Imidazolecarboxhydrazicles.
I. Chemistry and Biological Evaluation'
The Drug-Plastic Research and Toxicology Laboratories, ('oilege qt Phni tnac 11, I
riii ti \it!/ oj" ?'r.cas,
.I u s h a , 7'cxas
Interaction of dimethyl imidazole-4,S-dic.arbosylate Kit ti hydrazirie mid ,wnie alkyl- niid aryl-suhstitiit ed liydrazines provided a s e r k of imidazole acid hydrazides;. The molecular structure of 1heae compoilnds was elricidated by chemical and/or spectroscopic methods. RIost of theae wmpoiinds together with so~iieiiit ermedialc': were screened for monoamine oxidase (11.40)inhibitory avt ivity. Lncier oiir test (widit ions, two of these coinpourids were foiuid to be significantly active for inhibiting the enzyme. They \\-we irnidazole-4,5-dic:~r~osylii~ acid 1-methylhydrazide 2-methylhydrazidr i I V ) mid I - m e t t i ~ l i i t i i t l a ~ o l e - l , ~ - c ~ i ~ acid ~ : i r ~dihydr:izitlr ~~~s~li~~ (XII).
Since the discovery of biological activity of isonicotiriic acid hydrazide? arid its therapeutic use as an antituberculous and antidepressant agent, numerous carboxylic acid hydrazides4-'j have been synthesized for biological evaluiition. At the present, there are a few heterocyclic. acid hydrazides that are potential monoamine oxidase (JIAO) inhibitors arid are being utilized in medicine. Our interest in the synthesis of iniidazole-4,.i-dicfiarboxyliracid hydrazides was for their value as possible intermediates in the syrithesis of bicyclic pyridazine: ~ i r d imidazole-coiitairiiiig systcnis. However, also of vhief interest WLS L: study 011 the chemistry of tliesr hydrazides :tiid their hiological properties. The present paper is LI report on the researclh carried out in the chemistry of these acid hydrazides niid screening methods for deterniiriirig their 1ZAO iiihibitory properties. Attempts were also made t o relate the rariation in tlic l~iologicalactivity :IS :L furictioii of cheniic:il structure. Chemistry.- - X qu:mtitatiw yield of imidazole-4,5dicztrboxylic acid dihytlrazide' (IIa), obtained at room temperature as a rwult of tlicl interaction of dimethyl i1iiidazole-4,5-dicarboxylate(I) and hydrazine, suggested that p o 4 d y I could be employed for the preparation of a series of substituted acid hydrazides desirable for our study. The reaction of inorlosubstituted hydrazine with I, where the forniatioii of positional isomers was possible, aclvaiiced ilie idea for studying thv factors influencing the ratio of these isomers iri a givcri reaction stid their relative physical and biological properties. ?'lie intcractiori of methylhydrazine with I in methanol Itas 11ec~1 attcliipt c(18 arid :issunwd to give o111y 111) I I I it low yirlti without proof of tlic structural f o r n d t , whicali also c~ouldco~icclivablybe III or IIT (Chart I ) . Our iiivehtigatiorr, howver, revc&d that the re:iction product i t as otic cmnpouiid i r i the i h x w c c of a solvc~nl and two i i i the presmc*eof a solvcwt such as 1-butanol. I'reliniinary study of these product.: by elemental an:ily( 1 ) This investigation \\'as s u g p u r t e d in part by research Grant C'..\-OG120 from the Xatiunal Cancer Institute, I3etliesda, RId. ( 2 ) 13:. A . Zeller. .J. Barsky, J. R. Foiits, \V. I'. Kirchheimer. a n d I.. S. Van Orden, h'zperientu, 8, 349 (1952). ( : 3 ) 13. 13. I3rudie, A. Pletscher, a n d P. .\. Pliore. J . I ' h a r m n c o l . l ? r p l / . 7 ' t l ? T G j J . , 116, 9 (1956). ( 4 ) TI. L. Yale, K . Lusee, .J. Martins, h l . Ilolsiny, F'. 11. Perry, a n d .1. H?rtistein, .I. Am. Chtm. Sac., 76, 1933 (1953). ( 5 ) .'E Zeller, A . Pletscli iitmann, 13. Ilegedus. and 0 . Strarib, A n n . S. Y . Acad. (6) T. S . Ctardner, E. Wenis, a n d J. Lee, .I. .Ired. J ' h f i r r n . Cttrm., 2, K K i (l9tjO). ( i ) K. G . .Jones, J . A m . Ctiem. Soc., 78, 159 (1956). ( 8 ) I{. S. ('astle and \ \ . S. Peesr, J . Ory. Ctirrn., 23, 153.1 (l!J58).
( ' I T \12 I.
H
+
HzNHHR R = H. CH,
I
-
I
H
H CONHNIIR &CONRNH2
b & ,,
CONRNHZ IIa, R = H b, R=CHa '
111
CONRNH, CONHNHR IV
and infrared spectroscopy showed that they I\ (w tlihydriizides of type 11, 1x1, or IT', and that the highmelting isoiiier, which was obtairicd in the prewicch of' :I solvent, w:iq identical with the product obtaiiied iii the :hscwce (Jf it solvcwt. Attcnipts \vwc iii:idc t o identify these c.onipouncls by c~oiivertiiig tlieni to t l i ( corresponding :miides by cleaving the rr~trogcrrnitrogen bond with IiaIiey nickel, by the method rcported by Sirisworth, and coniparing the3e :iinitlcs n i t h the authentic samples obtained as n result of reacting I n-itli methylamine or ammonia. This method, houever, WLS riot ~uccessful; riclither :i produd iior the \tartirig vompouiicl could be isolated froni t h c I ~ i i ~ i c y iiicl\el--c~th:tiiol4urry. This failure might he t l u t~o thct for.ni:it 1011 of a i 1 iniidnzole :wid hydrtLzid~iiic~lie1 (WIIIples. S n i r spwt rohcopy was employed nvxt for t h r d u c3id:ttiori of tht. qtructure of these isoiiier5. The strwtuix* iiiiitl~zolc-l,.i-clic:~rboxylic:wid 1-nictl~ylhydrxzidc 2methyl1iydr:izidc (IT-) was :issigned to the lom-nieltiiig. w:Lt cr-soluhlc isoincr 1)ernuse its m i r spcctruni r c ~ c d c c l tlic p r ~ w r wof t u (i rcsonn7ice pe:tks for methyl protoiis :tt b 3.0 aiicl 2.9. 1)ue to tho cleshieltling iiifluorire ot thc(8:Lrhonyl group, thew resoriarice pr:tbs were :Lssigiied t o methyl groups iiexl to and :twag from the c:irhonyl group, respectivdy. C'oiiiparison of the positioii of t hehe resoIiaiiw peaks with the single resonawr p d :it 6 3.0 for the methyl protoris of the high-nieltiiig i'oiiier established the structure of this compound t o 1 ) ~ iiiii(lazole-4,.i-dic.:vboxylic :toid bis(2-methylhytlrazitlr) (1Ib) rather tliari 111. Ll f her mnfirmation of t hc, riiolecular strurtiirc of ILb acc~omplishdby treating it with acetone. Viililr on(' yidtl* :I clihytlr
I ~ ~ I n A Z O L E C A R R O X H Y n R ~ 4 Z I D E S1 .
September 19BG
compound was obtained after heating I I b with acetone for 48 hr. I n a like manner, the reaction of I with phenylhydrazine was examined and found to be inconsistent with the literature report8 The reaction product in the absence of a solvent mas not analogous to the methyl derivatives IIb, 111, or IV, but rather a high yield of a half-ester, which by chemical and physical methods, discussed for TI,proved to be V (Chart 11). However, when the reaction was carried out in the presence of 1-butanol, the major product was imidazole-4,5dicarboxylic acid bis(2-phenylhydrazide) (TI) although a very sniall quantity of V, together with another substance which was not identified, was also isolated. The assignment of the structure VI. to the major product of the reaction was made first on the basis of its infrared and nnir spectra. A single resonance peak for phenyl protons at 6 6.9 indicated the existence of equivalent magnetic environments for phenyl rings. This assignment was further substantiated by treating the product with acetone analogous to the treatment given to methyl derivative IIb. In this case, likewise, no hydrazone was formed and only the starting compound was isolated. Similarly, the molecular structure of T‘ W L S proved.
66 1
dicarboxylic acid bis(2-isopropylidenehydrazide) (1’111) as a possible route for the synthesis of X was explored. Prolonged heating of I I a in acetone and methanol afforded VIII, and catalytic hydrogenation of VI11 at high temperatures afforded X. The isolation of a halfhydrogenated product, imidazole-4,5-dicarboxylicacid 2-isoprop ylidenehydrazide 2-isopropylhydrazide (IX), after a short period of hydrogenation, suggested that this reduction occurs stepwise, according to Chart 111. CHARTI11
H
CONHN=C(CHJ, CONHN=C(CH3),
PtO
A H,
VIII H
CONHNHCH(CH3)I
H?
CONHN =C(CH3)* CHIKT I1
IX CONHNHCH(CH3),
Is “zCH3
+
CONHNHCH(CHJ2 H2NNHC&,
V
L
( E-.C O z C H 3
X
H CONHNH C&5
I
CONHNHC&, VI
The reaction of 1,l-dimethylhydrazine with I occurred under a somewhat strenuous condition to give imidazole-4,5-dicarboxylicacid bis(2,2-dimethylhydrazide) (T’II). The lorn rate of formation of T’II could H
CONHN(CH,), CONHN(CH,&
It was desirable to synthesize 1-methylimidazole-43dicarboxylic acid dihydrazide (XII) for its value as an intermediate in the synthesis of some heterocyclics and also for the purpose of comparison of its physical and biological properties with IIa. Interaction of hydrazine with diethyl l-methylimidazole-4,5-dicarboxylatehas been reported by Jones’ to yield only 1,4-dihydroxy-5methylpyridazino [4,5-d]imidazole (XIII). Exploration of the possibility of synthesizing XI1 revealed that, by adding hydrazine to dimethyl 1-methylimidazole-4,sdicarboxylatell (XI),XI1 could be obtained without any difficulty (Chart IV). Unlike IIa, XI1 is a relatively low-melting, water-soluble conipound and manifests pronounced biological activity.
VI1 CHARTIV
possibly be ascribed to the influence of the steric hindrance. The steric factors together with a relative lessening of hydrogen bonding in the molecule might be the elements which contribute to the relative water solubility and low melting point of VI1 as compared with the other structures in the series. Because of pronounced biological properties manifested by some aliphatic and heterocyclic acid isopropylhydrazides,6t10it was also of interest to prepare imidazole-4,5-dicarboxylic acid bis(2-isopropylhydrazide) (X). Due to the difficulty in obtaining isopropylhydrazine, synthesis of the intermediate imidazole-4,j(10)F. H. bIcMillan, F. Leonard, R. I. Meltzer, and J . -4.King, J . A m . Pharm. Assoc., Scz. Ed., 42, 457 (1953).
OH
/
CHJ
OH
\
XI11
XI
XI1 (11) R. A. Baxter and
r. 5. Spring, J . Chem. Soc., 232
(1945).
IMIDAZOLECARBOXHYDRAZIDES. I TABLE I IMIDAZOLECARBOXYLIC ACIDHYDRAZIDES X
NO.
X
IIa IIb 11; I17 1-11 VI11 IX
H
H H H H H
X
H
H H
Y
Z
HNNHi HSNHCH, HXNHCH, HNNHC6Hs HNNHCsHs HNN( CH3)n HNX;=C(CHB)i HNXj=C(CH3)2 HSNHCH( CH,)n NHNHz
HNNHz HNNHCH3 CHsNXHz OCH3 HNNHCsHj HNN( CH3)2 HNN=C(CH3)2 HN?rTHCH(CH,)i HNNHCH(CH3)Z HNSHz
XI1 CH3 Isocarboxazid ... a &.leltingpoints vary somewhat, with the rate of heating, point was determined at a rapid rate of heating.
hlp,
b
Formula
OC"
>300 2 10-2 12 195-197 247-250 219-220 179-183b 245-25W 253-256 210-212 232-23jd
CjHS?;602 CTHiiN6Oi C7H12N60i CizHnN403 C17H16N6Oi C9Hi6X6Oi.HzO CiiHi6N6Oi.0.5HiO CiiHisNd32 CnHzON6Oi C6HioN6Oi
...
...
&felting point before crystallization.
was separated. This compound was crystallized from methanol and water to give 0.9 g (50Y0)of X I I : mp 232-235'; urnax 3345, 3285 (NH), 1665, 1645 cm-l (C=O). Anal. Calcd for CsHloN6Oi: C, 36.36: H, 5.09; N, 42.41. Found: C, 36.40; H, 5.29: N, 42.54. Pharmacological Methods.-The compounds listed in Table I were screened for possible monoamine oxidase (MAO) inhibitory activity by the method of Aceto and Harris.15 I n addition to this test, a study was made to det'ermine the degree of sleeping time potentiation by some of the compounds in mice. Finally, the effect of the series on rabbit blood pressure, respiration, EKG, and EEG, following intravenous administration, was iiivestigat ed. M A 0 Inhibition.-A dose of 1.1 mmoles/ke was administered by the oral route to mice. Since some of' &e compounds were relatively insoluble in water, a 37, suspension of gum acacia in distilled water was used as a vehicle. Accordingly, mice used in the experiment were removed from food for 24 hr prior to oral intubation of the test compounds, while water was available ad libitum. Five mice in the weight range of 20 to 28 g were intubated with the calculat'ed dose and observed for general signs of activity over a 2-hr period. Reserpine was then administered a t a dose level of 2.5 mg/kg by the intraperitoneal route and allowed to remain undisturbed for the next 3 hr. At this time, each mouse was evaluated as to the degree of pt,osis by at least tIvo individuals. The resdts of the ratings were compared with t w o sets of control animals, one receiving the vehicle and reserpine only, the other receiving only aqueous reserpine. The dose of I\' and XI1 had to be reduced due to extreme symptoms of toxicity demonst>rated a t the 1.1-mmoles/kg level. Isocarboxazid, a therapeutically employed M A 0 inhibitor, was administered in analogous manner for purposes of comparison of activity (see Table I). Sleeping Time Potentiation.-AIost M A 0 inhibitors potentiate the sleeping t,ime of mice under hexobarbital narcosis. Consequently, 10 mice each for IV and XII, which manifested significant inhibitory activity in the ptosis test, were orally administered a t a dose level of 0.275 mmole/kg and 0.525 mmole/kg, respectively. Two hours later the animals were given 60 mg/kg of hexobarbital sodium intraperitoneally. Ten mice, receiving only the vehicle orally and hexobarbital intraperitoneally were used as controls. Sleeping time w3s recorded as the time from administration of hexobarbital until the mice regained the ability to return to a righted position three times within 20 sec. Effect on Rabbit Blood Pressure, Respiration, EKG, and EEG. -Rabbits were anest,hetized with 30 mg/kg of pentobarbital prior to intravenous administration of the compound. The animals were then surgically prepared for direct cannulation into the right common carotid artery to record blood pressure while (15) M. D. Aceto and L. P. Harris, J . Toxicol. A p p l . Pharmrol., 7, 392 (InR?).
-Ptosis Oral dose, mmo1es;kg
1.1 1.1 0.275 1.1 1.1 1.1 I .1
inhibitionActivity prior t o reserpine
Mean ptotic score
Normal Normal Increased Increased Increased Reduced Reduced
1.0 0.5 2.6 0.8 1.0 0.5 0.5
...
...
... 1.1
Increased 0.8 0.525 Increased 2.2 0.275 Increased 3.8 See ref 14. When the melting
the left external jugular vein was cannulated for administration of the compounds. The diffuse cortical electroencephalographic effects were monitored through subdermally implanted electrodes. Electrocardiographic responses were recorded from needle electrodes in the legs and chest. Graded doses of each compoiind were administered a t various time intervals and the results were evaluated before the next administration.
Results and Discussion A summary of the results obtained in the ptosis study is recorded in Table I. Compounds IV and XI1 manifest significant activity as is shown by its ability to prevent reserpine-induced ptosis. If the activity of these compounds is compared on a mole to mole basis, it may be seen that isocarboxazid is approximately 1.5 times as active as IV and 3 times as active as XI1 in ptosis inhibition. These two compounds, together with T W , are soluble in water. Although water solubility seems to be a criterion for an enhanced biological activity in this series, molecular structure plays a more important role. I n VII both hydrogens in the N2 position of the chains are substituted by methyl groups, which in a sense confirms the postulate16 that, in order for an acid hydrazide to have a M A 0 inhibitory property, the presence of a t least one hydrogen on ?? is necessary. Except for I V and XII, the remaining compounds under our test conditions did not demonstrate M A 0 inhibitory property. Therefore, one may postulate that the hydrazide linkage in the less soluble compounds in this series is too stable to release the active hydrazine17 at the target site. However, VI1 is a soluble compound, but does not possess activity analogous to IV and XII. Compounds VI1 and VI11 both demonstrated some degree of toxicity which was manifested by reduced activity in mice before reserpine administration. A similar reaction was observed when isocarboxazid was administered. Compounds V, VI, and X caused the mice to show an increased activity 2 hr after administration, but were not able to inhibit the (16) J. H. Biel, A. Horita, a n d A. E. Drukker, "Psychopharmacological Agents," hlaxwell Gordon, Ed., Academic Press Inc., New York, N. T., 1964, p 359. (17) J. H.Biel, P. A. Nuhfer, and A. C. Conway, Ann. N . Y. Acad. Sci., 80, 568 (19.59).
