July 1966 105" and maintained a t this point for 15 hr. The residue, left after distilling the solvent under reduced pressure, was stirred well with ice water and the unreacted starting materials (1.9 g) were filtered off rapidly. The filtrate was adjusted to p H 8.5 (cooling) and the product was extracted with chloroform, washed with water, and dried (NazS04). Filtration and removal of the chloroform left a thick oil which solidified on rubbing with petroleum ether (bp 30-60"). Recrystallizations from chloroform-hexane (Norit) yielded 1.99 g (29YG)of 1- as white crystals, mp 106-108". Two additional recrystallizations from chloroform-petroleum ether (bp 30-60") gave an analytical sample of T' as white, needle-shaped crystals: mp 107-109"; infrared (CHCl,), 2.96 and 3.10 (NH), and 5.83 p (C=O); ultraviolet maximum (95yoethanol), 2.04 mp (log E 4.43), showing a bathochromic shift (10-3 S alcoholic KOH) to 2.37 mp (log e 3.28). .4naZ. Calcd for CljHZ0S2O2:C, 69.20; H, 7.74; S , 10.76; mol wt, 260.3. Found: C, 69.06; H, 7.82; S , 10.69; mol wt, 261.8.
NOTES
617 Experimental Sections
The a-ketoaldehydes were prepared from the corresponding a,a-dichloroacetophenones.6 N-Pyrro1idinoacethydrazide.-A mixture of ethyl N-pyrrolidinoacetate (15.7 g, 0.1 mole), hydrazine hydrate ( 5 g, 0.1 mole), and 20 ml of ethanol was refluxed for 4 hr. The solvent' n-as evaporated and the crude oil was distilled a t l05-11Oo (0.2 mm), yield 11 g (77%). AnaE. Calcd for C6H13?;30: S, 29.35. Found: X, 29.49. Treatment of an ethanol solution of the free base with HC1 gave the hydrochloride salt, which was recrystallized from ethanol, mp 203-206" dec. Anal. Calcd for C6HI8N3O.2HC1: C, 33.34; H, 6.99; CI, 32.82; N, 19.44. Found: C, 33.11; H, 6.96; C1, 32.34; N, 19.63. The N-piperidino-, N-morpholino-, and K-diethylaminoacethydrazides were prepared by the same p r o c e d ~ r e . ~ Phenylglyoxal N,N-Disubstituted Aminoacethydrazones. General Procedure.-A mixture of the a-ketoaldehyde (0.01 mole) and the corresponding N,N-disubstituted aminoacethydrazide (0.01 mole) in 10 ml of methanol or ethanol was stirred a t 20" for 8 hr. After cooling, the products were filtered and recrystallized. The yields, melting points, solvents of crystallization, Antiviral Compounds. XIII. Aminoand analytical data are summarized in Table I. Biological Testing. Maximal Tolerated Dose (MTD) in the acethydrazones of A r o m a t i c a-Ketoaldehydes Embryonated Egg.-The compounds were dissolved in saline solution buffered a t p H 7.2, containing 500 IG of penicillin G E. MASSARANI, D. NARDI,L. DEGEN,. 4 S D M.J. MAGISTRETTI and 0.5 mg of streptomycin/ml. Descending doses of each compound dissolved in 0.1 ml were inoculated into the allantoic Research Division, Recordali s.a.s., Xilan, Italy sac. Each dose was injected in three embryoiiat'ed 9-day old eggs. The highest dose which did not provoke mortality within j Received January 22, 1966 days was defined as the IITD. Antiviral Methods.-Embryonated 9-day-old leghorn hen eggs and influenza A viruh [allantoic fluid containing 108-109 EIDjo I n a previous paper we described the synthesis of (median egg-infectiiig dose) of egg-adapted PR-8 strain] were water-soluble derivatives obtained by condensing aused. Vaccinia mouse neurotropic virus [(ATCC) CAM ketoaldehydes and Girard reagent.' Since several (chorioallantoic membrane) homogenized and purified by aromatic a-ketoaldehydes have been shown to exhibit cent,rifugation cont'aitiing 106-107 ELDjo (egg lethal dose) of antiviral we have prepared a series of new egg-adapted WR strain] was used. A. Virucidal Tests.-For each dose, 0.5 NTI) dissolved i n 10 phenylglyoxal N,N-disubstituted aminoacethydraml of buffered saline solution was added t o lo2, lo3, or lo4 EID,, zones in order to study their antiviral activity (Table I). and the three solutions were kept in water baths a t 37' for 1 hr. All compounds were tested on embryonated eggs inThen the allantoic sacs of 5 eggs (for each dose) were inoculated fected with vaccinia virus and A-PR8 virus. They with 0.1 ml of one of the incubated solutions. B. Virustatic Tests.-For each dose, the allantoic sacs of were found inactive against vaccinia virus; the phenylfive eggs were inoculated with 0.1 ml of allantoic fluid containing glyoxal derivatives were also inactive against ,4-PR8 either 1, 10, or 100 EIDo5of virus, and the eggs were stored 1 hr virus. Some derivatives of biphenylglyoxal (4 and 5) at 37". Then the allantoic sac was inoculated with 0.1 ml of of p-phenylthiopheiiylglyoxal (9), and all derivatives buffered saline solution containing 0.5 3 I T D of each compound. of p-phenoxyphenylglyoxal exhibited virucidal acC. Evaluation of the Activity.-For iiifluenza virus, the eggs were stored a t 35" for 48 hr, then a t 4" for 12 hr, arid finally tested tivity against A-PR8 virus. for the presence of hemoagglutinin. For vaccinia virus, the eggs No activity was observed up to a concentration of 50 were stored a t 37" for 7 days and the mortality of chick embryos pg/ml when the compounds were tested for bacteriowas recorded. static activity on the following niicroorganibins : Antimicrobial and Antifungal Methods.-The compouiids were diluted in 1 : 2 ratios in Difco nutrient agar inoculated with E. Escherichia coli, Pseudomonas aemginosa, Proteus vulcoli 100, P. aeruginosa 112, P. vulgaris, R. subtilis, S.azirezis S.G. garis, Bacillus subtilis, Staphylococcus aureus, Xtrepto511, and i l l Ilifco brain heart, infusion agar inoculated with coccus pyogenes, JIycobacterium tuberculosis, TrychoStr. pyogenes huinanus A88, and the results were read after phyton, and Candida. incubation for 18 hr at' 35-37'. By the same procedure the The compounds were also screened for acute toxicity compounds were tested in Kirchner-Hermann medium 10yc beef serum inoculated with X . tuberculosis 37 Ra, and the results in mire, for smooth iiiuscle relaxing activity, for effects were read after incubation for 10, 17, and 24 days at 35-37'. on blood pressure and respiration, for coronary vasoThe compounds were tested also in Sabouraud broth inoculated dilatation, and for antiarrhythmic, antitussive, antiwith Trichophyton nzentagrophytes 1236 (the result,s were read convulsant, and antiinflammatory activity. A low after incubation for 4 days at 26'), and with Candida albicans 28 (the results were read after incubation for 18 hr at 35-37"). aritictoiivulsant activity was shown by 3-6; conipoundr Pharmacological Methods.-For all t'ests N I I R I albiiio mice 4, 8-10, arid 13 exhibited antiinflammatory activity on and Wistar albino rats were used. The acute to.cicity of each formalin edema, but were ineffective as analgesics. compound was determined by administering it intraperitoneally The data on acute toxicity, antiviral, anticonvulsant, to mice in descending doses. XIortality was recorded over 24 hr and antiinflammatory activities are summarized in and indicative LDso values were estimated. Smooth muscle relaxing activity was t,ested in vitro by hIagnus' Table 11. method8 o n the small intestine of a guinea pig stimulated by
+
i l l E. Massarani and L. Mauri. Boll. Chim.Farm., 103, 904 (1964). ( 2 ) R . B. hloffett, B. D . Tiffany, B. D. Aspergren, a n d R. V. Heinzelman, J . A m . Chem. SOC.,79, 1687 (195;). (a) C. A. De Bock, J. I3ruy, a n d J. S . Walop, .Vature, 179, 706 (1957). ( 4 ) G. Cavallini, J . X e d . Chem., 7 , 255 (1964).
(5) All melting points are corrected and Tvere determined on KoflerHeiztischmikroskop melting point apparatus. (6) L. Rlauri and D. Nardi, Farmaco (Pavia), Ed. Prat., 18, 651 (1963). ( 7 ) R I . Pesson and S. Dupin, Bull. Soc. Chim. France, 250 (1962). (8) R. l l a g n u s , A r c h . Ges. Phusid. Pfluuer's, 102, 123 (1904).
