Charge transfer mechanism for benzodiazepine (BZ) action

407

Bioelectroehemistry and Bioenergetics, 16 (1986) 407-426 A section of J. Electroanal. Chem., and constituting Vol. 212 (1986) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

923 - CHARGE ACTION

TRANSFER

MECHANISM

FOR BENZODIAZEPINE

CORRELATION OF REDUCITON POTENTIAL STRUCTURE AND DRUG ACTIVITY

PHILIP

W. CRAWFORD

Department

of Chemistry

NORMAN

W. GILMAN

Department

of Exploratory

MICHAEL

D. RYAN

of Chemistry,

manuscript

KOVACIC

l

WITH

.**

University of Wisconsin-Milwaukee,

Department (Revised

and PETER

OF BZ IMINIUM

(BZ)

Research, Hoffmann-LuRoche,

Milwaukee,

Inc., Nut&,

WI 53201 (U.S.A.)

NJ 07110 (U.S.A.)

* Marquette

received

University, Milwaukee,

WI 53233 (U.S.A.)

April 30th 1986)

SUMMARY A novel mechanism for BZ action is proposed in which a BZ is protonated by GABA or protein RNH: to yield an iminium species that is responsible for drug activity via charge transfer (ct.). The following evidence from our work and prior studies supports this concept: (1) binding sites for BZs and GABA are apparently on the same protein complex; data point to possible interaction between the two ligands; (2) recent theoretical studies propose reaction of basic imine of BZ with cationic RNH: of protein at the receptor site; (3) BZ is protonated by weak acids, such as acetic and GABA, to give iminium ions which exhibit appreciable and favorable increases in reduction potential in vitro; (4) the reduction potentials are of the same order of magnitude as for a number of other biologically active compounds; (5) reversible electron uptake has been shown to occur with some protonated BZ drugs; )UP - UP,z 1 calculations from the voltammograms of some BZs indicate the potential for reversible electrochemical processes; (6) correlations exist involving reduction potential of BZ iminium, structure, and drug activity; (7) a significant number of BZ agonists, inverse agonists, and antagonists incorporate the imine-type precursor of iminium; (8) the postulated c.t. pathway is in keeping with a variety of bioelectrochemical phenomena arising from active site binding.

l l

To whom correspondence should be addressed. * Electrochemical Society Active Member.

0302-4598/86/$03.50

0 1986 Elsevier Sequoia

S.A.

408 Iminium appears to be implicated in the action of several other drugs, such as PCP and MPTP, that are associated with brain chemistry. The iminium ct. theory is broadly applicable to physiologically active agents.

ADDITIONAL

BZ CNS DMF DMSO GABA MPTP PCP SAR

ACRONYMS

IN THIS PAPER

benzodiazepines central nervous system dimethylformamide dimethyl sulfoxide y-aminobutyric acid 1-methyl-4-phenyl-1, 2, 3, 64etrahydropyridine phencyclidine structure-activity relationship

INTRODUCTION

Since their discovery in the late 1950’s, the benzodiazepine (BZ) drugs, e.g. Valium@ (diazepam) and Librium@ (chlordiazepoxide), have enjoyed widespread commercial success as minor tranquilizers. The BZs exhibit a broad spectrum of pharmacological activity resulting in use as anxiolytics, anticonvulsants, sedativehypnotics, and muscle relaxants [la,2a,3-61. Some of the marketed compounds are depicted in Fig. 1 (l-9); some variations (10-16) are also employed in the present study. A tremendous volume of research has appeared dealing with new members, analogs, synthesis, activity, binding, and mode of action.

P

7H2 )_$.._~

c,&.J2N(C2Hrqj

C6H4F-o

C&i

Prazepam

c,>foH

Cl

Flurazepam

c,&oH

w-b

C6H4CI-o

Triazolam

c,iyoH C6H&l-o

C6”5

5

6

7

Alprazolam

Temazepam

Lorazepam

C6H5 6 Oxazepam

409

C8H4F-o 9

C6H4CI-o

‘gH5

10

‘gH5

11

Ro 22-3245

12

Ro 22-4213

Ro 5-3464

Cl ‘gH5 13

14

Ro 5-2921

c@‘,F-0

C6H4OH-p

‘sH5 15

Ao 5-2748

16

Ro 7-2900

Ro 5-6910

C6H5 18

19 21 20

P

Q---p

#T6H5QJ---& r

\ 22

N’

24

23

pgirr 6) 3

\

ribose

25

CH3

26

g_#J’“‘“’ F

\ CH3

0 27

Fig. 1. Structure formulae of benzodiazepines, agonists, inverse agonists, antagonists, and heterocyclic di-N-oxides.

The BZs are known to influence the activity of all the major areas of the CNS, particularly the limbic structures. Presumably, they exert their effects through stereospecific binding with protein receptors at several sites within the CNS, and are intimately involved with GABAergic neurotransmission. However, the precise mechanism of action at the molecular level is unknown.

410

Recently, it was proposed [7] that the iminium ion plays a vital role in physiological activity involving a number of important areas, e.g. brain chemistry, carcinogenesis, action of drugs, redox enzymes, and herbicides. In general, the iminium species (17) is believed to be generated metabolically in uiuo. Its major function is participation in c.t. processes, such as interference with normal electron transfer or generation of toxic oxyradicals. According to the theory, the mechanism of drug action entails electron abstraction from a substrate, e.g. protein, as schematized in equation (1).

e,

_&_ 11

-=

-_Ncc-

II

(1)

17

The positive charge enhances electron uptake from cellular materials. The c.t. is completed by electron donation to an acceptor. The concept has been recently applied to several agents which are associated with brain chemistry, namely, PCP [8] and MPTP [9]. A considerable amount of electrochemical work has been done on the BZ class of compounds, including assay methods. One objective of the present electrochemical study was to ascertain the reduction characteristics of the protonated forms. Emphasis is placed on interaction of BZ with GABA or protein RNHZ which is considered to be an important event. The proposal is advanced that BZ iminium, derived from protonation, plays a role in c.t. processes. Correlations are made involving reduction potential, structure, and physiological activity. Similarities to related CNS agents and other biologically active iminium species are discussed.

MATERIALS

AND METHODS

The BZs were obtained from the following sources: Hoffmann-La Roche (diazepam, flurazepam di-HCl, midazolam, Ro 22-4213, Ro 22-3245, Ro 5-2748, Ro 5-3464, Ro 7-2900, Ro 5-6910, and Ro 5-2921), Wyeth (oxazepam and lorazepam), Upjohn (alprazolam and triazolam), Sandoz (temazepam), and Warner-Lambert (prazepam). GABA was purchased from the Aldrich Chemical Co. Cyclic voltammetric and differential pulse polarographic data were recorded with a Princeton Applied Research Corp. (PARC) model 174A polarographic analyzer connected to a Hewlett Packard model 7035B X-Y recorder. All solutions were degassed for 15 minutes with prepurified dinitrogen that was passed through an oxygen scrubbing system. The working electrodes consisted of dropping mercury attached to a mechanical drop dislodger for differential pulse polarography and hanging mercury drop for cyclic voltammetry, whereas the reference and counter electrodes were saturated calomel (Sargent-Welch or Corning) and platinum wire, respectively, for both electrochemical procedures. An IBM aqueous Ag ]AgCl electrode in saturated KC1 was used as the reference for polarography of Ro 5-6910 and for some samples in DMF. The supporting electrolyte was tetraethylammonium

411

perchlorate (G.F. Smith Chemical Co.), and the solvents were a 50% aqueous ethanol solution (ethanol from U.S. Industrial Chemicals), DMSO and DMF (both from Aldrich Chemical Co. in the higher available purity). Test solutions were prepared by diluting to the mark with solvent in a volumetric flask to which pre-weighed amounts of test compounds and supporting electrolyte had been added in order to obtain the desired concentrations. Acid solutions were prepared similarly using water as solvent to obtain the indicated concentrations: perchloric acid (0.05 M), acetic acid (0.5043 M), and GABA (0.5043 M). Acid solution was added to test solutions during individual test runs in amounts that would provide the desired concentrations. pH measurements were performed with a Corning model 125 pH meter on aqueous control solutions, e.g. aqueous ethanol solutions with and without the added acids. RESULTS

AND DISCUSSION

Reduction potentials

and drug mechanism

Differential pulse polarography (d.p.p.) and cyclic voltammetry (c.v.) results for some of the more physiologically active BZs (l-11) in 50% aqueous ethanol are summarized in Table 1. All compounds were irreversibly reduced under the various conditions. Polarography of diazepam yielded a single wave with a reduction potential of - 1.34 V (Fig. 2) versus s.c.e. Voltammetry in the same system gave a single wave with a reduction potential of - 1.39 V (Fig. 3). This corresponds to the 2-electron reduction of the azomethine (imine) bond which for BZs is the major electroactive site [lo]. A number of the other BZs shows analogous results. Compounds 2, 3 and 9 gave single waves with similar potentials, - 1.29 to - 1.37 V for polarography, and - 1.32 to - 1.43 V for voltammetry. Compounds 6-8, each with a hydroxyl group in the 3-position, gave results similar to those of diazepam, except that 6 showed a second small wave at a more negative potential. Polarography of these compounds yielded reduction potentials from - 1.37 to - 1.41 V, whereas voltammetry showed values ranging from - 1.41 to - 1.47 V. Prior investigators have demonstrated the simultaneous 4-electron reduction of the imine bond in the 4,5-position and reductive dehydroxylation at the 3-position, which has been shown to occur over a broad pH range (111. More recent evidence suggests that dehydroxylation does not occur in neutral solution [12]. Some of the compounds gave somewhat more positive reduction potentials, on the order of 0.1 to 0.2 V, than diazepam. Compounds 4 and 5 reduced in single waves with potentials of - 1.21 and - 1.22 V, respectively, in polarography and - 1.26 and - 1.27 V, respectively, in voltammetry. Compounds [13] 10 and 11 showed several reductions, with the first waves appearing at - 1.15 and - 1.21 V, respectively, in polarography and at - 1.19 and - 1.24 V, respectively in voltammetry. Polarography of the BZs was carried out in the presence of strong acid (perchloric). In all cases, the reduction potentials became appreciably more positive, i.e., reaction became more facile. The values ranged from - 0.64 to - 0.94 V (Table 1).

412 TABLE 1 Voltammetry and polarography of commercial BZs with HClO, or GABA (aqueous ethanol) Compound

d.p.p. b

C.“. a

1 Diazepam 2 Prazepam 3 Flurazepam ’ 4 Triazolam 5 Alprazolam 6 Temazepam 7 Lorazepam 8 Oxazepam 9 Midazolam 10 Ro 22-3245 8 11 Ro 22-4213 g.h

[G~AI W

up (v)

[HCQ 1(m W

up (V)

0 0.048 0 0.048 0 0.048 0 0.048 0 0.048 0 0.048 0 0.048 0 0.048 0 0.048 0 0.048 0 0.048

-1.39 -1.18 - 1.43 -1.20 - 1.32 d - 1.09 d - 1.26 - 1.05 - 1.27 - 1.10 -1.41 t -1.17 f - 1.44 - 1.11 f - 1.47 - 1.16 ’ - 1.36 - 1.07 -1.19 f -1.00 r - 1.24 ’ - 1.11 f

0 0.5 0 0.5 0 1.0 = 0 0.5 0 0.5 0 0.5 0 0.5 0 0.5 0 0.5 0 0.5 0 0.5

-1.34 -0.80 -1.37 -0.81 - 1.29 - 0.80 - 1.21 - 0.83 - 1.22 - 0.78 -1.37 -0.88 -1.37 - 0.91 -1.41 -0.94 -1.31 - 0.80 -1.15 - 0.64 -1.21 - 0.73

d

f

f ’ f f

100 mV/s, tetraethylammonium perchlorate (0.1 M), substrate (0.5 m M), hanging Hg drop electrode, versus s.c.e. 2 mV/s, tetraethylammonium perchlorate (0.1 M), substrate (0.5 m M), dropping Hg electrode, 1 s drop time, versus s.c.e. Obtained as the dihydrochloride. Neutralized with 0.125 M NaOH prior to obtaining data. Acid present in dihydrochloride; no HClO, added. More than one reduction peak observed. Not commercial. Somewhat less active [13].

uvs s.c.e.w I

-2.0

-1.8

-1.6

-1.4

I

I

-1.2

-1.0

I

-0.8

-0.6

Fig. 2. Differential pulse polarography of diazepam in aqueous ethanol (50X), 2 mV/s.

413

-2.0 I

-1.8 I

-1.6 I

-1.4 -1.2 I I u vs s.c.e.(v)

Fig. 3. Cyclic voltammetry

-1.0 I

-0.8 I

1

of diazepam in aqueous ethanol (SO%), 100 mV/s.

A representative polarogram (d.p.p.) is shown in Fig. 4. Compounds 10 and 11 exhibited several waves. Compound 6, however, reduced in a single wave upon acidification. The effect of solvent variation on the reduction potentials was also examined. Data for some of the BZs in DMF are given in Table 2. Diazepam produced a single polarographic wave at a potential of - 1.89 V (uersus s.c.e.) in the aprotic medium. The other compounds behaved similarly. In all cases, the potentials were significantly more negative in the aprotic environment than in aqueous ethanol. However, in the presence of strong acid the reductions occurred at approximately the same potentials as for those in the aqueous system.

uvs s.c.e.(v)

-2.0

d

-1.8

-1.6

-1.4

I

I

-1.2

-1.0

Fig. 4. Differential pulse polarography (0.5 mM), 2 mV/s.

I

-0.8

-0.6

of diazepam in aqueous ethanol (50%) with added perchloric acid

414 -2.0

-1.8

-1.6

-1.4 uvs

-1.2

-1.0

-0.8

s.c.e.(V)

Fig. 5. Cyclic voltammetry mV/s.

of diazepam

in aqueous ethanol (SO%), with added GABA

(0.048 M), 100

Prior electrochemical studies [14-M] have been carried out on a number of the BZs. Reduction potentials from the literature for some of these compounds are listed in Table 3. As the acidity increases the reduction potentials also increase. The values obtained under various conditions, from acidic solutions (pH 2.17 to 4.80) to basic solutions (pH 8.92 to 12.0), range from -0.52 to - 1.50 V, respectively. Since prior investigators have determined the effects of variation in pH, we did not repeat this aspect. The results of our work are in keeping with the prior findings. However, there are some differences which may be attributed to changes in pH and solvent. Reduction potentials can be appreciably modified by alterations in the medium [19].

TABLE 2 Polarography Compound

of BZs with HClO, WQ

(DMF) a

1(mW

y (v)

Reference electrode b s.c.e.

1 Diaaepam

0 0.5

-1.89 -0.82

2 Prazepam

0 0.5

-1.84 - 0.77

Ag /AgCf

5 Alpraaolam

0 0.5

-1.71 - 0.70

Ag IAgCl

6 Temazepam

0 0.5 0 0.5 0 0.5

-

Ag lAga

7 Lorazepam 8 Oxazepam

1.70 = 0.81 1.72 0.87 1.76 0.87

s.c.e. s.c.e.

* 2 mV/s, tetraethylammonium perchlorate (0.1 M), substrate (0.5 m M), dropping Hg electrode. b Ag IAgCl references differ from s.c.e. by about + 0.05 V. ’ More than one reduction peak observed.

415 TABLE 3 Literature reduction potentials of BZs Solvent

PH

u, = (v)

Reference

1 Diazepam

B&ton-Robinson

27% EtOH + acetate buffer B&ton-Robinson buffer

3 Flurazepam

B&ton-Robinson

6 Temazepam 7 Lorazepam

0.01 M HCI 1.0 M HCI Britton-Robinson + 10% DMF Britton-Robinson

8 Oxazepam

B&ton-Robinson + 10% DMF B&ton-Robinson

-1.15 -0.74 -0.90 - 1.22 - 0.73 -1.10 -0.72 - 0.63 - 0.52 - 0.98 - 0.60 - 1.45 -0.74 -1.02 - 0.60 - 1.50 -0.76 - 0.99 - 0.94 - 1.26 -0.97 - 0.747

14

2 Prazepam

12.0 4.0 4.80 12.0 4.0 12.0 4.0

Compound

12 Ro 5-3464 13 Ro 5-2921

buffer

buffer buffer buffer buffer

27% EtOH + acetate buffer 27% EtOH + acetate buffer 278 EtOH + acetate buffer 0.1 M HC1+20% MeOH

a versus

buffer

8.9 2.2 12.0 4.0 8.9 2.2 12.0 4.0 4.8 4.8 12.0 4.8

16 14 14 15 17 14 17 14 16 16 16 18

s.c.e.

In application of the iminium theory of c.t. to the mechanism of action of BZs the protonated form of the imine bond in the 4,5-position is invoked. In diazepam and the 3-hydroxy analogs (7 and 8), this nitrogen is the preferred site of protonation [20,21]. One feature is the enhanced ease of the reduction of the iminium species (Table 1) [22]. Of course, strong acid, as used in this study, is probably not available at the active site. For this reason, investigations were made on the effects of protonation with weaker acids, e.g., carboxylic and RNHC, functionalities that are widespread in living systems. The two chosen were acetic acid, a simple model (Table 4), and GABA. In voltammetry of diazepam in aqueous ethanol with acetic acid, a single wave at a reduction potential of -0.86 V was observed, similar to the value obtained in the presence of strong acid in polarography. This is not surprising since the pH of both solutions is approximately the same, 3.5. The acetic acid concentration is much greater than that of perchloric. A similar result was obtained with alprazolam and oxazepam, although for the latter the difference is somewhat larger. The acid which might be involved with BZs at the active sites is GABA [l-3,5]. The voltammetry results from the BZs in aqueous ethanol with added GABA are shown in Table 1. Diazepam in the presence of 0.048 M GABA gave a single wave

416 TABLE 4 Voltammetry of BZs with acetic acid (aqueous ethanol) a Compound

lCWO,Hl

1 Diazepam

0 0.048 0 0.048 0 0.048

5 Alprazolam 8 Oxazepam

y (v)

(W

- 1.39 -0.86 - 1.27 -0.83 - 1.47 -1.00

,J

’ 100 mV/s, tetraethylammonium perchlorate (0.1 M), substrate (0.5 m M), hanging Hg drop electrode, ver~u.r s.c.e.

at a reduction potential of -1.18 V (Figure 5). Similar results were found for the other compounds with a few exceptions. Compounds 2-5 and 9 reduced in single waves with potentials ranging from - 1.05 to - 1.20 V. Compounds 6-8,10 and 11 showed more than one wave with reduction potentials for the first ones ranging from - 1.00 to - 1.17 V. It is~significant that in all cases definite increases (0.17 to 0.33 V) in the reduction potentials in the positive direction were observed, indicating that GABA is protonating imine to some degree. These reduction potentials are significantly more negative than those obtained with acetic acid due to the lower acidity of GABA. Although the reduction potentials of the BZs in aqueous ethanol in the presence of GABA are fairly negative, they are similar to those (Ur,*, V) of some other iminium type species possessing physiological activity, e.g. NAD+ ( - 0.98) [23], dioxidine ( - 1.06) [24], protonated O(6)-methylguanine ( - 0.98) [25], and 1-methyl-4-phenylpyridinium ion ( - 1.08) [9]. The results from a brief study of solvent effects (DMSO) with diazepam and GABA are summarized in Table 5. The drug alone showed a single wave at a reduction potential of - 1.91 V. In the presence of aqueous GABA there was a shift to a more positive potential, - 1.79 V. Addition of solid GABA caused electron uptake to occur at - 1.83 V. Evidently, protonation is considerably less effective in

TABLE 5 Voltammetry of diazepam with GABA (DMSO) a

[GABAl( W 0.0

up 09 - 1.91

0.048 b OX048 =

- 1.79 - 1.83

’ 100 mV/s, tetraethylammonium perchlorate (0.1 M), substrate (0.5 mM), hanging Hg drop electrode, versus s.c.e. b Added as aqueous solution. ’ Added as solid (5 mg).

417

this system, due partly to the observed lower solubility of GABA in the aprotic solvent. The relationship between GABA and the BZs at the active site undoubtedly differs from that in oitro. The alkylammonium entity from the GABA zwitterion might convert BZ to the salt form. Alternatively, protein RNH: derived from lysine (or protonated histidine) [26] could serve as the proton donor. The reaction should be readily reversible owing to the involvement of acid and base [27] which are comparatively weak. A recent theoretical study [28] addresses key aspects of BZ chemistry relative to the iminium concept. A large negative potential was associated with the imine nitrogen which in one model was considered to interact with a cationic receptor site, e.g. RNH: from protein lysine. The process could entail either complete or partial protonation. Calculations indicate that electronic rather than conformational differences are responsible for variations in relative receptor affinities. In another QSAR investigation involving a large number of BZs, data showed that N(4) made a statistically significant contribution to binding site affinities, presumably via reversible, complex ,formation with a cationic functionality, such as a protonated amine group [29,30]. Theoretical calculations [31] reveal that competition of imine and ammonia for proton (iminium-ammonium equilibrium) is importantly influenced not only by inherent basicity, but also by geometrical considerations as would pertain at the active site in a biological system. In an arrangement in which the lone pairs of the two bases point toward one another, the proton prefers the Schiff base. BZs bind stereospecifically with high affinity to CNS receptors [1,32]. There is appreciable evidence in the literature that GABA is also involved at the active site [33]. The BZs exert their effect only in the presence of endogenous GABA during the activity of GABAergic neurons [la]. On exposure to GABA the affinity of the BZ agonist binding sites for their ligands increases [2b,2c,3]. Conversely, GABA binding is enhanced by the presence of BZs [34]. These observations buttress the notion of intimate interaction between the two ligands in vivo. Initial results indicated that the BZ receptor site was located close to that of GABA [2e,35]. It is now known that those for GABA are coupled to high affinity BZ receptor sites and in fact both receptors are located on the same macromolecular protein complex [2e,2m,36]. In view of these findings, it is conceivable that GABA is in intimate contact with BZ, which could result in protonation. On the assumption that the iminium species plays an important role as a catalytic c.t. agent in ho, several possible modes exist whereby this could be translated into physiological activity. In some instances, particularly in the case of brain chemistry, an important aspect comprises interaction of iminium with the CNS. This would involve interference with normal electrical processes, e.g., shunting, blockage, or enhancement of nerve depolarization. Usually BZs specifically facilitate neurotransmission at GABAergic synapses and may increase the sensitivity of neuronal membranes to GABA effects [5]. The BZ receptor is thought to be part of a GABA receptor-chloride ionophore complex [2f,3,32,35]. BZs enhance the effect of GABA on the chloride channels, thus increasing the ionic conductance by facilitating

418

opening of the chloride gate and switching on the chloride electrode, a process involving transfer of charged ions [2h,3,5,33]. GABA agonists increases the uptake of chloride by cell-free brain membranes [37]. Also, changes are caused in the electrically excitable membrane properties of spinal neurons which involve elevation of the action potential threshold and alteration in the steady-state current-voltage relations in the membrane [2h]. It is also relevant that one set of GABA receptors regulates calcium flux [2d], whereas another governs chloride ion permeability. Some GABA receptors alter membrane polarity and others modify transmission by influencing a component of the neurotransmitter release process. [2d]. These and related [38] phenomena support the notion that the mechanism of action of the BZs could well involve charge transfer. What might be the source of the electrons which are proposed for the catalytic transfer? One reasonable possibility is the protein that comprises the active site. Prior studies lend credence to this approach. Disulfide linkages are capable of undergoing reversible reduction reactions in electrochemical processes with various proteins at pH 7 [39a]. Interestingly, the reported reduction potentials (-0.5 to -0.8 V) are similar to those observed with BZs in this and prior studies. Several theories have been advanced concerning the mechanism by which BZs produce their effects. The most supported one postulated that the drugs, after receptor activation, interact allosterically with the GABA receptors via a regulating protein that controls the activity or the formation of special receptors having a high affinity for GABA, thus increasing affinity of the GABA receptor for its ligand and potentiating GABA effects [la,2b,5]. Likewise, the BZs might improve coupling between GABA receptor activation and chloride channel opening [2i]. Our own approach embodies these concepts, but also invokes specific interactions at the molecular level with resultant electrophysiological consequences. Another less-supported theory involves facilitation by the drugs of GABA release from GABAergic nerve endings [5]. Correlation of reduction potential

with structure and activity

BZs of lower drug potency (12-16) (Table 6) were also studied for comparison with compounds of higher activity. There are similarities and differences in comparison with the results in Table 1. Compounds Ro 5-3464 and Ro 5-2921 reduced in single waves similar to diazepam, with the exception that the potentials were somewhat more negative under all conditions. Ro 5-2748 behaved in like fashion except that a small peak occurred at around - 1.0 V, which may be due to an impurity or an equilibrium reaction. In acid, the small peak was not observed. Ro 7-2900 reduced in two waves with first peak values slightly more positive than those of diazepam. However, in both weak and strong acid only one peak is observed with values somewhat more negative than those of diazepam. Ro 5-6910 showed no reduction under these conditions. The average values for the two categories of BZs are as follows: (Table 1) HClO,, -0.81 V; GABA, - 1.11 V; (Table 6) HClO,, -0.93 to - 1.35 V; GABA, - 1.27 to - 1.61 V. These findings are in broad

419 TABLE 6 Voltammetry and polarography of BZs with lower activities (aqueous ethanol) Compound

12 Ro 5-3464 13 Ro 5-2921 14 Ro 5-2748 15 Ro 7-2900 16 Ro 5-6910

d.p.p. b

C.V.s

[GABAl( W

up (v)

0.0 0.048 0.0 0.048 0.0 0.048 0.0 0.048 0.0 0.048

- 1.49 - 1.22 -1.56 - 1.28 -1.60’ - 1.30 - 1.33 c - 1.27 NRC NR’

lHt%l 0.0 0.5 0.0 0.5 0.0 0.5 0.0 0.5 0.0 0.5

@W

q? (V - 1.48 - 0.88 - 1.51 - 0.95 - 1.56 ’ - 0.95 - 1.28 = - 0.95 NRdS NR d*e

100 mV/s, tetraethylammonium perchlorate (0.1 M), substrate (0.5 mM), hanging Hg drop electrode, versus s.c.e. 2 mV/s, tetraethylammonium perchlorate (0.1 M), substrate (0.5 mM), dropping Hg electrode, 1 s drop time, versus s.c.e. More than one reduction peak observed. versus Ag 1AgCl. - 3 V estimated.

agreement with a previous study, in which several of the same compounds were examined [16]. Pharmacological results for the antipentylenetetrazol test [4,13,40-431 are given in Table 7. In general a fairly good correlation exists involving EDso, the clinical dose used in humans, and reduction potential. In conclusion, generally lower reduction potentials were observed for those BZs possessing decreased drug activity, in accord with the theoretical guidelines. Recent publications address SAR [4,28-30&l-46]. The aspect of structure-activity uersu~ reduction potential will next be considered with focus on a few salient structural features (18). The BZs that incorporate the Schiff base unsaturation are more active than their reduced counterparts [44]. The behavior of Ro 5-6910, which gave no reduction even in acid and which exhibits lower activity [41], shows the importance of this feature. Similarly, compound 19 was found previously to be non-reducible [18]. Imine to iminium conversion is a necessary feature for physiological activity according to the framework of our c.t. concept. It is pertinent that activity is retained with the nitrone functionality in chlordiazepoxide which can be metabolically deoxygenated [2k]. Most commercial BZs of highest activity have an aryl group at the 5-position [4]. One possible function could be resonance stabilization of the radical formed by one-electron reduction of iminium. Several of the drugs possess more than one imine bond permitting stepwise protonation with formation of a conjugated diiminium species. In some cases initial protonation might not occur at N(4). Therefore, the effects of variation in acid concentration on some compounds of this type, triazolobenzodiazepines (triazolam and alprazolam), pyrimidobenzodiazepines (Ro 22-3245 and Ro 22-4213), and

420 TABLE I BZ antipentylenetetrazol activity (ED,,)

a and IC,, values

Compound

EDse (mg/kg)

Reference

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1.4 4.1 1.6 0.06 0.3 0.7 0.07 0.6 2.1 0.59 17 800 800 175 800 5.4 =

4 40 40 4 4 40 40 40 41 13 13 42 4 4 43 41

Diazepam Prazepam Phlrazepam Triazolam Alprazolam Temazepam Lorazepam Oxazepam Midazolam Ro 22-3245 Ro 22-4213 Ro 5-3464 Ro 5-2921 Ro 5-2748 Ro 7-2900 Ro 5-6910

ICW (nW

b

5 110 14.8 4 20 16 3.5 18 4.8 1.9 60 90 145 11.5 130 130

a The ED, was calculated as the dose which would prevent convulsions in 50% of the mice tested after administration of 125 mg/kg of pentylenetetrazol by the subcutaneous route [43]. b Ref. 41. ’ Probably due to a metabolic effect.

midazolam, were investigated (Table 8). In almost every case, adding acid in more than equimolar concentration produced some shift in the reduction potential toward more positive values, but the effect was not appreciable, the largest amounting to only 0.07 V. Changing from the l-substituted benzodiazepin-Zone structure to triazolobenzodiazepine also increases activity [4]. Alprazolam and triazolam are both generally more potent than diazepam. The reduction potentials of both compounds are usually more positive than those of diazepam under similar conditions. In strong acid, however, the opposite was observed to a slight degree for triazolam. The substituent at the 7-position is important as well. Previously, it was observed that at least for the tranquilizing effect of various BZs the nature of this substituent plays a significant role [47]. Although controversy exists and additional work is needed concerning SAR [4,48] at this site, commercial BZs generally contain an electron-withdrawing entity at C (7). Electron-withdrawing substituents, e.g. Cl and NO,, in the aromatic ring are known to facilitate electroreduction, [49], thus lending support to the notion that electron uptake could be an important event in uiuo, as well as in uitro. Comparison of the reduction potentials of diazepam with those for Ro 5-3464 shows that the former reduces at more positive potentials in all cases, in line with the known substituent effects. Electron-donating groups in the 7-position generally exert the opposite effect. Alkyl substituents in the aromatic nucleus are reported to deter electroreduction [49]. Comparison of the reduction potentials of

421 TABLE

8

Effect of varying Compound 4 Triazolam

5 Alprazolam

9 Midazolam

10 Ro 22-3245

11 Ro 22-4213

acidity

on d.p.p. of BZs with more than one imine bond a [HClO,]

(m M) b

qJ (v) -1.21 -0.83 -0.76 - 1.22 -0.78 - 0.73 - 1.31 - 0.80 -0.76 - 1.15 -0.64 - 0.64 - 1.21 - 0.73 - 0.68

0 0.5 1.0 0 0.5 1.0 0 0.5 1.0 0 0.5 1.0 0 0.5 1.0

a 2 mV/s, tetraethylammonium perchlorate drop time, versus s.c.e., act. ethanol. b More than one reduction wave observed.

(0.1 M), substrate

(0.5 mM),

b b b b b b

dropping

Hg electrode,

1 s

Ro 5-2748, containing a 7-methyl group, to those of Ro 5-2921 shows that in general Ro 5-2748 is more difficult to reduce, although this was not observed during DPP with added HClO,. Ro 5-2921 is generally more active than Ro 5-2748 [4]. These studies lend support to the notion that a relationship exists involving ease of reduction, physiological activity, and BZ iminium structure. BZs with higher activity generally reduce at more positive potentials than those of lower activity. Significantly, the results with added GABA generally give a better SAR fit than those from perchloric acid. Absolute correlation between structure-activity and electrochemical potential is, of course, unrealistic. Many other important factors are involved in the mechanistic aspects of drug activity, e.g., active site binding. Compounds with lower activities usually exhibit lower in uitro binding in the [3H]-diazepam assay [5], which may relate to c.t. if binding involves protonation. The assay values for compounds 1-16 are shown in Table 7. For the most part there is reasonable correlation between IC,, and EDSo values. Other factors would include stereochemistry, diffusion, solubility, metabolism, and adsorption. Is it reasonable to expect that the reduction potentials obtained for BZ iminium could translate into physiological activity? There are prior reports which indicate such a relationship in other systems. For example, mitomycins (quinones) possessing less negative Ui,* values exhibit more powerful antibiotic activity [50], and evidence points to a relationship between reduction potential and antitumor activity of certain benzo- and naphthoquinones [51]. A similar correlation was observed for antibacterial heterocyclic di-N-oxides which include iodinin (20) [52] and dioxidine (21) [24,52] (diiminium type). An analogous situation exists between carcinogenic

422

potency and lJi,z values for ionic purines and pyrimidines which are generated during DNA alkylation [25]. Other cansiderations According to the iminium theory the protonated BZs act in uiuo as charge transfer entities. In this study, as well as in most prior literature, the reductions observed during voltammetry were found to be irreversible. The reaction pathways in electrochemical processes are known to be dependent on the type of medium used [8,19]. The nature of the solution, whether highly polar or aprotic, can produce profound alterations in the reaction characteristics of certain substances. Likewise, scan rate can affect the reversibility. The scan rate producing the backward sweep wave is related to stability of the anion radical [53]. For example, at faster scan rates (2 V/s) C.V. of quinoxaline-1,Cdioxide proved to be reversible, whereas at much ,slower rates (0.1 V/s) the opposite was true. Similar results were obtained for various phenazine derivatives [52]. Quite significantly, recent experimental work showed that for lorazepam and oxazepam in acid media the first electron uptake in reduction of the imine double bond was reversible [12]. In the present work UP- UP,z calculations from the voltammograms of the BZs in aqueous ethanol in the presence of GABA indicate that some have potentially reversible electrochemical reactions with very fast followup chemistry. Compounds 2-11 gave values between 50 and 70 mV, whereas for compounds 1 and 12-15 the range was SO-100 mV. In the absence of GABA the values for all BZs ranged from 70 to 100 mV. The theoretical value for a Nemstian wave is 56.5/n mV at 25OC, n being the number of electrons. The ability to participate in c.t. reactions in uiuo could be influenced by other factors, such as active site binding. The influence that conditions can exert is illustrated by comparison of the reduction potentials obtained in aprotic and protic solvents in the present study. Kaye and Stonehill pointed out that reduction potentials in uiuo may be more positive than in vitro due to better in uiuo functioning [54]. BZs are known to undergo hydrolysis in acidic media. Flurazepam cleaves to the extent of 70% in 0.1 A4 HCl with t,/, of 21 minutes, whereas diazepam under the same conditions does so to the extent of 24% with t,,, of over 24 hours [15]. Midazolam is also susceptible (55,561. In the present study, however, the amount of time the compounds were exposed to acid was quite minimal, 3-5 minutes for voltammetry, and S-10 minutes in the case of polarography. Therefore, hydrolysis is probably negligible under these conditions. Many prior electrochemical studies in acid media have been reported with usually no indication of hydrolysis. Appreciable numbers of compounds have been found to act as agonists, inverse agonists, or antagonists via binding at BZ receptor sites [2g,2m,2n,3,5]. Antagonists bind, but produce no response and have no effect on the affinity of GABA for the receptor complex. Inverse agonists bind, potentiate convulsions, and decrease the affinity of GABA [45]. Many of them incorporate the imine-type double bond, e.g.: j%carbolines (22), pyrazoloquinolinones (CGS-8216) (23), imidazopyridines

423

(EMD41717) (24), triazolopyridazines (25), purines (inosine) (26) and imidazobenzodiazepines (Ro 15-1788) (27). Theoretical studies indicate that, analogous to the BZ case, protonation of the pyridine nitrogen of /?-carbolines is effected by a cationic species at the active site [57]. However, differing physiological activities may reflect stereochemical (conformational) [58] variations in binding, fixation to different sites, differences in degree of protonation and acidity of the proton donor, or altered electrochemical properties. A question which has been raised is whether or not a natural ligand for the BZ receptor exists. Many drug receptors are known to function with endogenous chemical mediators [2j,3,5]. To date no such compound for the BZ receptor has been reported. It is conceivable that such a ligand, if found [59], might also possess an imine structure. In prior literature studies correlations have been made between in vitro electrochemical behavior and physiological activity of some other CNS drugs. One such investigation suggests that oxidative mechanisms of the dibenzoazepine neuroleptic drugs, e.g., loxapine, could play an important role in the activity [60]. A relationship to our study comprises the observation that in vitro oxidation generates an iminium species from the imine-containing precursor. There is similarity between electrochemical oxidations of phenothiazine or imipramine and those carried out in vitro or in uivo [39b,61]. Studies suggest that the mechanism of action for certain other CNS drugs is related to charge transfer [62,63]. A significant number of other compounds with physiological activity relating to the CNS also contain the imine functionality, particularly antihypertensive agents [64]. A few examples are: pralidoxime chloride (hypertensive agent) [65], 3-alkylsydnones (CNS stimulants) [66], guanabenz (antibypertensive) [64], St 587 (cui-adrenoceptor agonist) [67]. Table 9 lists various classes of additional compounds [68-711 which incorporate the iminium functional group along with their reduction potentials and physiological responses. Hence, one can reasonably infer that a relationship might well exist between electron transfer and physiological activity. Reviews deal with potentially

TABLE 9 Reduction

potentials

for iminium compounds

having physiological

activity

Iminium compound

Physiological activity

Reduction potential (V)

Ref.

Diquat

Herbicide Herbicide Antibacterial Antibacterial Antibiotic Antimalarial Carcinogen

-0.57 -o.64D +o.so -0.58 + 0.02 - 0.80 -_I a

68 68 69 52 70 71 25

Paraquat Crystal Violet Indinin Pyocyanine. H+ Mepacrine.H+ Purine Iminium ’ versus Ag/AgCl. b oersus Ag/AgBr. ’ uersu~ s.c.e.

a b = a ’

424

toxic iminium ions from oxidative metabolism of xenobiotics [72] and iminium-conmining alkaloids [73]. The iminium theory appears broadly applicable to agents involved in a wide variety of biological systems: carcinogens [25], antibacterial drugs [24,52], PCP [8], nicotine [8], spermine [8], MPTP, [9], antimalarials [74], mesoionics [75], and anticancer agents [76]. ACKNOWLEDGEMENT

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