, Ber., 65, 1192 (1932). ( 7 ) R I . Bergmann and L. Zervas, Biochcm. Z . , 203, 280 (1928). ( 8 ) H. Pauly, 2.physiol. Chem., 42, 508 (1904). (9) RL. Bergmann and L. Zerras, ibid., 175, 145 (1928). (10) T h . 'A'ieland, A n n . , 580, I59 (1953). (11) A. Kossel a n d E . Edlbacher, Z . physiol. Chem., 93, 396 (1915); E . Bamberger, A n n . , 213, 267 (1893). (12) H. D. Springall and H. D. Law, Quart. Revs., 10, 230 (1956).

ethanol, respectively, and were found to contain one molecule of alcohol. Direct esterification of I with methanolic hydrogen chloride or treatment of I with diazomethane and subsequent acidification with hydrogen chloride led to the methyl ester hydrochloride 111. The hydrochloric acid of I11 was removed by diazomethane to yield the free ester IIIa. Treatment of I with thionyl chloride yielded the corresponding acid chloride hydrochloride IV, which gave I11 on treatment with methanol. Unlike other N-carbobenzoxyamino acid chlorides, IV failed to cyclize to the respective N-carboxy-a-amino acid anhydride. l 3 Dicarbobenzoxy-L-histidine(I) is readily decarbobenzoxylated by nucleophilic reagents such as sodium methoxide and amines, yielding N-carbobenzoxy-L-histidine (II), With ammonia and aniline, the corresponding benzylurethans formed were also isolated. N-Carbobenzoxy-L-histidine (11) is an amphoteric substance owing to the presence of the acidic a-carboxyl group and the basic imidazole group in the molecule. Its solubility in water, sparing solubility in ethanol and insolubility in acetone, ethyl acetate and benzene suggest a zwitterionic structure. 1,N-Dicarbobenzoxy and N-carbobenzoxy-DLhistidine were prepared analogously to the respective L-histidine derivatives. The melting point and solubility of N-carbobenzoxy-DL-histidinediffered considerably from that of N-carbobenzoxyL-histidine. -4 comparison of the properties of both forms with those reported by Bergmann and Zervas for N-carbobenzoxy-L-histidine6 (see Experimental) suggested that the substance isolated by these authors was in fact the racemic form. For the purpose of comparing the properties of the 1,N-dicarbobenzoxy-histidine with those of other carbobenzoxyimidazole compounds, carbobenzosyimidazole hydrochloride (V) and carbobenzoxybenzimidazole hydrochloride (VI) were prepared by coupling imidazole and benzimidazole, respectively, with carbobenzoxychloride either in alkaline aqueous medium or under non-aqueous conditions. Both V and V I are decarbobenzoxylated analogously to 1,N-dicarbobenzoxyhistidine by nucleophilic reagents such as amines and sodium methoxide. On heating they split off benzyl chloride and carbon dioxide similarly to 1,N-dicarbobenzoxyhistidine methyl ester hydrochloride (111). (13) E. Katchalski, Advances i n Protein Chem., 6, 123 (1951).

Dec. 20, 1957

DERIVATIVES OF HISTIDINE, IMIDAZOLE AND BENZIMIDAZOLE 6417 CARBOBENZOXY

CH-NH

I1

CH-X-COOC7H.I

CH-NH

C-N

C-K

or

CH2 CH-COOH I

R ; CsHa; R’ == HCHI

1

\CH

C-XN

I

C7H7OCOCl

CHI

I 1 CH-COOH I

_____f

I CH-COOH I

CHz

XH*

or R‘OH

CH-N-COOC7H7

1 I CH-COC1 I CHz

or CHIOH, HCl

CH-K-COOC7H7

I/

ZCH C-N.HC1

1

-

CHIOH

I

NHCOOC7H7 IV

I

ZCH C-X.HC1 CH2

I

XHCOOC7H7 11

by an assumed cleavage of the C(2)-0 bond by the halogen acid leading to the formation of 1,3dicarbobenzoxybenzimidazolium chloride (X) . Similarly to the labile complexes of tertiary amines with acid chlorides, X decomposes readily into 1carbobenzoxybenzimidazole and benzyl chloroformate. COOC7H7

COOC7H7

COOC7H7 \‘HI or IX

COOCiH7

I I

CH-COOCHI NHCOOC7H7 I11

I n the case of benzimidazole, 1,3-dicarbobenzoxy derivatives could be prepared. When benzimidazole was coupled with two moles of benzyl chloroformate in the presence of aqueous alkali, 1,3dicarbobenzoxy-2-hydroxybenzimidazoline (VII) SH

,

I

ITHCOOC7H7 I

HCI

+ RXHCOOC7Hr 4-R’OCOOC7H7

x

K;-COOC,H,. . K x > C H

+ C7H7OCOCl

VI For the elucidation of the structure and reactivity of the various imidazole derivatives described above non-aqueous titrations15 proved of considerable value. The titrimetric properties of imidazole, benzimidazole and histidine and of some of their acyl derivatives are summarized in Table I. The data given demonstrate that the free imidazole groups are considerably more basic than their respective acyl derivatives. Thus while a free imidazole group requires one mole of HClO4 using VI VIII, R = CH3 thymol blue (TB) as indicator (yellow to red) (see IX, R = C7H7 no. 1 and 3), its acyl derivatives do not consume was formed. The pseudobasic hydroxy group of HC104 under these conditions (see no. 7). This VI1 is very reactive yielding with alcohols the cor- behavior explains also the observation that the responding 1,3-dicarbobenzoxy-2-alkoxybenzimid-hydrochlorides of the acyl imidazole derivatives azolines. The methoxy and benzyloxy deriva- impart a red color to T B (see no. 9). In the prestives, to which formulas VIII and IX are ascribed, ence of crystal violet (CV) in glacial acetic acid, resemble the corresponding dibenzoyl compounds which requires a higher acidity than T B for its described by Gerngr0~s.I~Unlike 1-carbobenzoxy- color change (violet to green), both the free and the imidazole derivatives, VI11 and IX are not decarbo- acylated imidazole groups are titratable with one benzoxylated by sodium methoxide in alcohol a t mole of HC1O4 (no. 7 ) . room temperature. Anhydrous hydrogen chloride The base-catalyzed alcoholysis of the acylimidaon the other hand causes rapid decarbobenzoxyla- zole group discussed previously, is evident from the tion with the formation of l-carbobenzoxybenzimid- titrimetric data of these compounds with HCIO, azole and benzyl chloroformate. The difference after previous exposure to catalytic quantities of in behavior toward nucleophilic reagents between methoxide in the presence of excess methanol (see the carbobenzoxy group attached to imidazole and no. 8, 10 and 12). Thus while benzoylbenzimidathose combined with benzimidazoline is not sur- zole does not absorb HC104 when T B is used as inprising considering that the pyrrole nitrogen of the dicator, one mole of HCI04 is consumed after expoformer is markedly acidic in character.2 The sensi- sure of the substance to methanol in the presence tivity of the 1,3-dicarbobenzoxy-2-hydroxybenz-of sodium methoxide (no. 9). For 111, V, VI and imidazoline (VII) and its alkoxy derivatives VI11 benzoylbenzimidazole HC1 (no. 10) one equivalent and IX toward hydrogen chloride may be explained N

N

(14) 0. Gerngross, Ber., 46, 1913 (1913).

K-COOC7H7 VI1

(15) J. S Fritz, “Acid Base Titrations in Nonaqueous Solvents,” T h e G. Fredrick Smith Chemical Co., Columbus, Ohio, 1962.

XBRAIIAM PATCRORNIK, XRIEH BERGERAND EPHRAIX KXTCIIALSKI

6418

Vol. 79

Experimental .\I1 melting points are uncorrected. 1 ,N-Dicarbobenzoxy-L-histidine(I;).--BenzJ-l chloroforina t e (17 ml.) rra.s added iii ten portions to an ice-corilecl s ~ ) l u t i i m of rA-liistidine h!.drticfiloridc liytlrate (10.5 g.) i i i 2 sodium hytlrositle (Xi 1111 ,! . .After the :tddition o f c,'ucIi Iiiirtion of tlie reagent t h e iiiisture was \-igorously shaken : ~ n t Iits pH adjusted t u $1-10 by the additioli of 2 sodium hytlroxide. total \-oluiue of approxiniately 60 nil. of alkali ~ r a consumed s and the coupling waq completed Trithin 20 minutes. T h e mixture was then acidified t o PH 2.4 wit11 cold 4 hydrochloric acid. Tlie white precipitate formed was caused t o coagulate by shaking, the supernatant w:ts discarded and thc crude product ( I 1 \ u s itnmetliati.lJ, rccrystallized from nietlianol. The l,S-tliearbobenzoxy-l,histidine crystallized with one molecule of tneth:inol ( L t ' i ; .p. 105-107" dec. when heated quickly, , in ~tcctonc-nietliaii(il 4: 1 b y volutiie). rteriiil \v:ib Liir-dried :it roonl t e m p e r a -

mole

so.

Comiii,un
1.

Imidazole o r benzimidazole

Titrant

HCIO, HClOi SaOAle 2 . 1midazole.HCI or benzi- HCIOI niidazole, HCI HClO4 h-aOMe :i. Histidine, free base He104 HCIOI SaOAIe 4. Histidine.HC1 HClOa HCIOh XaOlTe i.S-C.irbohenzaxyliistiHClO4 dine ( [ I j HCIOi SaOlIe ( i I1 1 mole SdOXle €ICIoI 7 I-BznzoylbenzimidaHClOr zrjle" o r 1 , s - d i c a r h u - I I C I O ~ benzox)-hi.;tidine SaOlle methyl ester ( I I I a ) S 1 Benzoylbenzimida€ICIO ioletLo r I I I ~ trace NaOAle (3IeOH: (1. 1,S-DicarbubenzoxySaOlIe hi5tidine methyl ester. IIYI ( I I I ) , I-cxrbobenzuxybcnzimidazoIe.HC1 ( \ ' I ) . Lcarbobenzoxyi midazole. IICl ( Y ) or I - b e n z o y l henzi midazolc. HCI 10 111, V ,V I iir I-benzoyl- HCIOI henzimidazole.HC1, I mole N a O l l e (lIeOH) I1 1.X-r)icarbohenzoxy€IC104 histidine (Ia: HCIOI

+

11.

1

1 I 2 1 1

1 2 trr I tr'

u \.

TU

Y V V T-B

TB CV TB TB

c\TB TB C\'

'rB TB CV '1.H '113 TB CV

I'

-

I1

G B K G

It Y-C Y-B Y -+ I< V- G Y-B U li

v U 13

---- r, -(;

1'1'

I3 1R

1

'TU

B

1

TB

R-Y-B

1

T13

TB CT'

HCIO:

trr I 1 2

HCIO: HCIOI SaOMe

trc trc trc

TB CV TB

Y

HCIO~

tr'

TB

I-

KaOAle

13.

2 1

----

TI3

cv

I'- B

-

Ia f 1 mole T a O l I e (hleOH) 1.3-Dicarbohenzoxy-Pmethoxybenzimidazoline ( V I I I ) or 1,3-dicarbobenzoxy-2-henzyloxybenzimidazoline ( I S ) \'III, I S , 4 trace S a O l I e (hIeOH)

1 1 tr' tr' trc 1

Culor change"

Indicatin

'Tt3

+-

12.

compound

TB TU

I '

-v-c; B

Y

Y

It

It

I
-l'l; neut. equir., 155 dcterrnined by anhydrous titration i i i Inet1i:inol with 0.1 S sodium niethoxitle using thyinol I ~ h c :is indicator, or in glacial acetic acid with 0.1 :Ir perchloric x i d in glacid acetic acid using crystal violet as indicator. S o titratable groups could be detected on titration in tlioxm e with 0.1 9 perchloric acid in dioxane using th>-molblue :LS indicator. A solution of 1,S-dicarbobenzoxy-L-histidinc in methanol shows an absorption maximum a t with :t molar ;xbsorption coefficient E = 3200. \$lien tlie crude 1 ,Xi bobenzosy-L-histidirle w:ii crystallized from etliaiic,l, cr ils with one nioleculc o f etlimol, Ib, n'ere obtained. d n d . Calctl. for C22H21SaOe.C2H50H: C , 01.4; 11, CcHsO, 9.6; neut. equiv., 469. Found: CzHjO, 9.5; neut. equiv., 171 dctcrminetl l a atid l b are solublp in acetoile a n d acct soluble in cold et11ani)l :tiid iiiioluble in ether and petroleurii etlicr. Metlianol a n t 1 ctlianr)l were tlle (itily tnrli .;olvc.llti found froin irhicli cr>-\tLilso f I could hc ohtaitietl. Tllu.: it ciiultl not hc crystnllizctl frcini propmiol, butanol, 1)enzcnL or ethyl acetat tnlliiie tlicarbobcnzosy-L-lli~tiilitieiirep:irutions containing methanol (Ia) or ethanol (Ib'l ivcre k ? t i n closed L-cssels at room temperature for several weeks d e compositiori occurred. %rom tlie resultiiig viscous matcrial S-carl~ohenzox~--i~-Iiisticliiie ci)ulrI he iso1:ited in a 70-85'; \-ielii h5- trituration with ether ani1 acetone anti cryst:illizaIW', Ilot tic.;ion of tlie solitl residue from e t ~ i a n o ~rn.11. ; prcsseti bl- admixture with a sample o f S - c , t r l ~ o h e ~ i ~ c ~ ~ ~ - i . liiitidine (in.p. 166-167"; prepared a s dcicribetl h e h v . .I similar decoiiipo~itioiioccurred withill several Iiours \ V ~ I C . I I z:ition of I from alcohol was III d s r i occurred when crud o n :L n:itcr-bath in ethyl :ice w i d for I i , L I f an hour. I n these case.; p:trtial r:iceinizatiilti

I< C B

R

Samples of 0.1 t o 0.2 ininole of tlie compound were tlissolved in 10 ml. of solvent a n d titrated with 0.1 stantlard solutions from :i2-ml. microburet. Thymol blue (TB! was used :is indicator :~iiddioxane as solwnt for titrations with lIC104 in dioxme and S a O M e in benzene-methaiiol. Crl-stal violet ( C l 7 served as indicator and glacial acetic acid a s solvent for titrations with HC104 in glacial acetic acid. 'I T = ?-ellon.; I< = red; \- = violet; G = green. t r = trace iahout 0.02 IIII.'~. ri Prepared according t o 0. Gerngrms, ref. 14,

of sodium inethoxide was added to neutralize the

hydrochloric acid present, prior to the titration with perchloric acid of the parent imidazole compounds formed as a result of the base-catalyzed alcoholysis. The titration data given in no. I 4 show that, as mentioned previously, no base-catalyzed nlcoholysis takes place in the case of dicarbobenzoxybenzimidazoliIie derivatives. The discussed alcoholysis reaction was also followed spectrophotometrically in the case of 1,Kdicarbobenzoxyhistidine (Ia) (see Experimental 1 .

Dec. 20, 1957

CARBOBENZOXY DERIVATIVES OF HISTIDINE, IMIDAZOLE AND BENZIMIDAZOLE6419

sium perchlorate was filtered off. Crystallization of N-carbobenzoxy-L-histidine in the filtrate started after the addition of acetone (10 ml.). T h e crystalline product was collected after two days. Yield 2.3 g. (go%), m.p. 164-165" dec.; after recrystallization from ethanol m.p. 166-167" dec.,I6 [a]'*~ -25.0' (c 6.0, in 6 N hydrochloric acid). Anel. Calcd. for CL4H16S304: C, 58.1; H , 5.2; N, 14.5; neut. equiv., 289.3. Found: C, 58.0; H , 5.2; N, 14.2; neut. equiv., 290, determined by anhydrous titration in ethanol n3th 0.1 N sodium methoxide or with 0.1 N perchloric acid in dioxane using thymol blue as indicator. When I1 (500 mg.) was hydrolyzed with boiling 6 N hydrochloric acid (10 inl.) for 1.5 hours, the hydrolysate gave a specific rotation of [a]*"D 4-13.0" calculated for a quantitative yield of histidine (26.8 mg.)." S-Carbobenzoxy-L-histidine is soluble in alcohol a n d water a n d is insoluble in acetone, benzene and ether. N-Carbo benzoxy-DL-histidine.-N-Carbobenzoxy-DL-histidine was prepared from 1,N-dicarbobenzoxy-DL-histidine analogously t o the L-compound; recrystallized from ethanol, m.p. 209-210' dec. Anal. Calcd. for ClnHljNaO~:C, 58.1; H , 5.2; N, 14.5; neut. equiv., 289. Found: C, 58.1; H , 5.4; N, 14.2; neut. equiv., 290, determined by anhydrous titration with 0.1 3 sodium methoxide. N-Carbobenzoxy-DL-histidineis soluble in boiling alcohol and boiling mater; it is sparingly soluble in cold water and alcohol and is insoluble in acetone, benzene and ether. Decarbobenzoxylation of 1,N-Dicarbobenzoxy-L-histidine. a. By Aniline.-1,S-Dicarbobenzoxy-L-histidine (Ia) (0.86 g.) mas dissolved in aniline (2 ml.) and the solution was heated on a water-bath for half a n hour. h'-Carbobenzoxy-L-histidine was precipitated b y the addition of acetone (20 ml.) a n d recrystallized from alcohol-acetone; yield 527; of the theoretical, m.p. 162" dec. After recrystallization from ethanol m . p . 164-165" dec., not depressed by admixture with I1 prepared as described above; neut. equiv., 290, determined by titration in alcohol with perchloric acid in dioxane using thymol blue as indicator. From the mother liquor S-phenyl benzylcarbamate was isolated in 855% yield, m.p. 75-76', after recrystallization from ethanol. T h e m.p. was not depressed on admixture with a n authentic sample of N-phenyl benzylcarbamate.'* b. By Ammonia.-1,N-Dicarbobenzoxy-L-histidine (Ib, 0.5 g.) was dissolved in concentrated ammonia (% ml.) whereupon crystallization of benzylcarbamate began. T h e mixture T Y ~ Sheated on a v-ater-bath for five minutes to bring the decarbobenzoxylation to completion. Benzylcarbamate (0.132 g., 82%) crystallized out on cooling, m.p. 85-86', not depressed on admixture with a n authentic sample of b e n z y l ~ a r b a m a t e . ' ~T h e mother liquor was brought t o dryness in m " o a n d the residue dissolved in ethanol ( 3 ml.). S-Carbobenzoxy-L-histidine (m.p. 163-164') crystallized on standing a t room temperature. c. By Sodium Methoxide.-In this case the extent of dccarbobenzor)-lation Jr-as followed spectrophotometrically a t 2380 A . where 1,s-dicarbobenzoxy-L-histidine (Ia) has a molar absorption coefficient of E = 3200, whereas the resulting ~-c:Lrbobciizosy-L-liistidiiie arid the methyl benzylcarhamate euch have molar absorption coefficients of about E = 200. In :t 0.014 11 solution of I a in methanol, in the prese ~ i c ci i f two equivaleiits o f sodium niethuxide, decarbobenztion a t i-ooni tcin1)erature was cornplcte within a few ~ninutcs. -~ ~__.__

(11;) Rergmann and Zervas (ref. I;) give 1n.p. ? l o o for N-carhoI,enz(j.uy~r.-histidine but d o not report a n optical rotation. I n the preparation o f this compound t h e authors used a procedure similar t o t h a t employed by us for t h e preparation of I, followed by heating of t h e intermediate in acetic acid. When a solution of I i n glacial acetic acid w a s evaporated t o dryness and t h e residue crystallized from alcohol we obtained a smali yield (15%) of material with m.p. 210' identical with S-carbobenzoxy-DL-histidine. I t seems therefore t h a t some racemization occurs during the decarbobenzoxylation of I in acetic acid and t h a t the DL-Compound only was isolated by the above authors owing t n its low solubility in alcohol. (17) M . S . D u n n , E . H. Frieden, M. P. Stoddard and H. V. Brown, J . Biol. Chem., 144, 487 (1942), give for L-histidine [a]% +13.3' (c 4.05, in 6.08 AVhydrochloric acid). (18) H. v . Soden and W. Rojahn, Be?., 3 4 , 2809 (1901). (1s) H. E. Carter, R I,. Frank and H. W.Johnston, O v g . S y i i i h r s e s , 23, 13 (1940).

T h e above quantitative decarbobenzoxylation reaction also could be demonstrated titrimetrically. Thus when a 0.1 M solution of Ia in methanol was neutralized with one equivalent of 0.1 N sodium methoxide to the blue end-point of thymol blue it consumed two equivalents of 0.1 N perchloric acid in dioxane on back titration t o t h e red endpoint (see Table I, no. 6). 1,N-Dicarbobenzoxv-L-histidvlChloride Hvdrochloride (IV) .-l,S-Dicarbobeizoxy-L-hi&idine (Ib, f . O 9.) was dissolved in thionyl chloride (1.0 ml.) a t 0' a n d the solution diluted with ethyl acetate (20 ml.). T h e crystalline 1,Ndicarbobenzoxy-L-histidyl chloride hydrochloride separated out within half an hour on seeding or scratching. T h e crystals were washed with ethyl acetate and dried i n vacuo over phosphorus pentoxide and potassium hydroxide; yield 0.5 g. 1n.p. 98-100" dec. Anal. Calcd. for CZZH210jK3C12:C, 55.2; H , 4.4; X, 8.8; C1, 14.8; mol. wt., 478.4. Found: C, 55.9; H , 5.0; X, 9.4; C1, 14.8; equiv. wt., 244 determined by anhydrous titration in aniline with 0.1 N sodium methoxide using thymol blue as indicator. This corresponds t o a molecular weight of 488 assuming the presence of two acidic groups per molecule. T h e presence of a n acid chloride group was demonstrated by titration with sodium methoxide as above after hydrolysis in moist pyridine a t room temperature. Three equivalents of sodium methoxide were consumed per mole of the starting chloride hydrochloride 11'. 11: is soluble in chloroform a n d is insoluble in ethyl acetate a n d benzene. 1,N-Dicarbobenzoxy-L-histidineMethyl Ester Hydrochloride (111).-Crude 1,K-dicarbobenzoxy-L-histidine (I) obtained from 10.5 g. of L-histidine hydrochloride hydrate, was dissolved in ethyl acetate (50 ml.), and the solution was washed with water and dried over sodium sulfate. T o t h e dried solution a n excess of diazomethane in ether was added. When evolution of nitrogen had ceased, d r y hydrogen chloride was passed through the solution, and the crystals formed were collected and dried in uucuo over potassium hydroxide; yield 17.1 g. (72Y,), m.p. 113' dec.; after recrystallization from acetone m.p. 117-118" dec. Anal. Calcd. for C23H2406S3C1:C, 58.3; H, 5.1; C1, 7.5; CH30, 6.5; neut. cquiv., 473. Found: C , 58.5; H , 5.4; C1, 7.6; CHBO,6.4; neut. equiv., 473, determined by anhydrous titration in ethanol with 0.1 1V sodium methoxide to the blue end-point using thymol blue as indicator. T h e ester hydrochloride gives a red color with the indicator which turns yellow when about half of the titrant has been added. This indicates that partial decarbobenzoxylation had OCcurred. I11 also can be titrated with 0.1 N tributylamine in dioxane to the yellow end-point of thymol blue, consuming one mole of base. I n this case no decarbobenzoxylation occurs. Alternatively I11 was prepared by direct esterification of 1,S-dicarbobenzoxy-L-histidine ( I a ) . I a (1.0 g.) was suspended in anhydrous methanol (20 ml.) a n d o t h e mixture saturated with dry hydrogen chloride a t 0 . After 12 hours a t room temperature the clear solution v a s brought to dryness in c'acuo a t a water-bath temperature of 35', and the crystalline residue mas triturated with anhydrous ether, collected a n d dried in mcuo over potassium hydroxide; yield 0.80 g. (77y0),m.p. 115-117' dec.; neut. equiv., 471, determined as abovc. 111 also was obtained from the chloride hydrochloride I\: (0.1 9.) by treatment with methanol (0.5 ml.) containing oiie drop of pyridine. T h e resulting solution was diluted with ether (10 ml.) and saturated with dry hydrogen chloride. T h e methyl ester hl-drochloride 111 which separated out was recrystallized from a c e t o n e e t h e r ; m.p. 118' dec.; neut. equiv., 470, determined as above. 1 ,N-Dicarbobenzoxy-L-histidinemethyl ester (IIIa) was prepared b y treatment of a n alcoholic solution of 111 with a n excess of diazomethane in ether. T h e reaction mixture was brought t o dryness and the oily residue left t o crystallize a t room temperature; m.p. 45-46', Anal. Calcd. for C23H23?;306:C, 63.1; H, 5.3; S,9.6; neut. equiv., 437. Found: C, 63.2; H , 5.4; N, 9.7; neut. equiv., 440, determined by titration in glacial acetic acid with 0.1 N perchloric acid using crystal violet as indicator. S o sodium methoside or perchloric acid was consumed when thymol blue was used as indicator (see Table I , no. 7).

6420

ABRAHAM PATCHORNIK, ARIEH BERGERAND EPHRAIM KATCHALSKI

T h e free ester is soluble in the usual organic solvents a n d is insoluble in petroleum ether a n d water. 1-Carbobenzoxyimidazole Hydrochloride (V). a. In Nonaqueous Medium.-Benzyl chloroformate (0.75 nil.) was added to an ice-cold solution of imidazole (0.68 g.) in anhydrous benzene (10 ml. ) a n d the mixture was left a t room temperature overnight. Imidazole hydrochloride, which had separated out, was removed by filtration, a n d the filtrate mas saturated with dry hydrogen chloride a t 0". T h e crystalline 1-carbobenzoxyimidazole hydrochloride formed was collected, washed with ether a n d dried in z " x o over potassium hydroxide a n d phosphorus pentoxide; yield l .05 g. (88%), m.p. 74-76' dec. Anal. Calcd. for CllH1102SZC1: C, 55.3; H , 4.65; N, 11.7; CI, 14.9; neut. equiv., 238.7. Found: C, 54.3; H , 3 . 8 ; K, 11.9; C1, 14.9; neut. equiv., 242, determined by titration with 0.1 N sodium methoxide in ethanol using thymol blue as indicator; neut. equiv., 246, determined by titration with 0.1 N tributylamine in dioxane using thymol blue as indicator. T h e change in color a t the end-point is from red to yellow. 1; is soluble in hot alcohol, hot acetone and is insoluble in ether. On heating or prolonged standing at room temperature V decomposes yielding benzyl chloride a n d carbon dioxide. Similarly t o the 1,S-dicarbobenzoxp-L-histidine ( I ) , V is decarbobenzoxylated by sodium methoxide. Thus imidazole could be isolated as the picrate (67 mg., m.p. 207-208') from a reaction mixture of V (72 mg.) in methanol (1 ml.) and 0.1 N methanolic sodium methoxide (3.0 mi.). b. In Aqueous Medium.-Imidazole (0.68 g.) dissolved in water (10 ml.) was coupled a t 5" with benzyl chloroforma t e (1.5 ml.) in benzene (10 ml.) at pH 9-10. Xpproximately 10 ml. of 1 N sodium hydroxide was consumed during the course of the reaction, which was completed within 20 minutes. T h e benzene layer was separated, a n d the aqueous layer extracted twice with benzene (40 m l , ) . T h e combined benzene solutions were dried over anhydrous sodium sulfate and saturated a t 0" with hydrogen chloride. T h e oil which separated was dissolved in acetone a n d crystals were obtained by the addition of ether (50 ml.); yield 0.3 g., m.p. 74-75' dec., not depressed on admixture with a sample of I' prepared as described in the preceding paragraph. 1-Carbobenzoxybenzimidazole Hydrochloride (VI). a . In Non-aqueous Medium.-Benzyl chloroformate (1.6 ml.) was added t o a suspension of finely powdered benzimidazole (2.36 g.) in d r y benzene (50 ml.). T h e mixture was shaken for one hour a n d the resulting benziniidazole hydrochloride was filtered off. T h e benzene solution was diluted with d r y ether (50 ml.) and saturated with dry hydrogen chloride a t 0'. T h e crystalline l-carbobenzoxybenzimidazole liydrocliloride (VI), was collectcd, washed with ether and dried in vacuo over potassium hydroxide and phosphorus pentoxide; yield 2.4 g. (83L?,), m.p. 99-100" dec. Anel. Calcd. for C15H130ZN~C1: C, 62.4; H , -1.3; S, 9.7; C1, 12.3; neut. equiv., 289. Found: C , 62.2; H , 4.2; S,9.2; C1, 12.2; neut. equiv., 289, determined by titration with 0.1 N sodium methoxide in ethanol using thymol blue as indicator. VI is soluble in ethanol and is insoluble in acetone and ether. b. In Aqueous Medium.-Finely powdered benzimidazole (1.18 g.j, suspended in water (20 ml.), was coupled at 0" with benzyl chloroformate (1.6 ml.) in the presence of 1 sodium hydroxide (10 nil.) and benzene (20 rnl.). The benzene layer was separated and the aqueous solution estracted with benzene (15 ml.). The combined benzene solutions were dried over sodium sulfate, saturated with d r y hydrogen chloride and left a t room temperature ovcrnight. T h e crystals of I-carbobenzoxybenzimidazolehydrochloride which had separated out were collected, washed with cther and dried in V ~ C U Oover potassium hydroxide a n d

VOl. 79

phosphorus pentoxide; yield 2.36 g. (82761,m.p. 97-99' dec.; neut. equiv., 287, determined as above.

1,3-Dicarbobenzoxy-2-hydroxybenzimidazoline (VII).-

Finely powdered benzimidazole (2.36 g.) v a s suspended in water (60 ml.) and coupled at OD, a t p H 8, with benzl-l chloroformate (6.4 ml.) dissolved in benzene (60 ml.). T h e p H mas maintained during the course of the reaction (1 hr.) by the addition of 1.0 N sodium hydroxide (approximately 35 ml. of base were consumed). T h e benzene layer was separated, and the aqueous solution extracted with benzene (40 ml.). T h e combined benzene solutions were dried over sodium sulfate a n d concentrated in z " o t o a volume of 10 ml. Petroleum ether was added (50 ml.) precipitating ail oil which solidified on trituration with petroleum ether; yield 5 . 5 g. (68Lj;,), m.p. 115-117'. Anal. Calcd. for C Z ~ H ~ O O ~C,N68.3; Z: H, 5.0; S , 6.9; active H , 0.25. Found: C, 68.6; H , 4.9; S , 7.1; active H , 0.28, determined by the Zerewitinoff method. A strong absorption band a t 3.0 g due t o the hydroxyl group W:LS observed in the infrared spectrum of the substance in chloroform.

1,3-Dicarbobenzoxy-2-methoxybenzimidazoline (VIIIj.-

Methanol (100 inl.) was added t o a concentrated benzene solution of VI1 obtained from benzimidazole (2.36 g . ) , and the mixture was refluxed for five minutes and allowed to cool t o room temperature. Crystallization was complctc mithin t\vo hours. T h e product was filtered, washed with cold methanol and dried in oacuo over sulfuric acid; yield I t was recrystallized from 6.1 g. ( 7 3 7 , ) , m.p. 125-129'. benzene before analysis. Anal._ Calcd. for C24H2z05S2: C, 68.9; H , 5.3; K, 6.7; CH30, i.4. Found: C, 68.8; H , 5.2; iY,6.6; CHaO,

7.6. When a methanolic solution of VI11 containing 0.1 equivalent of sodium methoxide was boiled for one minute, the intact starting material could be recovered in almost quantitative yield.

1,3-Dicarbobenzoxy-2-benzyloxybenzimidazoline (1x1

was prepared analogously t o the corresponding methoxy derivative VI11 using benzyl alcohol (30 ml.) instead of methanol. The benzyl alcohol solution was heated for five minutes on the water-bath whereupon the product crystallized. I t was collected, washed with methanol and dried in vecuo over sulfuric acid; yield 75yo, m.p. 135139'; after recrystallization from benzene m.p. 142-145'. .4nal. Calcd. for C30H260SS~: C, 72.8; H , 5 . 3 ; S ,