PAOSPHONYLATED AChE
Journal of Medicinal Chemistry, 1970, Vol. 13, N o . .7 435
Model Studies for the Reactivation of Aged Phosphonylated Acetylcholinesterase. Use of Alkylating Agents Containing Nucleophilic Groups GEORGE11.STEINBERG, CLAIREN. LIESKE,ROGER BOLDT, Physiology Department, Medical Research Laboratory, Rrscarch Laboratories, Edgewood Arsenal, Jlarylancl 21010
JOHN C. G O A N ,
E. P O D A L L
AND H.4ROLD
Rcsrarch Division, Melpar Inc., Falls Church, Virginia 22046 Received May 12, 1969 Using sodium p-nitrophenyl methylphosphonate as a model for aged (dealkylated) phosphonylated cholinesterase, a study was made of its rate of reaction with various alkylating reagents containing nucleophilic groups. I n DRIF the alkylation rate ranged widely between a second-order rate constant, kg = 190 M-1 hr-1, at 2 5 ” , to no observable reaction in 96 hr at 60’ corresponding to kg vc~l t o itirlude 11hcriiic OH :rnd :I Iii.tiditic.-tleri~.c,ci
PHOSPHONYI, \TI;L) ACHE
\'I
Journal of Sltrlirinal Chonistri/, 1.970, T'ol. It,?,.Yn.
0
.j
437
in its anionic form," a solution of pH -3.4 was used, An aqueous solution, pH 3.4, of VI was mixed with alkylating compound dissolved in dioxane to give a final solution containing 71.5y0 (v/v) H20 with the concentration of VI and alkylating compound each a t 5X M . The mixture was refluxed for 4 hr. A control mixture, without alkylating agent, was treated in an identical fashion. Analyses were performed by assay method A. The results are given in Table I. T A B L EI TRAXSIEST ALKYLATION OF SODIUM ~NITROPHENYL NETHYLPHOSPHONATE I X AQCEOUSSOLUTIOS
CH
v methylphosphonate, VI, and RT. The reactions involved are depicted in eq 5 and 6. Further, since the desired esters were prepared by an alkylation reaction similar to that envisioned for the aged inhibited enzyme, 11, this approach gave information on comparative alkylation rates. C. Convenience of Assay.-Hydrolysis of the model compounds T.' could be easily and conveniently measured spectrophotometrically a t near neutral or at elevated p H by measurement of absorbance of the anion of p-nitrophenol a t 402 mp16 (V and VI do not absorb at this wavelength). Two assay procedures were used to determine the concentrations of T' and p-nitrophenol that were produced in the reaction vessel. I n method A an aliquot of the in situ preparation was added to 0.1 111carbonate buffer, pH 10, and heated on the steam bath for 10 min. This treatment gives complete hydrolysis of mixed esters T' to p-nitrophenol and presumably ROPCH3(0)(O-Ka+), yet gives no appreciable hydrolysis of unreacted VI (see Experimental Section). Therefore, the absorbance a t 402 mp is a measure of V plus free p-nitrophenyl present in the sample. I n method B, the aliquot of synthesis mixture was mixed rapidly with 0.1 *ll Tris buffer of pH 7.15, 23", transferred quickly to the spectrophotometer cuvette (total time 20 sec) arid a continuous record made of the increase in absorbance a t 402 mp to completion of the hydrolysis reaction. With this method, the absorbance a t end point giveq a measure of mixed ester, V (assuming 100% hydrolyyis to p-nitrophenyl) , plus free p-nitrophenol present in the aliquot. lirom the slope of the linear plot of log A /(A --At) us. time (first-order reaction) one computes the hydrolysis rate constant. The yintercept gives a nieaSure of free p-nitrophenol present in the original aliquot, so that the concentration of V can be computed from the combined end point and 9intercept information (iv'rle infra). Results and Discussion Alkylation. (1) Validation.-Initial tests were made to determine whether the alkylation of VI to yield mixed esters of type ITwould take place in aqueous media. I n order to minimize the hydrolysis of V and also the alkylating compounds, yet to maintain VI (16) A . I . Rigqn. Trans. Fnradau Sor., 60, 800 (1954).
Alkylating agent
Per cent alkylation"
28.7, 22.5, 26.7, 24.6, 25 Phenacyl bromide (VII) Benzyl bromide ( X I I ) 34.4, 31 Phenacyl bromide oxime ( X X I I I ) 17.7, 21 33, 3.5 a-Bromophenylacetic acid (XXX) Styrene oxide ( X X X I I ) 0, 0 2-Pyridohydroxam> 1 chloride hydrochloride ( X X X I I I ) 61, 62 Phenyldimethylsulfonium methanesulfonate (XXXIV) a Represents extent of alkylation achieved under the experimental conditions. The mixed esters formed had decomposed completely under the conditionq of reaction. See text for conditioni and dizciiision.
These have been corrected for 13% hydrolysis of VI which was observed in the control experiment. Utilizing assay method B, i t was found that the reaction mixtures contained no hydrolyzable ester. l8 Accordingly, the mixed ester formed had decomposed completely under the reaction conditions. This was confirmed in the case of p-nitrophenyl phenacyl methylphosphonate 111, using a sample of the pure compound. Under the 0
0
I
C" 111
test conditions, pure I11 decomposed completely (to give 100% free p-nitrophenol). Further support for the ester formation-decomposition route is given by the marked differences in yield of p-nitrophenol (indicated as per cent alkylation in Table I) for the various alkylating compounds together with the close reproducibility of the replicate runs. It was shown, also, that mixed phosphate esters could be prepared by reaction of phenacyl bromide with diethyl and dibutyl phosphate9 in aqueous EtOH, albeit in low yields (see Experimental Section). The use of completely nonaqueous solvents gave superior results. I n particular. it was found that various mixed esters V could be readily formed at room temperature by reaction in NeCN or DAW. The alkylation reactions were 50-100 times more rapid i n DAIF than in 1IeCN. Accordingly, DLIF was used as solvent for the alkylations performed in the rest of these studies. (17) T h e pK. of phenyl methylphosphonic acid is 1.39 (J. 0. Edwards, unpublished d a t a ) . Hence the pKa of p-nitrophenyl methylphosphonic acid is probably somewhat lower, but certainly not higher t h a n 1.39. (18) T h e same result was obtained when alkylation mas performed at pH ,5.
TABLE I1 HYDROLYSIS RATESOF PRODL-CT OF l t r ~ ~ c ~OrFo s CYL BROXIDF; . ~ S D VI IS Inw, 250. OMPARISOS WITH ;\CTHI 7.99 w.7 0 0. l 3 J 4 I O , 73 7 .SI) \I:$ , 5 l1.01:~ 0 . 194 ) 22.7.; 7.33 92.2 0.022 0.266s; 6 29.7.i 7.9s 56.b 0.041 0.276 1 OS 0 . 1:34 0.2!)2 a Hydrolysis iii 0.1 -11Tris, pII 7.15>2.j". , the constaiicy of thc computed rate constantb. :ind their e with that for authentic 111. we conclude (1) that I11 formed upon incubation iu DlIF, ( 2 ) its formatiorl irred progreisively, and (:3) it n a 5 the only >\I\)st:inw fornicd ~t liich hydrolyzed to J ield p-nitrophenol under the cxyerimentul asbay conditions. Fin:ill> . it5 presence T\ as also confirmed by tlc (see Experirnciit a1 Section). It also cart be see11 from the progressive incre:~sei i i .Iu tlint both the authentic 111, and thnt prvpared i n D l 1 IT, wlvolyzed to give p-nitrophennl to sonic extent during prolonged incubation. I t i3 not knoivii Ivhether this represents "purt"' d v o l y 4 s or Iiydrolj bv trace, of E120 present in t h DAIl:. But, a t le, n i t h this compound, decompo4tion in DlIk' was sufficiently slow to permit determination of its hydrolysi;. rat c' i n aqnc~oussolutiou. Similar rciults werc oht ainecl c phen:icyl broniide (T'II) concentrut ioii TV:I 1. Siiicc VI is st:tblti in id(^ nisay condition\. o I I t ' ( ~ D I I c:ilcul:tte the swnnd-ord(~rrat,(\ cvnst:irit f o r
Journal of Medicinal Chemistry, 1970, Vol. 13, No. 9 439
PIIOSPHONYLATED AChE
alkylation of VI from the slope of the plot of 1/[R] U S . time (where [R] = concentration of phenacyl bromide and also of VI ; see Figure 3). It can be seen from Figure 1 that the alkylation reaction does not go to completion since A, for the 98-hr curve should approach that of the pure ester, 111. One can speculate on a variety of side reactions that might be involved, including alkylation of liberated p-nitrophenol and reaction with DAlk'.l9 However, the side reactions did not interfere with the determination of the hydrolysis rates and therefore were not investigated. The concentration of phenacyl bromide for each incubation time was calculated from the A , values (which measures the degree of conversion of VI1 into 1111, ie., [RY] = A , ( t h ) - A , ( o b s d ) , where A a ( t h ) is the pnitrophenol absorbance that one would obtain if all of the phenacyl bromide had been converted into I11 and A, (ob.d), the p-nitrophenol absorbance actually observed. The plot of l / [ R T ] us. time was found to be linear for approximately one half-life. The computed second-order rate constant was 12 J1-l hr-l. Upon repetition, using a larger number of short time points, shown in Figure 3, the rate constant was 18.8M-l hr-l. It appears therefore that the alkylation reaction rate can be estimated to well within one order of magnitude from initial rate data (where side reactions of the alkylating agent are minimized). Since in these experiments the hydrolysis of the product ester I11 was carried out as a second step (essentially independent of the alkylation step in DAIF), the extent of conversion of Y I into ester should be primarily a function of the concentration of the reactants. This was found to be the case. At 5 x 111 with respect to each VI and VII, a maximum conversion of 35% was obtained. When the concentration of VI1 was increased by a factor of 3, the conversion into I11 was incrcased to 5670;'0.20 Since I11 is a comparatively unstable phosphonate ester, achievement of such high conversion values suggests that the preparation of mixed phosphonate (phosphate) esters by alkylation of salts in DAIF should have synthetic utility. The starting materials are comparatively simple and readily available and the reaction conditions are mild. I n particular, for coniplex compounds of biological interest, where large quantities are not required and purification, if required, can be performed by chromatographic techniques, this procedure should be advantageous. When the reaction of VI with 1-bromo-3-phenyl-2,3propanedione (VIII) was examined in D N F , a fairly dark yellow-brown color appeared in the DiUF solution immediately after mixing. As before, the DAIF mixture was placed in the constant temperature bath a t 25" and aliquots were taken periodically for assay a t pH 7.15 (method B). Here, the curves indicating p-nitrophenol formation were straight lines parallel to the x-axis, ie., A . = A, for each line. However, the value of A0 increased progressively with time of incubation in DMF. Thus, all of the p-nitrophenol was present a t the time that the spectrophotometric recording was begun (approximately 20 see after mixing of the (19) G. hl. Cuppinger, J. Amer. Chem. Soc., 76, 1337 (1Y54). (20) Percent conversion was determined from the value uf A ,
- A a in assay B. I n each case this reached a maximum r a l u e and then declined. With t h e 1:l ratio of reactants, the maximum value was reached in 11.25 hr. and with t h e 3 : l ratio. in 7 lir. I n both cases, t h e same product was obtained, as determined frurn Iisdrolysis rate measurement.
60
120
180
240
300
360
430
TIME OF HYDROLYSIS (SECONDS)
Figure 2.-First-order plot for hydrolysis of 111,p H 7.15, 0.1 A1 Tris, 25": (0)pure 111,after G m i n incubation in D M F (Figure 1, curve 1);(1) range in values computed from raw data taken from curves 3 to 6 in Figure 1 (periods of incubation of VI1 and VI in DhIF ranged from 5 to 29.75 hr); (0) pure 111, after 62-hr incubation in D h l F (Figure 1 , curve 2).
I
280
-
260
-
240
-
I
I
c
r
7
K
1 MME [HOURS)
Figure 3.-Reactioii between phenacyl bromide and V I in I I l I F , 25'. Initial coiiceritratioii of each reactant, 5.0 x 10-3 X. 11 represents the concentration of each of the reactants determined "by difference" using assay A.
D M F solution with the aqueous buffer). Control experiments showed that the increasing values of Ae were not due to impurities in VI11 or to HBr that might have been produced by hydrolysis of VIII. Thus, incubation of 5 X M VI with an equal concentration of HBr in DAW for 6 hr gave no p-nitrophenol in the assay; nor did a reaction mixture of 2.5 X 111 each, VI,
Journal of Jftdicinal Chemistry, 1970, 1'01. l;, S o . .i 441
PHOSPHONYLATED AChE TABLEI11
-4LKYLITIUS O F SODIVM p-NITROPHENTL RIETHYLPHOSPHONATE,
HYDROLYSIS RATEOF -- hlkslating agentNO.
THE
RESULTIXT~ I I X EESTERS, D V,
-
VI1
R a t e of alkylation of 82 (M-1 l x - 1 ) 25' 60'
--VI?
18.8 (10.6) Approx 190 0.12 1.0 0.3 1.76 (113)
VIIId IX
X XI XI1
166 (0.6)
43
86
XI11
XIV
K.r. (150 hr ) 0.48 (417)
xv
VI,
IN
DIMETHYLFORMAIIIIDC.
IX ~ W E O U S SOLUTION, pH
-Hydrolysis
7.15, 25'
r a t e of the resultant mixed ester, V-
Conversion
k b s d (S8C-l)
7 . 6 x 10-3 4.63 x 10-4c Instantaneouse 2 . 1 2 x 10-418 3.86 x 10-4' 3.78 x 10-4' 1.4 X 4.90 x 10-41 3.70 x 10-4fs8 1.4 x 8 . 3 x 10-4' 5.22 x 10-6* (3.77 x 10-6)
3.27 x lO"/.Q 1.79 x 103 1.83 x 103' 5.0 x 105 1.44 x 103 1.87 x 1 0 3 f . ~ 5 . 0 x 106 8.4 X lo2' 1.33 x 105*
'
'
50 7 47 38 20
7.3
7.6 X
91 .2
27
YVI XVII
2.76 X 1.6 X l o T 3
231 440
50 11
XVIII
7.3
SIX
xx XSIt XXIII XXIIIk
x
10-4
hpprox 128
8..57 X 1.97 X l o w 3
27
1.31
4.2
0.3
x 10-J
x
10-4
8.6 X l o 2 80.9 355.
57
530
1.69 X lo3
13.8
XXIV XXVl
spprox 160
XXYI XXVII
(HO)nPOCHrCI
XXVIII
Br CHsCOOH
XXIX
a-Bromoruccinic acid
4OL&reaction in 24 hr 407, reaction in 24 hr 7% in 3 hr
Instaiitaneuuse Iiistantaaeouse
30
Instantaneouse
28
Instantaneous8
40
Instantaneouse
40
7
N.r.
XXX
(48 hr) N.r. (48 hr)
XXXI
K.r. (92 hr)
XXSII
K.r. (48 hr)
a Alkylation at 25": TI and alkylating agent, 5 X 31 each. At 60": 10-2 &' each. React,ioii half-t,imes are given in parenthesis. N.r. = no reaction for time given. b For conditions rited under a : ,4, x 100 !t,heoretical absorbance for complete react,iori. Hydrolysis at pH 4.6: 0.1 J I acetate. J. Wegmanii and H. llahn, Hdu.Chini. A c t a , 29, 1247 (1946); Calrd: C, 47.6: I€, 3.1; Found: C, 48.6; 11, 3.6. e kf>i,>d>O.l5 sec-l. f Hydrolysis at, pTI 10.0, 25'; carboilate bi:ffer (0.1 JI). Q Measwement made O I L isolated, purified, and characterized sample of t,he mixed ester. 1.1 X 10-2 31 XIV plus 10-3 -I2 S'I i i i l\leC?J, refluxed 3 hr. Hyl\lp 87-88'; Buchner, Ber., 25, 1160 drolysis rate measured at p H 7.29 in 0.1 Af Tris. kobsd computed for pH 7.15 in parenthesis. H . Korteri aud i J I p 133", .4beriius, J . Prakt. Chem., 40, 428 (1900); mp 131°, Beilstein, 12, 245 (1900). (1892), mp 86-88'. 12. Scholl, Ber., 34, 1901 (1901). "Compound" was mist,ure of bromide and chloride. See 111. l\Iaaaki, K. Fukui, arid 11. Ohta, J . Ory. Chcm., 32, 3564 (1967). Prepared by Ash-Stevens, Inc., mp 166-167.5"; Calcd: C, 32.57; 13, 3.08; Br, 64.18. Found: C, 32.51; H, 3.18; Br, 54.39.
drolysis mte by 3 or 4 order.; of magnitude; ( 3 ) fast, compounds which had liberated d l of the p-nitrophenol in ley5 than 20 sec under assay conditionz. Slow.- The.e included thc l f e , E t , benzyl. and oiiitrobenzyl ezters. It i? of ititerezt to note that in goiiig from 1Ie to o-nitrobenzyl the reaction rate is increased h) :I f'wtor of only 2 . This indicate. the comparatiw lack of misitivity of the rate of the hydrolytic reactioii to changes in tht, (iiucleophilic :it tack on phosphor noillcaving ester group. Hydro1 of the two benzyl esters corresponding to XI1 arid XI11 are each substantiall). first-order in phosyhonate and OH-.23 I t i.; striking that the CO-containing compound corresponding t o XI\' also falls into the .low group. The gemdimethyl groupq prevents eriolization of the carbonyl group mid c:Ln also sterically hinder acceas by OH- or H 2 0 to cither (or both) the phozphonate m d CO g r o u p It I i ~ t . beeti yhowii from deuterium incorporation data t h;Lt the ticeeleration of h> drol) (Jf 111 i* not due to enolisatiori, but most probably involves the formation of c:irboii> 1 hydrate. 2 4 The Stewart and Breigleb molecular model of the niixed ester corrcspoiiding t o XIT.' slio.itO~ little or no crowding around the P atom. Theril appc:trs to be ample space for entry of OH- for tlttack o n 1'. O n the other hand, thc t n o ,\le group? c~tusc cvii.itler:tblc cronding ahout thc a d j x e n t C'O. Thu., i t 11 ould q)pcar that thry eliiiiiriate C'O :meleratioii of I he liyclroly~isrwctioii b , ~iilterf irig n i t h OH- (01 H2O) :Lddition to the CO, ivhicli i h rcquired for its ptrticipation in the Intermediate.-These include t lic unwbatituted itiid ?J- and o-nicthoxyphenacj 1, 2-hetoc) clohexyl, acetonyl, .~-~ritro-~-h~droxybeiizyl, :itid tlic carbamoylmethyl (+tcr.. Iteduction i n the rate of hydrolysis by OM(. substitution on the benzene ring in the phenacyl esters is iii keeping vith the participation of the GO group in coricrntratiun ( i f O H - is 71.7 tinieb Kreater a t 111110 tiinn a t l i t 1 Ilence, tlie rate ratios n l i o i i l d give this valiie, for a reaction that in iirnt-order i n [Oil-]. If. however, as is often the case in ii)-drolysis reactiunh, 1 l i e 1,iiRt.r wrnliunerits wntribiite t o reaction velocity, modest deviation.f r u n i t l i r calrulatrd ratii) may be expected. See. f u r exaiiiyle ref 14. ( 2 4 ) C . N. Lierhe, to be putilialied. (2;i) T l i r
7.13.
react ion. 'I'hc slight a t l d i t i o d rct:trd:i- introduction of :L ,\le0 group into the
f the phenacyl moiety (esters corresponcliiig to compound XI'II 2's. XVI) niid a 10- to 15-fold retardation on passing from I11 t o o-metliylplieiiacyl p-nitrophenyl methylpho~plioii:Ite?" further support this suggestion. In vien of the misitivity of the hydroiysi,. of the CO-containing mixed esters Y to bulkirig ne:tr thc, CO group, the neitr identity in h~drolysi.;rates of the, phmacyl utid acetong 1 conipouiidq (ekters corre.poritlitip to T'II and XI) is somewh:it surpri4ng. A t pH 4.6, the predoniiiiaiit reaction in CO-s liydrolyiis iio longer involves the hydrosidc io1 stead of tlie calculated (bawd upon [OH-] reduction i l l rate of 330 times in paszirig from pH 7.15 t o pH 4.ti one observe? oiilv 16.4 :tiid 4.33 times, respectivelj., f o r the phenacyl und acetonyl comporind~. The CO-participatioii renctioii i~ wisitivc t o otlwi >tructur:d chmges about the ( ' 0 gi'oup. T h t i ~ .111 comparing tlie citer correapondirig to XVIII with t h i t of XTX, thew is it 12-fold reduction in ratc. Thc ])henolic OH in the cstcr corresponding t o XX is a l a1)lc ~ lo participate. iii thc hydrolysic reaction. Howvm.rr, it participate- much l e v eff (Jet 1~ than (loch CO. .21though it i- :ipproxirnatt.ly 30 ioiiizcd2si:it 11137. I !j,-0 t hst :ihigh eoricetitration of : o n 1 5 ;tv:iil;hle I'or i v i w tion, the d c g r c ~of acceltmtiori i. only of the s i i i m ~0 1 d ~ r carbnmoJ I group
p-nitrophcnol :it a r rnpid than that of the :tcc.toiiyl compound (ester corresponding to XIX). T l i r x particip:ttion of the :tinid(> group in displac.eirze~itO I I I' ;Lt r w r neutral p1-I i- particularly riotenwrthy b w n u + of poszible implicatioii oi the peptide bond in the c:it:tlytic function of enzymtis irivolvirig I)hosphonyl roup transfer. Previoudy, Pchrnir : i d Zioudrouz6 o t ) x ~ i v dthat i t ith amidc coli-
Journal of Medicinal Chemistry, 1970, Vol. 13, N o . S 443
~HOYPHONYL.YPED AChE
tainirig mixed esters of diphenyl and diberizyl phosphoric acid the amide group participates in cleavage of phosphate, but only through attack on the ester C atom with the formation of heterocycles. No P-0 cleavage was detected by them. The present result suggests that with a sufficiently good leaving group attached to P,such as p-nitrophenyl or possibly a n enzyme active site, the peptide group may also part,icipate by this simpler, less disruptive pathway for phosphate displacement. Fast.-These include the esters corresponding to VIII, X X I I I , XXIV, XXV, XXVI, and XXVII. I n each case the hydrolytic reaction to give p-nitrophenol was complete a t the beginning of the assay. With each, there was a progressively increasing amount of pnitrophenol found upon periodic assay of the DLIF synthesis mixture. It is probable that alkylation took place in D M F and that the mixed esters, being unstable, decomposed in the D X F . Alternatively, they might have hydrolyzed in the assay solution too rapidly to have been noted. A further possibility is that the pnitrophenol may have been formed in the D M F through a concerted displacement reaction. Support for the alkylation, rapid hydrolysis sequence comes from hydrolysis rate studies with the purified mixed ester corresponding to XXIII. The hydrolysis rate is exceptionally rapid. At p H 4.90, tl!, = 1.34 min; calculated t l / , a t pH 7.41 = 0.9 s e ~ . ~ 7Several attempts a t the synthesis of pure samples of each of the mixed esters corresponding to VIII, XXIV, and XXV were unsuccessful, suggesting their instability. The three compounds share as a common feature a strong electron sink, which greatly activates the CO g r o ~ p . ~Such ~ . ~activation ~ would be expected to enhance its overall contribution to the phosphonate hydrolysis reaction. (4) Hydrolysis of Alkylating Agents.-Hydrolysis rates of several of the alkylating agents in aqueoussolution are given in Table IV. At constant pH, the hydrolysis reactions were first-order in each case. The kobsd was calculated by the method of Guggenheim30 from the record of alkali titrant required to maintain constant pH. At the pH values recorded in Table IV, reaction end-points approach 1 mol of base consumed per mol of compound, indicating simple hydrolysis (eq 7). At higher pH values, the quantity of alkali 1tCOCH2Y
+ OH- +RCOCH20II + Y-
(7)
consumed increases. The increase is a t least in part due to further decomposition of RCOCH20H.31 Favorski rearrangement32may also be a contributing factor to the additional alkali consumption a t higher pH values. However, this matter was not investigated. (27) C . X. Lieske, J. IV. Hovaneo. G. hI. Steinberg, and P . Blumbergs, Chem. Cammun., 13 (1968). J. IF7.Hovanec, C. N. Lieske. G. bl. Steinberg, J . N. Pikulin. A . B. Ash, a n d P . Blumbergs, First Northeast Regional Meeting of the American Chemical Society, Boston, Mass., Oct. 1968, Abstracts, p 97. (28) D. L. Hooper, J . Chem. Soe. B , 169 (1967). (29) P.Pocker, J. E. RIeaney. and B. J. Kist, J . P h y a . Chetn.. 71, 4509 (1967).
(30) E. A. Guggenheim, Phil. Mag., 2 , 538 (1926). (31) Support for the assumption of simple hydrolysis a t “lower” p H , where 1 mol of acid is produced, was obtained with phenacyl bromide. Upon refluxing for 5-6 hr a t p H 8 in 1 J4 borate there was 7 6 7 5 % conversion into phenacyl alcohol (determined b y vpc). At higher p H , phenacyl alcohol decomposes. Thus, a t p H 13, the K band, Amax 248 rn# (in MeCN, Xmax 244.5 mcL; E, 1.13 X 104) disappears instantaneously. B t p H 11, a similar loss in K band occurs, b u t , it takes place over a period of several hours. (32) R . B. Loftfield, J . Arne?. Chem. Sac., 76, 35 (1954).
The much higher hydrolysis rate of XIV compared with that of VI1 is certainly indicative of an elimination reaction rather than OH replacement (eq 7 ) . It is particularly striking that the compounds containing “remote” electron-withdrawing groups (vide supra) such as VIII, XXV, and XXXVI hydrolyze so much more rapidly than does phenacyl bromide, YII. An increase of this magnitude is difficult to explain in terms of simple added inductive effect on the CH2 carb o The ~ mechanism ~ ~ of hydrolysis of a-halo ketones, which are among the most rapid of the S N ~hydrolysis reactions known, is still not settled.34 We would like to suggest that their hydrolysis may be similar in mechanism to that of the CO-acclerated phosphonate hydrolysis. This would involve OH- attack on CO to give either a preequilibrium hydrate or a concerted displacement reaction by the carbonyl 0. Such a mechanism would be compatible with the very large increase in hydrolysis rate in VI11 and XXV, since the CO group is greatly activated (marked increase in degree of hydration) by the adjacent CO group28and the pyridinium ring.29 (5) Enzyme Reactivation.-p-Sitrophenyl phenacyl methylphosphonate (111) provided a means for examining the premise that incorporation of a propinquity catalytic group, a, into the phosphonate moiety mould increase the rate of spontaneous reactivation (dephosphonylation) of the phosphonylated enzyme (reaction 4). Like other p-nitrophenyl phosphates and phosphonates, I11 ib a potent inhibitor of AChE. Inhibition is progressive (see Figure s),it i5 not diminished by dilution of the inhibited enzyme and reactivation occurs upon treatment with the oxime, TMB-4 (note ref 11). The inhibition reaction is depicted in eq 8.’ I n this case the propinquity catalytic group, z, is the carbonyl. It was reported earlier14that the hydrolytic 0 enzyme
+
0
@ C H - O ~ O ~ S O -
-
w 111
0
0 c n L > me-O--l’O(
I1 I CH
[I
I
-
c -(-J
+
FlO-+O
[\I
reaction of I11 to give p-nitrophenyl and phenacyl met$ ylphosphonic acid (Na salt) is approximately lo4 times faster than the corresponding reaction for ethyl p nitrophenyl methylphosphonate (ester corresponding to I X ) which lacks the CO group. A comparison of the rates of spontaneous reactivation, pH 7.4, 25”, of enzyme inhibited by the two phsophonates is shown in Figure 6. Both reactivations follow first-order kinetics. The ethyl methylphosphonylated enzyme reactivates with t l / , = 12,000 min, while the phenacyl methylphosphonylated enzyme (Bio-Gel 10 separated from impurities after inhibition) reactivates with t l / , = 180 57 min. Generally, spontaneous return of activity occurs more rapidly when the groups attached to P
*
(33) F. G . Bordwell a n d W.T. Brannen, Jr., ibid., 86, 4645 (1964). (34) See. for example: D. J. Pasta, K. Grtrves, and M. P. Serve, J . Org. Chem., 82, 774 (1Y67); K.Okamoto, H. Kushiro, I. Nitta, and H . Shingu, Bull. Chem. Soc., J a p . , 40, 1900 (1967).
INCUBATION TIME (MIN.),PRIOR TO ASSAY
Figure .i.-Reactiori of 111 with S(:hl I O eiizyme buffer mix; (B) compoiiiid I11 incubated for 1 hr a t pH 7.4 prior i o ndditiiiii of v r i ~ y m e ;( ( ‘ I iiiiLreaterl .iCkiK r o i i t i , i i l : ( I ) i return of activity iri the preseim of 10-3.11 TAIB-4. I 10
-
10
-
1
9 -
8 7 -
b -
__ 5 : 1-F
-
1’
Figure tj.-Spoiitaiieous reitctivatioii of iiihibited eel hChli;, pll Inhibitors: ( A ) cornpowid 111: (B) ethyl p-riitropheiiyl methylphosphoiiate. (F) represeiitn the fraction of i’estored enzymatic activity. In each case, the inhibited enzyme wan Sephadexed t o remove residual inhibitor pr,ior tii reactivatiiiii study. i . 4 > 2.-)’.
are m i i t l l . J 1 Thus, the properl) positioned CO group would appear to contribute considerably to speeding the reactivation process. 36 That propinquity catalyii. iy riot the only contributor t o rapid spontaiieous reactivation is evidenced by a recent report that his(@chloroethj 1)phosphorylated bovine erythrocyte cholinesteraw reactivates spontaneou4y with n half-tinit.
Journal of .Iltdiclnal Chemistry, 1970, Vol. 13, AYo.-3 445
ture. ( 2 ) Alkylation does take place, but the enzyme active site is no longer a "good" leaving group. This might result from a conformational change concurrant with the occurrence of aging or perhaps as a result of a separate nonspecific reaction of the alkylating agent with another site in the enzyme. If this were so, the group z activated hydrolysiy if it occurred a t all, would lead to self cleavage of the inserted R group rather than separation of the phosphonate moiety from the enzyme. Clearly, only further studies can establish the validity of any of these speculations. Experimental Section A11 reagelit.; w-ere analytical grade or equivaleiit. Most of the test compounds were obtained from commercial soiirces or were prepared by literature methods aiid gave the reported physical properties. Piirification by distillation or recrystallization was performed as required. Sodium p-Nitrophenyl Methylphosphonate (VI). A. Bis(pa rapidly stirred saspeiinitrophenyl) Methy1phosphonate.-To sion of 143 g (0.8 mol) of rigorously dried finely powdered sodium p-nitrophenolate in 1.4 1. of dry EtvOcoiitaiiiiiig 45 g (0.45 mol) of EtaN, 49 g (0.38 mol) of freshly distilled methyl phosphonodichloridate was added slowly. The mixture was stirred a t room temperature for 30 min, refliixed for 2 hr and allowed t o stand overnight. The solid was filtered and washed (H,O) until the wash was colorless. It was then siispended, with stirring, three times iii 1 1. of ice-water to remove residiial sodium p-iiitropheiiolate. ;2fter drying in V U C Z I O the material was crystallized twice ( l l e p c O ) to give 61 g (67',,) of product, mp 122-123" (lit,.38135'). B. Sodium p-Nitrophenyl Methy1phosphonate.-To 36 g (0.106 mol) of bi~(p-iiitropheiiy1)methylphosphoiiate iri 1 1. of MeCN, 1.S.i 1. of 1 KOH was slowly added with stirriiig. The reactioii mixture was acidified with iicOH to pH 5.8, the MeCN removed in vacuo, and the residue ext,racted (Et,O, 4 X) to remove the p-nitrophenol formed in the reaction. The remaining colorless liquid was evaporated to a small volume and passed over a I)owex-.jO (acid) ion-exchange resin. The free acid was recrystallized from CHCla-peiitane to mp 107-108" and converted into the N a salt by slow addition of an equivaleiit quantity of K a O l l e iii AIeOH. NeOH was removed and the product recrystallised fiom EtOH, mp 265-270°, yield, 28 g (90%). Anal. (C.;H;O,XNaP) C. H. P. . . Alkylation Reac'tion.-Pheiiacyl bromide is used to exemplify the staiidard procedure. D h i F (Baker Anal.) was purified by treatment with CaH, aud distilled a t 50 mm directly into a storage vessel containing type 4 h molecular sieve (Linde Div., Unioii Carbide Corp.). A. Standard Alkylation Procedure.-Phetiacyl bromide (Eastman Khit,e Label, a-bromoacetophenorie) and VI were mixed in U l I F in equal coiiceritrations of 5 X 10-3 M and the solution placed iii a coiistaiit temperature bath at, 2 5 " . Periodically, 25-pl aliquots were takeii for assay of the resultant mixed ester. With the less reactive alkylating compounds reaction was conducted at, 60". B. Assay of Mixed Esters. Assay A.-The sample was added to pH 10, 0.1 JI carbonate buffer, refluxed 10 min, cooled t,o room temperature and the p-nitrophenolate content determined from the absorbance a t 402 mp (Beckman DB-2 spectrophotometer). The absorbance gave a linear Beer-Lambert relationship over the useful concentration range. Under these conditions, VI gave a negligible blank (2Cc), while the most stable ester, ethyl p iiitrophenyl meth>-lphosphonate \vas completely hydrolyzed in less than 3 mill. Heiice, this assay gave a quaiititative measure of the converuioii of V I into mixed ester. Assay B.-The sample was added to temperature-equilibrated aqiieoiia 0.1 X Tris, pH 7.15, Go,rapidly mixed arid transferred to a cuvette mounted in the thermostated compartment of the spectrophotometer, and a recording made of the progressive hydrolysis of the mixed ester by measiiremeiit, of the absorbance at 40% mp (p-iiitrophenr)xide). The time elapsed from initial mixing to start o f the spectrophotometer recording could be rediiced to ii miiiirniirn of 20 see. From this assay, one coiild A\-
(38) H. Tolkrnitli. U.S. Patent 2 , 6 6 8 , 8 4 5 , Fell. 9 , 1954; Chem. Abatr., 49, 5519 [19.55),
determine (a) the initial p-nitrophenol content, (a measure of decomposition of mixed ester during the alkylation period), ( b ) the total p-nitropheriol available, Le., the total amount of mixed ester that had been formed via alkylation, and (c) the hydrolysis rate of the prepared mixed ester. I n every case, the hydrolysis reactions followed good first-order kinetics and thus strongly indicated the preseiice of a single hydrolyzable species. C. TICofMixed Ester.-To further substantiate the formation of I11 from the reaction of pheriacyl bromide with 17, in DlIF, an aliquot of the reaction mixture was spotted on a silica gel tlc plate next to atithelltic 111. The spots were moved with 93(;.;CHCl3-5y0 Me&O and developed with IZ vapor. Authentic 111 gave a single spot a t Rf 0.6. The r e a d o n mixture gave a large spot a t Rt 0.6, some unreacted phenacyl bromide (Rf, 1.0) and VI at Rr 0. There also were observed a few minor spots of unknown composition. D. Alkvlation in Aa Et0H.-To iEtObP1O)OH 13.051 n , 0.020 ,of), dissolved 'in 20 ml of 'HnO;- the& was addLd 30 ml of 0.2 X phosphate buffer, pH 7.0 and 50 ml of EtOH containing 4.073 g (0.020 mol) of phenacyl bromide. The clear mixtiire was refliixed for 3.25 hr, cooled (pH non- 1.9), and vacuum stripped to remove most of t,he EtOH. The remaining solution was extracted with three 30-ml aliquots of CHC13, the aliquots combined and vacuiini st,ripped. There remained 2.86 g of oil. Analysis by vapor phase chromatography ( 2 5 SE-30 on 100120 mesh Gas Chrom S, 6 ft X 14! in., column temperature 168", injector temperature 230", argon flow rate 75 ml, miii., detectorargon diode) gave 0.29 g (5.lyi) of a peak a t t = 13.75 miii which corresponds to that of pure diethyl phenacyl phosphate. With (BLIO)?P(O)OH,under the same conditions the yield of dibutyl phenacyl phosphate was 6%. KOeffort was made to maximize yields. Enzyme Studies. A. Inhibition and Spontaneous Reactivation.-To a solution of eel A4ChE(Sigma, type 111),approx 5 X 10-9 M in 0.3 ?il KC1, 0.157, gelatin, 1.5 X W Tris, pH 7.4, G o there , was added 11139 dissolved in dioxane t o give Jf I11 and dioxane. I11 was added last t o minimize its hydrolysis prior to reaction with the enzyme. Aliquots (0.2 ml) were periodically taken and assayed for enzymatic activity by adding to 2.8 ml of a mixture contained in the thermostated reaction vessel ( 2 5 ' ) of a Radiometer TTTl Titrator, fitted with Autoburette ABU-1 and Titrigraph SBR-2. The resulting 3.0 ml of .)I Tris (pH 7.4), assay solution had the composition: 5 X 0.3 X KC1, O.lcG gelatin, 0.037 X acetylcholine chloride. Enzyme activity was determined from initial slope of the record of rate of addition of 0.015 .V KaOH to maintain constant, pH. Xz was passed over the assay solution in the covered titration vessel to prevent CO? absorption. For measurement of reaction kinet,ics, the enzyme was incubated with inhibitor for 15 min and the inhibited enzyme separated from residual inhibitor by passage through Sephadex G-50 or Bio-Gel 10. Reactivation of the enzyme contaiiied in the protein fractions followed first-order kinetics. Controls of uninhibited enzyme were run concurrently and their values (which did not change by more than 20% from their initial activity values) were used in computing reactivation results. For the reaction A -+ B, where A represents inhibited enzyme and B, reactivated enzyme, the firat-order kinetic relationship is hi (B,/B, - B t ) = k t , where Bt and B , represent enzyme activities a t times t and a ,respectively. Actually, B , was determined from the activity of a control enzyme solution identical with the incubation mixture except that it contained no 111. To correct for changes in B,, let the fraction of activity restored, F = Bt:'B,. Then, In [ l / ( l - F)] = kt. Reactivation study of ethyl methylphosphonyl AChE was performed as above. Instead of 111, ethyl p-nitrophenyl methylphosphonate40 (ester corresponding to I X ) was used to inhibit the enzyme. B. Reactivation with TMB4-The incubation mixture contaiiiiiig 111, composition as cited above, was maintained for 13 AI ThIB-4. miii. Then, 0.53 ml was mixed with 0.06 ml of This solution was kept a t 2 5 O and aliquots were taken for assay. Correctioii was made for illhibition (reversible) of the enz:-:-,ie by ThiB-4. C. Alkylation of Aged Inhibited Enzyme. Test for Return of Activity.--AChE (Sigma, type 111) dihited 1 to 1000 (to ap-
lcc
(39) C . S . Lieske, J. IT. Hovanec, G . AI. Steinberg, J. S . Pikulin, IT. J. Lennox. A . B. Ash, a n d P. Blumbergs, J. Agr. Food Chem., 17, 255 (1969). (40) .'iR.I . DeRoos, Rec. Trac. Chim. Paus-Bas, 78, 145 (1959).
446 Journal oj Medicinal Chemistiy, 1910, ['ol. 13, N o . 3
proximately 2 X 10-8 -21)in 1.08 nil of staiidard Tris-gel buffer M Tris, 0.3';.', gelatin, 1 ilf KCl, pH 7.4) was mixed with X pinacolyl met~1iylpho;iph~~iiofliioridate (soman) 0.12 ml of arid the mixture incubated a t room temperature overnight. This treatment, yields aged inhibited enzyme, 1 1 . 3 The solution il rill) was placed on a Sephadex G-50 (11 X 1 cm) or Bio-Gel I-'-10 (14 X 1 cm; 60-180 mesh) column and eluted with st,andard Tris-gel buffer. Effluent (8 ml) containing the protein fractioil was collected. The effluent ( 2 . 5 ml) %-as mixed with 2.0 ml of Hd.3 and 0.5 ml of test compound (dissolved in H 2 0 or other I solutioii was maintained at. appropriate solvent j. The rea 25", at constant pH, usually with the Radiometer Aut.c,titrator. hliquots were taken, ed through t,he gel column ro separate the enzyme from residual test compound (anti its I drolysis products). Assays were then made for enzymatic tivity both at once and after iiiciit)ation for 2-2 hr a t the I employed during the first illcubation to permit additional time flJr possible self-reaction. "Control" Irieasurernent,s of inhibition of intact enzyme by test compounds were performed in the identical manner, except, that uninhibit,etl enzyme WVRS n-ed as the enzyme source. Test for Release of V - - - A 0.1-ml sample of lo-: -11 AChE was incubated with 1 fil of 32Plabeled 0.01 somaii (Defense ltesearch Board of Canada) in 10-3 J I Tris ipII 7.4), 0.3'; gelatin, arid 1 M KC1. The inhibited eniynie was allowed t o age overnight a i d the mixture was then passed through a Bio-Gel 1'-10 column to remove Iionproteiii bound 32P. A 0.5-ml aliqiioi of the inhibited enzyme was mixed with 0.3 nil of 2 x JI pherwcyl bromide (VII) in 50CC :ty itoetone and the mixture incubated for 4 days. The treated AChE was divided into two parts, one of which was treated for 1 hi. with 2 x 1 0 - 2 111 TMB-4 ian oxime reactivator of iuiaged phosphoiiylatecl AChE)." .2 control sample of inhibited untreated enzyme and each of the tn-o treated fractions were solvent extracted. Each was ma.de acidic with two drops of 5 3 H 2 Y O 4 and extracted three times with 1.0-nil portions of water saturated I : 1 BnOH-C61&. The entire organic phase was transferred to a 2.3-cni plaiichet and made alkaline to phenolphthalein with 10 'V KaOH then dried with moderate heat from a heat lamp. The planchet was coiuitetl with Tracerlab l o a background counter (Omni/'guard). Hydrolysis of Alkylating Compound.-Each of the conipouritls listed in Table IV was made up t,o 10-3 J1 in it mixture of 0.1 .If aq KC1 and EtOH. The quantity of EtOH was limited to t,he miriimurn required to insure complete solution of the test compourid. The rate of hydrolysis was determined from the rate of delivery of 0.0146 12' KaOH required to maintain constant pII with the Radiometer Autotitrator TTTI. Titration was performed 011 3 ml of test solution in a covered vessel using the 0.25nil capacity buret,te. Ni was passed over the solution to minimize ( 2 0 1 absorption. The reactioii in each case was first-order. The rate constant, kobsri, w:ts cslciil tl iising the (hggenheini pr~cedure.~O kinetic^.^^^^^ A. For Assay B. First-Order Kinetics.---'l'lit~ hydrolysis reactions of the mixed phosphoriate esters are typified by III.'+ The reaction is first-order each in 111, [OH-], arid buffer base. At coiistant pII, therefore, the reaction is firstorder (eq 9) since [OH-] arid [buffer base] remain constant). .
(41) A. A . Frost, and K. 0. Fearson, "Jiinetira and hleclianism," ,John Wiley & Sons. Inc., New York, S . Y . , 19 (42) W. J. hIoore. "Physical Chexni*tr. Prentice-Ilall, In
