THE ELECTRONIC STRUCTURE OF CARBAMATE DERIVATIVES AS THE INHIBITORS OF CHOLINESTERASE

THE

ELECTRONIC AS

STRUCTURE

THE

OF

INHIBITORS

OF

CARBAMATE

DERIVATIVES

CHOLINESTERASE

TAKASHI BAN Departmentof Pharmacology,Facultyof Medicine,Kyoto University,Sakyo-ku,Kyoto CHIKAYOSHI

NAGATA

NationalCancerResearchInstitute,Chuo-ku,Tokyo Received for publication July 30, 1965 The inhibition of many

of cholinesterase

researchers

in relation

by some of carbamates

to the clinical

(2, 3) of the compounds. In this paper the author's chief concern tionship

between

cholinesterase

the electronic

inhibitors

and

structure

usefulness

is mainly

has attracted (1) and

devoted to investigating

of several structurally

the inhibitory

potency

the attention

insecticidal

the rela

related carbamate

of these compounds

(acetyl) cholinesterase, on the basis of the experiments et al. (4) and Wilson et al. (5) and to trying to analyse

activity

against

type true

of Kolbezen et al. (2), Myers the inhibitory mechanism of

them by this means. METHODS As the bamate and

the

the total

of a series

of car

7r electron

density

superdelocalizability were

treated

methyl

carbamate

in the

phenyl

moyl

choline

by

LCAO here

and

of the its

the MO

are

series with

part

phenyl

N-methyl

and

carba

and

N,N

adopted

are listed

with the reports

of Fukui et al.

(7, 8). Sometimes finer differences of reactivity among some analogous compounds can be detect

隆 志 ・永 田

as also shown in this report.

親義

employed

N

as those of Fukui et al. (6). Concerning the meaning of the superdelocalizability the readers

伴

parameters

calculation.

some substituents

molecule

parameters

ed by this index

the

method.

the

in Table 1. These values are the similar values

might consult

Energy

in

variational

derivatives.

The energy

1.

for a nucleophilic

calculated

of the simple

Molecules

dimethyl

indices

compounds,

reactivity method

reactivity

TABLE

* The coulomb integral

of the atom

X is written as ax=a+axJS, where a and f are the coulomb integral and the resonance integral in benzene respec tively. t The resonance integral between the atom X and Y is written as (3x_Y=l(3. + In the carbamoyl choline series, the latter value was adopted. In this case only the coulomb integral of the carbon atom adjacent to the substituent is assumed to be changed and estimated as aci=a+0.5J3.

FiG. 1. Relationship between logarithm of median ChE (fly brain) inhibitory molar concentration (pI50) of substituted phenyl N-methyl carbamates and the total 7r electron density of the ethereal oxygen atom (I., q_o_), that of carbonyl oxygen atom (II., qo=), that of the carbonyl carbon atom (III., q,, 0-), the superdelocalizability for a nucleophilic reactivity of that atom (III., Sr(N), 0------) and logarithm of alkali hydrolytic constant (IV.) of these compounds. This last value (IV.) is taken from Ref. (3). The scale of q, is taken in inverse order, because the nucleophilic reactivity would be increased as the atom loses its 7r electron share and gets posi tive. The results of calculation of the approximate superdelocalizability for a nucleophilic reactivity of the carbonyl carbon atom were published previously (28). In this and subsequent figures, each line indicates a regression line.

As for the carbamoyl choline derivatives, the alkyl group was excluded from the calculations because it might not be conjugated with the it electron system of the car bamate ester group. RESULTS AND DISCUSSION We calculated philic reactivity and found that

the total it electron

density

and the superdelocalizability

for a nucleo

on various positions of several substituted phenyl N-metyl carbamates the total it electron density of the ethereal oxygen atoms and, though

in the lesser degree, that with the house-fly

brain

of the carbonyl cholinesterase

oxygen

atoms had

inhibitory

potency

(2) (Fig. 1-I and II) and with that of the rat brain by Myers et al. (4) (Fig. 2), while the positive charge or the superdelocalizability

almost linear

observed

correlation

by Kolbezen et al.

true (acetyl) cholinesterase

observed

for a nucleo

philic reactivity of the carbonyl carbon atoms was inversely related to them (Fig. 1-111 and 2) and roughly parallel with the hydrolytic ability in the alkaline solution measured by Kolbezen et al. (2) (Fig. 1-III and IV). The similar tendency shown in Figs . 1-I, III and 2 suggests that the enzyme of Kolbezen et al. and

that

of Myers

et al. are the same

kind. On the other hand, we also calculated

the

positive charge and the superdelocalizability for a nucleophilic reactivity of the carbonyl carbon atoms

of the

methyl and

carbamoyl

N,N-dimethyl

total n electron

density

choline

and

derivatives

its N and the

of the ethereal oxygen

FIG. 2. Relationship between logarithm of median ChE (rat brain) inhibitory molar concentration (pI50) of substitut ed phenyl N-methyl carbamates and the total it electron density of the ethereal oxygen atom (q_0 0-) and the superdelocalizability for a nucleophilic reactivity of the carbonyl carbon atom (Sr(N), •------) of them.

atoms of them. Seen in Fig. 3 is the parallel

relationship

between

those indices

of the carbonyl

carbon atoms and the rate constant (k,,: referred to later) of the formation of the irre versible enzyme inhibitor complex measured by Wilson et al . (5) and the inverse rela tionship between the total rr electron density of the ethereal oxygen atoms and the rate constrant The

k3'. parallel

the ethereal

relationships

or carbonyl

inverse relationships

between

the reactivities

for an electrophilic

oxygen atom and the cholinesterase

between

those for a nucleophilic

atom and the latter in the phenyl

carbamate

reaction

inhibitory

reaction

potency

of the carbonyl

of

or the carbon

series were also noticed by Kolbezen et al ., more

Metcalf, and Myers et al. in the papers cited above (2-4) though they were estimated quantitatively. By plotting

the I50 value to the reactivity

of the ethereal

or carbonyl

oxygen atom

FiG. 3. Relationship between logarithm of the rate constant k3 of carbamoyl choline derivatives and the total it electron density of the carbonyl carbon atom (q, , the superdelocalizability for a nucleophilic reactivity of that atom (Sr(N), •------) and the total r electron density of the ethereal oxygen atom (q-o-, 0-) of these compounds.

estimated

by Hammett's

sigma

values, the formers

(2, 3) suggested

that

as an initial

process either oxygen atom might react with the positive site of the enzyme receiving the attack of water, hydrolytic cleavage of C-O bond would occur. planation reversible

and then This ex

seems to be consistent with the finding that some of the carbamates are not competitive inhibitors but are competitive substrates of which the destruction

rate is very slow (9-13). There have been some indications such as prostigmine and furthermore,

requires

at its esteratic

which the dialkyl phosphates The above led Myers

but simple potency

involved

site of the enzyme

might form the carbamoyl

(14),

derivatives

similar

of

to that in

the enzyme.

et al. (4) to the supposition

inhibitory

reaction

in the esteratic

site (5, 11, 12, 15-18) in a manner

phosphorylate

not be substrates

the cholinesterase the inhibitory

the basic group

that some of the carbamates

the cholinesterase

mates might

that the binding of some of the phenyl carbamates

that these phenyl

competitive

N-methyl

carba

because the order

of

is the reverse of that which might be expected

if

a transesterification

inhibitors,

of the N-methyl

carbamoyl

group

from the phenol to the active center and is also the reverse of that observed with ana logous organophosphorus compounds. Though

the final answer

would be given by a more direct attack on this problem,

more recent works of Wilson et al. (5, 18) seem to us to give a more reasonable tion for our consideration The general reaction moyl enzyme According

proposed

of the electronic

structure

scheme for the cholinesterase by Wilson, Harrison

of the carbamates. inhibitor

and Ginsburg

to this scheme and their equation

interpreta

which

produces a carba

(5) is shown below.

(18), I5, value at the attainment

of the

steady states isapproximately equal to KkIk4-(K, = ki)assuming k2» k,,K,» I5,and

that

the E' complex

mainly

contributes

to the

inhibition. Therefore,

it is expected

that

the

rate constant

k3 is and the

smaller

tion

K,

rate

the

constant more

potent

and

the

will

be the

larger

the

the dissocia

constant

k, are , of the

inhibition

cholinesterase. The

magnitude

on the

nucleophilic

carbon

atom

ratic

of K,

may

with

the

basic

site of the enzyme.

tionship

shown

largely

of the group

Here

in Figs.

by the interpretation the inhibitory

depend

reactivity

carbonyl

in the este

the inverse

1-111 and 2, is explained

that the contribution

potency of the compounds

are not very strong The magnitude the comparative

rela

of the initial process may be negligible

probably

corresponding

for

to the finding that they

inhibitors. of k3 depends on the lability of C-O (C-X) bond.

values of this bond lability

by the magnitude

We could estimate

of the positive charges of

the ethereal oxygen atoms, because it is stated that as the electron deficiency of the atoms of susceptible

link is increased,

and the reaction

the activation

energy of bond cleavage will be decreased

rate (k3) will be increased

(16, 19, 20). is the reverse of the results in Figs . 1-I and 2. These seeming contradictory results are explainable when one considers This expectation

may exist an acidic group in certain

near the basic group of the esteratic

substrates might participate

the protonation

of the ethereal

with the hydrolytic

that there

site (16, 21-24) which

cleavage of this bond through

oxygen atom.

When this is the case, the smaller the positive charge of the ethereal oxygen atom is, the more easily the bond cleavage will be promoted through the formation of the stronger

hydrogen

bond

(16, 19).

From the above discussions, true (acetyl) cholinesterase electronic

structure

we might suspect that these relationships

inhibitory

indicate

that

potency of phenyl

N-methyl

between

carbamates

the

and their

they

are substrates , but, that their rate determining process is not the initial process (designated as k, and k2) but the second process (desig nated as k3). It must be stressed that the rate determining necessarily N-methyl

lie in the second process. and N,N-dimethyl

by the relation Whether

between

derivatives

Some of them such as carbamoyl may have it in the initial

may not

choline and its

process as suggested

their reactivity

the rate determining

to be dependent

process of all the carbamates

on the magnitude

indices and rate constant k3'. process is the initial process or the second process seems of the rate of the initial process , because it is known by the introduction of quarternary

that when the rate of the initial process is promoted ammonium

ion (to the substrate)

which interacts

with the anionic

site of the enzyme ,

the second step is also promoted

(25, 26) though

in the case of prostigmine

the acidic

group in the esteratic site rather seems to be deprived of its binding ability (14). This would be the case of carbamoyl choline inhibitors. But when the rate of this process is not accelerated as much, the second process which is not affected by the initial process and is by nature

a slower process, would become the rate determining

would be the case of phenyl

N-methyl

carbamates

process, and this

except for its quartenary

ammonium

salts. Recent

work of Metzger

of cholinesterase

by diphenyl

and Wilson (27) which carbamoyl

also suggests that the rate determining nary

investigated

process of these inhibitors

which have no quarter

salts seems to be in the second process.

The inhibitory

potency of some other carbamates

molecule

N-methyl

or alkyl

the verification

without

such as m-trimethyl

with some substituents quarternary

ammonium

on the other part of the

ammonium

ion may be useful

for

of this supposition.

Our consideration propriety matically

carbamates

carbamates

reaction

chloride and their fluoride

ammonium

salts of phenyl

the inhibitory

and methyl carbamoyl

of the electronic

structure

is interesting

as it is suggestive

of the

of the assumption of Wilson et al. that some carbamates are destroyed enzy through two intermediate stages and also it is suggestive of the functional

dependency

between

the esteratic

site and the anionic

site.

SUMMARY The total rr electron vity of several phenyl tives are calculated cholinesterase

density and the superdelocalizability N-methyl

inhibitory

The relationships

derivatives

and

carbamoyl

in relation

choline

reacti deriva

to their true (acetyl)

potency. found are as follows :

1. In the phenyl N-methyl 7r electron

carbamate

by the simple LCAO MO method

for a nucleophilic

carbamate

density of the ethereal

series the order of the magnitude

oxygen atom and the carbonyl

of the total

oxygen atom is almost

parallel to that of the inhibitory potency of the enzyme, while the order of the magni tude of the positive charge or the superdelocalizability for a nucleophilic reactivity of the carbonyl

carbon

atom is the reverse to that.

2. In the carbamoyl

choline series, the order of the magnitude

and the superdelocalizability is parallel complex,

for a nucleophilic

to the rate constant while that

reactivity

of the formation

of the magnitude

of the positive charge

of the carbonyl

of the irreversible

of the total rr electron

density

carbon

enzyme

atom

inhibitor

of the ethereal

oxygen atom is the reverse of that. From these results, and the possibility enzyme, it is suggested action

between

that in the latter

the carbonyl

carbon

esteratic

site of the enzyme supported

ion and

the anionic

atom

that these carbamates

might carbamylate

series the rate determining of carbamates

by the one between

site (the initial process), while

and

the

process is the re

the basic group

the quarternary

in the

ammonium

in the former series , it is the C-O

bond cleavage process through the hydrogen bond formation between the ethereal oxygen atom of the compounds and the acidic group in the esteratic site of the enzyme (the second process). It is stressed contribute

that

to clarifying

exploration

of the electronic

the inhibitory

mechanism

structure

of the compounds

may

of the enzyme.

Acknowledgement : The authors wish to acknowledge that a part of the calculation were carried out on the computers in Department of Public Hygiene, Faculty of Medicine, Kyoto University and National Cancer Research Institute, Tokyo. We also wish to express our thanks to Dr. Lowy and Mr. Robert Ratcheson, of the Center for Brain Research, University of Rochester, Rochester, N.Y. for reading the manuscript and for their kind advices. REFERENCES 1) STEMPEL, A. ANDAESCHLIMANN, J.A. : MedicinalChemistry,Edited by BLICKE, F.F. ANDCox, R.H., vol. III, p. 238, John Wiley, New York (1956) 2) KOLBEZEN, M., METCALF, R.L. ANDFUKUTO,T.: J. Agr. Food Chem.2, 864 (1954) 3) METCALF,R.L. : OrganicInsecticides, p. 317, Interscience Publishers, Inc., New York (1955) 4) MYERS,D.K., KEMP,A. JR., TOL,J.W. ANDDEJONGE,M.H.T. : Biochem. J. 65, 232 (1957) 5) WILSON,I.B., HARRISON, M.A. ANDGINSBURG, S. : J. Biol. Chem.236, 1498 (1961) 6) FUKUI,K., MOROKUMA, K., NAGATA, C. ANDIMAMURA, A. : Bull. Chem.Soc.Japan 34, 1224 (1961) 7) FUKUI,K., YONEZAWA, T. ANDNAGATA, C.: J. chem.Phys.29, 1247 (1957) 8) FUKUI,K., YONEZAWA, T. ANDNAGATA,C.: Bull. Chem.Soc.Japan 27, 423 (1954) 9) GOLDSTEIN, A. ANDHAMLISCH, R.E. : Arch. Biochem.Biophys.35, 12 (1952) 10) MYERS,D.K.: Biochem. J. 52, 46 (1952) 11) MYERS,D.K.: Ibid. 62, 556 (1956) 12) CASIDA, J.E., AUGUSTINSSON, K.-B. ANDJONSSON,G. : J. econ.Ent. 53, 205 (1960) 13) FELLMAN, J.H. ANDFUJITA,T.S. : Fed. Proc. 23, 384 (1964) 14) WILSON,I.B.: Biochem.biophys.acta 7, 466 (1951) 15) MYERS,D.K. ANDKEMP,A.: Nature,Lond. 173, 33 (1954) 16) BERGMANN, F. : Advancesin Catalysis12, 130 (1958) 17) AUGUSTINSSON, K.-B., FREDRIKSSON, T., SUNDWALL, A. ANDJONSSON,G. : Biochem.Pharmacol.3, 68 (1959) 18) WILSON,LB., HATCH,M.A. ANDGINSBURG, S. : J. biol. Chem.235, 2312 (1960) 19) BERGMANN, F., RIMON,S. ANDSEGAL,R. : Biochem. J. 68, 493 (1958) 20) PULLMAN, A. ANDPULLMAN, B.: Proc.nat. Acad. Sci., Wash.45, 1572 (1959) 21) WILSON,LB. ANDBERGMANN, F. : J. biol. Chem.186, 683 (1950) 22) WILSON,LB.: The Mechanismof Enzyme Action,Edited by MCELROY, W.D. ANDGLASS,B., p. 642, Johns Hopkins Press, Baltimore (1954) 23) BERGMANN, F., SEGAL,R., SHIMONI,A. ANDWURZEL,M. : Biochcm.J. 63, 684 (1956) 24) BAN,T.: THISJOURNAL13, 225 (1963) 25) WILSON,I.B. : Discussions Faraday Soc.No. 20, 119 (1955) 26) WILSON,I.B. ANDCABIB,E. : J. Amer.Chem.Soc.78, 202 (1956) 27) METZGER, H.P. ANDWILSON,I.B. : Fed. Proc.23, 316 (1964) 28) BAN,T. ANDSHINAGAWA, Y. : SeibutsuButsuri2, 23 (1962)