Imidazoles. 11. 5(or 4)-(Moiiosubstitu ted Triazerio)iiiiidazole-4(or 5)-carboxainides'
5(or 4)-(Monosubstituted triazeno)imitiazole-4(or S)-carboxamides ( \' or tautomeric struc.1ures) w r e synthesized by the reaction of 5-diazoimidazole-4-carbosamide x i t h primary aliphatic and aromatic amines. The himpler alkyltriazeno derivatives were shown t o decompose to 5( or 4)-aminoimidazole-4(or 5)-carboxamide (AIC) in aqueous or methanol solutioiis; they niay, therefore, function as latent diazo compounds in which AIC is a carrier molecule. Triazenes obtained from two arylaniines, ethyl glyciiiate, and ethyl phenylalanate decvmpose more slowly, and the mode of decomposition may he more complex. 5( or 4)-( h1ethyltriazeno)imidaxole-4(or 5)-carboxamide is active against mouse leukemia L1210. Moderate :ict.ivity was also observed aniorig a f e n other derivatives in tests for antineoplastic efi'ect,s.
Diazotizat io11 of .>(or -I.)-ariiiiioiniidxzole-l(or 5 ) varboxamide (I) origirially gave a com1)ouiitl dehcribed :i- 2-azahypoxanthine (iinidazo [4,.i-d]-z~-triuzln4(8H)-one, II).2 Subsequently, it x i s s l 1 0 ~ 1 that 1 ~ the diazotizatioii niay be performed in such a way that 3c~i:Lzoiinidazole--I.-c.nrboxainicle(111) niay be i3olatetl aiid characterized, even though it cyclizez readily in ,olutioii to 2-azahypoxarithine (11). The diazo tlerivativc. vouples with aroiiiaiic subst rate- under conditioii. t ypit~al of diazoriiuiii cvxipling r e a c t i o ~ i s . ~Furtherinore, reactions of I11 with secondary :tiiiiiic- prodiirc triazeries (IV), some of whirl1 Iin\-c been shon 11 to exert antitumor effect\.' Thew rezult- led t o n b t d y of the preparation :tiid propert it.. of sonip :iiialogoii(or 4)-(iiionowb,tituted triazeiio)imidazol~~--I.~or ,>)carboxaiiiides (V or tantonieric ~ t r i i ( ~ t u w ~ ) .
iiiethaiiol o r i l l a 1 1 cwect of the amiiie; yields UN(' wriahle. -1ftc.r thc inztability of these triazench iii iiiethanol had been demon.trated, a 4 described below, ethyl :ic.etate n :I- employed a5 tlie solvent, u i d the pure triazeiies J7 >eparated i i i high aiid reproduciblc yit\lds. d l l re:tc*tion iiiixtiires w ( w routincly protectml from light. 1)i-ubstitutc~ct-trinzeiio derivative, (IV) li:ivc> b e c ~ i i hoviii previou4y to he itable (for :it least 1-2 day.) i nqueou' sollitions protected from light.4 111 holuoils expored 10 light thry w r c tmisfornied iiito 2:~zah~l)osarithiiic (TI), prc-uiiiably 1 ia the diazo roiii1 m i i i i d 111. Ultr:n.iolet ah+orl)tion studie- -ho\vetl that the riioiio:ilkyl triazerics : t r ~uiist:ible in solutioii c ~ e i 111 i the ahwicdc, of light. I n the example illuitrrttccl l)y I igiire 1. iii;~xiiii:i:it 320 :tiid 232 nip ( ( * u i * v1)~ oh~
0
I
I11 IT
L.
0.0
220
240
~
Figure l.--I)ec.oniporitioii
The sinipler 5jor -I.)-(iiioiiosubstituted triazeiio)iniidazole--2(or .i)-czarboxamidcs werct initially obtained by procedures4 used t o pre1)art: the disubstituted triitzcIioimidazoles. Reactions of 111 with the all~)ropriateaniines were allowed to proceed in anhydrous (1) This investigation was supported b y the Cancer Chemotherapy Na-
tional Service Center, Sational Cancer Institute, Piational Institutes of Health, Contract No, SA-43-pli-1740 and Ph-43-64-51, and hy the C. F. Kettering Foundation. (2) D. TV. FTooley and E. S h i v , J . Biol. Chem. 189, 401 (1931). ( 3 ) Y.F. Shealv, R . F. Struck, I,. 13. IIolrim, and J. .L Montgomery, .I. O r g . Chem., 26, 2396 (1961). (4) T.F. Siiealy, C'. . \ , Kraiitls, ; i n i l . I , .\. .\Ziintgiimeiy, il,,d.,27, 2l,-Al 11 9 6 2 ) .
( 5 ) 1.. F. Rhealy, J . .\, M o n t g o m e r y anti \\.. R . Laster, J r . , Rioihtm. I'harmacol., 11, 674 (1962).
L_;L.--
260 280 300 320 K a v e length, niM.
340
''
.-\ 360
380
of 5(or 4)-(but~itriazeiio)iniid:~z~~le-
4( or 5)-carbosaniide (1-c) to 5(or 4)-aminoiniidazole-i( or 5 ) cmboxamide (1) in methanol (7.36 X 10-j Jf) at 25'. Tinw int,ervals in minutes between the addition of methaiiol :rnd thr beginning of the tracing of curves 1--9 were as f(~1low~: 2.n,
7 . 5 , 12.5, 21.5, 30, 45, GO, 130, 240.
served nilliiii a fen- iiiiiiute- of the additioii of nictlian01 to the butyltriazene (T'c) decreased in interi\ity even though the btock qolutiori was protected from light in the same manner that iolutionq of the disubstituted triazerioiiiiidazole. h:td 1)een. The Iwoducat resulting froni di-\ociatioii Iloncvc~r, 1s I l O t 2 IC', but *II(' (I) :tb-or])tloii a t 268 inp roiicwiiiit :iiit \\ i t 11 the clcc*re:iw of tlic illit i d 1ii:ixima
IMIDAZOLES. I1
January 1966
is identical, except for slight residual absorption ill the 320-mp region, with the spectrum of AIC free base in methanol. I n like manner, it wab shown that AIC is formed in either phosphate buffer (pH 7 ) or, more slowly, in methanol solutions of Va-c and in buffer solutions of Vf-g. The latter compounds were not studied in methanol. Isoabsorptive points were observed a t 247-249 and at 280-282 mp, unless the formation of AIC mas essentially complete when the first spectra were recorded. The formation of AIC (I) from the morioalkyltriazenes mas confirmed by isolation. -4specimen, isolated following the dissociation of the butyltriazerie in mater, was shown by its melting point, infrared spectrum, and paper chromatographic behavior to be identical with authentic AIC free base. Since the absorbance of pure h I C free base in niethaiio1 or buffer is zero above 315 nip, the rate of disappearance of the siniple triazeiies may be estimated from the rate of disappearance of the absorption maxiniuni in this region. The straight-line plot (Figure 2 ) of the logarithm of the absorbance at 320 nip us. time shows that the decomposition of the butyltriazerie (Vc) follows first-order kinetics. I n methanol, the half-life (to.&)of the butyltriazerie at 25" was determined from Figure 2 to be 32 min.; in phosphate 1.15 1
0.46 1 0
I
10
I
I
30 Time, min. 20
1
I
40
50
60
Figure 2.-First-order decomposition of Va and T'b to I at 25'. A, Tc (7.36 X M ) in methanol, absorbance at Amax 320 mp; B, Va (13.6 X A4) in phosphate buffer ( p H T), absorbance a t A,, 320 mp.
buffer a t p H 7, t o 5 of the niethyltriazene (Va) was similarly determined to be 8 min. Other observations of the dissociation of Va-c in methanol or buffer solutions were made a t uncontrolled laboratory temperatures (near 25") and give, therefore, only rough approximations of to.i; the differences were sufficiently great to permit qualitative comparisons. Thus, in methanol the approximate half-life values observed in this way for Va and Vb were 8 and 1 hr., respectively; in buffer, Vb and Vc had been converted almost completely to AIC when the first curves were traced a t 7 and 2 min.,'j respectively. The cyclohexyl (Vd) and t-butyl (Ve) derivatives proved to be less stable than the straight-chain alkyl derivatives. A specimen of ( 6 ) These time intervals included periods of slower decomposition in methanol since the buffer solutions were prepared by first dissolving the specimens in methanol (because of their sparing solubility in water) and then diluting to the final concentrations with buffer.
35
Vd was obtained, but it decoriiposed 011 staiidiiig ill the solid state. Addition of the diazoiniidazole (111) to a solution of t-but'ylamine caused the evolution of a gas, which was assumed to be nitrogen, and 2-butene formed by immediate decomposition of the t-butyltriazeno derivative (Ve) ; AIC was demonstrated by paper chromatography to be present in the crude residue from the reaction. Three additional structural variants (Vf and g, Vh arid i, and Vj and k) mere prepared, and their stabi!ity and biological activity were investigated. Basic groups were int'roduced into the triazerie moiety as a possible means of stabilizing the triazene grouping. The diethylaminoethyltriazerie (Vf) was, indeed, niore stable than the siniple alkyltriazenes, its half-life in buffer solution a t 25' being approximately 3 hr. Separat'ing the basic group from t'he triazene group by four carbon at'oms decreased the stability; Vg appeared to form XIC in buffer at least as rapidly as the methyltriazene (Va). Both the ethoxycarboiiylniethyl (Vh,i) and the aryl derivatives (Vj,k) decomposed in buffer solution more slowly than the alkyltriazenes. The spect'ral changes mere more complex than those of Va-c and Vf,g, arid interpretat,ion was further complicated by the presence of ultraviolet-absorbing aryl groups in Vi-k. However, it was apparent that the p-methoxyphenyl derivative (Vk) was more stable than the methyltriazene, that the r:ite of disappearance of t'he phenylalariate derivative (Vi) approximated that of t'he diethylaniinoethyl derivative (Vf), that the glyciriate derivat'ive (Vh) W.J more stable thaii Vi, and that the p-bromophenyl derivative (Vj) was the most stable of the derivatives prepared. :\laxima near 270 nip, suggestive of AIC, eveiit'ually appeared in the spectra of solut'ioiis of the glycinate and aryl derivatives, but it has not been established whether AIC is a decomposition product of these triazeries at' pH 7. Whereas the ethyl (Vb) arid butyl (Vc) derivatives rapidly decomposed iri 0.1 N sodium hydroxide to XIC (A,, 277 nip in base), Vh and i were converted immediately by 0.1 N sodium hydroxide into products having maxima at 254 and 262 mp, respectively. In methanol, Vh and i displayed a very slow decrease in their absorption maxima during a period of several days, and a methanol solut'ion of the p-broniophenyl derivative showed no change during an 8-day period in which the solution mas protected from light only during the first day. The stability of the product of I11 arid p-bromoaniline suggested that it might be an azo compound formed by rearrangement of the initially formed triazene.' This possibilit'y was eliminated by treating the pbromophenyl derivative at pH 1.5 with S,S-dimethylaniline. The principal product obtained was 4-bromo4'-(diinethylaniino)azoberizerie (VI), which was ideiitified by comparison with an authentic The p-broinobenzenediazonium ion necessary for the formation of the azo compound VI must have come from the p-bromophenyltriazerie (Vj). Paper chromato( 7 ) T h e preparation of 5(or 4)-amino-2-(p-bromophenylazo)imidazole4(or 5)-carboxamide by coupling of .\IC and p-bromobenzenediazonium chloride has been claimed: I-.Hirata. I . Teshima, and K . Iuashita, R e s . R e p t . S a u o y a I n d . Sci. Res. Iiist., No. 8 , 70 (1955); Clirrn. .4hstr., 61, 57581 (1957). (8) H. Goldschmidt and f3. Ilarilach, Ber., 26, 1374 (18Y2); I. S . Zlimrirova, Zh. Ohslich. Khim., 27, 2704 ( 1 9 5 i ) ; J . Geii. Cliem. l.SSR, 27, 2 i 4 7 (1957).
Experimental SectionI2 5(0r 4)-(Substituted Triazeno)imidazole-4(or B)-carboxamides (Va-k).-The general procedure used to synthesize the triazenes Va-k is illustrated by the preparation of the 2-diethj-1aminoethyl derivative (Vf). During all operations involved i n the preparation and purification of the triazenes, they werr protected from light as much as possible by wrapping the rraction flasks, filter funnels, or other equipment used to contain the triazenes with aluminum foil. 5-Diazoimidazole-4-carboxamide (111) (400 mg., 2.9 mmoles) was added in small portioiiq during 1.5 hr. to a well-stirred solution of 2.05 ml. (14.6 mmoleh) of redistilled X,N-diethylethylenediamine in 10 ml. of dry ethyl acetate. The reaction mixture was kept under nitrogen and was protected from moisture. The mixture was stirred for a n additional hour, and the precipitated product was collected on a filter, washed well with ethyl acet,ate, and dried in a high vacuuni -
(12) Solvent systenis used t o develop paper chromatograms were: (1) I-butanol saturated vitli water, (2) I-butanol-acetic acid-water ( 5 : 2: 3 b y volume), (3) 2-propanol-water-concentrated aqueous ammonia (70: 2 5 : 5 IJYvolume), and (4) acetate buffer (pH 6.1 or 0.7). Spots were detected with illtrar.iolet light a t 254 and 365 my.
January 19GG
IMIDAZOLES. I1
37
a t room temperature over P206; yield, 694 mg. (94%). Alarger amount of Vf that, according to its infrared spectrum, contained a small amount of the diazo starting material was purified by slurrying 5-10 min. with water at p H 9, washing with water, and drying rapidly; yield, 80%. Data on the preparation and characterization of the individual triazenes are recorded in Table 11. Because of the instability of the triazenes, purification procedures, other than washing or slurrying the crude products with ethyl acetate or with ether, were usually not practicable. An exception, in addition to T'f, was the p-bromophenyl derivative ( T j j, which was recrystallized from 2-methoxyethanol by the addition of a mixture of benzene and hexane. Generally, however, the preparative procedure was designed to cause precipitation of the triazene in analytically pure form. All of the triazenes are stored in brown bottles over a drying agent. Slthough they appear t o be stable a t room temperature when stored in this manner, they are routinely kept a t 0-5'. 5(or 4)-Aminoimidazole-4(or 5)-carboxamide (I) from 5(0r 4)(Butyltriazeno)imidazole-4(or5)-carboxamide (Vc).-A suspension of 200 mg. of Vc in 15 ml. of water was stirred a t room temperature. A gas, presumed itoj be nitrogen, was evolved, and the mixture became homogeneous within 1.5 hr. The ultraviolet spectrum of an aliquot indicated that only AIC ( I ) was present. Lyophilization of the reaction solution left 144 mg. of product (theoretical yield of AIC monohydrate, 138 mg.). Recrystallization of 100 mg. of this material from water and drying of the recrystallized product in vacuo over PZOS at room temperature gave 58 mg. (adjusted yield, 60%) of crystals melting at 172-174" dec. (capillary), lit,.13 liO-lilo. The infrared spectrum was identical with that, of authentic AIC monohydrate, and paper chromatograms showed only AIC. Acidic Dissociation of 5(or 4)-(p-Bromophenyltriazeno)imidazole-4(or 5)-carboxamide (Vj).-A mixture of 400 mg. of Vj, 3.3 ml. of SIX-dimethylaniline, 20 ml. of ethanol, and 20 ml. of water was acidified to p H 1.5 with concentrated HCl and stirred a t room temperature for 42 hr. A red precipitate was collected by filtration, washed with water, and dried; 316 mg. (8070 calcd. as VI). Paper chromatography showed that this material was chiefly 4-bromo-4'-( diniethy1amino)azobenzene containing small amounts of two impurities. Paper chromatograms of the filtrate were complex, containing several spots one of which was VI. Recrystallization of the red solid from ethanol gave 180 mg. (46%) of red crystals, m.p. 159-160". The infrared spect,rum and paper chromatograms of this material were identical wit,h those of a n authentic specimen of VI (m.p. 159160') prepared by coupling p-bromobenzenediazonium chloride with N,N-dimethylaniline.8 Paper chromatograms of a second crop (60 mg.) contained spots corresponding t o VI, the starting material T'j, and 5(0r 4)-(p-dimethylaminophenylazo)imidazole4(or 5)-carboxamide4 (VII). The latter compound is the expected product if dissociation of V j gives the diazoimidazole I11 and p-bromoaniline. Ultraviolet spectra were recorded with a Cary Model 14 recording spect,rophotometer. Solutions of the triazenes were prepared by rapidly dissolving a weighed specimen in 5-10 ml. of methanol and quickly diluting to the final concentration of about 10 mg./l. with either methanol or p H 7 phosphate buffer except, for Vh and i, which had to be dissolved initially in a small quantity of dimethyl sulfoxide before the final dilutions were made. The time ( t ) was recorded when a tracing was begun a t 400 mp; about 80 see. elapsed between the beginning a t 400 mp and the recording of maxima near 320 mp. Solutions were protected from light during their preparation, and stock solutions were stored in the dark either at uncontrolled room temperature (near 25') or, where stated, at 25' (thermostated bath). During studies on the decomposition of a triazene, a fresh portion of the stock solution was rapidly transferred to the cuvette for the tracing of each curve a t a given time. 111addition, observance of the dissociation of the methyltriazene (\-a) and diethylaminoethyltriazene (Vf) derivatives in phosphate buffer was repeated n-ith a Perkin-Elmer AIodel 202 spectrophotometer; the triazene solution was left continuously in the thermostated ( 2 5 ' ) cuvette, readings were made at the long wave-length maximum, and the solution \vas shielded from the light beam between readings. The half-life values were in agreement with those found by the method described above. (13) J. A. Montgomery. K. Hewson, O r y . Chem., 24, 256 (1959).
R. F. Struck, and Y. F. Shealy, J .
Acknowledgments. -The authors expreis their appreciation l o Dr. W. C. Coburn, ,Jr., for helpful discussioiis, to Dr. J. A. ,\loiitgoniery for enrouragemeiit iii this work, to A h . W. E. E'itzgibboii arid associates of the Organic Preparatioiis Sectioii for prepariiig largc
yuaiit itics of iiit(~riiiediatt~i, 1 o A \ L i i . 1 ran be easily separated by thcii differing acidity. Iri three other c8n-e- we cnrried out a reduction of S-nitrosoaiithranilic acids XI i t h sodiuiii hydrowlfite following a procedmcx 1)reviou4ydescribed. The :3-1iydroxp-lH-iiid:izoles weir' traiiiforinetl t o the cwrresponding hodiuiii salt\ a i d nlloir-ed t o rea(-t I I I i w r t \olveiits M ith tlic 1)rot)or chloroalkgldialkyl:iiniiic+, ubing differelit bn-e., -olveuts, ratioq of ic:ictiiig substancei, teiiilmttture-, aiid periodi of heating Through a11 these varying coiiditioiiq, the formation of the derivatives of type I was always :iccompanied by side products. Only i i i a few c a v s were these isolated and identified : generally, we limited ourselves to sep:~rating compounds I froiii the mixtures. This could bc easily : ~ c ~ i ~ o ~ ~ i ~1)y ~ l (i 's~llil ~' (~J it ill ~ L t O ~ l ' (~i1c~e) ~1~3~~ p cI-~ i \
(1) L l : ~ i u i < 111 1Yfi'li
(1
(
or-1, ,tud C,
i'aia/~u l r o t
( h , m . ( I t u i n t I , 66, I I t )
