BESZYL ~ I O I E TIS Y~IEDICIN CHEMISTRY AL
Journal of Medicinal Chemistry, 1970, Vol. 13, -Vo. 5 957
Role of the Benzyl Moiety in Biochemical and Pharmacological Processes' CORWINHAKSCH AND RICHARD KERLET Department of Chemistry, Pomona College, Claremonl, California
91 711
Iircrivrd June 16, 1970 The structure-activity relationship is examined for several sets of drugs containing the tieiizyl moiety. The reaiiltx indicate that substituents with radical delocalizing ability have the niost pronoilliced eflect 011 activity. This suggests that radical abstractioii of benzylic or allylic hydrogens yields radicals which are quite toxic to a variet,y of oxidative biochemical processes.
I n extending our studies of extrathermodynamic biochemical structure-activity correlations, interest has developed in reactions which may be influenced by substituents which are effective stabilizers of free radi c a l ~ . ~ The - ~ radical parameter ER in linear combination with the hydrophobic parameter 7 was found4 to give a quite good structure-activity relationship for chloramphenicols of the type : HOCHCHCH,OH q€€COCHC12
X The quality of the correlation is seen in eq 1. I n
+
log A = 3 . 0 7 E ~ 0 . 2 3 ~ 0.77
+
n
r
S
(1)
eq 1, A represents relative activity as determined by the microbial kinetic method by Garrett.j Vsing u in place of E R in eq 1 gave a much poorer correlation with r = 0.441. The chloramphenicol data, because of the good choice of substituents, are particularly well suited to bring out the differences between the two parameters, E R and u. For many of the functions commonly used in medicinal chemistry such as the halogens, alkyl, and alkoxy, there is little difference in the values of E R and u . 111a previous study of bactericidal action of benzyl alcohols,6 good correlation of structure with activity was obtained using log P and u. In reexamining4 these data, E R was found t o give a higher correlation than u . In reexamining the data the assumption was made that ER for ortho substituents would be the same as for para substituents. This allowed the use of 18 data points in the correlation. For a more rigorous test we have omitted 5 derivatives with ortho substituents to obtain eq 2. Although the correlation with log 'I alone was good (1' = 0.946), adding the term in ER is statistically significant ( F l , l 0 = 3.32; F1,,oaO.10 = 3.28). For a limited set of substituents containing mostly halogens and alkyl derivatives there may be (1) This work was supported by Grant CA 11110 from the Sational Institutes of Health. (2) C. Hansch, J . M e d . Chem., 11, 920 (1968). (3) C. Hansch and E. J. Lien, unpublished results. (4) C. Hansch, E. Kutter, and A. Leo, J . M e d . Chem.. 12, 746 (1969). (5) E. R. Garrett, 0. K. Wright. G. H . Miller, and H.L. Smith, rbsd., 9, 203 (1966). (6) E. J. Lien, C. Hansch. and S. M. Anderson, abzd., 11, 430 (1968). I n ronverting the original experimental data used in deriving eq 23 and 61 into moles/kg, a systematic error of 3 log units was units of log l / C where C made. This does not affect the correlation coefficient or the coefficients with ?r or u in these equations. It does lower the value of the intercept by 3.
-
+
n 1
= 1.234( zk 1 5 ) E R C 0.709(*0.15) log P 0.774( .to. 39)
log
-
r
S
+
+ 13 0.960 0.244
(2)
a close parallel between E R and 01. Using UI in eq 2 in place of ER does not result in a significant reduction in the variance. Using some of the same benzyl alcohols6 against a different set of microorganisms yielded data correlated by eq 3. Log P alone gave a good correlation ( r =
+
Benzyl alcohols us. P. vulgaris Escherichia coli Pseudomonas sp. n
8 0.954 0.140
+
Benzyl alcohols us. Staphylococcus aureus S. albus S. faecalis
1 log-C
=
r
+
S
+
~.SC%(*~.~)ER
0.578(*0.11)1og P 0 . SOO( 0.28)
+
11 0.976 0.164
(3)
0.942); however, addition of the term in ER resulted in a significant reduction in variance (F1,*= 11.3; FI,~ a ~ =. 11.3). ~ ~ The use of u in place of ER in eq 3 does not result in a significant reduction in variance (F1,s = 2.1). Another example of the exceptional toxicity of benzyl alcohol turns up in the study of the action of a set of 7 aliphatic alcohols and PhCHZOH. I n our first attempt to correlate the toxicity of these alcohols to Salmonella typhosa17a poor linear relation between log 1/C and log P ( T = 0.826) was obtained. At the time the correlation was made there was no reason to assume that PhCHzOH operated in a different mode and therefore drop it from the correlation. Doing so now, we obtain the much better correlation: 11
1
log -
C
=
0.818 log P - 1.301 7
1'
S
0.934 0.098 (4)
PhCH,OH is about 3 times as toxic as eq 4 predicts. The allylic group is also well known for its ability to stabilize a radical: CH2=CH-CH2.t.CH2CH=CHz. One might look for exceptional toxicity in this function. If its toxicity is of the chloramphenicol type, this should be most apparent in rapidly dividing cells where the rate of protein synthesis is
(7)
J. M. Schafferand F. W. Tilley, J. Bacten'ol., 14,259 (1927).
most triiport:mt ; t hi5 i i i t l r w l :ippcars t o tx, t rut,. lt(bcentlj , the toxicity of a group of niiscellariroui compounds t o developing chick t>mbryos wa. reported ' The 4tructure-:tcti~ity relationship i h coiit:~irir~ci~ in It
log
1
('
=
-0
0 i f i log I'
1A(log P I 1
I
\
+
+ 2 OX
IO
0 0x2 0 1 12
(.i)
13rcaus:e of its extremely poor fit the log 1 C' foi, allyl alcohol was not used in deriving eq 5 . Using t h e valur of 0.17 for log P of allyl alcohol in f q 3, it i:, ierri that dly1 alcohol is 100 times as toxic as predicted by simple lipophilic character alone. This same exceptional toxicitj. for ally1 :tlcohol was first noted by I'icaud.q Hr observed that allyl :tlcohol was more toxic than i1mJ.I of ~ ( itlcohol to goldfi9h. From the linear rel~~tiori I T out' would expect -1mOH (log P 1.34) to hr mol t~ ii:ircotic :I- :ill> I alcohol. It1 considering other drugs which cont:iiri xctli t' benzylic hydrogens, the antimalarials come to niiiid. Quinine itself falls into thii category. -\Iore iiiteresting i.: tht. fact that :,omt' of the most potent antimalarials :trv the 2-phei1yIqui1ioli11~~metharlols. lo The enliancecl :ictit ity of the 2-pheiiplquiiiolirle tlcrivatiw- ovcr v:trioil- other function3 at this poiition m:ij of cource btx t l i w to :I varictj of re i t i q ; I i o n t ~ v e r ,it i- of intrre-t t o note that while u for the. 1'11 group I S cloie to ztiro, I'h i b oiic of the more eficctive substituent- i n thr. t l t 3 1oc;tlizatioii of r:tdiculs.'l l'hr phototosicit). of the.(. drug\ m:q. also 1i:ir.t. :I tlqwridciico 0 1 1 rdic:il stabiliz:it ion. ThrL recriit ~ v i d r ~ i i tlxit c e ~ cluiniiie ~ irihibits mitrij e3 t1i:it chloranipl.it.nico1 doe. also hat both Inolwule- :ire tlrriv:itiwof PhC'H?OH and thitt the btxnzj I i c h \ tlrogetii in:\\ well bt. the critical toxophoric group. utions a s TT-~II :I> :I iiumbei. ot iti:rted th(1 w i t c h for tho rsnmpl(+ cwn4dercd in this p i i p ~ r
1
Method The radical parameter ERis ariulogou:, t o the Hunimett-type parameter u + formulated h j Rrov 11 :rritI i s defined as follows:1".14 ,CH
,CH
In t y 6, €3. is a styrene polymer radical arid iii eq 7 . C, i- the chain transfer constant of the substituted (8) J . MoLaughlin. J. P. Marliac, 11. .J. V e r r e t t , A f , li. hfutrhler, and 0. 0.Fitzhugh, I n d . H y y . J., Z5, 282 (19643. (9) M . Picaud, C. R . Acad. Sci., 114, 108 (1887). (10) A. J. Saggiomo, K . Kato. and T. Kaiye, J . M e d . Chem., 11, 277 (1968). (11) G. H . Williams, Chem.Ind. (Londonj, 1286, (1961). (12) K . A . Conklin, S. C. Chou. and S. Ramanathen. Pharmacology, 1, 247
(1069). 118) T. Yamamotoand T . Ocsu. C h e m . l a d . ( L u n d o n ) ,787 ( 1 9 6 i j . 114) T. Otrii, T . I t o , Y. Fuji, and h?.l m o t n . Bull. C h ~ m,Sw. . , ? , I , , , , 4 1 , 204 IlHtiU1.
log
= tr/:K
+h
(IO)
If polar t#ects :ire niuch differelit than in the rt:ferenctb teni (eq 6). ttirii c.(i $1 is itial. For work with :tlcohols it. \vas fouiid chlornmphenicols :iritI be that the addition of it tterm iii u did not improve tht, corrclnt ioii d t h o for t h e w biochemical proce term in s is iicc ry for good correlation. T h that the u ttart11 in these correlat ioiis suggest,s the biochr. es are very rlost. i t i nature to thosr of t q Ci. While there is little correlation between ERaiid u I , there isq unfortmat itly good correlation,20:it l a s t for :I (15) F. R.Mayo. J . d i n e r . Chtm. Soc., 65, 2324 (1943). (16) C. C. Price, J . Polgm. Sct., 9, 772 (1948). (17) C. H. Bamford, . A . D. .Jenkins,and R..Johnston, T r o n s . F n r a d a y Roc 59, 530 (1963). (18) J. E. Lefflerand E . (ininn-alii, "Rates and Equilibria of Organic Heac. ricins," Wiley, S e w York, S . \ ~ .1963. , p 211. (18) C. G. Swain and E . ( ' , I.upton, J. A m e r . Chem. Soc., 90, 4328 I l M i U i (201 C. Flanwh and t< l i e r i ? y , ('hem. Tiid. (1,ondon). 204 illrti(0.
T'iuL>;
I
\ N D ANTISPASMODIC A C T I O N OF H K N Z Y I , ~ ~ l , ( Y ~ f f O I , S
HF.'I'IC,
Log relative bacterioidal action' Ohsd Calcdu
Log relative anesthetic action' Obsd Calcdk
Log relative antispasmodic action' Obsd Calcd'
2hld z oc 2u+E Derivative ZEKa 2 *h 3,5-12-2-OH 0.41 1.76 0.33 1.16 -0.20 1.96 2.040 1.48 1.715 1.70 1.823 3-1-.5-Br-2-OH 0.41 1.55 0.37 1.19 -0.16 1.72 1.908 0.41 1.34 0.41 1.22 : a
En
-0.83 -0.66 -- 0. 37 ~-0 . 27 I),01;
0.24 0.24e i J . 17 I).1 I 11. 0:; -- 0 07,
11.2;;
11. 1 0
- 0 . 17
44‘1
a*
-1.70 -1.30 -0.92
0.18d - I . 6Sd -0.61 - 0.04
-0.7s -0
Obsdb
Ira
. :;I
.- :j.
--
CalcdC
2 . 80 1 82
- 1.04 lI.lill 0,s;;
11.70 I1 00 I I .-I I 0 12 0 1:j 0 76 0 . 1I
(I (jl 0 R!)
A I,or I’,,;,,l
- :3 .47 1 -2.963 -- I . 650
52
ll,.-J2 I) I
.-,
. . O , 117 0.11 0 , 011
-
v,,,,
’
0 . 03 0.16 I1 17 I).G
0.YtiS 0 I .Y!)
I) 4 4
I . lli7’
l),;;4
0 . ,iWl
I). 02
I).00 0 . 00 0 *-I41 0. :ysteni. \VI?have assiimed the same valiic~of El4 because of the lack of E R values. for ?;ITy as reported for S ( C H , )) f These points not included in the regression.
II :3-CH3 3-OCII3
radical stabilizing character of the Fubstituent may be involved. The anesthetic action of the benzyl dcohols as measured by testing them 011 frog slcin. The results are summarized as follow: I1
1
s
0.984( 1 0 26) O.051(fO020)
16 0.905 0.337 (14)
log R B R = - 3 . GO7 ( f 4 . 0 ) E ~ 1.329 (zt0.45) log P 0 086 ( f 0 92)
16 0 927 0.305 (15)
log R B R logP
=
+
+
+
The additional term in eq 15 is significant with respect to eq 14 = 3 . 8 ) . ,Ilthough the confidence intervals on the h’R terms in eq 13 and 1; are broad, it seems safe to say that the coefficient in eq 13 is very likely positive while that in eq 13 is very likely negative. Thus, while radical delocalization by the substituent appears to improve bactericidal action. it teiids to decrease anesthetic action in frog skin. ‘l’his might be the result of greater ease of oxidation in the frog skin. Adding i~ term in (log P)‘ to eq 15 results in :t further improvement in the correlation ( r = 0.932; s = 0.287); however, again the correlation i, riot good enough to place confidence limits on log P(,. The use of U I in erl 13 instead of ER results in :I slightly poorer corre1:ttion ( s = 0.31’7). The coefficients with the log I-’ termb in ey 13 niid 15 are quite different. The values in e q 14 :tnd I5 are near 1, which is what is uiiislly found with iionspecific hypnotic i~ctivity.?~The value of 0.63 of eq 13 compares with the coefficic.nt of 0.73 found for a number of equations correlatiiig :intibacterial activity for a varicty of drugs with Gram-positive org:misms.2j Sixteen of the benzyl alcohols were also tested on cat intestine for antispasmodic action. One derivative (3,5-C12-2-OH) wab verj- poorly correlated and was omitted in the derivation of eq 16: I/
log KBR
P
+
0.701(*0.25)10g 0.589(*0.lS)
r
c
=
1.5
0.S64 0 300 (16)
12.1) C. Hsnaoh and 9. M. Anderson, J . M e d . Chem.. 10, 745 (1967). ( 2 5 ) E.J. Lien, C Hanscb and S. 31,Anderson, r h d . , 11, 430 (1968).
S o improvement in correlation was obtained by adding electronic ternis or a term in (log P)’ to eq 18. Thi may be due to the noise in the data. Equation 1 is a poorer corre1:ttion than those obtained for bactericidal or anesthetic action. While the above correlations are for quantitative structure-activity work a satisfaction in themselyes, they are only partial support for the special role of the benzyl moiety in biochemical systems. The espected effect is seen in the bactericidal action; however, essentially the same rationalization of the data can be obtained with a]. One would not expect thf sanie effect to be present in anesthetic arid nntispasrnodic action :tiid, in fact, it is not. One of the reasons that the role of ER is iiot more clcarly heen in the bactericidal activity of the benzyl dcohols is that all of the molecules contain :L ring OH which means that as a group they are quite activated to begin with; that is, abstraction of an a-H is onl) wildly rate-limiting. By far the more i’:Ltelimiting factor is relative hydrophobic chitracter. LilsO, the selection of substituents was very poor for t h t a purpose of nial than t h r w time> I/ = 1.1481f0.L‘O)log I-’ C +0.339(fO 1 1 )
log
I*
\
1
X
0 983 0 106 (17)
as effective as eq 1i would predict. While the primswy iiitrcotic activity of the alcohols is undoubtedly due t o a physical mechanism, it may well be that relatively high concentrat ions of radical-forming molecules could contribute to the narcotic action by interfering with oxidative processes on which the transmission of nerve impulses in the CNS depend. I n the case of the anesthetic action of the hydroxybenzyl alcohols correlated by eq 14 arid 15, we find little or no evidence for tht>
Journal of Jfeclicinal Chemistry, 2970, Vol. 13, S o . 5 961
B1;NZYL ;\IOIETY IN ? r I E D I C I X I L CHEMISTRY
radical substituent effect. Presumably because the benzylic hydrogens are already so active, it is difficult t o discriminate between small substituent effects (no groups with large ERvalues were employedj. However, in eq 17, where one is comparing very active benzylic hydrogens with those of the aliphatic alcohols, a clear difference can be seen. Enzymic Action of Amino Acid Oxidases.-A structure containing benzylic H atoms similar to the benzyl alcohols are the phenylglycines. Several studies of the enzymic oxidation of such compounds have been made. One of the most extensive is that of Keims, et aZ.,26in which hog kidney D-amino acid oxidase was employed. I n studying the correlation of their data, the following parameters were studied singly and in all reasonable combinations: T, u, u+, U R , ER,a,5 . Of the large number of equations considered, the following are essential for consideration. The highest single variable n log T', ,:, log T',,
log l',,,,
=
1'
s
"000(k0.49)u+
+ 0.133(f0.38)
11 0 . 9 5 2 0.499 (1s)
+
-3.287(j=0.279)~ f1 . 69)uf 0.539(k0.45) 11 0 . 9 7 5 0.3% (19)
=
2.988(*0.48)u
=
data constitutes an excellent example of the complex set of possibilities which one encounters in interpreting reaction mechanisms and underlines the importance of carefully considering the interrelationships between the various substituent constants now in use. Of eq 18-20, there are several reasons for selecting eq 20 as most likely describing the situation besides the fact that it gives the highest correlation. It fits in with the other examples discussed earlier in this paper that benzyl hydrogens are implicated in biochemical processes. il most important fact supporting eq 20 is that considerable evidence has been found that radicals are involved in the oxidations of amino acid oxidases. a 9 -d 1 If we accept eq 20 as most likely, we must then ask about the significance of the signs of the coefficients associated with the two variables, u and ER. The positive coefficient with u indicates electron withdrawal by the substituent promotes oxidation and the negative coefficient with E R indicates that delocalization of an odd electron by substituents inhibits hydrogen abstraction of a benzylic hydrogen atom. Oxidation could be formulated as
+ 3.946(
- 6.383(*1.99)E~
+ 0.541(*0.34) log 1',,,
=
11 0 . 9 % 0.293 (20)
- 2 . 7 0 5 (*1.43)u2
+ '2.592 ( h 0 . 7 7 ) ~ + 0.170 (h0.43)
+
11 0 . 9 6 6 0.446 (21)
correlations were obtained using u (r = 0.880) or u+ (r = 0.932). The resonance parameter 6l (r = 0.865) was less successful. The addition of the hydrophobic parameter ir did not improve the above correlations. Equations 19 and 20 represent the extended Hammett equation developed by Yukawa and TsunoZ7 and applied by Otsu and Yamamoto to radical reactions via Equation 20 yields the best correlation and indicates that radical effects appear to be most important. However, as we have pointed outlZ0it is hazardous to use only equations 19 and 20 to diagnose a reaction mechanism as radical or polar. Equation 21 was included partly because Cammarata28has recently shown that there is a high correlation between uz and ER and partly because Neims, et al., emphasized the parabolic dependence of oxidation on u. Other 5, u uR, etc. two-variable equations such as gave much poorer correlations than eq 19-21. The high correlations with eq 18-20 emphasize the probability that direct resonance interaction between substituent and reaction center is involved in the transition state. Equation 21 can be interpreted to mean that there is an optimum electron density at the point of side chain attachment as Neims, et al., emphasized, or that a radical effect is involved because of the relationship between u2 and ER. This set of
+
A AH,
- Dc-coo..k$Bc-cooI
r"j
--L
X
x
+
NH 1
AH,>
I
I
--a mc-cooc-coo-
x
/
xs
I
0
.I--
c1
+
(26) H.H. Keims, D. C. DeLuca, and L. Hellerman. Biochemistry, I,203 (1966). (27) Y. Yukana and Y. Tsuno. Bull. Ckrm. SOC.J u p . , S I , 965, 071 (19.59).
(28) A. Cammarata, S . J Yau, J. A. Collett, and A . N. Martin, Mol. Pharmacal., 6 , 61 (1970).
B
1
In the above sequence, J3. represeiits a free radical containing enzyme or a radical generated by such an enzyme. The general process has been formillatfedby Massey and Curt'i.30 (29) H. Beinert, J . B i d . Chem., 111, 463 (1957). (30) V. JIassey and E. Curti, ibid., 944,1259 (1967). (31) J. L. F o x and G. Tollin. Biochemistry, I,3893 (1966).
I1
I n this symbolism FAID= fluviii adellitit. diiiucleotidtL. ;Zti = :imino acid, :itid JA = imiiio acid. 111 thv first step, tjhe amino :icid is bound to the enzyme complex; then 2 H atoms :we removed stepwise to yield the imino acid which is theii desorbed arid hJ~drolyzed t o the keto acid. 111sequence I . step h \voultl bc, piwiiotecl bj. +Lit substituents mid step I3 \vould bt, hiiidertd by mcli substitueiits. It seems utilildy that step A ivoultl bc so critical that r:idic:tl substituent fft'wts could br pasily detected i n this ~)rocess. I3otli t h e beiizeiiis riiig :inti IICOO- tire present to stabilize :I riidic:il o t i tliv a-C. However, oiice such :i ixclicd is formed, cxteiisivch delocnlizatioii of it \vould hiiider step 13. thc :tbstruction of the seconcl 1-1,. 1'01:~~eff'ects iiiiplicated by thc positivci coeficieiit u i t h u \\auld prob)rt;iIit i i i s t q w .I :1ii(1 B th:m in tlit~ Thr> iiiterrel:ttioii.hil) bet\\ t i fiJ
