16-AZASTEROIDS
Xlarch 1967
method of Shore, et c ~ l . employing ,~ a later modification6 where one-tenth the uqual aniount of o-phthalaldehydc was ubed. Duplicate 1-nil aliquots of plasma were analyzed; fluorescence of the final solutions was measured on a Turner fluorometer' equipped with 7-GO primary and '3-A secondary filters. The lower limit of sensitivity was 0.003 pg of histamine/ml of initial sample, arid the precision of reading samples was +O.OOO.j p g . Itccovcrieq of added histamine from plasnin nverngcd 9'3o/c (range 90-96%). =\\rerage deviations of single determinations from their means did not excccd 47$. Serotonin did riot contribute in this ns~ny.
Experimental Section Physical and analytical data are listed in Tables I1 and 111.
A. N,N'-(l-Benzyl-4-phenyl-4-piperidylmethyl)terephthalamide Dihydrochloride (XIV).-To a chilled mixtiire of 2.0 g (7.1 mmoles) of 4-phenyl-4-aminomethyl-1-benzylpiperidine (Aldrich Chemical Co.) in 15 nil of anhydrous methylene chloride was added (dropwise) a mixture of 0.73 g (3.6 mmoles) of terephthaloyl dichloride iii 10 ml of anhydrous CHZC1,. The mixtiire was htirred at room temperature 1 hr, then allowed to stand at room temperature overnight. T h e white solid was collected by filtration and recrystallized from absolute methanol, giving 1.82 g (67Co) of white crystals, m p 283-290". A sample was roriverted to the free amine (partitioned between CHCI, and 1 0 ' ; S a O H ) and recrystallized from 2-methoxyethanol. B. N,N'-(4-Phenyl-4-piperidylmethyl)terephthaIamide(VIII). -To 500 mg of 100yo Pd catalyst were added 20 ml of glacial acetic acid and 1.3 g (1.7 mmoles) of XIV. The mixture was hydrogenated at TO" and atmospheric pressure, taking up the
177
theoretical amount of hydrogen in 1 hr. The mixture was diliited with 25 nil of water, the catalyst was removed t)y fi1tr:itioii, aiid the filtrate was evaporated in vacrio to drJmesa. The gummy material was taken up in water aud alkalized (pH 10) with 10% NaOH to precipitate a white solid. The solid was collected by filtration and washed thororighly with water to leave 0.74 g. Recrystallization from 2-methoxyethariol afforded 0.56 g (657;) of white crystals. C. l-Carbomethoxy-4-cyano-4-phenylpiperidine (XIX).-To a mixture of 5.0 g (22.5 mmoles) of 4-pheiiyl-4-c?.anopiperidiiie hydrochloride, 100 ml of water, 2.2 g (54 ninioles) of SaOIT, and 50 nil of CHCl:, wa:, slc~wlyadded 2.0 nil (27 mmoles) of methyl chloroformate. The mixture was vigorously stirred at room temperatiire for 2 hr, chilled, aiid acidified to pTT 2 with 6 - \ ITCI. The mixtiire was stirred (chilled) for 1 hr, and the chloroform layer was removed and washed with water. The CHCI, extract was dried (3lgSO4) aiid evaporated in vucuo to dryness to give an orange syriip which crystallized upon standing to yield 5.0 g (91%) of white cryst,ala. .4n analytical sample was ohtained from cyclohexane.
D. 1-Methyl-4-aminomethyl-4-phenylpiperidineDipicrate (XXII).-To a cold suspeiision of 7.7 g (0.2 mole) of LiAlHl i n 1'75 ml of anhydrous tetrahydrofuran was slowly added 5.0 g (22.3 mmoles) of XIX. The mixture was refluxed 13 hr. Excess LiAlH4 was decomposed by the careful addition of absolute ethanol. The reaction mixtiire T V ~ Sthen treated \vit,h water, stirred briefly, and evaporated in Z J ~ C Z L Oto near dryness. The pasty material was extracted with 1-butanol, which was dried (SIgS04) and evaporated in uuczm to yield 3.64 g (SO',) of orange syrup. ,4 sample u-as converted to the picrate aiid recrystallized from 2-methoxyethanol.
l-Methyl-4-hydroxymethyl-4-phenylpiperidineTerephthalate (XXIX).-To a chilled mixture of 1.02 g ( 5 mmoles) of l-met,hyl-4phenyl-4-hydroxymethylpiperidine3 i l l 10 ml of anhydrous CI-T?Cl, was added a suspension of 0.505 g ( 2 . j mmo1e.s) of terephthaloyl chloride in 10 nil of of anhydrous CH?Cl?. The mixture was stirred a t room temperature (25') for 18 hr, and the white crystals were collected by filtration. Tritiiration with acetone gave 1.2;i g of criide material. I~ecrystallizatioiifrom 2-propaiiolabsolute methanol yielded 0.292 g (ll?;), mp 276-270". Anal. Calcd for C34H,10?;504.2HC1.HYO: C, 64.6; IT, 7.03; pu', 4.43. Found: C, 64.6; €1, 7.09; X, 4.50.
( 5 ) P. A . Shore, A. Buckhalter, and V. H. Cohn, Jr., J . Phnrmncol. E z p t l . Therup., 127, 182 (1959). (6) 11. r o n Redlich and D. Glick, A n a l . Biockem., 10,459 (1965). ( 7 ) hIodel 110 was equipped Trith a high-sensitirity conversion kit, No. 11&865, and a microcurette adaptor, E o , 110-66.
-
l6-Aza Steroids R. W.KIERSTEAD, ,4.FARAONE, AWD A. BORIS Chpimrnl R w n w h Depnitiiwnt, Hofmann-La Rorhe, Inc., S u t l q ,
\-pi!-
. J E ~ S P I J 071 10
Rpceived October 3, 1966 The preparation of reveral 16-axaandrostenes is described, ntilizing 3~-hydrour-l6,17-seconlldro~t-3-eae-l6,17The latter compoiind wa9 transformed, u t a i t i diester IIa, half-eyter IIt), acid chloride IIc, aiid iwcyanate 111, into 3P-h\.dro\\.-lB-azaaiidrnit-~-eli-17-oiie (V) In a similar maiiiier 10azaestroiie was prepared from the methyl ether of marrianolic acid. The resiilt. (Jf -ome preliniinarv biological tests are reported. dioic acid ( I ) as .tarting material
The introduction of a nitrogen atom into the steroid nucleus has led to conipounds exhibiting diverse biological activity.' Although most of the ring D aza steroids previously described2 are analogs of D-homosteroids, the preparation of various 17-aza steroids has been reported3 in which the nitrogen is incorporated i n a five-membered D ring. Studies on the corresponding 16-aza series were initiated by Bachmann and Tiamirez, who described4 the synthesis of dl-1G-azade(1) 51. Martin-Smith and AI. F. Sugrue, .I. Pharm. Phnrmacol., 16, 569 (1964). (2) L. Tokes in "Steroid Reactions," C. Djerassi, Ed., Holden Day, Inc., San Francisco, Calif., 1963, pp 502-531. ( 3 ) (a) S. Rakhit and h l . G u t , J . Org. Chem., 29, 859 (1964); (b) S. Rakliit a n d hI. G u t , Sleroids, 4, 291 (1964). ( 4 ) W .IC, liic.(V). 111 H Inure co~ivenientprocedurc, the iwcyanate I11 \\:la converted directly : ~ n din high yield to the lactarn 1' oil treatiiient nith dilute 11 0xid:Ltic)ti of V hy t l i c ()ppeii:iiicr method g u v ~ the cixpected 3-1ieto 1:lrt:Lm T I . Yigorouy reducation of thr I:ic.tam V with lithium :t1i1nii1iurn hydride iri dioxaric. g:iw the aininc "11. ?'reutnient of the lattcr compound with acetic anhyt i l idch i l l pyridine gave the S-acetyl acetate VIII, whicah w:i\ hydrolyzed to the cwresponding alcohol I S . 1te:lc'tioii of the amine VI1 with formaldehyde :itid formic. wid gave the S-methyl derivative X. Oppeiiaii(~ositlation of I X and X gave the correspondirig I\etoti(+ X I and XII, respectively (ree Chart 11).
WtPr XIIIb"'
01 t 11C 1:lttt.r ~YJIll~~Olllld l \ : i y IlythOl) Z(Y1 n-ith potassium (*art)oii:ite mlutiori to givc tlic L i i o \ \ I I half-ester XIIIcll 11 hich c*onvertetl, i 1~ t l i v : t i . i t l (Bhloride, to t lie c*orresp ing azide S I 1 Id. ' I ' h c t cartide azide rearranged o n heating i n 1)cliizetic' >olut ioii t o afford the iwcyanate XIT', whivh on t rc:itnicwt \\ i i 11 dilute potassium hydroxide was converted directly t ( 1 the methyl ether of 16-azaestrone (XVa).; The kit1 PI compound IT-as deniethylated readily witli pyridiiiv hydrochloride to give 16-azaestrorie (XT'b). 1letluc~tion of XVa with lithium aluminum hydride 1ti c~10x:11ic solution gave the cwresponding amine, 11 hivli 11 :I\ iwlated as the hydrochloride (XVI). h r : w 11:~- j>i Pviously reported the iiolation of this anlilic :L* the. hydrobromide salt. (10, [a) \I 1 m i r / I t m ('hem. h o c , 1 5 , 6362 (1 11 1. ('uderre, ani1 I' < i iiilcksliank. i h i i l . , 75, 6231 i 1 9 i O ( 1 1 ) I irst i , i r i > i r t c l 1,) I I l w r xnd l i \ I w w l i ~ r I W i (IWO)
JInrch 1867
~R-AzASTEROIDS
Biological Results.-Xone of the 16-azaandrostenes described herein exhibited any significant antigonadotropic activity when asiayed orally for 10 days in immature niale rat:, at 1 mglratlday. The compounds were evaluated for progestational activity in the JIcPhail assay and were found inactive.12 The compounds in this series did not exhibit any significant uterotropic activity when assayed orally for 3 days in the immature female rat at 1 mg/rat/day. When assayed in ovariectomized both 16azaestrone (XVb) and its methyl ether XVa exhibit very weak estrogenic activity, having less than of the activity of estradiol benzoate. Both compounds exhibit antiestrogenic activity14 and cause a 42 and 38% inhibition, respectively, of the uterine weight increase due to estradiol benzoate.
178
chloride was then added, and heating and stirring were continued for an a d d i h n a l 30 min. The reaction mixt,nre was cooled aud t'he solvents were removed under reduced pressure, care beiiig takeii that' the bath temperature did not rise above 43". The residue was diluted with benzene and the solution was evaporated t,o dryness under reduced pressure in order to remove the last, t,races of oxalyl chloride. The crude product was slurried with 300 ml of hexane (no attempt ihorrld be made to heat or "cry*talliae" at^ this point*)and the resiilt,ing precipitate was filtered and washed with hexane to give 197 g (917)of the criide acid chloride IIc, mp 114-122",which is siifficiently pive for the next step. A small portion was crystallized from hexane to give the analytical sample, mp 130.3-132' (vac.), [ C Y ] ~ ~ D -74.5" ( c 1.0, CHCI,),"":::A: 3.54 and 5.86 fi. Anal. Calcd for C?3H&10:: C, 65.00; H,7.83; C1, 8.34. Found: C, 65.27;H, 7.93: C1, 8.09. Acetate of 1,2,3,4,4a,4b,5,6,7,8,lO,lOa-Dodecahydro-7-hydroxy1 -isocyanatomethyl-2,4b-dimethyl-2-phenan threnecarboxy lic a vigorously stirred arid cooled Acid Ethyl Ester (III).-To (5-10")solution of 197 g (0.463mole) of I I c in 4 1. of acetone was added a solution of 154 g (2.37moles) of N a y 3 in 620 ml of water over a 20-min period. The reaction was stirred in the cold Experimental Section15 for an additional 20 min and was then diliit,ed with 3.3 1. of wat,er. Diethyl Ester of 3~-Hydroxy-16,17-secoandrost-5-ene-16,17- The mixt,ure was extracted four times with benzene and the beirzene layers were washed twice with water and once with brine. dioic Acid (&).-To a suspension of 10.0 g (0.028 mole) of The combined benzene layers a e r e dried ( Na&04) and aboiit 230 3p-hydroxy-l6,17-secoandrost-5-ene-16,17-dioicacid (I) ( m p ml of beiizeiie u-as evaporated urider reduced presstire in order to 246-249" dec)I6 and 100 ml of benzene was added 15 g (0.10 remove the last traces of water. mole) of the diethyl acetal of dimethylformamide and the reThe remaining benzene solut~ion (of crude azide I I d ) was siilting stirred mixture was heated under reflux for 3 hr (the mixheated under reflux with stirring for 1.5 hr, whereupon the evolutiire became clear after about 40 min). T h e cooled reaction tion of gas had ceased. The reaction mixt'ure was cooled and the mixt,ure was filtered through a bed of Celite (made up in ether) solvent was removed under reduced pressure t,o give a yellow and the filtrak was diluted with an equal volume of ether. The oil which crystallized upon addition of 200 ml of hexane. The orgaiiic solutioii was washed twice with 2 A- HCI, twice with 2 -l' material was filtered and crystallized from ether-hexane (1: 10) XaOH, and once with water. The dried ( S a 2 S 0 4 )solvent was to give 131 g (70%) of isocyanat,e (III), mp 122-127" (vac). removed under reduced pressure and the resultiiig semicrystalline Crystallization from ether-hexane gave the analytical sample, oil was crystallized from ether-hexane to give 8.84g (78%) of I I a mp 127-128" (vac), [ 0 1 ] ~ 7 ~ -.i4.6" ( c 1.0,CHCI,), X',fJ."I3 4.39 as an off-white solid, mp 93-95.5' (vac), lit.8 mp 103.5-104.5". Monoethyl Ester of 3,9-Hydroxy-16,17-secoandrost-5-ene- and 5.80 p. Anal. Calcd for C23H33NOj: C, 68.46: H, 8.24: K, 3.47. 16,17-dioic Acid (IIb).--4 solution of 34.0 g (0.61mole) of KOH Foiirid: C,68.19:H, 8.03; N, 3.62. in 340 ml of water was added t,o a solution of 34.0 g (0.087mole) The correspoiiding methyl erter had mp 161.5-163.5",[ ~ Y I ~ ~ D of I I a in 680 ml of methanol and the resulting mixture wa5 heated - 5.5.2' (c 1.0,CHC13),::A: 4.41 and 5.81 p . under reflux for 1 hr. The reaction mixtiire was cooled immeilnal. Calcd for C2?H:$,SO:: C, 67.84; H, 8.02; N, 3.60. diately arid most, of the solvent, was removed under reduced Fonrid: C,68.11;13, 8.30: N, 3.66. pressure, care being t,aken that t,he bath t,emperature did not Hydrochloride of 1,2,3,4,4a,4b,5,6,7,8,lO,lOa-Dodecahydro-7ribe above 4.5'. The residue was diluted with about, 1 1. of water hydroxy-1-aminomethyl-2,4b-dimeihyl-2-phenanthrenecarboxylic and was then extracted with ether-methylene chloride (3:1). Acid Ethyl Ester (IV).-A solution of 22.0 g (54.5 mmoles) The aqueous layer was cooled in ice wat'er and acidified to congo of the isocyanate I11 in 220 ml of t-butyl alcohol was heated red with about 500 ml of 6 HC1. The resulting precipitat,e under reflux for 22 hr and then was cooled and evaporated to was filtered and washed with water. The wet, compound was dryness under reduced pressure. The residue was dissolved i n crystallized from acetorie-water (1:l)to give 24.7 g (76%) of 150 ml of 4 X ethanolic HC1, and the solution was heated under IIb, mp 176.3-178" (vac), lit.8 mp 176-177.3'. reflux for 10 min. The cooled reaction mixt'iire was evaporated Acetate of 1,2,3,4,4a,4b,5,6,7,8,10,1 Oa-Dodecahydro-7-hydroxyto dryness and the residiie crystallized from ethanol-ethyl acetate 1 -chloroformylmethyl-2,4b-dimethyl-2-phenanthre~ecarboxylic to give 7.68g (38%) of t'he hydrochloride of IT, mp 239-240.3' Acid Ethyl Ester (IIc).-To a solution of 185 g (0.51mole) of the dec (vac). Crystallization from ethanol-ethyl acetat,e gave the dry half-ester I I b in 1280 ml of anhydrous pyridine was added analytical sample, mp 239.5-240.5' dec (vac),::A: 5.83 p . 785 ml of freshly distilled acetic arihydride and the reaction mixAnal. Calcd for C?,H3,C1N03: C, 64.38: If, 9.21; C1, ture was allowed t,o stand a t room temperature for 48 hr. The W,3 . i 7 . Found: C, 64.53;H, 9.46:C1, 9.70;N, 3.91. reaction mixture was poured into 7 1. of water and v-as extracted 3p-Hydroxy-16-azaandrost-5-en-17-one(VI. A. From the three timer with ether-CI-I&12 (3:1). The organic layers were Isocyanate 111.-A solution of 100 g (1.79moles) of KOH in 100 washed three time9 with 2 >Y HC1 and once with water. The ml of water was added to a suspension of 100 g (0.248mole) of combiiied organic layers were dried ( Na2S04) and evaporated the isocyanate 111 ( m p 122-127" (vac)) in 940 ml of methanol. wider reduced pressiire to give 206 g of crude acetate, which The reaction mixture became warm and clear and was allowed to was used directly for the next step. stand at ambient' temperature overnight. It, was evaporated Oxalyl chloride (300 ml) was added rapidly to a stirred solution under reduced pressure to about one-quarter of the original o f the crude acetate (206 g ) iii 2 1. of benzene, and the temperavolume and was diluted with 2 1. of water. T h e resulting pretnre of the reaction mixture was then maiiitairied at 66-69' cipitate was filtered, washed with water, arid crystallized directly for l..i hr by heating iir ari oil bath. An additional 50 ml of oxalyl from et,haiiol (charcoal), whereiipoii 50.8 g (71%) of lactam \-, mp 272-274' (var), came ont of the boiling solnt~ion. An addi(12) Assayed at a dosage of 1 me/day sc in the rabbit. tiorial 9.4g of product ( m p 266-272" (vac)) was obtained upon (13) Assayed a t a dosage of 1 mg/day PO in the rat. concentration of the mother liquor:. Crystallization from chloro(14) .Administered orally to rats at a dosage of 20.000:1 by weight against form-acetonitrile gave the analytical sample, mp 272-274" estradiol benzoate. the latter being administered subcutaneously. (vac), [01]23n -73.9' ( e 1.0,CHClr),: : :A 2.98 mid 3.93 p . (1.5) 1Ielting points mere determined in capillary tubes and are corrected. The infrared spectra were recorded on a Beokman Instrument Model IR-9 Anal. Calrd for CI8H2,KO2: C, 74.70; H, 9.40: N, 4.84. and the ulrraviolet spectra on a Cary recording spectrophotometer Model Foulid: C,73.02;H,9.23;N,4.89. 1411. Celite is a diatomaceous silica product (Johns-Alanville Company), B. From the Amine Hydrochloride 1V.-To a solution of Alerck-Darmstadt silica gel (0.2-0.5 mm) was used for column chromatogra100 nig (0.27mmole) of the hydrochloride IV in 1.0 ml of methp h y . Florisil (60-100 mesh) is a synthetic magnesium silicate (Floridin a w l was added a solution of 150 mg 12.7 mmoles) of KOH in C'u., Hancock, \\ , Va.) 0.15 ml of water. T h e mixt,ure was heated on the steam bath (16) Prepared by the method of \V. J. Adams, D. I(. Patel, V. Petrom, %nil I . A . Stuart-\\ebb, .I. Chem. S o c . , 29T (195fi). for 10 miri and was then evaporated to dryness. The residue was
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). Crystallizat8ionfrom 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 Grant T-337A from the American Cancer Society, by a Public Health Service research career program award 5-KX-CA 18718 from the National Cancer Institute and by Training (+rant 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
