NOTES
May, 1964 The resulting pale yellow solution was poured into 600 ml. of ice and water and XIV precipitated out (yield 957,). It was dried and sublimed a t 3 mm. pressure. White crystals of XIV were obtained which melted at 104-105.5". The infrared spectrum exhibited the expected strong secondary amide linkage at 1665 em.-', strong acetate absorption at 1220 cm.-', and C-C1 absorption from 700-800 cm.-'. Anal. Calcd. for CI4H11Cl&O: C1, 23.32. Found: C1, 25.19. Biological Results.-Screening of the compounds prepared was done with 2-month old Swiss Albino mice.26 The compounds listed in Table I were screened in the Biology Department of Hunter College.*' The procedure used was the established technique of the Sloan-Kettering Institute,28except that instead of injecting 24 hr. after implantat,ion of tumors, injections began between 72 and 96 hr. after implant,at,ion. The vehicle used was Planters Peanut Oil. The mice were sacrificed the eighth day after implantation. Carcinostatic activity was obtained with 11, VIII, XII, and a combination of 11 and SI1 at the specified dosages. Since we have had regression with 2-fluoro-9,lOphenanthraquinone ( S V ) , * jit W ~ L Sof interest to try a combination of XV, XII, and 11. In a test using VI11 at, 125 nig./kg./day, the tumor on one of the test mice, which was definitely measurable, completely regressed by the eighth day. Dr. H. Christine Reilly of Sloan-Kettering Institute has obtained a tumor diameter measurement of T I C in em. of 75% for a testing of XI1 at 250 at 50 m g . / k g . / d a ~ . ~ ~ mg./kg./day and 927, for IT
Acknowledgment.
The aut'hors wish to thank Dr.
H. Christine Reilly and Dr. C. Chester Stock for helpf u l discussions and commuiiicatioiis as well as Phillip Pfeffer and Steven Roseiiberg for their technical assist'ance. (26) Mice, (18-20 g . ) were obtained from Daries and Kagle Inc., New York. N. T. (27) T h e Sarcoma 180 was obtained from Dr. H. Christine Reilly. T h e screenings were assisted by Steven Rosenberg, Henry Clayman, and Virginia Wevurski. (28) Cancer Res., Suppl. N o . 1. 91 (1953); Suppl. No. 2 , 179 (1955); Cancer Res., 18, No. 8, 49 (1958). (29) Private communication from Ilr. H. Christine Reilly.
The Antileukemia Activity of Tylocrebrine
Uepurttuent of Cheiiiistry, Il'ollongong Cnicersity College, lt70110ngony,.V.S.W., .1 ustralza
Received Soveinber 16- 1963
The isolation, structure determination, arid synthesis of the alkaloid, tylocrebrine, from the S o r t h Queensland slender vine, Tylophora crebii$oi-a, has been reported previously.' This levorotatory alkaloid was shown to have a pherianthroindolixidirie structure (I), OCH,
36 1 TABLEI SARCOMA 180
Dose, nig./kg.
12.0
Survivors,
,Y o u t of Y
4
6
I)lJbf3,
nip./kg.
10.0
1881
Survi\ ors. Y out of Y
62
10
9
Average tumor ueiglit i l l 1 1 1 ~ . Test Control
735
1138
co1s2a governing their screening program. iiig is a report of the results obtained.
"/, test/ control
64
The follow-
Methods Test animals used as hosts were noninbred albino niice (Swiss) in the case of Sarcoma 180,*"and BDFI hybrid mice in the cases of Adenocarcinoma 'it15*~and Lymphoid Leukemia L1210.Zd All injections were administered intraperitoneally once a day except in the case of Sarcoma 180 when two injections per day were given. The solvent employed as vehicle of drug administration was carboxymethylcellulose with the exception of one series of tests involving Lymphoid Leukemia L1210, when another solvent (two drops of Tween 80 diluted to 20 ml. with saline) was used. Mice were sacrificed after fourteen doses, i.e., on the eighth day of administration in the case of Sarcoma 180; after eleven doses, Le., on the twelfth day in the case of Adenocarcinoma 755; while in the case of Lymphoid Leukemia L1210, administration of the drug was continued until death.
Results Inactive Tests.-Against either Sarcoma 180 or h d eriocarcinoma 755 administration of tylocrebriiie at nontoxic dose levels showed insufficient decrease in the tumor weight of mice in comparison with the control animals. Active Tests.-Administration of tylocrebrine to the Lymphoid Leukemia L1210 system considerably increased the survival time of the test animals. There are variations in the increases observed which appear to depend mainly upon the size of the administered dose, but occasional variations within the same dose range were also observed. The criterion of reproducTABLEI11 LYMPHOID LEUKEMIA L1210 Dose, Ing./kg.
10.0 6.ti
I
( 1 ) E. Gellert, T. R. Govindachari, 11.V. Lakshmikrtntham, I. S. Ragade, 12. Rudzats, and N. Viswanathan, J . Chem. Soc., 1008 (19132).
1185
test/ control
TABLE I1 AUESOCARCINOMA 755
Survivors for at least I3 days o u t of Y
x
15 .o
Tylocrebrine has been tested against three tumor systems by the Cancer Chemotherapy Kational Service Center, U. S. Department of Health, Education, and Welfare, Bethesda, Md., in accordance with the proto-
R,
Average tumor weight in mg. Test Control
4.4 2.2
6 6 6 6
1; 1:
6
Average survival time of animals in days Test Control
11.o
6 6 6 6 6 6
15 .o 14 0 13 7 14 .O 13.8
6
11.6
6
11.7 12.5 11.7 10.6
ti
6 6
9.1 9.9 9.9 8.7 8.3 9.1 9.9 9.1 9 .9 9.1 9.9
x, test/ control
120 151 141 157 168 151 117 128 126 128 107
( 2 ) (a) Protocols for Screening Chemical .\gents and Natural Products against Animal Tumors and other Biological Systems, in Cancer Chemothera p y Rept., N o . 25 (1962): (b) Protocol 1.301; ( c ) Protocol 1.302; (d) Protocol 1.303; (e) Protocol 12.303.
iblc activity in this tuiiior is a T j C 5 l5Oo/l :it the optimal dose.2e Conclusions
l'ylocrebrine shows high activity against Lymphoid Leukemia 1,1210 in mice, and thebe tests indicate that hest results are likely to be obtaincd at a dose level of about 10 mg./kg. The activity ascertained in tlicsc tests is sufficient for scheduling this coinpouiid for picclinical pharmacology and, iii the ahseiice of prohibitivc toxicity, for large-scale clinical testiiip. Acknowledgment.-The author5 n.bh t o express thanks t o Dr. .J. L. I-Iartwell, Chief of the liesearrh Communications Branch, Caiircr Chemotherapy S a tioiial Scrviw Ceiitc'r, Xatioiial Canwr Iiistit ut(,. Bethesda 14,31d.,for his int(wst iii this project.
1,2-L)isubstituted Naphth [2,3-d]imidazole-4,9diones and Corresponding Quaternary Salts'
Hoover and Day? described t lie pi,epai,atioii aiid properties of a number of 2-alkyl-1H-iiaphth [2,:3-d]iniidazole-4,9-dioiies. Some of these compounds were reported t o have inhibitory activity agaiiist Escherichia coli 113-3 and B9G.2 The present wo1.k deals with thc preparat'ion of I-alkyl- aiid 1-aryl-2-methyliiaphth[2,:3-tH]imidazole-l,'i)-dioiies ( I ) . quaternary salts of these compounds, and a st,udy of various properties of these subst'ances. 2-ilcetamido-3-alky1- or -;hryl-l ,I-naplitlioyuiiioncu wci.e prepared according t o the directions of Truitt, d These compounds w r o converted to tlie imidazoles (I) by t'he actioii of 2 S sodium hydroxide as directed hy I'ries aiid Rillig4 and utilized by Hoovcr aiid Ilay.? It is iiit~ei&iiig t>oiiotc i i i our \voi.k that t,lic cxpectctl 2-acetaniido-:3-aiiiliiio-l,l-iiap~it~io(~uiiioii(, \vas ohtairied if aii cthaiiol solutioii of ariiliiic and 2act~tamido-K-cIiloro-l,4-iiaplithoc~uino1i~~ ( 2:1 molc ratio) was refluxed. H o \ v c \ ~ i . ,if the reactants w r c ~ iisctd i i i a 1 : 1 inole ratio, 2-iiietliyl-l-phenyl~iaplitli[ L',:~-d]imidazole-l,!)-dioiie\\-as produced iii good yield. p-Bromoanilinc and p-t,oluidiiie ga7.c siinilai~ result;. but, with lower yields. .Ilkyl ainiiies did not gi1-c: iniidazoles uiider similar conditioii. Wheii the imidazoles (I) w r w lieated with nietliyl iodide (or other reactive halidtts) tlie quateriiaiy salt:: (I I) m r e obtained. Thesr compounds melted nit ti vigorous evolution of a gas. T h e pyrolysis of I1 (I3 ( I I S i ~ i i ~ ~ o r ticnv part l 111-n g r a n t (('\..j$lO8) froiii tlir Satioii:il ('niiver 111mal Institiires of IIraitli, 1,elr:irtrrirnt of Ileait a r i i l \Velfnre, lietliestla, >Id., n P'ar,ult> G r i t i i t f r u m ZJurtli Te x.?rsity, and a Pnrkr. Ila\-is ;anti ( ' ( 2 . l ~ ' e l l < , i v \ l i i ~ ~ . and A . 11. I):ty, .I, . l m . C I I I , ~A .o c 3 76, 4148 (l!l,ji!, AI. IVood, ,Jr., n n d R. 1.. Ilall, . I . 01.".Chrni., 2 5 , 1 I t i j l
(l!lUl). ( 4 ) Ii. l'rit'a :ind li. Uilliu. B L ) . , 68, 11% \1$)23j.
