NOTES
January 1968 (nonbonded interactions of e-Me groups in 8-CYand -y are raised in boat forms, and models show that the cis-1,3-diaxial AIe-lIe interaction of 8-P is not relieved in the corresponding boat), the sole active member (y) being about as active as the y-1,2,5-trimethyl isomer.'j Ethyl 3-a-phenyltropane-3-P-carboxylate, which is somewhat more potent than meperdine,'e would be expected to have a significantly large skew-boat 10 population because the chair conformer 9 is destabilized
191
Effects of Certain Arylhydroxaniic Acids o n Deoxyribonucleic Acid Synthesis b y Ehrlich Ascites Tumor Cells in Vitro' GLENR.GALE Velerans Administration Hospztal and Ueparttrient of Pharmacology, Jledzcal College of South Carolina, Charleston, South Carolina AND
JOHN B. HYNES
Hynes Chemacal Research Corporalton, Durham, Sorth Carolina Received J u l y 7 , 1967 Ph
Acknowledgment.-The author acknowledges Drs. 11. A. Iorio and P. Pocha for assistance in this work.
A recent report2 described a select'ive inhibition by salicylhydroxamic acid (I) of deoxyribonucleic acid (DSA) synthesis in Ehrlich ascites tumor cells. Characteristics of the inhibition were similar in some respects to the actions of hydroxyurea3 and of oxamylhydroxamic a ~ i d ,the ~ , ~hydroxaniic acid analogs of carbamic acid and oxamic acid, respectively. Effects on the synthesis of ribonucleic acid (RSA) and of protein were nominal and were considered to be of a secondary nature as a consequence of the lowered rate of DSA formation. The inhibit'ion by I was further evident immediately upon adding the compound to the cells; that is, no preincubation was necessary to evoke the effect. The rate of DSA synt'hesis resumed the cont,rol rate upon removal of the inhibitor by washing the cells, indicating no firm binding to the cells and no irreversible alteration of the cells by the compound. The work herein described was initiated to determine the ways in which structural features of compounds related to I may influence the course of nucleic acid synthesis in a tumor-cell test system. Biological Data.-Table I shows the 50 and 90% inhibitory concentrations of each compound on DXA synthesis in Ehrlich ascites tumor cells. With one exception, data are presented as obt'ained with no preincubation (ie., inhibitor and isotopic precursor were added to the cell suspension simultaneously) and, also, following 1-hr preincubation of the cells with each compound prior to addition of the isotopic precursor. The relative potency of 9 of the 12 compounds was increased by the 1-hr preincubation period, the most striking example being that of XII. Slopes of the regression lines were fairly closely grouped when inhibitor and precursor mere added simultaneously but varied erratically following the 1-hr preincubation period. Figure 1shows the effects of each of the 12 compounds on DSA, RNA, and protein synthesis in Ehrlich ascites tumor cells. The selectivity of action of I on DNA synthesis was confirmed; that is, t,he rate of DNA synthesis was depressed almost SO% after 1-hr exposure of the cells to the compound with no appreciable diminut'ion in the rate of RKA or protein synthesis. Com-
(15) 0. I . Sorokin, Izu. Akad. Nauk. SSSR. 460 (1961). (16) M .R . Bell and 5.Archer, J . A m . Chem. Sac., 82, 4638 (1960). ( l i ) h l . R. Bell and S..kcher, ibid., 82, 151 (1960). (18) P. S. Portoghese, J . Pharm. Sei., 6 6 , 865 (1966). (19) .I. H. Beckett. .L F. Casy, G. Kirk, and J. \Talker. J. Pharm. 1'ham"of.. 9 , 939 (1957). ( 2 0 ) .\, .\lbert a n d E. P. Sergeant, "Ionization Constants of .kids and bases." hlethuen, London, 1962.
(1) This work was aided b y Grant GM-13958 from t h e National Institutes of Health, U. S. Public Health Service. (2) G. R. Gale, Pmc. Soc. Ezptl. B i d . M e d . , 122, 1236 (1966). (3) (a) J. W. Yarbro, B. J. Kennedy, and C . P. Barnum, Proc. S a t l . A c a d . Sei. L'. S., 63, 1033 (1965); (b) J. IT. Yarbro, W.G . Niehaus, and C. P. Barnum, Biochem. B i o p h y s . Res. Commun., 19, 592 (1965); ( c ) R. L. P. .\dams, R . Abrams, and I . Lieberman, J . B i d Chem., 241, 903 (1966). (4) G. R. Gale, Concer Res.. 26, 2340 (1966). ( 5 ) G. R. Gale, J . X u t l . Cancer Inat., 38, 51 (1867).
9
10
by a-Ph-bimethylene bridge interactions. Spectroscopic evidence supports this contention for related P-ethyl and phenyl ketones." I n a recent analysis of stereochemical factors in narcotic analgetics, Portoghesels stated that the conformational requirements for most of the 4-phenylpiperidine analgetics appear to be minimal. From the present evidence, however, it is probably more accurate to state that although a fairly wide range of 4-phenylpiperidine conformations are compatible with activity, those in which the aromatic and piperidine rings approach coplanarity (as in the skew-boat with phenyl in the bow-sprit position) may be most effective in evoking a response. Experimental Section Where a n a l p e s are indicated only by symbols of the elements, analytical results obtained for those elements were within 1.0.47, of the theoretical values. The pmr zpectra were recorded on a Varian A-60 spectrometer operating at the normal running temperatiire with TATS az ztandard (internal with CDCl, and external with DzO as solvent). a-1,3-Dimethyl-4-pheny1-4-propionoxypiperidinehydrochloride (a-prodine), mp 222" (lit.18 220-221 "), and the corresponding p isomer (p-prodine), mp 199-200' (l1t.I9 195-196'), were obtained by heating the a- and p-piperidinols l a with propionic anhydride and pyridine.'@ New esters prepared in this way were as follows (uncorrected melting points determined with a BuchiTottoli apparatus in capillary tubes). a-4-Acetoxypiperidine ( l b ) hydrochloride, mp 216-218", from i-PrOH-EtZO. Anal. (C1,H~ZClNOZ)C, H, N. p-4-Acetoxypiperidine (Ib) hydrochloride, mp 211-213", from EtOH-Me&O. A4nal.C, H, N. 0-4-Butyoxypiperidine (Id) hydrochloride, mp 196-197', from EtOH-MeCOEt. Anal. (C1~Hz6ClI\'02) C, H, X. p-4-Butyroxypiperidine (Id) hydrochloride, mp 202", from EtOH-MeCOEt; umax 3400 em-'. Anal. C, H, ?: (low C value due to water of crystallization). All of the esters had v&"' near 1720 cm-' (ester C=O). The pK,' values of the prodine isomers in 5OcJc EtOH-HZO, determined by hlbert's and Sergeant's method,Zouere a , 7.68 i 0.06, aiid p, 7.75 f 0.06.
pounds 11, VIII, nrid XI 11ere initiull~ inhibitory t ( I DXJ1.yiithesih. but activitj \\ :L\ reduced upon preincubatioii, iridicatiiig ;L posd)lc converbiori of ail i ~ c tivc ugerit to :tri inactive one. Tlic oii5et of tlepressioii of RS.1 s\mthebi> 11) I1 nhich n a > c o u p l d ivith 1 0 s of DSA1-iiiliibitiiigc a p a c i t j 11:i\ reproduciblc (threc. PXptlriment.), suggesting coiiver:,ioii of I1 to :m inhibitor of 11s-1iyntheiis. Corideririg the possibility t1i:it IT 11i:iy he reduced to the amide, bcnznmide T V ~ Zteatrcl for it5 inhibitory :iction on IIX-1 lourid to be totally inactive i i i tl pounds VI aiid VI1 di-played relativ I)sLlsyritheqis but only after expowre IJf the cell:, for 1-2 lir. Compo I s h o n etl a marked d e c t i v i t j arid thc clateiit of inhibition :igaiii-t D S X sy 11 :L\ greatrr th:m )tainecl I\ itli I at the wnie coiiccvitrntioii. Compound IX T irtudly completely iiihibitcd DSA iynthe~ii.hut thi\ n :tccomp:iriied I>\ ~
i 0
p. 5110'M
-
0 2
0
e
$5~10%
0
I
2
0
I
2
0
1
2
January 190s
-
1
25000-
193
NOTES
IL
125001-
X IO'H
-
25000
1OOOOr
0-1.75110"M X S x IO'Y
0 2 1lO"Y
22500
-
20000
-
32000
17500
-
28000
11250
-
36000
-
22500
20000
F
P
12500f-
PI
O~I.5xlO'M X.61l0'Y
-
w
I-
3
E
I K
w
a
PIL
u)
I-
z
20
25000-
H
o * IO-3u 15000
xa
014 1 IO-' M
X.2x10aM
20000
-
MINUTES
Figiire 2.--Ihmxibility of the inhibitory actioiis of arylhydroxamic acids 0 1 1 D N A synthesis in ascites tumor cells: 0 , no inhibitor 0 and x , cells exposed to inhibitor for 30 min at indicat,ed concentrations; solid lines, cells then washed with fresh XIEM; dotted lines, cells then wabhed with MEA1 containing t,he inhibitor at the same concentration as during the initial 30-min exposure. Following the washings, isotopic thymidine was added, and its incorporation int,o DNA was assessed a t 20 and 40 min. Other experimental details are in ref 6.
the cells to each compound for 3 hr a t the 90% inhibitory concentration.6 N o depolymerization whatsoever of preexisting D S A could be demonstrated. Experimental Section Chemistry.-The previoiisly unreported hydroxamir acids were prepared by the method of Scott and \\'ood7 modified according to the solubilities of the esters and of the resrilting plodiicts. The methyl or ethyl esters used were obtained commercially with the exceptiolis of methyl 2,6-dihydroxyheii~oate, which \vas prepared by the method of Tomillo,* methyl 3-amitio-2pyrazitioate, prepared according to Ellitigsoii, et aL,g and methyl
( 6 ) G. R. Gale, J. G. Simpson. and A. B. Smith, Cancer Res., 27, 1186 (1967).
( 7 ) A. W. Scott and 13. L. Wood, J . Org. Chem., 8 , 515 (1943). (8) K. Tomino, Y a k u g a k u Zasdiz, 78, 1419 (1958). (9) R . C. Ellingson. R. L. Henry, and F. G. McDonald, J . A m . Chem. Soc., 67, 1711 (1945).
2,3-dihydroxybetizoate, synthesized according to King, et aZ.'@ The melting points (Fisher-Johns apparatus, uncorrected) and analytical data (Galbraith Laboratories, Inc., Knoxville, Tenn.) are presented in Table I1 along with the literature values for the known compounds. With the exception of VII, each of the hydroxamic acids was recrystallized from HIO using activated carbon. Compound VI1 was recrystallized from absolute AleOH by the addit,iori of petroleum ether (bp 30-60"). As indicat'ed in Table 11, it was often necessary to recrystallize twice in order t,o obtain arialytical purity. A tiiiniber of rinsiiccessfiil attempts were made t o prepare pure i,c-hydroxybetizoylhydroxamic acid. Becaiise of its tendency to form intractable oils, the react,ion was performed in the following manner which yielded the product actlially tested. A 16-g sample (0.4 mole) of S a O H was dissolved in 100 ml of H 2 0and 16.4 g (0.2 mole) of KH,OH 0.3H2S04 was added with cooling and stirring in a N, atmosphere. Methyl m-hydroxybenzoate (13.2 g, 0.1 mole) was added, and the resulting mixture was left for 2 days with a slow S, purge. After neutralization with 3 *V HzS04, the solution was evaporated to dryness under vacuuni. The solid was extract,ed with N e O H and dried twice over 1\IgS04. (10) F. E. Iilng, J. H. Gilks, and Al. 11.' Partridge, J . Chem. Soc., 4206 (1955).
