Enzyme inhibitory activity of certain phosphonate esters against chymotrypsin, trypsin and acetylcholinesterase

BIOCHIMICA ET BIOPHYSICA ACTA

441

BBA 65029 ENZYME I N H I B I T O R Y ACTIVITY OF CERTAIN PHOSPHONATE ESTERS AGAINST CHYMOTRYPSIN, TRYPSIN AND ACETYLCHOLINESTERASE BETTY J. BOONE, E L M E R L. B E C K E R AND DONALD H. CANHAM*

Division of Biochemistry, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D.C. (U.S.A.) (Received January 7th, 1964)

SUMMARY

O-p-nitrophenyl-O-ethyl-o~-chloroalkylphosphonateswith the

alkyl group varying in length from 4 to 7 carbons have been synthesized. Of the four chloroalkyl compounds tested, the 6-chloroheptylphosphonate showed peak anti-chymotrypsin (EC 3.4.4.5) activity, but gave minimum anti-trypsin (EC 3.4.4.4) activity; the greatest activity against red cell acetylcholinesterase (EC 3.1.1.7) came with the 7-chlorobeptylphosphonate. This pattern of inhibitory activity differed distinctly from that reported for the corresponding alkylphosphonates. INTRODUCTION

We have previously reported on the inhibitory activity of a series of 0-p-nitrophenyl0-ethyl alkyl and phenylalkylphosphonates against chymotrypsin (EC 3-4.4-5), trypsin (EC 3.4.4.4), and human red cell cholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) (ref. I). Here it was shown that quite small changes in structure were capable of causing quite large changes in inhibitory activity against a given enzyme. The detailed results were considered to lend further support to the idea that the structural features important for the activity of the substrate are also important for activity of the organophosphorus inhibitor against that same enzyme. To further test the effect of various structural factors on the inhibitory activity of organophosphorus compounds, we synthesized a series of co-chloroalkylph0sphonate esters having the general formula O

11

C1(Ckla) x - - P - - O - - C , FI6

The specific purpose was to ascertain the effect, if any, of the substitution by the larger, more electronegative chlorine atom for a hydrogen atom on the terminal carbon * Present address: Montana State University, Department of Pharmacy, Missoula, Mont.

(U.S.A.).

Biochim. Biophys. Acta, 85 (1964) 441-445

44 2

It. .[. B O O N E , E. L. B E { ; K E R , I}. H. { ; A N t t A M

of tile c o m p o u n d {m the ability of the phosphonates t{} inactiwtte trYt)sin, chym{)trypsin, and ch{}linesterase, under the same c(mditions as t)reviously used t{} s t u d y the action {}f the c(}rrest}on(ling alkylph(}sphonates.

I C X P E I { I M E N T A L I'I{:OCE D U Rt'2S

The O-p-nitr(}phenyl O ethyl-o-chloroalkylphosphonates were synthesized by methods previously described", except that the usual ARBUZOV method a was supplanted b y a reverse addition reaction 4 using (C2HaO)~-P-Na, which led to larger yields. Oxalyl chloride 5 was used in place of PC1 a to form the corresponding chloridate. P,ecause of their lability to heat and their tendency to hydrolyze, the esters were distilled in a falling fihn molecular still with a mean free p a t h of I.o cm.

TABLE

1

PltYSICAL CONSTANTS (IF ()-Y, T H Y I , - ( ) - p - N I T R O P H E N O L - r ~ ) - C H L O R O A I ~ K Y I ,

Alkvl ~roup

I }i ;t!ltation*

.*1nalvsis

X D""

Formula empirical

{).{3c}2 llllll

1.5280

Ct,+,Ht+NO~ PCI

C t +.9~} t t 5. to X t.3o CI r o . o l

tt.S9 5.27 ~-45 7.20

[3"; ~

{}.or} I llllll

I ..52{} t

Cl3lll~,N()al'Cl

C t{}.St lI 5.1,', N |.l 7 CI t o . 5 5

t<,.~7 "-It 3.!}I 11.88

t3S'

{}.{}{}tmm

t.5z48

CttlI2tN()fl'(21

C t S-t ~ lI 0 . o ; N t.ot CI t o . o 3

t7.q/ +~.t/ 3.9c}

{" }0-5 ° lI 0.35 N 3.q'~ ('1 O.t}5

t q-~ t <~.: 3.S3 to.t t

Te,np.

Press. (ram It¢)

Butyl

t38~

Amyl

11cxyl

tleptyl

PIIOSPttONAT1,;S

i.t 1 -'

o . o 0 4 lllnl

1.523o

CtsIt.,~NO~['CI

* Distillation temperatures listed are the minimum at f a l l i n g filnl m o l e c u l a r still h a v i n g a 1.o cil/ p a t h .

necessary

fl}r d i s t i l l i n g

9.75

t h e pl'odlll;I

ill

Acctyl cholinesterase was assayed by the method of MICHEl. II and chym{)trypsin and trypsin by the previously described modification of this technique 1. The inhibitory activity of the various compounds against the three enzymes was determined as described previouslyL The inhibitory activity was recorded in terms of the I50, the molar concentration of inhibitor required for j,=o°,o inactivation when inhibitor and enzyme reacted for 15 rain at 25 °. For convenience, these values were transformed into the pI~o, the negative logarithm of the Ia0. In all cases, conditions were such t h a t the I50 could be taken as inversely proportional to the secon{t order reaction constant of the inhibitor and enzyme t. 13zochim.

13iophys.

_ t c/a, 83 ( l {jiq} 4 t t -t43

443

PHOSPHONATES AS ENZYME INHIBITORS

The first order hydrolysis constants were determined in o.o66 M phosphate buffer, pH 8.3, at 37 ° as previously described 2. RESULTS

The results of the inhibition studies as well as the hydrolysis constants are given in Fig. i. The hydrolysis rates showed only a slight decrease from 4.11.Io-*min -1 to 3.55" io 4 rain-1 in going from the 4-chlorobutyl to the 7-chloroheptylphosphonate. The anticholinesterase activity (solid line, Fig. I) increased 1.8 fold in going from the 4 carbon chloroalkylphosphonate to the 7 carbon chloroalkylphosphonate. -3

4ydrolys[s

........

~ ......

"8"- . . . . . .

Constants

J

~Acetyl • Cholinesterase o

-e.

...o._.______.-- o

o'""""'"

6.0

0.

s ~ s af

5.0

Chymotrypsin ,, ,..,."~ r** 'a

o :>, o

4.0

.c

3.0

trypsin

-~" Air.p ~

~

~"" ~.

(u-Chloro Alkyl Phosphona)e Esters .....

n-Alkyl

Phosphonole

Esters

I

1

!

I

4

5

6

7

Number

of

Carbon Atoms

in

Aiky[ Chain

Fig. I. A c o m p a r i s o n of h y d r o l y s i s c o n s t a n t s a n d i n h i b i t o r y activities (plso) t o w a r d acetylcholinesterase, c h y m o t r y p s i n , a n d t r y p s i n , vs. t h e n u m b e r of carbon a t o m s in t h e alkyl c h a i n of O-p-nitrophenyl-O-ethyl-o~-chloroalkylphosphonate a n d O-p-nitrophenyl-O-ethyl-n-alkylphosp h o n a t e s . T h e h y d r o l y s i s c o n s t a n t s a n d i n h i b i t o r y activities for t h e n - a l k y l p h o s p h o n a t e esters were t a k e n f r o m BECKEE, FUKUTO, BOONE, CANHAM AND BOGF.R (I963).

The antichymotrypsin activity of the 4 compounds is also pictured in Fig. I (solid line). The antiehymotrypsin activity of the 4 compounds (Fig. I, solid line) increased 3.6 fold in going from the 4-chlorobutyl to the 6 chlorohexylphosphonate, but the addition of one more carbon decreased the activity of the 7-chloroheptylphosphonate to 73% of the activity of the 6-chloro compound (Fig. I). The changes in antitrypsin activity among the 4 compounds are relatively little, but there is discernible a small, but reproducible, minimum in activity with the 6chlor ohexylphosphonat e. Biochim. Biophys. Acta, 85 (1964) 441-445

444

B . J . BOONE, E . L . BECKER, D. H. CANHAM DISCUSSION

There is not only little, if any, difference among the hydrolysis rates of tile chloroalkylphosphonates studied here, but in this respect, there is no real difference between the chloroalkyI compounds and the corresponding alkylphosphonates (Fig. t). FU'KVrO AND METCaLV2 measured the hydrolysis rate of the O-/5-nitrophenyl-O-ethyl-3-chloropropylphosphonate and the propylphosphonate under the same conditions used here. They found k -- 7.53' IO 4 min ~ for the chloro compound, and k = 4.7" I°-1 for the propylphosphonate. This is ascribable to the effect of the strong electron withdrawing activity of the chlorine atom in increasing the lability of the P--4) -~/Y : ~ - N ( ) , , bondL From the results with the longer chain members of the series, it is evidm~t that the chlorine atom does not exert its influence in this regard beyond 3 carbon atoms. When the number of carbons in the alkyl chain is between 4 and 7, neither the number of the atoms in the alkyl chain, nor the presence of a chlorine atom ~m the terminal carbon afft:cts appreciably the rates of the reaction of these phosphonates with water. However, a glance at Fig. I will demonstrate that both factors affect the reactivity of these compounds with enzymes. FUKUTO AND METCALF~'showed that the rate of reaction of the 3-chloro-t)ropylphosphonate with fly brain cholinesterase was only 8°:o of that of the propylphosphonate. They attributed this decrease in activity to the repulsion of the strategically placed electron rich chlorine atom by the anionic site of the enzyme. Fig. i demonstrates that this same effect is operating in the reaction of the chloroalkylphost)honates with the hnman red cell cholinesterase. As the carbon chain is lengthened, however, this effect of the chlorine atom1 is progressively lessened and the activities of alkvl and clfioroalkyl compounds tend more and more t~, approximate each other. WALLACE et al. a have postulated that a negative charge is present in chymotrypsin close to one of the loci for attachment of the aromatic group. The activity of the chloroalkylphosphonates does not seem to be affected by such a putative negative charge on the enzyme, suggesting that the negative charge is too far from the binding site of the alkyl chain to interact with the terminal chlorine of the ehloroalkylphosphonates. The effect of the terminal chlorine atom seems rather to be due to its size since it seems to act similarly to an added methylene group. Thus, the optimum inhibition by the chloroalkylphosphonates is given bv the 6 carbon compound instead of the 7 carbon phosphonate as demonstrated for the alkyl seriesL The small but reproducible minimum in antitrypsin activity flmnd with the 6chlorohexylphosphonate contrasts with the optimum given by the corresponding hexylphosphonate. Positively charged amino acids, such as lysine and arginine, are required in the preferred substrates for trypsin suggesting that this molecule possesses an anionic binding site at a definite distance from the esteratic site. If this is so, the minimum antitrypsin activity given by the 6-chlorohexyl compound might be due to the repulsion of the electron rich chloro group by this putative anionic site. Thus, the results of this study further demonstrate that small structural variations within the framework of the phosphonate ester will produce large changes in the inhibitory activity of a given enzyme, and that not only the magnitude, but the direction of change in inhibitory activity will depend on the enzyme being inhibited as well as the nature of the structural variation. Biochim. Biophys..-tcta,

85 (lO64) 44~-445

PHOSPHONATES AS ENZYME INHIBITORS

445

REFERENCES 1 E. L. BECKER, T. R. FUKUTO, B. BOONE, D. H. CANHAM AND E. BOGER, Biochemistry, 2 (1963) 72 • T. R. FUKUTO AND R. L. METCALF, J. Am. Chem. Soc., 81 (1959) 372. A. E. ARBUZOV, in N. ALEXANDRIA, On the Structure of Phosphorous Acid, I9o 5, as cited in: J. Am. Chem. Soc., 66 (1944) lO9. A. ToY, Victor Chem. Co., personal c o m m u n i c a t i o n to E. L. BECKER, 1962. 5 Z. PELCHOWlCZ, J. Chem. Soc., (1961) 44. e H. O. MICHEL, J. Lab. Clin. Med., 34 (1949) 1564. 7 T. R. FUKUTO, in R. L. METCALF,Advances in Test Control Research, lnterscience, New York, p. 172. 8 R. A. WALLACE, A. N. KORTZ AND C. NIEMAN, Biochemistry, 2 (1963) 824.

Biochim. Biophys. Acta, 85 (1964) 441-445