Chemical Reviews 2000

8/3/2019 Chemical Reviews 2000

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Quantitative StructureActivity Relationships of Mutagenic and CarcinogenicAromatic Amines

Romualdo Benigni,*, Alessandro Giuliani, Rainer Franke, and Andreas Gruska

Istituto Superiore di Sanita, Laboratory of Comparative Toxicology and Ecotoxicology, Viale Regina Elena 299, I-00161 Rome, Italy,and Consulting in Drug Design GbR, Gartenstr. 14, D-16352 Basdorf, Germany

Received November 10, 1999

Contents

I. Introduction 3697II. Toxicology of Aromatic Amines: Epidemiological

Evidence3698

III. Mechanisms of Action 3699IV. Qualitative Notions on the StructureActivity

Relationships of Aromatic Amines3700

V. QSARs for Mutagenicity 3700VI. QSARs for Carcinogenicity 3703

VII. Original Model for the Carcinogenic Potency ofNonheterocyclic Aromatic Amines in Rodents

3704

1. Data and Methods 3704A. Carcinogenicity Data of Aromatic Amines 3704B. Chemical Structures and Chemical

Parameters3704

2. Results 3707A. Modeling the Carcinogenic Potency in

Mice3707

B. Modeling the Carcinogenic Potency inRats

3708

3. Discussion of the Carcinogenic PotencyQSARs

3710

VIII. The QSARs of Aromatic Amines in Perspective 3711VIII. Acknowledgments 3713

IX. References 3713

I. Introduction

Among the various toxicity endpoints, chemicalcarcinogenicity is of primary interest because i tdrives much of the current regulatory actions on newand existing chemicals a nd i ts experimental deter-mination involves t ime-consuming and expensivea n i m a l t e s t in g . H ow ev er , on l y a r e la t i ve ly s m a llpercentage of the chemicals in commerce have cur-rently un dergone testing, so the support of structure-

activity relationship (SAR) an d quantitat ive struc-t u r e-activity relationship (QSAR) ap proaches (astools for both predictive toxicology and mechanismelucidation) in t his field ar e of part icular interest. Inrecent years, there has been strong pressure fromsociety, in general, and from government agencies,in particular, to develop general prediction models

in order to cope with the thousands of chemicalspresent in the environment, for which experimentaldat a a re n ot available and likely will never exist. Tworecent compara tive exercises on the prediction of chemical carcinogenicity using different methods oralgorith ms provided extr emely import an t evidence onthis subject.1,2 It was demonstrated that the presentlevel of SAR knowledge permits the identification ofmany potentially carcinogenic chemical functional-it ies. Thu s, application of t he SAR knowledge is

already reliable for an efficient use in priority setting,as demonstra ted by t he successful priorit izationperformed by the U.S. National Toxicology Program,which found 70% carcinogens among the suspectchemicals, whereas only 10-20% of the chemicalsselected on exposure/production considera tions (hen cewithout any bias in terms of biological activity) werecarcinogenic. 3 However, a common weakness of theapproaches was the difficulty in correctly predictingthe noncarcinogens with alerting functionalities, i.e.,the presence of a structurally alerting feature couldbe negated by other structural factors modulatingpotency or eliminat ing activity. Hence, althoughcurrent prediction methods are reasonably successful

at discerning major, structurally alerting classes ofcarcinogens, greater uncertainty is associated withthe predictions for individual chemicals, becausem e t h od s d o n ot a d e qu a t e ly d is cr i m in a t e a ct i vi t ywithin these classes.

A possible way to at least partially overcome thisdifficulty is to develop QSAR models for differentclasses of chemicals an d to use th e resu lting modelssafter assigning the compounds t o be evaluat ed to thecorrect classsfor predictions. To be able to do that itis, of course, necessary to make such QSARs avail-able. Wher eas collections of QSARs of individualclasses of toxic chemicals are largely available forsome end point s (e.g., a quat ic toxicity4), QSARs forclasses of carcinogens are quite limited and sparse.O n e o f t h e r e a son s is t h a t q u a n t it a t ive d a t a oncar cinogenic potency a re largely missing. A rema rk-a b le e xce p t ion i s t h e cl a ss of a r om a t i c a m i n es .Obviously, the level of use and industrial importancehave determined such large experimentation. In fact,a r om a t i c a m i n es a r e w id e sp r e a d ch e m ica l s w it hconsiderable industrial and environmental impor-tance: for example, aromatic amine-derived dyes aresynth etic organ ic colorant s, widely used in the textile,paper, leather, plastics, cosmetics, drugs, and food

* To whom correspondence should be addressed. Telephone: +3906 49902579. F ax: +39 06 49387139. E-ma il: rben [email protected]. Istituto Superiore di Sa nita . Consulting in Drug Design GbR.

3697Chem. Rev. 2000, 100, 36973714

10.1021/cr9901079 CCC: $35.00 2000 American Chemical SocietyPublished on Web 08/17/2000


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indust ries. Moreover, several t ypes of ar omatic aminesare generated during cooking.5-7

This paper, first, illustrates the toxicological prob-lems posed by the aromatic amines, together withevidence on their mechan isms of a ction. A sh ortoverview of th e qualita tive stru cture-activity notionsderived from the above evidence is a lso provided.Then we review in detail the available QSARs for theexperimental results on the mutagenic and carcino-genic properties of th e am ines. Given t he pa ucity ofQSARs for carcinogenicity, we developed ad hoc forth is paper QSAR models for t he car cinogenic poten cyof nonheterocyclic aromatic amines. Finally, thevarious QSARs are put into perspective.

II. Toxicology of Aromatic Amines:Epidemiological Evidence

T h e a r om a t ic a m in e s a r e on e of t h e ch e m ica lclasses in which the structural and molecular basisof carcinogenicity is most clearly un derstood.5 Thisclass of molecules offers th e un ique possibility of

covering all the investigation levels, ranging fromphysicochemical properties to epidemiological find-ings in human populations, with rational explana-tions.

Exposure to a romatic amines occurs in differentindustrial and agricultural activities as well as intobacco smoking. Aromatic amines have been usedas antioxidants in the production of rubber and incutt ing oils, as int ermediates in azo dye ma nufactur-ing, and as pesticides. They are a common contami-nan t in several working environment s, including thechemical and mechanical industries, and arylamines-based dyes are widely used in textile industry andin cosmetics.8 T h e w id e u s e of a r om a t ic a m in e s

Romualdo Begnigni received his education in chemistry at the Universityof Rome La Sapienza. He then joined the Istituto Superiore di Sanita(Italian National Institute of Health), where he got a permanent positionin 1977 and remained except for a sabbatical at New York University in1988. He worked experimentally in the field of molecular biology andenvironmental chemical mutagenesis. In the 1980s he turned his attentionto the statistical analysis and modeling of toxicological data and to thestudy of the relationships between the structure of organic compoundsand their toxicological properties (mainly mutagenesis and carcinogenesis).

Alessandro Giuliani received his degree in biological sciences at theUniversity of Rome La Sapienza in 1982. Until 1977 he worked at theSigma-Tau Laboratories; then he moved to the Istituto Superiore di Sanita,where now he is Senior Scientist in the unit directed by Dr. Benigni. Hisresearch interests focus on the exploration of soft modeling approachesto quantitate biological experimental results. To this aim, he appliedmultidimensional techniques to a variety of fields ranging from classicalQSAR to physiology, behavioral sciences, cardiology, and signal analysis.In recent years he has collaborated on the development and applicationof a novel signal analysis technique, recurrence quantification analysis(RQA), to physiology, molecular dynamics, and complex systems physics.Recently the technique was demonstrated to be promising in developingquantitative sequence/activity studies in proteins.

Rainer Franke received his Dr. rer. nat degree in physical chemistry fromTechnical University Dresden and his habilitation from Martin-LutherUniversity Halle/Wittenberg. After working in a district hospital in Dresdenand at the Humboldt University in Berlin in the field of biochemistry, hejoined the Academy of Sciences of the GDR in Berlin doing research inmedicinal chemistry. Since 1991 he has been Director of Consulting inDrug Design GbR. His primary interest is in the relationship between thestructure of organic compounds and their biological properties.

Andreas Gruska studied chemistry at the Ernst-Moritz Arndt University inGreifswald. After working with R. Franke at the Academy of Sciences ofthe GDR in the drug design field for four years, he joined ChemiekombinatBitterfeld doing pesticide design. In 1982 he joined the ParmacologicalInstitute of the Ernst-Moritz Arndt University in Greifswald. Since 1992he has been Vice-Director of Consulting in Drug Design GbR in Basdorf.His research interests include QSAR and computer-assisted drug design.

3698 Chemical Reviews, 2000, Vol. 100, No. 10 Benigni et al.


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together with th e presen ce of relatively specific, veryhigh exposures perm itted t he development of epide-miological knowledge unparalleled for other chemicalclasses.

Bladder cancer in men is the m ost stu died t umort y pe : a l a r ge n u m b e r of s t u d i es (s ee Vi n ei s a n dPirastu 8 for a comprehensive review) relating pro-fessional exposure to arylamines (both complexmixtures and single chemical agents) and bladder

cancer h ave been pu blished. The odds r atios (theratio between the tumors observed in exposed pop-u la t ion a n d t h e t u m or s ob se r ve d in a ca r e fu llymat ched cont rol population) for arylamine exposur eg o fr om a r ou n d 2 (i .e ., a 2 -fol d i n cr e a s e i n t h eprobability of developing bladder can cer) for verymild exposur es up to aroun d 100 for extr emely highexposures. 8-11

Most of the above studies r efer to exposures tomixtures of aromatic amines. For 2-naphthylamine,o-toluidine, benzidine, and 4-aminobiphenyl, it hasbeen possible to select cohorts of individuals experi-encing exposure that can be reasonably consideredas single-agent exposure,8 thus providing formaldemonstra tion of the carcinogenic potent ial of theseagents for humans. In the case of 4-aminobiphenyl,there are molecular epidemiology studies8,12 t h a twere a ble to identify a specific DNA addu ct ident ifiedas a derivative of 4-aminobiphenyl. This same DNAadduct was present in exfoliated bladder cells of smokers;13 the presence and concentra tion of DNAaddu cts was correlat ed str ongly with 4-aminobiphe-n yl-hemoglobin adducts. The 4-aminobiphenyl-hemoglobin addu cts in both smokers an d nonsmokersi s m o d u l a t e d b y t h e N-acetylation phenotype: ir-r e s pe ct i ve of t h e s m ok i n g s t a t u s of t h e s u b je ct s ,the genetically based slow-acetylator phenotypewas associated with high concentra tions of the ad-duct.8

The evidence regarding th e carcinogenic potent ialof aromatic amines in animals was available beforeform al epidemiologic stu dies were condu cted: in th issense, arylamines ar e one of the best examples of thepredictivity of animal experiments for hum an risk.14

T h e e vi de n ce i n e xp e r im e n t a l a n i m a ls h a s b ee ncrucial in the classification of some aromatic aminesfor their carcinogenicity to humans. Benzidine-baseddyes an d MOCA (4,4-meth ylene bis-2-chloroaniline)w er e cl a s si fi ed b y t h e I n t e r n a t i on a l Ag en cy forResearch on Cancer (IARC) as probable carcinogensb a s ed on t h e s t r on g e vi de n ce i n a n i m a ls b efor e

epidemiological evidence was available.8

III. Mechanisms of Action

T h e a r o m a t ic a m i n e s h a v e t o b e m e t a b ol iz ed t oreactive electrophiles in order to exert their carcino-genic potential . Scheme 1 provides a simplifiedpicture of the main metabolic steps. For aromaticamines an d am ides, this typically involves an initialN-oxidat ion to N-h y d r ox ya r y l a m i n e s a n d N-hy-d r ox ya r y la m i d es , w h ich i n r a t l iv er i s m e d ia t e dprimarily by cytochrome P-450 isozyme c (BNF-B)a n d d (ISF-G).15,16 The initial activat ion of nitroaro-mat ic hydrocarbons is likewise thr ough the format ion

of an N-hydroxyarylamine, a reduction catalyzed byboth microsomal and cytosolic enzymes. 5,16 Microso-mal nitroreduction also appears to depend on cyto-

chrome P-450 complex, in pa rticular ra t liver isozymesc, d a n d b (P B-B ) a n d e (PB-D). Cytosolic nitrore-d u ct a s e a ct i vi t y i s a s s oci a t ed w it h a n u m b e r of enzymes, including DT-diaphorase, xa nt hine oxidase,aldehyde oxidase, and alcohol dehydrogenase.16 Inaddition to the reactions of nitrogen oxidation andreduction (main activation pathways), certain aro-matic amines and nitroaromatic hydrocarbons areconverted into electrophilic derivatives through ring-oxidation pat hways. N-Hydroxyarylamines, imino-quinones, and epoxide derivatives are directly elec-trophilic m etabolites, while N-hydroxyarylamidesrequire esterification before becoming capable ofreacting with DNA.17

A ca s e in p oin t of t h e cr u cia l r ole p la ye d b ymetabolism in determining biological activity of aromatic amines is the case of 1-naphthylamine. Thischemical was originally considered to be a humanbladder carcinogen: the results of subsequent epi-demiological studies coupled with the failure todemonstr ate a carcinogenic response in an imal mod-els indicated that this is not the case. 18,19 This lackof carcinogenicity a ppears to be du e to t he failur e of1 -n a p h t h y la m i n e t o b e m e t a b ol iz ed t o a r e a ct i veelectrophile.20 Although 1-napht hylamine has notbeen found to be carcinogenic, its N-oxidized deriva-tive, N-hydroxy-1-naphthylamine, is strongly tum-origenic.18,21,22 N-Hydroxy-1-naphthylamine readily

S c h e m e 1 . P a t t e r n o f Me t a b o l i c Ac t i v a t i o nP a t h w a y s o f A r o m a t ic A m i n e s . Th e S c h e m eS k e t c h e s t h e M o s t R e p r e s e n t a t i v e P a t h w a y s ( s e ed e t a i l s i n t h e t e x t )

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binds to DNA, and the reaction results in the forma-tion of a major DNA adduct through reaction of thea r yl n it r oge n a n d or t h o c a r bon a t om s a t O6 ofdeoxyguanosine.23 Evidence ha s also been presen tedthat a minor adduct is formed by N-substitution atC8 of deoxyguanosine.24 This last reaction is typicalfor N-hydroxyarylamines, whereas r eaction with th eO6 position, which is normally associated with SN1-type reactions, seems to be u nique to N-hydroxy-1-

naph thylamine. The r eaction ofN-hydroxyarylamineswith DNA is proposed to proceed through a proto-nat ed nitrenium ion pair;17 thu s, the relative sta bilityof this reaction intermediate appears to be a crucialpoint in determining the biological activity of aro-matic amines.

The DNA adducts generated in a nimals ar e similart o t h o s e fou n d i n v i t r o a n d h a v e a v er y v a r ia b lep e r si st e n ce i n t i ss u e s for t h e d iffe r e n t a r om a t i camines. This difference in persistence may resultfrom the fact that different structural distortions ofthe DNA are recognized with different efficiency bythe DNA repair enzymes that operate the excisionof the adduct.16

The polymorph ism a nd different ial distribut ion ofthe enzymes responsible for the metabolic activationof aromatic amines ha s a crucial role in determ iningthe organ specificity observed with these substances.For instance, if N-acetylation precedes N-oxidation,the concentration of N-hydroxyarylamine availablefor transport to the bladder decreases. 16 Thus, indi-viduals with a rapid acetylator phenotype should beat a lower risk for bladder cancer from exposure toaromatic amines, which is what has been actuallyobserved.25 Likewise, t he inability of dogs to N-acetylate aromatic amines is consistent with theirsusceptibility to bladder tumors. While acetylation

appears to afford protection from bladder tumorinduction, t he opposite ma y be tr ue for other tissues.Thus, a higher incidence of colon cancer has beenfound in low-risk individuals with a rapid acetylatorphenotype.16

There is evidence26 in rats that the expression of acetyltran sferase in t issues of the central nervous,gastrointestinal, urinary, and reproductive systemsis highly regulated, as it is in other organs commonlyassociated with aromatic amine carcinogenicity. Thesubtleties and specificities of such complex and highlyorgan-specific toxification/detoxification balance pro-d u ce a h ig h va r ia b ilit y in t h e t a r ge t or ga n s of aromat ic amines t hat in fact exert t heir carcinogenicpotential at many different sites.

IV. Qualitative Notions on the StructureActivityRelationships of Aromatic Amines

The large amount of data on animal carcinogenesisallowed for the sketching of some basic SAR require-ments for the carcinogenesis induced by a romatica m i n es . T h es e q u a li t a t iv e r u l es a r e cl ea r l y s u m -marized by Lai et a l .5 The basic requirement is thepresence of an aromatic ring system (a single ringor more than one ring forming a conjugated system,fused or nonfused) and the amine/amine-generating

group(s). Amine-gener at ing groups (due t o meta bolicinterconversion) are typically the hydroxylamino,nitr o, an d nitr oso groups. In some cases, replacementof an amino group with a dimethylamino group doesnot result in a significant loss of th e carcinogenicactivity of aromatic amine compounds since metabolicN-demeth ylat ion readily occurs in vivo. Oth er im-p or t a n t s t r u ct u r a l f ea t u r e s a r e ( 1 ) t h e n u m b e r a n dna tur e of aromatic rings, (2) the na tu re an d position

of the a mine/amine generating groups, (3) the na tur enumber and position of other ring substituents, and(4) the size, shape, and polarity of the molecules.Interestingly, many of the structural features thata r e i m por t a n t for t h e ca r ci n og en i ci t y a l so h a v eimportant influences on their bioactivation mecha-nisms.

The number and nature of aromatic rings modu-lates the carcinogenic potential of aromatic aminesvia the modulation of the leaving potential of theacyloxy anion that is the rate-limiting step of thebioactivat ion process. The force of conjugation, fa-cilitating the departure of acyloxy anion, increases

fr om p h e n yl t ow a r d h i gh e r a r y l g r ou p s . T h i s i scon s is t e n t w it h t h e fi n di n gs t h a t a n i li n e (s in g lephenyl ring) is a weaker carcinogen than benzidineor -n a p h t h y la m i n e (t w o p h e n y l r i n gs ) a n d m or elikely with th e presence of th e term nu mber of ringsi n t h e Q S A R s o f t h e a r o m a t i c a m i n e s .27 E v e n t h en a t u r e a n d p os it i on of t h e a m i n e o r o f t h e a m i n e -generat ing group influences th e carcinogenic poten-tial at th e level of bioactivation step: for example,for dialkylamino groups with bulky or long alkylsubstitution, N-dealkylation does not readily occurto allow furt her bioactivation. Replacement of th edimeth ylamino group of 4-dimeth ylaminoazobenzeneby a diethylamino or a higher dialkylamino group h as

been shown to lead to a marked attenuation of i tscarcinogenicity28 and muta genicity.29

R in g s u b st i t u en t s ot h e r t h a n a m i n o or a m i n o-generating groups have been reported to modulatearomatic a mines carcinogenicity mainly by stericeffects: th e larger th e substitu ents (especially in theortho position), the less potent the chemical. 5 O n t h econ t r a r y , t h e s u b st i t u t ion of a ch l or o g r ou p or ameth yl/meth oxy group ortho to th e am ino group oftenenhances activity.30,31

V. QSARs for Mutagenicity

Because of the shortcomings of the rodent carci-nogenicity bioassay (long times, high price, sacrificeof large numbers of animals), the aromatic amineshave repeatedly been tested in short-term mutage-nicity assay, notably with the Salmonella typhimu-rium (Ames test) bacterial a ssay.32,33 This assay is areliable tool for qualitatively predicting rodent car-cinogenicity (hence for extrapolation to huma ns),since chemicals which are positive in the Ames testha ve a high probability of also being rodent car cino-gens (80% for the general universe of chemicals,with differences from class to class). I t should beadded that the reverse is not true: unfortun ately, anegative Ames test does n ot provide useful informa -



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t i on , s in ce i t h a s b ee n s h ow n t h a t a n Am e s -t e s tnegative chemical ha s about the same probability ofbeing a car cinogen or a noncarcinogen.34-37 The largen u m b e r of e x p er i m en t s on a r om a t i c a m i n e s p e r -for m e d w i t h t h e Am e s t e s t h a s p r ov id e d a l a r ged a t a b a s e o f m u t a g e n ici t y r e s u l t s t h a t h a v e b ee nstudied with QSAR a pproaches by several aut hors.Two reviews have a ppeared on such QSARs.38,39 Th efollowing is a presentation of the individual QSAR

studies.Trieff et a l.40 studied the Salmonella mutagenicity

o f 1 9 a r o m a t i c a m i n e s t e s t e d i n t h e s t r a i n s T A 9 8(fram e-shift mut ations) and TA100 (base-pair mu ta-tions), with the addition of S9 metabolizing fractionfrom Aroclor 1254-induced rat liver. Separate QSARmodels were found for the two strains by multiplelinear regression.

The bacterial mutagenic potency was defined asBR ) 1 + NR/nmol, where NR is the net revertantn u m b er . T h e r e v er t a n t s a r e t h e ce lls t h a t u n d er -went muta tion. The indicator variable I1 was 1.0 ifthe amine or acetamido group was proximal (adja-

cent ) to the junctur e (i.e., th e carbon at om connectingthe substituted ring with the rest of the molecule).I2 r e la t e d t o w h e t h er t h e a m in e gr ou p w a s fr e e(I2 ) 1) or acetylated (I2 ) 0). Equations 1 a nd 2 a requite similar and show that mutagenicity increasedwith lipophilicity. On the other hand, mutagenicityw a s r e d u c e d w h e n t h e a m i n e o r a c e t a m i d o g r o u pw a s in or t h o t o t h e ju n ct u r e , b e ca u s e of s t er ichindrance in i ts biotransformation. Mutagenic po-tency was also decreased by the acetylation of thea m i n o g r ou p , p r ob a b ly b eca u s e t h e a ce t yl g r ou pneeds t o be first split off prior to oxidation of th eamine group.

Ford and Griffin 41 related the mutagenicity of a

variety of heteroaromatic amines present in cookedfoods with th e sta bilities of the corresponding n itre-n iu m ion s (s ee S ch e m e 1 ). T h e s t a bilit y of t h en i t r en i u m i on s w a s m e a s u r ed b y t h e ca l cu l a t edenergy (H) of the process

H w a s ca l cu l a t e d u s i n g t h e s e m ie m p ir i ca lAM1 molecular orbital procedure. It appeared thatt h e m u t a g e n i c p o t e n c i e s (m ) i n t h r e e S a l m on e ll as t r a i n s (T A9 8 , T A1 00 , a n d T A1 53 8 ) cor r e la t e dw it h t h e H values according to the following

equations.

Ford and Herman 42 stu died the r elative energetics(H) of arylamine N-hydroxylation a nd N-O h e t -erolysis (ArN H 2 fArNHOH fArNh+) for cond ensedsystems of two, three, and four rings using semiem-pirical AM1 molecular orbital theory. Limited cor-relations between the energetics of nitrenium ionformation and experimental TA98 and TA100 mu-tagenicities wer e found.

An important contribution to the QSAR modelingof aromatic and heteroaromatic amines mu tagenicitywas pr ovided by Debnat h et al.,43 who collected da taon a wide number of chemicals with largely differentbasic structur es (e.g., aniline, biphenyl, ant hra cene,pyrene, qu inoline, carbazole, etc). Th e experiment aldata referred to Salmonella TA98 and TA100 strains,with S9 metabolic activation. The mutagenic potencyis expressed as log(revertants/nmol). The AM1 mo-lecular orbital energies are given in electronvolts. Themutagenic potency in TA98 + S9 was modeled by

where HOMO is the energy of the highest occupiedmolecular orbital, LUMO is th e ener gy of the lowestun occupied m olecular orbita l, an d IL is an indicatorvariable that assumes a value of 1 for compoundswith t hree or m ore fused r ings. The electronic term sHOMO and LUMO, though statistically significant,accounted for only 4% of variance, whereas log Palone accounted for almost 50%. The most hydrophilicamines (n ) 11) could not be tr eated by eq 6 an d were

modeled by a separate equation containing only logP, t h u s s u g ge s t in g t h a t t h e s e a m i n es m a y a c t b y adifferent mechanism. The mutagenic potency in theSalmonella strain TA100 + S9 was expressed by

Also in this case, a different equation was neces-sary for the most hydrophilic amines (n ) 6). Overall,the principal factor affecting the relative mutagenic-

log BR-TA98 )-1.639 ((0.399) + 0.816 ((0.127) log P -

0.752 ((0.174) I1 + 0.377 ((0.174) I2

s ) 0.78 n ) 19 r2

) 0.78 (1)

log BR-TA100 )-1.559 ((0.282) + 0.784 ((0.090) log P -

0.735 ((0.123) I1 + 0.496 ((0.123) I2

s ) 0.80 n ) 19 r2) 0.88 (2)

ArNH 2 + P h N+

H f Ar N+

H + P h N H 2

log(m ) TA98 )

-0.181 ((0.043) H+ 0.227 ((0.2792)

s ) 0.966 r2) 0.593 n ) 14 (3)

log(m ) TA100 )

-0.147 ((0.024) H- 0.1619 ((0.450)

s ) 0.540 r2)0.770 n ) 13f (4)

log(m ) TA1538 )

-0.2417 ((0.0353) H- 0.801 ((0.765)

s ) 0.245 r2)0.922 n ) 6 (5)

log TA98 )

1.08 ((0.26) log P + 1.28 ((0.64) HOMO -

0.73 ((0.41) LUMO + 1.46 ((0.56) IL + 7.20 ((5.4)

n ) 88 r) 0.898 s ) 0.860 (6)

log TA100 ) 0.92 ((0.23) log P +

1.17 ((0.83) HOMO - 1.18 ((0.44) LUMO +

7.35 ((6.9)

n ) 67 r) 0.877 s ) 0.708 (7)



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i ty of the aminoarenes was their hydrophobicity.Muta genicity increased with increasing HOMO val-ues: this positive correlation seems reasonable sincecompounds with higher HOMO values are easier tooxidize and should be readily bioactivated. For thenegative correlation with LUMO, on t he other h an d,no simple explanation could be offered by the authors.A remar kable difference between t he m odels for th etwo Salmonella strains was that the TA100 QSAR

lacked th e IL term present in the TA98 model. It washypothesized tha t larger a mines ar e more capable ofinducing frame shift mutations (TA98 is specific forframe shift mutations, whereas TA100 is specific forbase pair subst itution m uta tions) and t hat this effecti s n ot a ccou n t e d for b y t h e i n cr e a s e of l og P a tincreasing size of th e molecules.

The above paper deserves two m ore comments.First , in a parallel work the authors 44 modeled themuta genicity of nitroarenes. The main metabolicpathway of the nitroarenes is supposed to include theformation of the hydroxylamine by cytosolic reduc-tase; th en t he fate of the activated compound sh ouldbe identical t o tha t of amines.5,16 As to be expected,

the equations reported for the nitroarenes are quali-tat ively very similar to the amine equat ions, with t hemajor difference being th at the HOMO term (relatedto the oxidative step of the amines) is missing. 44 Thisi n di ca t e s t h a t s u ch e qu a t i on s n ot on l y p r o vi de am e a n s for p r e di ct i n g m u t a g e n ici t y, b u t ca n a l soreveal as pects of th e activation mecha nism. A secondcomment concerns inactive compoun ds. While th eQSARs for the aromatic amines are quite good inmodeling mutagenic potency, they are less satisfac-tory when one wants to predict the activity of thenonmu tagenic amines: in man y cases inactive com-pounds are incorrectly predicted to be highly mu-tagenic. 43

F or t h e s a m e s et of com p ou n d s con s id e r ed b yD eb n a t h e t a l .,43 the discrimination between mu-tagenic and nonmutagenic amines was studied morein deta il by Benigni et al.45 It appeared that lipophi-licity alone ha d no discriminat ing power in TA98 andTA100, which is at odds with the major role playedin the modulation of potency within the group of active compounds. Though statistically significant,discriminant functions separating muta genic fromnonmu tagenic amines sh owed a r eclassification ra teof only about 70% accuracy. They were based mainlyon electr onic and steric hindra nce factors. The sam ew a s t r u e for t h e n it r oa r e n es m u t a ge n icit y. I n a

second paper, the same group46

tried to improve thediscriminant models for the mutagenic activity of theamines in Salmonella. The best discrimination wasob t a in e d b y s p l it t i n g t h e a m i n es i n t o s t r u c t u r a lsubclasses. The single-ring amines were best sepa-rated by electronic factors (first HOMO and secondLUMO, in decreasing order of importance) (correctreclassification rate around 70%). This result con-firmed the central role of metabolic transformationin the muta genic activity of these chemicals. Thediphenylmethan es were m odeled by the contr ibutiont o m ol a r r e fr a c t iv it y of t h e s u b st i t u en t s i n or t h oposition to the functional group, thus indicating thenegat ive effect of steric hindra nce on t he accessibility

of the metabolizing system (correct reclassificationrate: 87% for TA98; 93-100% for TA100). Stericfactors, as measu red by a similarity index, were alsoa key factor in the discrimination of biphenyls. Thefused-rings amines were all mutagenic, so no dis-criminant model was necessary. The authors con-clu d ed t h a t t h e m in im u m r e qu ir e m en t s for t h emut agenicity of the a romatic amines (as modeled bythe discriminant functions) were different from the

factors r uling t he modulation of potency.Using their computer program CASE, Klopman et

a l.47 analyzed a set of approximately 100 aromaticamines. Th e CASE m ethodology is a softwar e pack-age that selects its descriptors automatically from alearning set of molecules. It identifies single, con-tinuous structural fragments that are embedded int h e com p le t e m ol ecu l e a n d s e le ct s t h os e t h a t a r estatistically associated with activity or nonactivityor with increasing potency. Normally, the programscreens th e molecules for all th e possible fra gmentsra nging from 2 to 10 hea vy (nonhydrogen) atoms. Theprogram was u sed to examine mut agenicity in Sal-monella stra ins TA98 and TA100 (with S9 activation)

and yielded a number of structural features associ-ated with mutagenicity and nonmutagenicity. Thiswork wa s extended by Zhang et al.,48 who studied 61heterocyclic amines formed during food preparation.I n b o t h s t u d i e s , t h e m a j o r f e a t u r e l e a d i n g t o m u -t a g en i c a c t iv it y w a s t h e a r om a t i c a m i n o g r ou p .Electronic parameters were also calculated, and theLUMO ener gy was foun d to corr elate n egatively withthe mutagenic potency of the molecules. A modelbased on a number of fragments (the amino groupin different combinations of atoms) together with theLUMO attained r2 ) 0.857.

Lewis et al.49 stu died a noncongeneric set of foodmut agens, th e ma jority being het erocyclic amines (n) 17). This study was in l ine with other studies of the sam e group aimed at highlighting the st ructurald e t er m i n a n t s t h a t m a k e t h e ch e m ica l s g ood s u b -stra tes for cytochr ome P4501 (CYP1). For the TA98strain (frame shift mutations) of Salmonella, the bestcorrelation of mutagenicity was with molecular di-ameter r) 0.91, hence with planarity. For the TA100stra in (base pair mut ations), the best correlation waswith the difference between the LUMO and HOMOe n er g ie s: h i gh m u t a g e n ici t y w a s r e la t e d t o l owvalues of the difference, h ence to high chemicalreactivity.

B a s a k a n d G r u n w a ld50 explored the suitability of

r ou g h a n d fa s t Q S AR m od e ls b a s ed on e a s il ycalculable th eoretical indices. For a set of 73 ar omaticand heteroaromatic aminesspreviously studied byDebnath et al.43sthe a uth ors calculated a wide range(n ) 90) of topological indices. Then th ey const ru ctedfive similarity spaces based on (a) count s of at ompairs, (b) principal components (PC) from the topo-logical indices, (c) P Cs from topological indices plu sphysicochemical para meter s used by Debnat h et al.,43

(d) PCs from physicochemical parameters, and (e)p h ys icoch e m ica l p a r a m e t e r s. I n e a ch of t h e fi vesimilarity spaces, the mutagenic potency of everychemical was est imated by averaging th e potency ofit s k-nearest neighbors (k ) 1-5). I t appeared that



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the easily computable method based on atom pairswas almost as reliable (r ) 0.77) as the similaritymethod based on physicochemical properties (r )0.83). The disadvant age with th is type of descriptorsis, of course, that interpretability is very limited.

Hat ch et al.51 stu died the m ut agenic potency (fra mes h ift m u t a t i on s i n T A9 8 or T A1 53 8 S a lm on e ll astra ins) of a series of heteroaromatic amines formedduring the cooking of t he food from two classes:

amin oimidazoazaar ene (AIA) (n ) 38) and aminocar-boline (AC) (n ) 23). For the AIA compounds, thefeatures relevant for the mutagenic activity were asfollows: num ber of fused rings, num ber of hetero-a t om s in r in gs 2 a n d 3 , m e t h yl s u bs t it u t ion onimidazo ring nitrogen atoms, and methyl substitutionon r in g ca r b on a t om s (r2 ) 0.78). The relevantfeatures for the AC compounds were as follows:position of the pyridine-type nitrogen atom in ring1, position of the exocyclic am ino group in ring 1, an dmethyl substitution at ring carbon atoms (r2 ) 0.80)The goodness of fit values referred to models includ-ing all the relevant features. In a further analysis,H a t c h e t a l .52 considered several molecular orbital

properties calculated at different approximations,togeth er with s tru ctura l factors, for 16 AIA muta gensa n d t h e ir n i t r en i u m i on m e t a b ol it e s . T h e m a j orfindings were as follows: (1) the potency increasedwith the size of the aromatic ring system, (2) potencywas enh anced by the pr esence of an N-methyl group,(3) introduction of additional nitrogen atoms inpyridine, quinoline, a nd quinoxaline rings supportedpotency, (4) potency was inversely related to theLUMO energy, (5) potency was directly (althoughweakly) related to the LUMO energy of the derivednitrenium ions, (6) th e calculated thermodynam icsta bility of th e nitr enium ions wa s directly correlatedwith nitrenium LUMO energy and with the negativecharge on the exocyclic nitrogen atom. The authorscommented about t he lack of a clear explan ation forthe role of LUMO energy, since the oxidation of thea m in e g rou p w a s e xp ect e d t o b e t h e m a in r a t e -limiting step in the m etabolism of the amines. Ha tchand Colvin 53 reconfirmed the above results in a widerset of 95 aromatic and heteroaromatic amines, to-gether with the puzzling role of the LUMO energy.

M a r a n e t a l .27 reevaluated the data set collectedby Debnath et al.43 with a very large set of descriptors(n ) 619), including various constitutional, geo-metrical, topological, electrostatic, and quant umchemical descriptors. A final model with six descrip-

tors was established (r2

) 0.8344). The most impor-tant descriptor was the number of aromatic rings,followed by (in decreasing order of import an ce) -po-larizability (second-order hyperpolarizability), hydro-gen-acceptor surface a rea, hydrogen-donor sur facearea , maximum t ota l inter action ener gy for the C-Cbond, and maximum total interaction energy for aC-N bond. Maran et al.27 concluded tha t t he leadingd e scr i pt or i n t h e ir m od e l ( n u m b er of r i n gs ) w a sapproximat ely proportional to t he a rea of the hydro-phobic aromatic hydrocarbon part of these moleculesand was thus directly related to the hydrophobicityof polycyclic and condensed ar oma tic compounds(correlation coefficient between number of rings and

log P r2 ) 0.3715). This correlation is only weak, andthe authors stressed that they could not add log P t ot h e ir m od e l. T h er e i s p r ob a b ly a h i gh m u l t ip lecorrelation between the entirety of their variablesand log P which was not investigated. However, thenumber of rings was preferred by the authors to logP based on the argument that i t is not an empiricalparam eter. The HOMO an d LUMO energies did n otappear in th e model.

VI. QSARs for Carcinogenicity

Although the ma jor concern posed by th e ar omaticamines derives from their carcinogenic potential, thenumber of QSAR studies is quite limited.

Yu t a a n d J u r s 54 applied their ADAPT (automaticdata analysis using pattern-recognition techniques)software system to a set of 157 aromatic amines; tob e in cl u de d in t o t h e d a t a s et , a com p ou n d w a srequired t o h ave biological activity data reported(either positive or negative) in at least three organs i t e s , i t h a d t o b e a r o m a t i c a m i n e , a n d i t h a d t obelong to one of five common structural classes

(biphenol, stilbene, azo-compounds, fluorene, meth-ylene). Topological a nd geomet rical descript ors wer eused, a nd to avoid chan ce separations, multicol-linearities were checked and the number of descrip-tors was reduced to 31. Particularly important werethe molecular connectivity environment descriptors,based on structural features related to the theory onthe mechan isms of action of the aromatic amines(e .g ., p r im a r y or s e con d a r y a m i n es , p r e se n ce of bridging groups, etc.). The ana lyses were repeatedwith severa l pat tern -recognition meth ods (Bayesianquadratic discrimination, Bayesian linear discrimi-n a n t , K-nearest neighbor classification, i terativeleast-squares linear discrimina tion, s implex discrimi-

nating algorithm, l inear learning machine). E achcompoun d was considered to be either a ctive (at leastthr ee active sites) or inactive (negative in all sites).The chemicals were divided in 11 possible subsets,a ccor d in g t o or g a n s a n d r ou t e of a d m in i s t r a t ion .S e ve r a l Q S AR a n a l ys e s w er e p e r for m e d , on t h edifferent subsets and on the entire set of chemicals,with the various pattern -recognition methods. Theiterative least-squares program enjoyed the mostsuccess (classification rates around 90%). Overall, theanalyses indicated that the number of rings (relatedto molecular volum e or bulk) is an import an t descrip-tor relating aromatic amino str ucture to carcinogenicpotential . Other importa nt descriptors were th ose

related t o size and shape (e.g., sm allest principalmoment). Several subsets of descriptors supportedlinear discriminant functions tha t could separa tecarcinogens from noncarcinogens.

Loew et al.55 challenged the capabilities of theoreti-cal chemistry to characterize the chemicals as wellas the physical and chemical interactions with thebiological tar gets. Eight aromatic am ines were se-le ct e d for t h e s t u dy; t h e s a m ple w a s s m a ll b u tconsist ed of four p air s of isomeric am ines. On e of eachpair wa s a n active carcinogen, while th e other wasinactive or of doubtful activity. Mutagenic potencydata, even though not obtained with the same bacte-rial strain, paralleled the carcinogenic activity; the



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weak mu tagens were t he inactive or more mar ginallyactive carcinogens. These pairs of isomers wereselected as ideal tests of the abili ty of calculatedelectronic parameters alone to predict relative bio-logical activity, since effects such as transport ande li m in a t i on s h ou l d b e m or e n e a r l y t h e s a m e forboth isomers of a given pair than for the group as awhole. Electronic reactivity parameters relevant tothe relative ease of metabolic transformation of each

parent compound to hydroxylamine by cytochromeP -4 50 , a s w el l a s t o ot h e r com p e t in g m e t a b ol icproducts involving ring epoxidation and hydroxyla-tion, were calculated. Comparing the results for pairsof i s om e r s , i n e a ch ca s e t h e v a lu e of t h e N -a t omsuperdelocalizabilityschosen as an indicator of theextent of forma tion of hydroxylamine from pa rentcompoundsswas larger for the more potent mutagen/carcinogen. Moreover, the less potent isomer in eachpair had the ring carbon which was most reactive(i.e., larger values of ring carbon superdelocalizabil-ity) to direct ph enol formation, which appea red to bean effective detoxification pathway. Ring epoxidation(as measured by -bond reactivity) appeared to be

more activating than detoxifying. In addition, twomeasu res of covalent addu ct forma tion ability of thehypothesized intermediate reactive species (arylni-trenium ion) paralleled the biological activity withineach pair (electron density on N an d C atoms in th elowest en ergy empty molecular orbita l of the a rylni-trenium ion).

For completeness, the work of Vracko56 and of Giniet al.57 should be ment ioned, wh ich will, however, notbe discussed in detail a s it is n ot specifically con-cerned with aromatic amines and is thus out of thescope of this r eview. QSAR models ba sed on th eoreti-cal descriptors were derived for noncongeneric setsof benzene derivatives, including different propor-

t i on s of a r o m a t ic a m i n es , u s in g a r t i fi ci a l n e u r a lnetworks. The models devised by Vracko56 were ablet o d e scr i be t h e t r a i n in g s e t , b u t t h e ir p r e di ct i onability of carcinogenic potency (TD 50) was l imited.Gini et al.57 performed a retrospective study on 104N-containing benzene derivatives that resulted in aquite good correlation after removal of several outli-ers.

VII. Original Model for the Carcinogenic Potencyof Nonheterocyclic Aromatic Amines in Rodents

Whereas several QSAR models h ave been gener-ated for the mut agenicity of the a romatic amines, wehave found in the litera tu re only two models specific

for their rodent carcinogenicity;54,55 moreover, onlyth e yes/no activity was m odeled. Vracko56 consideredt h e ca r ci n og en i c p o t en cy of a n u m b e r of a m i n e swithin a model for noncongeneric aromatic chemicals.For th is reason an d for t he sa ke of completeness, weperformed ad hoc for this paper a QSAR analysis ofthe carcinogenic potency of the nonheterocyclic aro-matic amines.

1. Data and Methods

A. Carcinogenicity Data of Aromatic Amines

The carcinogenic potency data used for this studywere the TD50 (mg/kg/day) values calcula ted by Gold

et al .58 T h e T D50 is the daily dose rate required tohalve the probabili ty of an experimental animal of remaining tumorless to the end of i ts standard l ifespan. We used the TD 50 values for r at and mouse asr e p or t e d i n t h e C a r ci n og en i c P ot e n cy D a t a B a s e(CPDB), available at the Internet site http://poten-cy.berkeley.edu/hybrid.html. These are har monicm e a n s o f t h e T D50 values for the different tumortypes, averaged over th e rodent s pecies. For t he scope

of the QSAR analyses, carcinogenic potency wasdefined a s follows: mice, BRM ) log(MW/TD50)mouse;r a t s , B RR ) log(MW/TD50 )ra t, w her e MW is t h emolecular weight .

B. Chemical Structures and Chemical Parameters

T a b l e 1 s u m m a r i z e s t h e s t r u c t u r e s o f t h e c o m -pounds (anilines, biphenylamines, naphthylamines,and aminofluorenes) for which carcinogenic potencydata were available. Chemical str uctures a re pr e-sented as substituted anilines according to the con-ventions outlined below.

To describe the chemical properties of the com-

p ou n d s , g lob a l a n d l oca l p a r a m e t e r s w er e u s e d.Global electronic properties were characterized by theEHOMO (energy of the highest occupied molecularorbital) and ELUMO (energy of the lowest emptymolecular orbital) calculated by the semiempiricalmolecular orbital method, AM1, after optimizings t r u ct u r e s a t t h e s a m e l ev el of t h e or y (p r og r a msystem SYBYL, Tripos). Overall hydrophobicity ise xp r e ss e d i n t e r m s of l og P computed from theprogram Tsar (Oxford Molecular).

In an attempt to gain some insight into possiblelocal effects, ring substituents were characterized byhydrophobic, electronic, and steric substituent con-stant s. Of the m any param eters tr ied, the followingappear in the resulting QSARs: I a n d R (inductiveand resona nce-polar electronic substituent consta ntsaccording to Swain and Lupton), MR (molar refrac-tivity; values scaled by 10-1), and Char tons ES valuesto characterize steric properties of substituents R atthe functiona l amino group (all data from r ef 59). Todescribe ring substituents, positions must be defined.The following conventions wer e u sed: (i) the func-tional am ino group is always in p osition 1sadditionalamino groups are t reat ed as su bstituent s; (ii) if morethan one amino group is present, we considered thefunctional group to be the one which has a substitu-ent in an adjacent position (ortho substituent). Otherconventions have also been tried but led to poorerresults. (iii) If only one ortho su bstituen t is present ,this substituent is placed in position 2.

Biphenylamines, naphthylamines, and aminofluo-renes were treated as substituted anilines. For thebiphenylamines (see Figure 1), substituents in thea n ilin e p a r t a r e ch a r a ct e r ize d a s in s u bs t it u t e da n i l i n e s . I n c a s e s 1 a n d 2 , t h e s e c o n d p a r t o f t h emolecule (second phenyl ring plus su bstituen ts a t t hisring) is then treated as a para su bstituent where thebridge X may be present or absent. Only MR valuesare available here. In case 3, the non-aniline partappears as the ortho substituent and is fully param-etrized with I , R , a n d MR. I n t h e ca se of t h e



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naphthylamines (see Figure 2), two situations arepossible. They are treated as anilines substituted by-C4H 4- with an estimated MR of 0.8. This amountis equally distributed over th e positions of substitu -tion so that MR2 ) MR 3 ) 0.8 in case 1 and MR3 )MR 4 ) 0.8 in case 2. To characterize electroniceffects, the I a n d R values of CHdCH 2 a r e u s edfor the two r espective positions. If additional sub-

stituents occur, the MR values are correspondingly

corrected; n o values of electr onic su bstituen t con-stants are then available. For the aminofluorenes,finally, only steric effects (MR) could be parametrizedfollowing the scheme presented in Figure 3.

All chemical parameters appearing in the QSARsas well as carcinogenic potencies are summ arized inTable 2. Table 3 summarizes the correlation matrixfor the entirety of compounds in Table 2. There areno serious collinearit ies between the independentvariables. However, multicollinearities occur whichwill be discussed in the context of th e respectiveQSAR equations.

Some special features of the compounds are char-acterized by the following indicator variables: I(Bi)

T a b l e 1 . S t r u c t u r e s o f C a r c i n o g e n i c C o m p o u n d s a

n o. r in g An X br idge X R

1 N 3-C4H 4-4 H2 B 4-P h -4-NH 2 H3 F 3,4-Me2 COMe4 B 2 -C l,4 -P h -3 -C l,4 -N H 2 CH 2 H5 A 2-Me H6 B 4-C(dNH)-Ph-4-N(Me)2 CdNH 2 Me 27 B 2-P h H8 A 2,6-Cl2,4-NH 2 H

9 A 2-NO2,4-N(C2H 4OH )2 Me10 B 4-CH 2-Ph-4-NH 2 CH 2 H11 A 4-Cl CONMe212 B 4-O-P h-4-N H2 O H13 A 2-OE t ,5-NH COMe H14 F 3-Me,4-NE t H15 A 3-NO2,4-OH H16 A H H17 A 2-OMe H18 A 4-Cl H19 A 2-Cl,5-N H2 H20 A 2-NH 2,4-Cl H21 A 2-Me,4-OMe H22 A 2-OMe,5-Me H23 B 4-SO2-Ph-4-NH 2 SO 2 H24 A 2-OMe,5-N H2 H25 B 4-CH

2-Ph-4-N(Me)

2CH

2Me

22 6 B 4 -C O-P h -4 -N (M e)2 CO Me227 N 2-C3H 3C(NH 2)-3 H28 A 3-NO2,4-OE t COMe29 A 2-OMe,5-N O2 H30 A 2-NO2,4-NH 2 H31 B 4-S-P h-4-N H2 S H32 A 2,6-(NO2)2,4-CF 3 (nPr)233 A 2,4,5-Me3 H34 B 4-P h H35 A 2-OH ,4-N O2 H36 A 2-OH ,5-N H2 H37 B 4-P h COMe38 B 4-P h -4-F H39 B 4-P h -4-F COMe40 F 3,4-Me2 COCF 34 1 B 2 -C l, 4-P h -3 -C l, 4-N H 2 H

42 B 4-SO2-P h-4-NH COMe NH 2 COMe43 A 4-OE t COMe44 A 4-F Me,NO45 A H Me,NO46 A 2-NH 2 H47 B 2-NH 2,4-Ph-3,4-(NH 2)2 H48 A 2,4,5,6-F 4,3-NH 2 H49 A 2,4,6-Me3 H50 A H Me251 A 4-Me H52 A 2-OH ,5-N O2 H53 A 2,4,6-Cl3 H54 A 3-Me H5 5 B 2 -O Me ,4 -P h -3 -O Me ,4 -N H 2 H5 6 B 2 -Me,4 -P h -3M3,4-NH 2 H57 A 2,5-Cl2,3-COOH H58 B 2-Me,4-CH 2-Ph-3-Me,4-NH2 CH 2 H

a A ) anilines; B ) biphenylamines; N ) na phthyla m ine s;F ) aminofluorenes. Bridge: bridge between the phenyl ringsin biphe nyla m ine s if pr e se nt. AnX: r ing substitue nt (a llcompounds described as substitu ted anilines; for definitions,see text). R ) substituent at the functional amino group.

F i g u r e 1 . Treatment of biphenylamine.

F i g u r e 2 . Treatm ent of naphth ylam ines .

F i g u r e 3 . Treatment of aminofluorenes.



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T a b le 2 . Ch e m i c a l D e s c r i p t o r s a n d C a r c i n o g e n i c P o t e n c i e s ( B R R , r a t s ; B R M , m i c e ) o f t h e C a r c i n o g e n i cC o m p o u n d s i n T a b l e 1

BRR BRM

n o. MR3 MR 2,6 I 2,6 R 2,6 ES (R) E H OMO E LUMO log P obser ve d pr e dicte da obse r ve d pr e dicte db

1 0.8 0.2 0 0 0 -8.3724 -0.3671 2.27 0.37 0.31 0.59 0.972 0.1 0.2 0 0 0 -8.2341 0.0005 2.16 2.03 2.21 0.97 0.863 0.56 0.2 0 0 3 -8.4704 -0.3989 2.61 2.26 1.58 1.47 0.854 0.1 0.7 0.42 -0.19 0 -8.5595 0.0261 3.60 1.14 1.655 0 .1 0.66 0.42 -0.19 0 -8.5528 0.4059 1.73 0.39 0.13 -0.89 -0.456 0.1 0.2 0 0 2 -8.3483 -0.0286 3.02 1.39 1.45 0.63 0.59

7 0 .1 2.64 0.12 -0.13 0 -8.5626 0.1005 2.95 -0.82 -1.118 0.1 1.2 0.84 -0.38 0 -8.2613 -0.089 1.52 -0.66 -0.329 0 .1 0.84 0.65 0.13 1 -8.7212 -1.0742 0.34 -0.44 -0.36 0.47 0.14

10 0.1 0.2 0 0 0 -8.4373 0.3141 2.56 1.00 1.29 0.79 0.2911 0.1 0.2 0 0 5 -8.7767 -0.1195 1.64 0.18 0.0912 0.1 0.2 0 0 0 -8.3253 0.1629 1.91 1.32 1.06 0.78 0.4513 0.1 1.35 0.26 -0.5 0 -8.717 -0.1664 0.2 -1.03 -1.3914 0.56 0.2 0 0 0 -8.0133 -0.2496 2.39 0.57 1.51 0.74 1.0415 0.74 0.2 0 0 0 -9.039 -0.7543 0.93 -0.30 -0.1516 0.1 0.2 0 0 0 -8.6088 0.4153 1.26 -0.46 -0.0417 0.1 0.89 0.29 -0.55 0 -8.5707 0.2834 1.01 0.62 -0.13 -0.89 -1.1918 0.1 0.2 0 0 0 -8.5906 0.1051 1.78 0.15 0.4219 0.1 0.7 0.42 -0.19 0 -8.3803 0.1761 1.00 -0.34 -0.13 -0.94 -0.4420 0.1 0.64 0.08 -0.74 0 -8.3819 0.111 1.00 -0.18 -0.13 -0.97 -0.321 0.1 0.66 0.01 -0.18 0 -8.5193 0.2607 1.48 -0.53 0.0422 0.1 0.89 0.29 -0.55 0 -8.5439 0.2527 1.48 0.15 0.0423 0.1 0.2 0 0 0 -9.0261 -0.3937 1.31 1.04 0.85

24 0.1 0.89 0.29 -0.55 0 -8.1651 0.3817 0.23 -0.12 -0.4 -0.82 -0.8325 0.1 0.2 0 0 2 -8.2965 0.3429 3.71 1.19 1.69 0.09 0.3926 0.1 0.2 0 0 2 -8.6024 -0.2779 2.85 1.68 1.39 0.50 0.5127 0.8 0.9 0 -7.9895 -0.3544 1.48 0.36 0.04 -0.01 -0.0728 0.74 0.2 0 0 3 -9.5438 -1.1895 0.94 -1.01 -1.2929 0.1 0.89 0.29 -0.55 0 -9.1996 -1.1264 0.96 -0.34 -0.330 0 .1 0.84 0.65 0.13 0 -8.5709 -0.9914 0.43 -0.60 -0.3331 0.1 0.2 0 0 0 -8.5894 -0.1632 2.25 1.77 1.18 0.81 0.632 0.1 1.48 1.3 0.26 6 -10.2645 -1.997 4.25 0.01 0.433 0.1 0.66 0.01 -0.18 0 -8.361 0.3996 2.67 0.60 0.45 1.34 0.6434 0.1 0.2 0 0 0 -8.4687 -0.0638 2.95 1.91 1.8235 0.1 0.38 0.33 -0.7 0 -9.1425 -1.0677 0.93 0.14 -0.1536 0.1 0.38 0.33 -0.7 0 -8.0935 0.3428 0.20 -0.06 -0.1537 0.1 0.2 0 0 3 -8.6681 -0.2232 2.58 2.25 2.3538 0.1 0.2 0 0 0 -8.5644 -0.3045 3.09 2.22 2.1239 0.1 0.2 0 0 3 -8.9676 -0.6448 2.72 2.36 2.4040 0.56 0.2 0 0 5.4 -9.1706 -0.8891 3.73 2.23 1.9741 0.1 0.7 0.42 -0.19 0 -8.341 -0.3259 3.242 0.1 0.2 0 0 3 -9.1575 -0.5674 0.57 0.78 0.5943 0.1 0.2 0 0 3 -8.6972 0.1085 0.99 -0.84 -0.14 -1.08 -1.0344 0.1 0.2 0 0 -9.7645 -0.4175 1.83 2.78 2.9145 0.1 0.2 0 0 -9.2655 -0.183 1.69 2.98 2.8646 0.1 0.64 0.08 -0.74 0 -8.3321 0 .4 0.48 -0.36 -0.31 -0.83 -0.7547 0.1 0.64 0.08 -0.74 0 -8.1015 -0.009 0.60 -0.26 0.1348 0.54 0.18 0.9 -0.78 0 -9.0632 -0.7425 1.04 0.32 -0.1349 0.1 1.12 0.02 -0.36 0 -8.3657 0 .4378 2.67 1.42 0.45 0.74 0.150 0.1 0.2 0 0 2 -8.4447 0.4541 1.84 -0.01 0.1651 0.1 0.2 0 0 0 -8.4698 0.393 1.73 0.11 0.1752 0.1 0.38 0.33 -0.7 0 -9.0747 -1.026 0.93 -0.74 -0.1553 0.1 1.2 0.84 -0.38 0 -8.7178 -0.3013 2.82 -0.12 0.6154 0.56 0.2 0 0 0 -8.5625 0.4141 1.73 -1.13 -0.4955 0.1 0.89 0.29 -0.56 0 -8.2224 -0.1597 1.66 2.37 2.0356 0.1 0.66 0.01 -0.18 0 -8.1338 0.00173.1 2.53 2.54 0.8757 0.69 0.7 0.42 -0.19 0 -8.9882 -0.6344 2.0058 0.1 0.66 0.01 -0.18 0 -8.3558 0.3482 3.50 1.49 1.62

a Predicted from eq 20. b Predicted from eq 13.

T a b le 3 . Co r r e l a t i o n M a t r ix ( a l l c o m p o u n d s i n T a b l e 2 i n c l u d e d )

BRM BRR log P E HOMO E LUMO MR 2,6 MR 3 Es (R) I 2,6 R 2,6

BRM 1 0.717 0 .496 0.208 0.012 -0.267 -0.095 -0.095 -0.274 0.369BRR 1 0.603 0.234 0.126 -0.374 -0.026 0.256 -0.392 0.302log P 1 0.008 0.018 0.014 0.0900 0.253 0.112 0.311E H OMO 1 0.386 -0.128 0.133 -0.362 -0.245 -0.099E LUMO 1 -0.028 -0.030 -0.105 -0.072 -0.048MR 2,6 1 -0.166 -0.196 -0.475 -0.329MR 3 1 0.212 -0.114 0.154Es (R) 1 -0.063 0.398I 2,6 1 -0.200R 2,6 1



11/18

) 1 for biphenylamines; I(BiBr) ) 1 for bipheny-lamines with a bridge between the ph enyl rings; I(F )) 1 for aminofluorenes; I(NO2) ) 1, if a NO2 groupi s p r e se n t ; I(RNNO) ) 1 , i f t h e a m in o g r ou p issubstituted with (Me)NO; I(monoNH 2) ) 1, if onlyone amino group is present; I(diNH 2) ) 1, if morethan one a mino group is present

Compounds 22 and 57 behaved as outliers in thecase of BRM and compounds 18, 41, and 47 in the

case of BRR; these compoun ds were n ot included inthe analyses.

2. Results

A. Modeling the Carcinogenic Potency in Mice

The following equation is obtained for BRM

The two most importan t variables in eq 8 ar e logP a n d MR 2,6 accounting for 32.8% and 15.6% of thedata varian ce, respectively. Carcinogenic potencyincreases with increasing hydrophobicity an d increas-ing ener gy of the h ighest occupied m olecular orbitaland decreases with the energy of the lowest emptymolecular orbital and with bulk in the ortho posi-tions. Some ster ic effect is also evident for su bstitu-t i on s a t p os it i on 3 (M R3 t e r m ). T h e ES(R) term,fi n a ll y, i n di ca t e s t h a t s u b st i t u t ion a t t h e a m i n o

nitrogen is un favorable for potency and tha t thise ffe ct b ecom e s s t r on g er a s t h e b u lk i n es s of t h esubstituent(s) increases.

Though significant, eq 8 is of only moderate sta-tistical quality, explaining only 71.4% of the datavarian ce. An an alysis in subgroups of compoun ds wasconsidered a strat egy to gain deeper insight. Thereare two possibilities to select subgroups: (i) accordingto th e ring system (anilines and aminobiphenyles( t h e r e a r e n o t e n o u g h c o m p o u n d s t o t r e a t a m i n o -fluorenes or n aph th ylamines separa tely)), (ii) accord-ing to functionality (compounds with only one andcompounds with more than one (substituted) aminog r ou p ). T h e fi r st p os s ib il it y d id n ot r e s u lt i n a nimprovement, but separating compounds with onea n d m or e t h a n on e a m i n o g r ou p l ed t o i n t e r es t in grelationships.

For the monoamines, eq 9 is obtained.

T h e m o s t i m p or t a n t q u a n t i t ie s a g a i n a r e l og Pa n d MR 2,6 , w h ich a l on e a l r ea d y e xp la i n 6 1.6%of t h e d a t a v a r ia n c e; t h e l ea s t i m por t a n t i s M R3,which contributes only 7%. In contrast to eq 8, noES( R ) t e r m a p p e a r s i n e q 9 . T h e r e a s o n m i g h t b ethat in this subgroup there are only four N-substi-tuted compounds with either R ) H,COMe or R )(n-Pr)2 so that the var iation of properties in R is verylimited.

Equation 9 can be improved by adding an indicatorvariable accounting for the occurrence of NO2 a s aring substituent.

T h e m e a n i n g o f t h e I(N O2) t e r m i s n o t c l e a r . I t

could represent an electronic correction of the EHO-MO/ELUMO t erms but could also indicate a special(potency increasing) r ole of th e NO 2 group.

EHOMO and ELUMO in eq 9 can be replaced byelectronic substituent constants for substituents inthe ortho positions. If Swain-Lupton constants areused, eq 9 t ransforms into eq 11.

E ve n t h ou g h t h e M R3 t e r m (w h ich i s of on l ymarginal importance in eq 9) is no longer significantat the 95% level, eq 11 shows a better fit than eq 9.Obviously electron-releasing substituen ts in t he orth oposition enhance carcinogenic potency. It is unusualsbut not without examples in the QSAR fieldst h a telectronic substituent effects occur for substituentsin only one position. The r eason probably is th at thevariat ion of electr onic properties in t he ortho positionis greater than in the other positions (considerablyhigher variances of electronic substituent constants);in a ddition, some position dependence might also beoperative.

For compounds with more than one free or substi-t u t e d a m i n o g r ou p , t h e fol low in g r e la t i on s h i p i sobtained.

BR M ) 0.56 ((0.18) log P +

1.03 ((0.74) EHOMO - 1.19 ((0.58) E LUMO -

0.79 ((0.37) MR 2,6 - 0.93 ((0.90) MR3 -0.22 ((0.19) ES(R) + 8.51 ((6.31) (8)

n ) 37 r) 0.845 r2) 0.714 s ) 0.485

F) 12.5 p < 0.001

BR M ) 0.74 ((0.31) log P +

2.60 ((1.27) EHOMO - 1.65 ((0.97) E LUMO -

0.85 ((0.46) MR 2,6 - 1.46 ((1.20) MR 3 +21.77 ((11.19) (9)

n ) 17 r) 0.936 r2) 0.877 s ) 0.394

F) 15.7 p < 0.001

BR M ) 1.03 ((0.31) log P +

3.37 ((1.11) EH OMO - 0.97 ((0.89) E LUMO -

0.96 ((0.36) MR 2,6 - 1.41 ((0.92) MR 3 +2.21 ((0.89) I(NO2) + 27.73 ((9.48) (10)

n ) 17 r) 0.968 r2) 0.937 s ) 0.281

F) 25.0 p < 0.001

BR M ) 1.45 ((0.36) log P -

1.30 ((0.78) I 2,6 - 2.45 ((1.37) R 2,6 -1.13 ((0.47) MR 2,6 - 2.32 ((0.76) (11)

n ) 17 r) 0.940 r2) 0.883 s ) 0.384

F) 22.6 p < 0.001

BR M ) 0.32 ((0.22) log P +

0.83 ((0.82) EH OMO - 1.39 ((0.47) E LUMO -

1.21 ((0.58) MR 2,6 - 1.07 ((1.06) MR3 -0.31 ((0.29) ES(R) + 7.41 ((7.21) (12)

n ) 20 r) 0.923 r2) 0.852

s ) 0.283 F) 12.5 p < 0.001



12/18

The most important contribution in eq 12 comesfrom MR 2,6 accounting for 50% of the data variance

followed by ELUMO account ing for 13%; log P

accounts for only 10% of the data variance. Replace-

ment of EHOMO and ELUMO by electronic substitu-

e n t con s t a n t s i s n o t p os s ib le i n t h i s c a s e a s t h e

additional amino group(s) also is a potential reactioncenter.

The equations obtained for the subgroup of com-pounds with only one or more tha n one (substituted)

amino group show a considerably better fit than the

relationship obtained for the entirety of compounds(eq 8). Obviously the separation of the series into

compoun ds with only one a nd compounds with m ore

than one amino groups does make sense. Equation

12 tells the same story as the relationships obtained

for the monoamines: bulk in positions adjacent toa n a m i n o g r ou p i s u n fa v or a b le for ca r ci n og en i c

potency, potency decreases with the energy of the

lowest empty molecular orbital and increases with

the energy of the h ighest occupied molecular orbital

a s w el l a s w it h h y d r op h ob ici t y, a n d b u lk y s u b -s t it u e n t s a t t h e n i tr oge n a n d in p os it ion 3 a r e

unfavorable.

There are, however, also differences between eqs

9-11 on the one hand and eq 12 on the other hand:the coefficients of the log P- and of the E HOMO terms

differ (the latter affords the difference of the inter-

cepts). EHOMO sh ows a mu ch smaller var iance (0.25in the monoamines and 0.07 in the compounds with

more than one a mino group) than log P (0.97 in the

monoamines a nd 1.23 in the compounds with more

than one amino group), so that the difference in thelog P term s is of primary importa nce. Obviously th e

depend ence of car cinogenic potency on h ydrophobicity

is much stronger in the case of the monoamines. Thisr e fl ect s i t se lf n ot on l y i n t h e h i gh e r r e gr e s si on

coefficient of log P in eqs 9-11 as compared with eq

1 2 , b u t a l s o i n t h e m u c h h i g h e r p a r t o f t h e d a t a

variance explained by the log P term in these equa-tions. Thus, the poor fi t of eq 8 is mainly due to a

different relationship of potency with log P for

compounds with only one and with more than one

(substituted) a mino group, respectively. If t his isaccounted for by allowing different slopes for the log

P t e r m , c om p ou n d s w it h on e a n d m or e t h a n on e

amino group can be treated in one equation (plot ofregression results in Figure 4).

Instead of EHOMO and ELUMO separately, thedifference ELUMO - E H O M O r e p r es e n t in g t h ehardness can also be used.

T h e h a r d n e ss is r e la t e d t o t h e b a r r ie r for t h ereaction of an electrophile with an aromatic com-pound. Debnath et al .43 found a similar relationshipfor the mutagenic potency of aromatic amines.

The statistical fit of eqs 13 and 14 is much bettertha n tha t for eq 8. They explain more t ha n 80% ofdata variance, which is an acceptable result for thetype of biological dat a considered. The log P t e r m sin eqs 13 a nd 14 account for about 33% of the da ta

variance, MR 2,6 for about 15%, and the electronicterms (EHOMO and ELUMO) for about 20%. Theseterms reflect the most importan t effects. Thus, inagreement with eqs 9-12, eqs 13 a nd 14 sh ow tha tthe key factors for th e carcinogenic potency in miceare as follows: (i) potency increases with increasinghydrophobicity (this effect seems to be more pro-nounced in compounds with only one amino group),(ii) increasing values of the energy of the highestoccupied m olecular orbita l an d decreasing values ofthe lowest empty molecular orbital enhance potency,(iii) potency decreases with increasing bulk in thepositions adjacent to t he amino group. In addition,bulk in position 3 and at the amino nitrogen inhibit

carcinogenic activity. Th e I(diNH2) term in eqs 13 and14 is of only marginal importance indicating that,other things being equal, compounds with more th anone amino group t end to be intrinsically somewhatmore active than monoamines.

B. Modeling the Carcinogenic Potency in Rats

With the results for BRM in mind, analyses forBRR were a lso perform ed for t he compoun d with onlyone and with more than one amino group separatelyas well as for the entirety of compounds. The follow-ing relat ionsh ip is obtained for compounds with oneamino group (plot of regression results Figure 11).

F i g u r e 4 . Plot of observed values of BRM against pre-dicted values from eq 13.

BR M ) 0.88 ((0.27) log P I(monoNH 2) +

0.29 ((0.20) log P I(diNH 2) +

1.38 ((0.76) EHOMO - 1.28 ((0.54) E LUMO -

1.06 ((0.34) MR 2,6 - 1.10 ((0.80) MR3 -0.20 ((0.16) ES(R) + 0.75 ((0.75) I(diNH 2) +

11.16 ((6.68) (13)

n ) 37 r) 0.907 r2) 0.823 s ) 0.381

F) 16.3 p < 0.001

BR M ) 0.88 ((0.26) log P I(monoNH 2) +

0.30 ((0.19) log P I(diNH 2) -

1.27 ((0.54) (ELUMO - EHOMO) -

1.08 ((0.31) MR 2,6 - 1.09 ((0.79) MR3 -0.22 ((0.12) ES(R) + 0.77((0.75) I(diNH 2) +

10.25((4.85) (14)

n ) 37 r) 0.907 r2) 0.822 s ) 0.382

F) 19.2 p < 0.001



13/18

T h e m os t i m por t a n t v a r ia b le i n e q 1 5 i s l og P,explaining 44% of the data variance, followed byI(RNNO)(36%), whereas only 12% of the data vari-ance is explained by I(Bi) and I(F), indicating thatthese par amet ers ar e of less importa nce. The MR 2,6term, finally, is of only marginal importance (3.5%).

In agreement with the results obtained for BRM,potency increases with hydrophobicity a s the keyfactor. There are, however, neither electronic termsnor effects of bulk in positions 3 or for th e subst itu-tion at the nitrogen (significant factors for BRM). TheMR 2,6 term has a positive sign in contrast to ther e la t i on s h i ps ob t a in e d for B RM , i n d ica t i n g t h a ts u b st i t u t ion i n t h e or t h o p os it i on m i gh t s u p p or tcarcinogenic potency in rats. Since this term contrib-utes very l i t t le to the explained da ta variance, thisconclusion is only tentative. These differences in theQSARs for BRM and BRR highlight specific featuresthat may account for the poor correspondence be-tween the BRM and BRR quantities (see discussion).

Th e I(R N NO ) t e r m i n e q 1 5 a ccou n t s for t h eunusu ally high potency of compounds 74 a nd 75,which possess the N(Me)NdO moiety instead of asimple amino group. Finally, the positive I(Bi) andI( F ) t e r m s l e a d t o t h e c o n c l u s i o n t h a t l a r g e r r i n gsystems tend to be more carcinogenic.

For compounds with m ore t han one (substituted)amino group, eq 16 is obtained.

In contrast to the result obtained for the monoam-ines, eq 16 seems to indicat e th at for compoun ds withmore than one amino group hydrophobicity is notimporta nt for car cinogenic potency wh ile electronicproperties (expressed by EHOMO) come into play.However, even t hough eq 16 shows very good stat is-t i cs , i t i s n o t t h e on l y p os s ib le r e s u lt d u e t o t h e

internal dat a structure. For the compounds consid-ered in eq 16, a m ultiple relationsh ip between log P,EHOMO, I(Bi), and I(BiBr) exists with r) 0.863. Itis, therefore, possible to replace EHOMO in eq 16 bylog P:

According to eq 17, carcinogenic potency does notdepend on the electronic properties but on hydropho-

bicity. Even though eq 16 shows a somewhat betterfit th an eq 17, th ere is no way to decide between t hesetwo possibilities. This illustrates how difficult it isto arr ive at un ambiguous resu lts if mu lticollinearitiesoccur. As will become clear , th e rela tionsh ip with logP is more l ikely to reflect the real si tua tion (seebelow).

Th e I(Bi) and I(F) (the latter significant at only P) 94%) terms in eq 16 indicate that biphenylamines

an d am inofluorenes are int rinsically more active thanpredicted by their log P or EHOMO values, respec-tively, which agrees with eq 15 for the monoaminocom p ou n d s . I n e q 1 7 , t h e I(F ) t e r m i s n o l on g ersignificant. If a bridge between the phenyl rings inbiphenylamines occurs, activity is decreased as fol-lows from the I(BiBr) term in eqs 16 and 17.

If all compounds are to be considered, eq 15 is tobe combined with either eq 16 or eq 17. Combinationof eqs 15 and 16 yields

E q u a t ion 1 9 i s ob t a in e d i f e q s 1 5 a n d 1 7 a r ecombined.

According to eq 18, carcinogenic potency in rat sincreases with hydrophobicity (in this case, only ofthe monoamino compounds) and the energy of thehighest occupied molecular orbital , and bipheny-lamines as well as aminofluorenes are intrinsicallymore active than th e other compounds. The electronicterm , th ough sta tistically significant, contr ibutes onlyvery little to the explained data variance. The pres-ence of a bridge between the phenyl rings of biphe-nylamines is unfavorable (negat ive I(BiBr) t erm).TheI(RNNO) term indicates the high values of BRR of

the two compounds with R ) NO. Equation 19, onthe other hand, does not show any electronic effect:potency increases with hydrophobicity in such a waythat this effect seems to be more pronounced in themonoamino compounds, analogous to eq 13 for thecarcinogenic potency in mice. Again, as in the caseof eqs 16 and 17, there is no way t o decide whethereq 18 or 19 is to be preferred.

B o t h , e q s 1 8 a n d 1 9 , s e e m t o i n d i c a t e t h a t t h ehydrophobic effect is more pronounced in the com-p ou n d s w it h on l y o n e a m i n o g r ou p , w h ich i s i nkeeping with the different coefficients of the log Pterm in eqs 15 and 17. As, however, the confidenceintervals of the regression coefficients associated with

BR R ) 0.46 ((0.35) log P + 2.00 ((0.76) I(Bi) +

1.70 ((0.67) I(F ) + 0.93 ((0.71) MR 2,6 +2.99 ((0.63) I(RNNO) - 1.10 ((0.57) (15)

n ) 20 r) 0.969 r2) 0.939 s ) 0.317

F) 43.3 p < 0.001

BR R ) 0.70 ((0.49) EH OMO +

2.43 ((0.38) I(Bi) - 0.77 ((0.40) I(BiBr) +

0.56 ((0.57) I(F ) + 5.58 ((4.13) (16)

n ) 21 r) 0.972 r2) 0.945 s ) 0.230

F) 68.4 p < 0.001

BR R ) 0.22 ((0.18) log P + 2.14 ((0.47) I(Bi) -

1.08 ((0.40) I(BiBr) - 0.34 ((0.26) (17)

n ) 21 r) 0.960 r2) 0.921 s ) 0.275

F) 66.2 p < 0.001

BR R ) 0.37 ((0.15) log P I(monoNH 2) +

0.61 ((0.43) EH OMO + 2.27 ((0.33) I(Bi) +

1.32 ((0.51) I(F ) - 0.56 ((0.47) I(BiBr) +

3.23 ((0.70) I(RNNO) + 4.79 ((3.72) (18)

n ) 41 r) 0.947 r2) 0.896 s ) 0.358

F) 48.7 p < 0.001

BR R ) 0.41((0.18) log P I(monoNH 2) +

0.22((0.21) log PT I(diNH 2) + 2.07 ((0.47) I(Bi) +

1.15 ((0.58) I(F ) -0.85 ((0.48) I(BiBr) +

2.67 ((0.61) I(RNNO) - 0.51 ((0.38) (19)

n ) 41 r) 0.942 r2

) 0.887 s ) 0.373F) 44.6 p < 0.001



14/18

log P I(monoNH 2) and log P I(diNH 2) in eq 19overlap, it is possible to reunite these two variablesinto a common log P term (plot of regression resultsFigure 5).

An electronic term cannot replace log P nor cansuch a term be added to eq 20. The conclusion is thathydrophobicity is the most important factor. Becauseof t h e d a t a s t r u ct u r e , h o w ev er , i t ca n n e it h e r b eproven nor be ru led out tha t some effect of EHOMOand ELUMO does exist in addition to the hydropho-bic effect. The key variable in eq 20 is log P whichexplains 42% of the data variance followed by I(RN-NO) with 20%. As already mentioned, the I(RNNO)term is afforded by compounds 74 and 75, which showvery high potency. Elimination of these compounds

does n ot affect eq 20 (the IRNNO term is, of course,no longer needed).

In t his equat ion log P accounts for 53% of the datavarian ce, clearly demonstra ting t hat hydrophobicityis indeed t he k ey factor for BRR. Again, no electr onicterm can be added to eq 21.

3. Discussion of the Carcinogenic PotencyQSARs

Significant Hansch equations are obtained for bothBRM and BRR. The key factor for the gradation ofcar cinogenic potency is hydrophobicity: both BRMand BRR increase with increasing log P. The influ-ence of hydr ophobicity is st ronger for compounds withone amino group in comparison with compounds withm or e t h a n on e a m i n o g r ou p . T h is e ffe ct i s m o r epronounced for BRM than for BRR. In the case of BRM, the different dependence of potency on log Pfor com p ou n d s w it h on e a n d w it h m or e t h a n on e

amino group explicitly has to be taken into accountw h e n for m u l a t in g Q S AR s for a l l com p ou n d s b yallowing for different regression coefficients of log Pfor mono- an d diamines. Th is is not necessary in t hecase of BRR. For BRM, in addition to hydrophobicity,electr onic factors a lso play a r ole: potency increaseswith increasing energy of the highest occupied andwith decreasing energy of the lowest empty m olecularor b it a l . As t h e s e or b it a l e n e r gi es a r e r e la t e d t o

electronic substituent constants, they can be replacedby electronic substituent constants in the group of monoamines where the reaction center is clearlydefined: potency is decreased by electron-att ractingsubstituents in the positions adjacent to the func-tional amino group (ortho positions). For BRR, elec-tronic effects are much less importa nt; wheth er su cheffects are operating cann ot una mbiguously be de-cided because of multicollinearities in the data struc-tur e. Carcinogenic potency also depends on the typeof ring system: aminobiphenyls (an d, in th e case ofBRR, also fluorenamines) are intrinsically moreactive tha n anilines or napht hylamines. A bridgebetween t he r ings of th e biphenyls decreases potency.

Steric factors are involved in the case of BRM butare not important in the case of BRR. BRM stronglydecreases with bulk in the positions adjacent to thefunctional amino group, and bulky substituents atthe nitrogen an d in position 3 also decrease potency.The latter effects are, however, not as important.Deeper insight into the role of substituents at thenitr ogen would require tha t n ot only the size, but alsothe nature of such substituents is taken into consid-eration, which, however, is n ot possible with thelimited variat ion in t his position present in t he series.In the case of BRR, R ) (Me)NO strongly enh ancespotency (compounds with this substituent have nomeasured value for BRM).

The QSAR models for BRM and BRR show commonfeatures as well as specific differences. The mostimportan t common feature is th e principal role of hydrophobicity, whereas the differences mainly re-side in st eric and electronic factors t ha t a re importa ntfor BRM but not for BRR. This situation reflects itselfalso in t he corr elation between BRM an d BRR, whichis statistically significant but explains only 58% ofthe data variance (r) 0.764).

Table 4 summarizes the situation for noncarcino-genic aromatic amines. If their carcinogenic potenciesare predicted from the QSARs, they all appear asw ea k ca r ci n og en s (T a b le 5 ). I n ot h e r w or d s , t h e

Han sch equations permit the recognition of strongcarcinogens and the estimation of the gradation of potency within active compoun ds but cannot separ at eweak carcinogens from inactive compounds. This isnot an uncommon situation with H ansch equationsas the properties connected with the gradation of potency need not be identical with those discriminat-ing between active and inactive compounds: thereca n b e m a n y r e a s on s ou t s i de t h e p a r a m e t e r s p a c erelated to potency which can render a compoundinactive. What is significant, however, is that thenoncarcinogens ar e placed in the region of very lowto low potency with the only exception being com-pound 70 which shows high values for both BRM and

Figure 5. Plot of observed values of BRR against predictedvalues from eq 20.

BR R ) 0.35 ((0.18) log P + 1.93 ((0.48) I(Bi) +

1.15 ((0.60) I(F ) -1.06 ((0.53) I(BiBr) +

2.75 ((0.64) I(RNNO) - 0.48 ((0.30) (20)

n ) 41 r) 0.933 r2 ) 0.871 s ) 0.398F) 47.4 p < 0.001

BR R ) 0.35 ((0.18) log P + 1.93 ((0.48) I(Bi) +

1.15 ((0.61) I(F ) -1.06 ((0.47) I(BiBr) -

0.48 ((0.30) (21)

n ) 39 r) 0.918 r2) 0.843 s ) 0.398

F) 45.7 p < 0.001



15/18

BRR. The situation is much better than in the caseof the mutagenic potency of aromatic amines where

the predicted potency for most of the nonmutagenicamines spans the entire range of values up to veryhigh potency.45 To separate active from inactivecompounds, classification methods such as discrimi-nant analysis or SIMCA have to be applied. This willbe the subject of further investigations.

VIII. The QSARs of Aromatic Amines inPerspective

Despite the very complex nature of the processesinvolved, the QSARs obtained by the various authorsare generally in good agreement with already pub-lished observations pertaining t o m echan isms of

car cinogenicity an d mut agenicity, respectively, ofaromatic amines. In general, aromatic amines requiremetabolic activation to yield the ultimate carcinogenor m u t a g e n , a n d t h e p r in ci pa l p a t h w a y(s ) o f t h i sbioactivation involves formation of a hydroxylaminewhich d ecomposes to a reactive nitreniu m ion int er-mediate5,16 (see Scheme 1). This bioactivation mech-anism for aromatic amines is believed to be the same

i n ca r ci n og en e s is a n d m u t a g e n es is , a n d for t h i sr e a s o n , t h e p r e s e n t r e s u l t s c a n b e c o m p a r e d w i t hQSARs for the mutagenic potency of aromatic amines.

In the most mechanistically oriented QSAR an aly-ses, the t oxic activity of th e amines was demonstr at edt o c or r e la t e w it h t h e e a s e o f f or m a t i on of t h e N-hydroxylamine,55 with the stability of the nitreniumion,41,52 and with the ease of formation of epoxideson the aromatic ring.55 Loew55 a l so fou n d t h a t t h e

ease of formation of phenols (a detoxifying pathway)is actually negatively correlated with the carcinogenicactivity.

All studies40,43 considering lipophilicity, includingthe present investigation, confirmed its centra l role.In part icular, Debnat h et a l.43 foun d t ha t lipophilicityis the main determinant of mutagenic potency, witha linear increase in potency observed with increasinglog P. Electronic effects were of seconda ry impor-tance, with potency increasing with EHOMO a nddecreasing with ELUMO. It was also found that thistype of relationship is only valid for compounds withlog P > 1 and does n ot hold for more hydrophilican alogues. This finding is in k eeping with th e resultobta ined for th e carcinogenic potency in a mouse inthe present investigation, i .e., that potency of themonoamines shows a much stronger dependence onlog P than potency of the diamines (our analysis ofcarcinogenic poten cy). The m ajority of compounds inthe paper by Debnath et al .43 for which the QSARshold consists of monoamines, and practically all ofthe too hydrophilic compounds are diamines. Forthe latter, an inverse relationship with log P wa ssuggested which, however, is not well supported bythe data. I t would be worth trying to find out whatt h e r e s u lt for m u t a g e n ic p o t en cy i s i f m on o- a n ddiamines are treated separately.

T h e H O M O a n d L U M O e n e r g i e s w e r e f o u n d t oh a ve a r ole b ot h for t h e m u t a ge n ic a ct ivit y inSalmonella43,46,48,49,52 and for the carcinogenic potencyin a mouse (our analysis). The role of the HOMOe n er g y c a n b e e a s il y r a t i on a l iz ed i n t e r m s of t h epropensity of the toxic amines to form the intermedi-ate metabolite N-hydroxylamine. The role of theL U MO e n er g y i s q u it e p u z zl in g . D e b n a t h e t a l .43

T a b l e 4 . S t r u c t u r e s o f N o n c a r c i n o g e n i c C o m p o u n d s a

n o. r in g An X br idge X R

59 A 3-Cl COOiP r60 A 2-Me,3-NH 2 H61 A 2-COOH H62 A 4-COCH 2Cl COMe63 A 2-Cl,4-NH 2 H64 A 2,4-OMe2 H65 A 2,6-Me2,4-OCONMe Me266 N 2-C4H 4-3 C2H 4NH 2

67 A 2-COOH ,5-NO2 H68 A 2-NH 2,4-NO2 H69 A 4-NH 2 H70 B 4-NH-Ph-4-NH 2 NH H71 A H CSNH 272 A 2-Me,4-NH 2 H73 A 2-Cl,4-Me H

a A ) anilines; B ) biphenylamines; N ) na phthyla m ine s;F ) aminofluorenes. Bridge: bridge between the phenyl ringsin biphe nyla m ine s if pr e se nt. AnX: r ing substitue nt (a llcompounds described as substitu ted anilines; for definitions,see text). R ) substituent at the functional amino group.

T a b le 5 . Ch e m i c a l D e s c r i p t o r s a n d P r e d i c t e d C a r c i n o g e n i c P o t e n c i e s ( B R R , r a t s ; B R M , m i c e ) f o r t h eN o n c a r c i n o g e n s i n T a b l e 4

predicted carcinogenic potency

n o. MR3 MR 2,6 I 2,6 R 2,6 ES (R) E H OMO E LUMO log P BR Ra BR Mb

59 0.6 0.2 0 0 4 -9.162 -0.1543 2.79 0.50 -0.50

60 0.54 0.66 0.01 -0.18 0 -8.3607 0.4333 0.95 -0.15 -1.2061 0.1 0.79 0.34 0.11 0 -8.8284 -0.455 0.96 -0.14 -0.5462 0.1 0.2 0 0 3 -9.3254 -0.8105 0.80 -0.20 -0.8963 0.1 0.7 0.42 -0.19 0 -8.1632 0.1491 1.00 -0.13 -0.1164 0.1 0.89 0.29 -0.56 0 -8.3083 0.2602 0.76 -0.22 -1.0265 0.1 1.12 0.02 -0.36 2 -8.9385 0.0892 2.25 0.31 -1.0166 0.8 0.9 0.13 -0.17 3 -8.5284 -0.4132 1.69 0.11 -1.0367 0.1 0.79 0.34 0.11 0 -9.4286 -1.5938 0.92 -0.16 0.0468 0.1 0.64 0.08 -0.74 0 -9.0498 -1.0257 0.43 -0.33 0.0769 0.1 0.2 0 0 0 -8.0719 0.411 0.48 -0.31 0.0770 0.1 0.2 0 0 0 -8.046 0.119 2.38 1.23 1.6771 0.1 0.2 0 0 3.2 -8.6991 -0.735 1.86 0.17 0.7772 0.1 0.66 0.01 -0.18 0 -8.0712 0.4025 0.95 -0.15 -0.2773 0.1 0.7 0.42 -0.19 0 -8.5409 0.1441 2.25 0.31 0.31

a From eq 20. b From eq 13.



16/18

discussed several possibilit ies. On e is t hat the twoterms LUMO and HOMO could be l inked togetherthrough the concept of hardn ess ( ) (LUMO-H O M O)/2 ) a s a m e a s u r e of ch e m ica l r e a ct i vi t y.Another hypothesis is that LUMO energy account sfor the reduction of the n itro group pr esent, togetherw i t h t h e a m i n o g r o u p , i n a n u m b e r o f t h e i r s e t o f amines. However, Zhang et al .48 a n d H a t c h e t a l .52

found a LUMO term in data sets without nitroarenes.

Another explanation could rely on a very recentfinding by King at al.60 They found a new enzymaticmecha nism of carcinogen detoxificat ion: a m icroso-mal NADH-dependent reductase that rapidly con-v e r t s t h e N-hydroxyarylamine back to th e parentcompound. In this case, a low LUMO energy coldfavor the detoxification. However, the LUMO energyof the metabolite is not necessarily coincident witht h a t of t h e p a r en t a m in e ; t h u s , t h e e n t ir e m a t t erneeds fur ther clar ificat ion.

A number of qualitative rules have been proposedr e ga r d i n g t h e p r op e r t ie s of a r om a t i c a m i n e s t h a taffect carcinogenic potency.5 Bulky substituents atthe nitrogen of the amino group generally inhibit

b ioa ct i va t i on . T h is i s i n k e ep in g w it h t h e ES(R)contribution found by us for N-substituents in thefu n c t ion a l a m i n o gr ou p i n a m ou s e a n d w it h t h efindings of Trieff et al . (inhibiting effect of theacetylation of the amino group).40 A g e n e r a l r u l esta tes t ha t carcinogenic potency decreases with stericbulk in the ortho position 5 (see also Trieff et al.40 a n dBenigni et al .46). T h is r u l e i s con s is t e n t w it h t h enegative MR 2,6 term observed by us for the carci-nogenic potency in a mouse. A mechanistic ra tionalefor these observations is that steric bulk preventsenzymat ic access to the n itrogen an d form at ion of th ereactive intermediate.

As was also found in our analysis of the carcino-genic potency, ring subst ituent s h ave been proposedto exert electronic and steric effects. In fact, accordingto Vracko,56 substitu tion of a chloro group or m ethylor m e th oxy g r ou p or t h o t o t h e a m in o gr ou p isconsidered t o often enha nce potency. This stat ementrequires clarification. Our present QSARs for thecarcinogenic potency show that ortho substituentscan operate t hrough at least three effects: directsteric, electronic, and hydrophobic. Thus, potency canincrease or decrease depending on the natu re of anortho substituent. Electron-donating ortho substitu-ents would sta bilize a positive nitrenium ion, wher easelectron-withdrawing substituents would destabilize

such an intermediate.It is more difficult to put into context work based

on topological a nd substructural param eters, as ther e s u lt s a r e d iffi cu l t t o i n t e r p r e t a n d d o n o t l en dthem selves easily to comparison an d genera lizat ion.However, a finding common to various authors wasthe correlation between activity a nd the number of aromatic r ings,27,49,52,54 which has been interpretedin differen t wa ys: (a) indicat or for the p lana r system sapt to induce frameshift mutations in TA98 Salmo-nella strain;49 (b) indicator for the hydrophobicity ofpolycyclic and conden sed ar omatic rin gs;27 (c) in dica-tor for th e presen ce of extended conjugated systemsthat favor the formation of reactive intermediates.5

Obviously t he simple empirical correlat ion betweenthe number of rings and toxic activity cannot suggestwhich (or wh at combina tion) of the a bove hypothesesis correct. A thoughtful insight into this issue wasp r ov id e d b y D e b n a t h e t a l .;43 t h e y s h ow ed t h a tbesides log P, a n a d d it i on a l con t r i b u t ion t o t h emuta genic potency in TA98 was given by the pres-ence of three or more fused rings. This effect wasa bs en t in T A1 00 s tr a in a n d w as r ela t ed t o t h e

specificity of TA98 for frameshift mutations.The latter result is a brilliant demonstrat ion of the

importance of using a common language for QSARmodeling. A common langua ge is t he only approachthat can tell us if and to what extent QSAR models(and the underlying chemical biological interactions)are similar. In the present review, the models basedon t h e H a n sch a p pr oa ch a n d on t h e u s e of t h eoperat ional definition of hydrophobicity

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