..
Reduction of IV in Acetic Acid.-Tiyenty-four grains (0.073 mole) of I V in 200 ml. of glacial acetic arid was hydrogenated in :i Parr hydrogenator in the presenre of 2 g. of p1:ttinurn ouitit. catalyst :it 4.2 kg./cm.2 pressure :it, 33--60° for 20 hr. T h e cxtalyst \vas filtered and the solvenl wits reinoved n n a steaiii bath in riccico. I )illite (10%) Iiyilroc*hloric acid (300 1111.) \+-:is :ttlded and the solution WVBJextracted with ether. The avid soltition !vas niatle basic: with :ininioniuni liydroside, extr:ic.teti with c~hlorofornr,:tiid the rrsidue aft>er rernov:tl of the solvent \vas distilled, I).p. 145-200" ( 1 mm.), IS.6 g. i s 1 lion of the ether solution ga\ 10,lI-dihydro-6H-dibenzo [u,rl] petroleum ether. .4nnl. C:~lcti. I ' o ~ C~lH2.j:C1, 91 2 5 : 11, 8.73. F O U I I ~c',: 91.20: 13, S.90.
A Viscometric Study of Hydrogen-Bonding Properties of Carcinogenic Nitrosamines and Related Compounds'
l'lie viscmity of tlie hepatic, c a r h o g e n s , dirnetiiylnitrosaniine a n d dioxanr, in binary mixture with eitiier nat,er or propionic a c d : shows a very large increase a h o w the v:ilurs of idral mixing. So sucsli inrarease is ohserved in the absence of a proton donor, as with mixtures of tiimetliylnitrosaniine-diosane, dimethylnittosariiinc. propionic anhydride, or ditnetliylnitrosanirie-benaene. The nonrarcinogenic: 1,l-dinlethylhydrazine is c.onsicierably more potent in forming hydrogen bonds wit,ti water or propiiinir acid as measured by tlie extent. of imixii i i i i i i i viscosity increase. However, while the viscosity of dimetli~-lnitrosarriirie-~~ater is independent of tlie pH, rieutralization of bot,h basic, groups in the hydrazine coinpound, as shown by its titration cqrve, causes c-onsiderablr decrease in t h e viscosity of its water solutions. Hydrogen bonding with propionic acid approximat,elj-parallele rarcainogenic activity of :L small series of nitrosamines and dioxane. The varbi 1 group of proteins appears 10 be the niairi participant' i n the hydrogen bonding of nitrosamines in the priicess of protein denaturation by these compounds. Hydrogen bonding with nit'rosamines is through t,lie nitroso oxygen and involves displarenient of tlie amino electron doublet, resulting in lack of basicity of the amino nitrogen. .Aryl subst,ituents decrrase 1ij.tlrogen bonding by rrdirrcting tlie dkplacement of t,he doublet..
HYU~~OG H OENS DISG
July, 1964
IS C A R C I N O G E N I C
piperidineJ4 acetamide,6 S-acyldialkylamines,' thioacetamide,B thioureaJ8t9 ethylcarbamate,lO,ll and dioxaneJ7 that have been termed "hydrogen bond reactors,"lza were actually shown to be powerful agents of protein d e n a t ~ r a t i 0 n . l Urethane ~~~~ and the hydrogen-bonding carcinogenic aminophenols, 1 2 b 2-amino1-naphthol and 1-amino-2-naphthol, were shown to produce lomeriiig of the thermal denaturation temperature of DXA.15116 The effect of carcinogenic azo dyes on the conformation of proteins17 and the enhancement of heat denaturation of DSA by carcinogenic polycyclic hydrocarbons18 have been recently reported, although these interactions may involve valence forces other than hydrogen b ~ i i d i r i g . ' ~These ~ ~ ~ ~findings are in line with the hypothesis, set forth by Roiidoiii as early as 1938,z0that carcinogenesis and the denaturation of proteins are closely related processes; this hypothesis may now be extended to include nucleic acids. The increase of viscosity of certain binary liquid mixtures has been known for some time, e.g., ref. 21-23, and has been attributed to the formation of hydrogenbonded molecular associatioiis extending over many molecules. 2 4 a Because of these transient polymeric chains, radial distribution calculatioiis may riot be applied to hydrogen-bonded liquids. Furthermore, hydrogen bonding raises the mutual potential energy of pairs of molecules passing in contact with one another. 1;or these reasons the viscosity of no hydrogen-bonded liquid is a t the present time accessible to rigorous mathematical treatment. z5 Severtheless the viscometry of liquid mixtures has been used recently by Bello and Belloz6as an effective practical tool for studying t'he enhancement by L i t of hydrogen bonding bet'ween monomethyl- and dimethylacetamide and water . I n the present report the nature of viscosity increase of iiitxosamirie carcinogens in mixture with certain solvents has been investigated, the interaction of dimethylnitrosamine with amino acid side-chain models has been studied, and the comparative hydrogen bonding ability of various nitrosamines and related agents has been measured. The study of the pH dependence of viscosity of dimethylnitrosamiiie, 1,l-dimethylB. Jackson and F. I. Dessau, Lab. Invest., 10, 909 (1961). C. Hoch-Ligeti and h l , F. drgus, unpublished results. Nelson. Science, 108, 626 (1948). 0. G . Fitzhugh and A Cancer Res., 17, 302 (1957). A . Rosin and H. Ung (10) A . Nettleship, P. S.Henshaw, and H . L. Meyer, J . Natl. CancerInst., 4, 309, 5 2 3 (1943). (11) C. D. Larsen, z h i d . , 7, 5(1946-1947): 8, 99(1947-1948). (12) J. C. 4 r c o s and R I . Arcos in "Progress in Drug Research," Vol. IV, E. .Tucker. Ed., Interscience. New York, ?i. Y., 1962: (a) p. 305; (b) p. 468; ( e ) pp. 439, 485. (13) RI. F'. . \ r p u s , C. .J. Leritae. and .J. F.Kane. Brperientia, 17,367 (1961). ( 1 4 ) 11. I:. .\rgiis, .J. C. .\rcos, .J. H. Rlathison, and .L Alam, submitted for publication. . h l . Kaye, Biochim. B i o p h y s . Acta, 61, 615 (1962). Belnian, E. Levine, and W.Troll, Federation Proc., 21, 374 (196?)). . TVatters and Cantero, P r o c . A m . Assoc. Cancer Res., 3, 370 (1962). (18) E. Boyland nnd B. (ireen, H i m h e m (19) .I. C . .\rcus and A l . .\reus. ArrneimztteZ-~o,sch.,8 , 486, 843 (1068). ( 2 0 ) P. Rondoni in "Advances i n Cancer Research," Vol. 3 , J. P. Greenstein and =\. Haddow, Ed., Academic Press, New York, N. Y.. 1955, p. 171. ( 2 1 ) 8. English and W. E. S. Turner, J . Chem. Soc., 106, 1656 (1914). (22) N. Madsen, Science, 114, 500 (1951). ( 2 3 ) P. Pascal, "Chiinie GenBrale," Vol. 111, Rlasson & Co., Paris, 1951, p. 7R. (24) (:, C. Pitilentel and h. L. McClellan, "The Hydrogen Bond." W. H. Freeman B Co.. San Francisco, Calif., 1960: (a) p. 61: (b) p . 201. ( 2 5 ) .I. Hondi in "Rheology," Vol. I , F . R . Eirich, E d . , .\cademic Press, I n c . , New Tork. N.T., 1956, p. 321. ( 2 6 ) .J. Bello, and H . R. Bello. ,Vatuw, 190, 4 4 0 (1961).
(6) (7) (8) (9)
.\.
SITKOSAbIIK.ES
46 1
hydrazine, and related amines, and of the titration curve of these amines permitted delineation of the molecular site of hydrogen bonding and of the role of the amino doublet in hydrogen bonding with nitrosamine carcinogens. Materials and Methods.--rlll compounds used, except Nmethylindole,*' were commercially available reagent grade products and were used without further purification. Binary or ternary mixtures were prepared in the proportions indicated in the figures and exactly 1-ml. volumes uere used in all viscosity determinations. Viscosities were measured a t 30, 45, or 65" with Cannon-Manning28 Ostwald-type semimicro viscometers (sizes 50, 7 5 , 100, and 150) in a constant temperature ( &O.OZO) wat'er bath using a "Bronwill" circulator. The kinematic viscosity ( v ) values obtained were plotted against the volume per cent of the mixtures. Viscosities representing ideal mixing were defined as the values along a straight line connecting the viscosity values of two single coniponents.*9 Because of the nature of the conclusions drawn from this investigation it was not considered necessary to determine the densities of the various liquid mixtures for calculation of the absolute viscosities. Potentiometric titrations were carried out at 23-25' with a Beckman G p H meter fitted with a glass electrode and a calomel reference electrode. All values in this report are averages of four to six determinations.
Results and Discussion Viscosity Increase and Hydrogen Bonding With Dimethylnitrosamine (DMN).--Figure 1 indicates that DMN is a potent agent, to form hydrogen bond-linked molecular aggregates with both water and propionic acid, as shown by the large increase of viscosity above the values of ideal mixing. The maximum increase with dioxane-water is close to twice as large as with DAIS-water which niay be due to the presence in dioxane of two electron-donor oxygen atoms. That the interaction responsible for the viscosity increase is due to hydrogen bonding is shown by the observation that DAIS and dioxane, both potent in forming hydrogen bonds with proton donors, fail to give increase of viscosity upon mixing. Actually, the curve (27) Custom synthesis by the Chernicals Piociirement Laboratories, College Point 56, N . T. (28) Cannon Instrument Cu., State College, Pa. (29) N o satisfactory fuririiila relating tlie viscosity of binary liquid t i ixtures t o the coniposition of tlie tijixrure and the viscosities of the two COIIIponents has been obtained as yet.30 This is especially the case for nlixtures of markedly dissiniiiar niclecular species and/or n-hen the two components interact u i t h one another causicg departure from ideality. Certain mixture fluidity equations30131 are in good agreement with selected experimental d a t a : however, it must be emphasized t h a t the correction term applied was arrived a t empirically and therefore the observed correlation between calculated and experimental values is not entirely unexpected.3' While i t is true t h a t several binary systems are knou-n where viscosity is not a simple additive property, the assumption t h a t a negative deviation relative t o tlre line representing the ideal linear mixture lau increases a s the difference of the viscosities of tlir two coniponents increascs32a has not been substantiated in later work,32b and does not appear t o be snpported by viscosity d a t a reported herein. I t niay be concluded, consequently, t h a t uhile the positive increase of \-iscosity in certain binary mixtures can be accounted for by the formation of niolecnlar associations.?4~ the particular paranieter(s) of \.iscosity deterniining negative deviation from the linear mixture l a a has nut been clearly determined. I t is supnested tentatively t h a t in certain binary systems mliere the iriolar \ olrriiie of the two conrponents (containinr centers of high electron density) is not very different. iiiarked negative deviation may be attributed t o repulsion between the two niolecular species. Xloreover, the effect of such repulsion will necessarily enter into t h e correction term of t h e mixture fluidity equationsot 81 since the "excess free energy of mixing" is calculated from the vapor pressures of the mixtures. (30) R. E. Powell, W. E . Roseveare, and H. Eyring, I n d . Eng. Chem., 33, 430 (1941). (31) S. Glasstone, K. J. Laidler, and H . Eyrinp. "The Theory of Rate Processes," XlcCraa-Hill Book Co., Inc.. New Tork, K. T., 1941, u p . ,516517. (32) (a) ,J, Kendall and Ii. P. Monroe, J . Am. Chrm. Soc., 39, 1787 (1917); (b) i b i d . , 39, 1802 (1917).
50 401
6.0
\\
4
\p
Dioxane
5.0 4.01
spionic
/ /
\
I
n
i
Anhydride
. >-n-Prooanoi 1
I DMH I 100 0
40 60
20
80
60
20
40 80 Volume Percent
100 0
0
I00
of viscosity chaiigc in the latter iiiixturcs shows a11 appreciable iiegative iiicrerncwt, of’ viscosity relativc t,o the values of ideal misiiig.29 That hydrogeii bonding is impoiisible for the viscosity increase is further supportc.d by the fact that “eliiniiiatioii” of the protoil as i i i D~\IS-propioiiic* aiihydi-idc mistiirw c a w ’ s almost total disappearaiico o f t h r p i n i ) u s l y 01 ~ i ~ v c largcx d 1.iscosity iiicrcascj. r . 1 Iic valueis slightly glicii, tliaii viscositi(+ of idoal inisiiig may likcly 1 ) ~due, to tlic prcw~iicc~ of sinall ainouiits ( i f pivpioiiic acid. Siniilai,ly. t1ici.c is i i o chaiigr or v~1.ysmall iic>g?:ativc, iiiciwnciit of viscosity relativtl to the! v a l u c ~of idcal iiiisiiig with mixturw 01’ 111I S aiid l)eiizciic, iiidicatiiig absciice of appreciabl(1 iiitcmctioii br%wceiithe two componelit#s.
Hydrogen Bonding Ability of 1,l-Dimethylhydrazine (DMH).-I*’iguw 2 shows iiiisturc viscosity r i i r ~ w of TI .\ I H , t hc red urcd , i IO I I ra rri I I ogc 11iv de lira t i VP ( f )
I > l I S . l‘hc’ f i g u i ~iitdic+atrsthat i.cplatemeiit of thc so by a11 aniiiio substit i i r l i i t docs not lead to leuset, sity i i i v i w w whcii i i i niisfwr ivith either watcl, opioiiic~acid. hui 011 t h coiitiwy gi1.m a (milsiclei.ahly 1aiyq.i. i i i c ~ ~ a of s r visvosity a h v e thc. \-alues of idrd niisiiig thati (low IIJIS. I I I I>:\II3-\vater a i d I).\IEl--pi.opioiiic acid mistuws Jl:\IH appears to bc ail oxclusivcly c~lcctrori-doiiat,iiig partnrr. This is SURgcstcd hy thc ahsciicc of h y d i ~ g c ihonding i as indicated by 1 1 0 viscosity t!liaiigc> \\-it11niistures of DlIH--I)ALS aiid hy iic>gativchviscosity iiic~iwncwtn-itli niisturt I).\II-I dioxaitc,. Tho absoiict. of positivo dcvia 1’roni va1iic.s of i t l w l niisiiig \\-it111>1IH-l-pi~)paiiol t l t c b
I
80 20
I
I
60 40 60 40 Volume Percent
I
20 80
Dioxane
‘--‘t+ Propionic Acid retw a, / -‘-DMN ‘n-Butonethiol 1
0 100
July, 1964
HYDHOGES
BONDISG IS CARCINOGENIC "-0
r0
3.6, NP Viscosity
468
~ITR06.4:\1IXES
the base. Seutralization in D J I H of only the dimethylamjno group, or neutralization of isopropylamine, does not drastically affect the respective viscosities since the onium group may establish the same number of hydrogen bonds as the amino group from which it was formed. CH3,
/CH3
,,H
CH3,
1 :p
132
0.8
4
I i # I
,
,
,
,
2
3
4
5
I
6
, 7
/CH3/H
0 N:H---O-H
N:---H-0
,
,
,
,
, I
8
9
IO
II
12
PH Fig. 4.-The pH dependence of kinematic viscosity of npropylamine (?;P)-water and isopropylamine (1P)-water mixtures as compared to the pot'entiometric titration curve of the amines. For the viscosity measurements ( a t 30") the volume proportion was ti0c; of either amine and 40',; water. This is the proportion to obtain riiasiniuni increments of viscosity above the values of ideal mixing, as established in experiments not presented in the figures. These experinients showed that at 30" the visrosit,y of S P singly was 0.52 centistokes and the viscosity of IP singly 0.45 centistokes. The maximum viscosity increment above the value of ideal mixing FYas 1.69 centistokes for TPwater mixtures and 1.80 centistokes for IP-water mixtures, a t pH 12.5. The pH was adjusted with HCl and account was taken of the water t'hus introduced when establishing the final proportion. Titration of each amine ( 2 ml. in 50 ml. of water) was carried out potentiometrically with 1 S HC1.
water (Fig. 2 ) and of 1-propanol-propionic acid (not shown here) gave, on the other hand, viscosities appreciably higher than the values of ideal mixing. These results are consistent with observations indicating that for maximum stability of intermolecular hydrogen bonding between two different molecular species, the "bridgehead" atoms must have similar proton affinities (Le., electr~iiegativities).~~!~~ If the "bridgehead" atom of the proton donor partner is much less electronegative than the "bridgehead" atom of the electrondonor partner (e.g., 1-propanol-DAIH mixtures), then polymolecular association composed of molecules of the proton donor alone will predominate: if, 011 the other hand, the relative electronegativity of the proton-donor "bridgehead" is high (zk.,stroiig acids), onium-type salt linkages will be established. 3 3 Effect of pH on Viscosity; the Molecular Site of Hydrogen Bonding.-Figures 3 and 4 show the pHdependence of "maximum viscosity" water solutions of DAIH, DAIS, n-propylamiiie, and isopropylamine, and the potentiometric titration curves of DMH, n-propylamine, and isopropylamine. Comparison of the DAIH titration aiid DAIH viscosity curves shows that high viscosity is maintained to the eiid of the neutralization of the dimethylamiiio group; then viscosity steeply decreases between about pH 8.6 aiid 6.6, leveling off a t the iieutralizatioii of the primary amino group. The viscosity of isopropylamiiie is, on the other hand, almost completely iridepeiideiit of the pH. Totally neutralized DAIH has a viscosity comparable to that of isopropylamine. The high viscosity of nonneutralized DAIH-mater mixtures is consequently due to the stroiig bifunctional hydrogen-bonding ability of (33) B Eistert, "Tautom6rie e t l\I&oni6rie," Presses TniX ersitaires de France Paris, 1949, p 169 (34) J C. Dearden and W. I . Eurbes, Can. J . Chem., 38, 896 (lY60)
The small viscosity peak toward the eiid of neutral izatioii of the dimethylamino group in DAIH and toward the end of iieutralizatioii of isopropylamine (and n-propylamine) may be ascribed to somewhat greater hydrogen-bond strength in the water-onium than in the water-amine associatioiis ( c j . ref. 3 5 ) . That addition of acid beyond the point of iieutralization causes disappearance of these viscosity peaks may be the result of competition by excess acid for hydrogen boiidiiig. The observation that neutralization of the primary amino group in DAIH causes a drastic decrease of viscosity and establishment of a viscosity level comparable to neutralized isopropylamine indicates that total neutralization causes loss of bifunctioiiality of hydrogen bonding by DAIH. This in turii indicates steric hindrance of the formatioii of double oniumtype, hydrogen-bonded, molecular associations. CH3,
,CH3
/H
N:H- -- 0-H
Figure 3 shows furthermore that the viscosity of the DAIS-water mixture is independent of the pH. The dimethylamino group in D l I S iii aqueous solutions is totally devoid of basic properties, but the formation of crystalliiie DAIS HCl by introducing dry hydrogen chloride in concentrated ether solution of DAIS has been described. 36 Carbon tetrachloride and beiizeiie have nom been found to be highly favorable for the formation of the hydrochloride from dilute solutions, but dioxane, acetonitrile, aiid propioiiic acid are iiicreasiiigly inhibitory iii that order. All these observations suggest that the viscosity increase of DAIS-water and DAIS-propionic acid mixtures is due to hydrogen bonding uza the electron doublet(s) 011 the nitroso oxygen and not 011 the amino iiitrogeii as is the case with DAIH. The hydrogen-bonding ability of the nitroso oxygen is further eiihaiiced by the increased electron density due to displacement of the amino nitrogen electron doublet in polar solveiits and predominance or stabilization of the polar resonant limit structure. (35) L Pauling. "The Xature of the Ciieiiiical Bond 1 , 1960 p 4 5 2 (36) E Renouf, Be, , 13, 1170 (1880)
sit> Press, Itliaca. N
"
Cornell U n i i er-
.. .
-Hi4 CH? \ /
('Hi
,?
N-N=O)
50% Phenol in Dioxane
I
.'
28..
, , , I
I
2.6,
'l'h, iinportaiice ton art1 tlic iiitroso iiiti~osainiiii(~s is furthcr hupportcd hy results ~ l i o ~ v i i in P-ig, 5 . The figure show- tlic rnasimuni iiicrerneiit-
I
8 I
2.4-
I
2.2-
/IPropa
..'t':
I501 4
I
I
I.8,
25% Phenol
5
9 4 4
,*A
in Dioxane
1.61.4-
,
8
1.2,
0.8 DEA DEF NP DEN DMN DPrN DO MPhN EPhN 60% 50% 70% 50% 40% 70% 60% 80% 80% Fig. 5.-lIasiiiiuiii increments of viscosity :hove the values ( i f ileal riiising in binary mixtures of propionic w i d with diethyliwetarnide (DEA), diethylformamide (DEF), S-riitrosopiperidirie (Sl'),diethylnitrosamine (])E?;), diinethylnitrcistrrriine (I>T\ISj . di-n-pri)pyliiitrosariiine ( I I P r S ) , dioxane ( I N ) ) , rnethylphenylnitrosamine ( I l P h S ) , or ethvlphen?-lnitrosaniiiie( EPhX). Thc, percentage values iri the figure indicate the volunie fracdtioiis : i t which the respective agents gave the ruasiiiiurii positive vis(-osit)' iiicmmierits i i i niisture with propionic. :irid. \%irosity detrriiiiiiations were cm-rietl o u t at 30".
of viscosity above the values of ideal rnixiiig iii binary
tnixturc- of propionic acid with diethylacetamide, diethylformamide, S-nitrosopiperidiiie, diethgliiitro-aiiiiii(1, D l I S , di-Ti-propylnitrosamine, dioxane, methylpliriiyliiitrosamiii~~.arid ethylpheiipliiitrosamiiic.. I t may br s(wi that, irlieli aii alkyl group in di-u-alkyliiitroaartiiii(- i- replaced hy a pheiiyl, hydrogeii-t~oiidlllg ability i+ roiihiderahly decreased lwcausc~ of pal ticipatioii ot thv aniiiio cioublrt i i i tlio i ~ w i i a i i wof the, aronnatic.huhstitii(~iit
c HS
CH?
\3
\r;-N=o\
/
-
0. CJ 0 ('Hi
\
N=N-Glr
.cf
+
N-N=O)
cf
...
IT-
Interaction of Dimethylnitrosamine with Model Compounds for Amino Acid Side Chains.--\'iswsit ies of h~iiarymixtures of I > l I N with ageiits which may 1)c cwisidrred as model compouiids for arniiio acid hidp rhaiiis have been 1111-estigatedto delineate the fun(.t ioiial groups which may be interacted with by meails of hydrogen boiidiiig i i i proteiii denaturatioii. E1.idc1ic.r for strong livdrogeii boiidirig betweii the cbai.hoxyI gi'oup aiid iiitrosamiiies, and for abseiiw of iiitciwtioii b ~ t ~ v e c hciizeiitl ii ( f o r phenyl) a d D l l S. liah t w ~ i givc~i i almvc~(I'ig. 1 aiid 5 ) . 1;igure (i~ h o u ~
0.s-l-
-\
25% Phenol in Benzene or Carbontetra. chloride p-Bulanelhiol n-Propylamine
HYDROGEPI' BOSDING IS
July, 1964
1 -
N-Methylindole
100 0
80 20
60
40
20
40 60 80 Volume Percent
0 100
Fig. T.-Kinematic viscosities of mixtures of dimethylnitrosamine ( D M K ) with compounds representing amino acid side chains. Viscosity determinations were carried out a t 30°, except pure phenol-DMS at 46' and pure indole-DMT a t 65'. The end points of the curves represent the viscosities of the pure (tompounds or their concentrated solutions as indicated. The proportions of the mixtures are given in volume per cent on two opposite scales in the abscissa.
ideal mixing when in combination with DAIS. The viscosity behavior of indole was similar to that of phenol except that the maximum positive viscosity increment in benzene was appreciably lower. The positive viscosity increment observed with N-methylindole in benzene is surprising since the only proton which may be involved in hydrogen bonding in indole is assumed to be the one linked to the nitrogen atom, on the ground that it can be replaced by alkali metals. Therefore, the possibility that carbon-linked hydrogen of indole may partake in weak intermolecular association with DAIS in certain conditions must be considered. The results with amino acid side-chain model compounds indicate that carboxyl groups are overwhelmingly the main participants in hydrogen bond-mediated interactions of proteins with nitrosamine carcinogens. Relation between Hydrogen-Bonding Ability and Carcinogenic Activity.-In view of the relationship between protein denaturing ability and varcinogenic a~tivity,'~-1~ it ~is~ 0of significance that the hydrogenbonding abilities of S-nitrosopiperidine, diethylnitrosamine, DAIS, di-n-propylnitrosamine, dioxane, and methylphenylnitrosamine decrease in the same order (Fig. 5 ) as their approximate carcinogenic activi-
C.4RCISOGENIC S I T R O S A h I I X E S
465
ties.Z--jJJ7 The carcinogenic activity of ethylphenylnitrosamine remains to be tested. It may be recalled that the ability of dioxane to act as a mitotic disorganizer a t levels comparable to that of the lung carcinogen, ethyl carbamate, has been attributed to its surface active proper tie^.^^ Diethylacetamide and diethylformamide, which are structural analogs of the nitrosamines, appear to possess only low carcinogenic activities when tested a t levels equimolar to DAIS,7 yet they show greater viscosity increase with propionic acid than the more carcinogenic nitrosamines or dioxane. These two apparent exceptions may possibly be due to only partial electron displacement toward the acyl groups and, thus, to contribution of a nitrogenlocalized electron doublet to hydrogen bonding. It is significant in this respect that the increment of viscosity was notably greater with diethylacetamide, containing the less electronegative acetyl groups, than with diethylformamide, containing, the more electronegative formyl group (Fig. 5 ) . Comparison of the maximum viscosity increases observed with DAIH (Fig. 2 ) and those found with nitrosamines (Fig. 1 and 5 ) shows, zv fact, that hydrogen bonding via an amino electron doublet produces a much greater viscosity increase than hydrogen bonding ilia a nitroso or acyl group. This is further indicated by the considerable viscosity gain (of respective mixtures with propionic acid) when the intranuclear -CH,- in the 4position of S-nitrosopiperidine is replaced by a monomethylamino group, resulting in l-methyl-i-nitrosopiperazine. Thus, the maximum viscosity increment of 0.79 centistoke with the noiibasic S-nitrosopiperidine rose to 21.97 centistokes with the monobasic piperazine compound, which is roughly half the value found with the dibasic D l I H (E'ig. 2). With respect to the relation between hydrogeiibonding ability and carcinogenic activity (Fig. 3 ) it must, of course, be remembered that in some instances breaking of hydrophobic bonds, dipoledipole interactions, etc., may be more important in bringing about deiiaturation and cellular alterations than interactions by means of hydrogen bonding. The apparent paradox that the powerful hydrogenbonding agent, D l I H , is inactive both as a carcinogen3 and as an agent of protein d e n a t u r a t i o r ~may ~ ~ ?be ~ ~the consequence of three mutually not exclusive situations : (a) in the physiological pH range bifunctionality of hydrogen bonding responsible for high viscosity is almost totally lost; (b) the pH sensitivity observed with DJIH, but not with DAIK, may cause considerable instability of hydrogen bonding with the former agent because of local p H changes in the microenvironment of the functional groups involved; and (c) orientation of the hydrogen bond(s) with respect to the S-S axis in DAIH may result in steric hindrance of interaction with functional groups. (37) H Druckrel, R Preussniann, D. Schmahl, and G . Bluin, \ nlui 48, 7 2 2 (1961) (38) K. Hohl. Ezperzenlza, 8 , 109 (1947).
ULSS
,
