ENZYME INHIBITORS.XV
)[arch 1967
ether layers were washed with water, dried (Na?S04),and evaporated to give 18.1 g of colorless oil which was chromatographed Eliitioii w i i h ethyl acaetatcI I I I 270 g of ~ i l i c agel (0.2-0..i i n i n ) . benzene (1:4) aiid finally with piire ethyl acetate gave 16.1 g of colorless oil. Crystallization from ether-hexane gave 11.56 g of half-ester (XIIIc), nip 9S-101.5°. Air additioirnl 2.51 g o f prodiict, mp $)M)i.5', wah recovered from the mother liquors (total yield 735; ). Crystallixation from ether-hexane gave the aiialytiral sample: mp 100..i-102.5°: [ c Y ] * ~ D + i 0 . 4 " ( c 1.0, CrTI:,OII); 210 mp ( e 8470), 2 i S (2060), 2S7 (19.50): aiid 3 . X 1 p. This compoiind was desriihed previoiidy a ] % + G 1 i 4' ( c l1.1!1, alwhol)." .[nul. Calcd f o r C?,,H,,O:: C, 69.34: H, i..ii. Foiirid: C!, G!).64: H, i.69. 16-Azaestrone 3-Methyl Ether (XVa).-To a solution of 12.5 g (0.0362 mole) of XIIIc in 63 ml of benzene was added 12.5 ml of oxalyl chloride, and the resulting mixture was heated a t 65' for 50 min. An additional 6.0 ml of oxalyl chloride was then added and heating was continued for 30 min. T h e react'ion mixture was evaporated to dryness and the residiie was crystallized from ether-hexane to give 11.68 g of crude acid chloride, mp 88.5-92', CHCll A,,,, .i..54 and >.SO p , This cornpoiitid has beeir described pi,evioii.-ly a i an oil.11 A soliitioii of 9.0 g (0.133 mole) of N a y 3 in 35 ml of water was added over a 10-min period to a cold ( 0 - 5 " ) solution of 11.38 g of critde acid c.hloiide in 228 ml of acetone. The mistmiirewas allowed to stir in the ice bath for an additional 15 miri and was then diliited with 1 1. of water. The mixture was extracted with ether mid the ether extracts were washed with water, dried (Na?SO4), mid evaporated to give the crude azide XIIId as an oil, 4.67 (sharp) aiid 5.81 p , A soliitioii of this azide in 400 r n l of 1)eiri.eiiewas heated iuider refliix for 1 hr at which time the evoliitioii of gas had ceased. The benzene was then removed iiiider rediiced pressiire to give the critde isoryaiiate X I \ - as ail oil, 3':A', 4.40 (broad) and 5.S1 p . A soliition of 11.3 g (0.2 mole) of KOH in 11.3 ml of water was added t,o a solution of t,he crnde isocyanate in 115 ml of methanol. The reacation mixt,ure was heated under reflux for l.*5hr and was theri diluted with ,500 ml of water. The resilltirig precipitat,e was filtered, washed with water, and dried to give 7.53 g ( i 3 5 yield from the half-ester XIIIc) of the lactam XI.a, mp 210.5212.5" (vac). Crystallizat8ion from CH?CL-ether gave the analytical sample: nip 211-212" (vac); [ c Y ] * ~ D+78.4" ( e 1.0,
Enzyme Inhibitors. XV.
181
C2HjOH); 220 mp ( E S600), 279 (1080), 2S7 (1780): 2.92 and 5.92 p ; 1it.j nip 202-205", [ o ] D +io.>" (C!1TCl3). . I n d C a l d f o r C,,I12:JV02: C', 7 5 . 7 5 : I I , S.12: S , 4 , ! ) l . Found: C, 75.56: H, 8.15: 1, 5.08. 16-Azaestrone (XVb).--A mixture of 4.0 g (0.014 mole) of S V a aud SO g (0.79 mole) of pyridiire hydrochloride was heated with st,iiriiig a t 210" for 40 mill iii a i l atmosphere of nitrogen. The react,ion mixture was then cooled aiid diluted with 600 ml of 2 S HCl. The result,ing preeipitate was filtered, washed with wat,er, and dried t,o give 3.40 g of critde prodiict,, nip :3D2-:264° (vac). Crystallization from chl~~i~ofor~n-ethatiol gave 1.8s g of S V b , nip :260-~362.5°(vac.). -4 fiirther O X ) g of X1-h i m p 362-364") war recovered from the mother liyiitrrs ( t o t d yield, 2 . T g, 7 3 5 ; ) . Crystallization from CHC13-ethanol gave I he analyt'ical sample: mp 362.-5-364.,5" (vac-): [ c Y ] * ~ D f82.5" ( e 0.1, CyHjOH): 220 mp ( e i 5 0 0 ) , 2x0 (20!)0),288 ( l S l 0 ) : A,, KBr 2.93, 5.95, and 6.01 p . Anal. Calcd for C r i H n N O ~ :C, 75.24; H, 7.W; Ii, 5.16. Found: C, i5.31: H, 7 . 3 ; X, 5 . l i . 16-Azaestra-1,3,5(10)-trien-3-013-Methyl Ether Hydrochloride (XVI).-A solution of 1.38 g (4.84 mmoles) of XVa in 46 in1 of dry dioxane was added rapidly to a boiling solution of 1.38 g (36.4 mmoles) of LiA41H4in 46 ml of dry dioxane and the resulting mixture was heated under reflux for 20 hr in a nitrogen atmobphere. Water ( i . 4 ml) was added slowly to t'he ice-cold reaction mixture which was then heated under refliix for 30 min. The hot mixture was filtered through a bed of Celite, the latter beiiig washed with hot dioxane. The filtrate was evaporat,ed to dryiies. and the resulting colorless oil was dissolved in benzeiie and pawed throiigh a .qhort column of Florisil. Evaporation of the elllatea gave an oil which was dissolved in CH,Cly, washed thi,ee times wit,h 2 .Y HC1, dried (Na&O4), and evaporated. The reqidiie was crystallized from CH2ClP-ether to give 1.16 g ( i 8 ( ' ; ) of XT.1, nip 298.5-301' ( v a r ) . Crystallizatiou from ethaiiol gave the analytical -ample: mp 298-301' (vac.); [ a ] % +?Go ( c (1.5, ethanol): 219 mp ( E 8050), 2779 (20(10), 2 S i (1810). -4nal. Calcd for CI8H&lXO: C, i 0 . 2 2 ; H, S.51: C1, 11.52: N, 4.3.5. Foiind: C, 70. h;,H:l3
AzzoH
Acknowledgment.-We are indebted to Dr. AI Xteyermark arid his staff for the mic:roanalyses arid t o Dr. V. Toome arid I I r . S. Traiman for the ultrnviolrt and infrared spectra, respert,ively.
A New Irreversible Inhibitor of Adenosine Deaminase'
HOWARD SCHAEFFER A N D EL-GEVE ODI~ qJ.
D c p n i h r r n l of .Ifrdir inn1 Chrinislrt/, Srhool of Phnrniacy, Statr 77niurrsil?~ of ,lim York n f Ruffnlo, Biiffirlo, .YPWFork
1421 /i
Receizwl Srptrniber 28, 1966 Previoiis stiidies have shown that, 9-(p-bromoacetnmidobenzyl)adenine (XT?) and %(m-hromoacetamidobenzy1)adenine ( X I T I ) are both good reversible inhibitors of adenosine deaminase but that XVI causes an irreversible inactivation of this enzyme a t a much higher rate than does XVII. I n a continuat,ionof studies on the effect of isomers on the inhibition of adenosine deaminase, a variety of 9-(ortho-substituted benxy1)-6-su.bstituted pnrines have been synthesized. These compounds were weaker reversible inhibitors of adenosine deaminase t,han was 9-benzyladenine. However, 9-(o-bromoacetamidoberizyl)adenine,even thoiigh it was a weaker reversible inhibitor of this enzyme than XVI or XTII, was found to be a good irreversible inhibitor of adenosine deaminase. Evidence is presented that this irreversible inactivation of adenosine deaminase proceeds through an initial reversible enzyme-inhibitor complex and not by a random bimolecular reaction between the enzyme and the inhibitor,
Recent studies have shown that 9-(p-bromoacetamidobenzy1)adenine and 9- (in-bromoacet'amidobenzy1)adenine are both good reversible inhibitors of adenosine deaminase obtained from calf intestinal mucosa. 2--4 However, when these compounds were (1) This investigation was supported by G r a n t T-337A from t h e American Cancer Society, by a Public Health Service research career program award 5-KX-CA 18718 from t h e National Cancer I n s t i t u t e a n d by Training ( + r a n t 5 T I G.\I 55 from the Division of General Medical Sciences. (2) H. J. Schaeffer and E. Odin, J . Pharm. Sei., 54, 1223 (1965). ( 3 ) H. J. Schaeffer and E. Odin, J . M e d . Chem., 9, 576 (1966). ( 4 ) I f . .I. Sriineffer nnrl I?. N. .lol>nnrm,.I. Phnrm. Rci., 65, 920 (1066).
evaluated as irreversible inhibitors of this enzyme, it was found that the para isomer was a good irreversible i n h i b i t ~ r ,whereas ~ the ineta isomer was a poor irreversible inhibitor of adenosine deaminase.4 Since the chemical reactivity of the ineta isomer is greater than that of the para isomer when 4-(p-nitrobenzyl)pyridirie is used as the nucleophilic reagent,4 it mould appear that the differences in the rates at which these two compounds irreversibly inactivate adenosine deaminase is due to the difference in the environment on the enzyme in which the alkylatling group of the inhibitor
March 19.37
En-zum INHIBITOIZS. XV
188
scribed in the lit,erature.10-12 Equal volumes (10 ml) of the following preheated (37') solutions were mixed: (a) 5% 4-(pnitmrobeiizyl)pyridinein 2-methoxyethanol, (b) 0.05 iZf phthalat,e buffer (pH 4.2) in water, and then ( c ) 0.81 m X solution of the alkylating agent in 2-methoxyethanol. The reaction mixture was incubated at 37" aiid at appropriate t,ime intervals, a 3-ml aliquot was removed and cooled briefly in ail ice bath. Then 1 ml of triethylamine was added to generate the colored quinoid-like free bare, and the absorbance was immediat,ely determined at 373 mp against, a blank which had been treated in an identical maiiner and contained all of the reagents except the alkylating agent,. -4comparison of the initial rates of react,ion are given in Figure 2. Reagents and Assay Procedure.-Adenosine deamiiime (Type I, calf intestinal mucosa) was purchased from the Sigma Chemical Go. The =say procedure for the reversible inhibitors has beeii dehcribed previo~isly~ and is a modification of the procediire developed by Kalckal.13 and by K a p l a i ~ . The ~ ~ measurement of the initial rat,es of the eiizl-mic reactions were performed at 2.5" in 0.06 J I phosphate buffer at' pH 7.6. Those iiihibit,ors which were only slightly soluble ill phosphate buffer were evaluated iii solutioris of phosphat,e buffer containing 10% dimethyl sulfoxide as previously d e ~ c r i b e d . ~ The method used to st,udy the irreversible iiiactivat'ion of adenosine deaminase at 37" has been described earlier3z4and is a modificat,ion of a published proced~ire.1~
Results and Discussion When the 9-(ortho-substituted benzyl)-6-substituted purines were evaluated as reversible inhibitors of adenosine deaminase, it n.as found that they were weaker reversible iiihibitors than 9-benzyl adenine (see Table 11). This weaker reversible inhibition of adenosine deaminase by the g-(ortho-substituted benzy1)adenines suggests that adenosine deaminase has limited bulk TABLE I1 REVEI~SIBLE INHIBITIOX OF ADENOSIXE DEAMINASE BY SOME 6-SUBSTITUTED '3-(OVthO-SLBSTiTUTED BEXZYL)PURINES
I m M concn for
Kl
Compda
v
50% inliibb
([Il/[Sl)~l.6
YO,
0.13 f O.0lc 1 . 9 i 0 . IC 0.31 f 0 . 0 1 VI NO2 4 . 7 i0 . 1 SH? 0.27 i 0.01 4 . 1 f 0 . 1 X SId NHCOCH~BI 0.41 i 0.01 6 . 2 i0 . 1 0.53 f 0.03 8 . 0 0 . 4 XI1 SHCOCH, 5 . 3 f0.3 SI11 KIICOOCsHs 0 . 3 5 i 0.0'2 13 0.10 lk 0 . 0 1 XV 1 . 5 i0 . 2 e 5 None of t,hese compounds served as a substrate of adeiiosiiie deamiiiase. The coiiceiitrat,ion of adenosine in all experiments was 0.066 mJI. In no experiment of reversible inhibition did the concentration of inhibitor exceed 0.12 mJI. In those cases where a higher concentration is shown for 50yc inhibition, the value was obtained by extrapolatioii of a plot of T ' a l T I vs. [I] where ITc,= iiiitial velocity of the iininhibited reaction, I' = iiiitial velocity of the inhibited reaction at varioiia inhibitor concentrations, aiid [I] = the various concentrations of inhibitor. c Average deviation. d Ki = 44 X 10-5 31, competit,ive inhibihr as shown by the double reciprocal plot method. e Data taken from ref 3.
*
(10) J. Epstein, R. vi. Rosenthal, and R. J. Ess, Anal. Chem., 47, 1136 (1955). (11) T. J. Rarcios, N. Datta-Gupta, P. Hebborn. and D. .J. Triggle. J . M e d . Chem.. 8 , 167 (196.5). (12) B. R . Baker and J. H. Jordaan, J . Heterocyclic C h e m . , 4, 21 (1965). (13) H. RI. Kalckar, J . B i d . Chem., 167, 461 (194i). (1-1) S . 0. Kaplan, M e t h o d s E n r y m o i . , 2 , 473 (1955). (15) U. R. Baker, Biochwn. I'hormucol., 11, 1155 (lYti2).
.-C
E
\
w
. c
v w Z J
U U
gz
,"
0
m S
a-
0,
NHCOCH,Br
NHCOCH,Br XVI
XVII
ccntratioii of the reversible E * * .I complex. Baker17 hits derived arid employed ey 2 to calculate the concaeiitration uf the E...I complex in terms of total enzyme, Et. 111 eq 2, K , is the dissociation constant of
:':I I .40
the [ E . . . I ] complex and [I] is the concentration of the inhibitor. In the case of XI, when [I] = 0.04 mJ1, [E...I] = 0.083[Et] and when [I] = 0.08 mJ1, [E. . . I ] = 0.15[Et]. Therefore, increasing the concentration of XI results in an increase of the [ E . . . I ] complex by 0.15[Et]/0.0S3[Et] or 1.8 times. Since the rate of irreversible inactivation is dependent on the cwicentration of the [E * * * I ] complex, it follows that hy increasing the concentration of XI from 0.04 to 0.0s mill, the rate of inactivation should increase by :1 factor of 1.S. Xn examination of Figure 1 reveals that the ratio of the rates of inactivation is, in fact, 1.8 i n excaellerit agreement with the calculated ratio. IVe believe that these data offer strong support to the suggestion that the irreversible inactivation of adenosine deaminase proceeds through an initial reversible E . . .I complex through which covalent bond formation occurs as outlined in eq 1. Additional evidence supporting the mechanism of irreversible inactivation of adenosine deaminase by XI was obtained in the following manner. When adenosine deaminase was incubated with :t mixture of XI and 9-(2-hydroxypropyl)adenirie, a reversible inhibitor of this enzyme, the rate of irreversible inactivation was lower (Figure 1). This protection of the enzyme from irreversible inactivation may be r:itionalized by assuming that the effect of the reversible inhibitor, g-(%-hydroxypropyl)adenine,is to lower the c*oncwitratiori of the reversible E . . .I complex between the enzyme and XI which in turn would result i i i :t reduction of thc rate of irreversible inhibition. It hah been suggested that buch protection from irrevcr4ble inhibition indicates that the active site of the enzyme is involved. 17n1* E'inally, the interesting observation has been made that for reversible inhibition of adenosine deaminase, the activity decreases 111 the following order: XVI(para) > XVII(meta) > XI(ortho). However, for irreversible inhibition of adenosine deaminase it has been found that the ortho and para compounds (XI and XVI) are much more effective than is the meta derivative (XVII). Comparative chemical reactivities using 4(p-nitrobenzyl)pyridine"-'2 as the nucleophilic reagent reveal that the order of reactivity is XI = 1.9, XVII = 1.4, itrid XVI = 1.0 (Figure 2 ) . Thus, even though the chemical reactivities of the three compounds ivith 4-(p-iiitrobeiizyl)pyndille :ne not dramatically different, the rates o f irreversible inactivation of adeno. hine deaminase are quite different. (1i) B. R. Baker, IT. TT. Lee, a n d E. Tong, J . Theoret. B i o l . , 3, 459 (1962). (18) J . A. Thoma a n d D. E. Koshland, Jr., J . M o l . Bzol., I,169 (1960).
10
20
30
40
50
60
70
00
TIME (rnin)
Figure 2.--Comparis;aii of the initial rates of reartioil of some alkylating agents with 4-(p-nitlobeiiz~l)p~ridiiieH t pI-1 4.2: 0, 0.2'7 m M iodoacetamide; A, 0.27 m31 S1-I; 0 , O.2'7 nlJ/ X I T I ; E, 0.27 mil[ X I
We have previously calculated that the amount' of the total enzyme Et in t'he initial reversible E . . . I cvmplex when X I is employed at 0.08 m31 concentration is 0.15[E,]. When XVI is employed at 0.03 mJ1 concentration, t'he amount of Et in the initial reversible complex is 0.70[EtI3and when XVII is employed a t 0.10 m31 concentration, the amount of E, in the init'ial reversible I complex is 0.73 [E,].4 Under these conditions the half-life period for the irreversible inactivation of adenosine deaminase is 58 miri for XI, 94 min for XVI,3 and >GOO min for XVIL4 Therefore, even though the caoncentration of the initial reversible E . . . I complex is lower with XI than wit'h XVI or XVII, the half-life period of irreversible inactivation is shorter. These data establish that the alkylation reaction between XI and the enzyme i n the E . . . I complex must be faster t'hari the same step for XVI and much faster than the same step for XVII. Because of these differences in the rates of alkylation of adenosine deaminase, it is possible that X I and XVI are alkylating different amino acids on the enzyme.'$ The meta derivat'ive (XVII) in the reversible E . . I complex is not held as close to a nucleophilic group on the enzyme :ind therefore has a much lower rate of irreversible inactivation. A liinetic analysis of the irreversible inhibitioii of adenosine deaminase will be the subject of B future paper. (19) l t is also possible t h a t sfa n d SF'I are alkylating tile sa1118 alilino
-
acid on the eniytne b u t that the alkylating group of S I is poaitioneil in the E ' . '1 complex closer t o the amino acid t h a n i t is with XVI. Therefore, the alkylation step is more rapid v i t h S I t h a n \\-it11 XVI. Experiments are in progress to differentiate these two ideas.
