Nucleosides of 2-Fluoroadenine'
The preparation of the anomeric (J-(9-cleox\.-u-r,!/lhi.o-peiitofiir.aiio.!.l)-.'-flii(iroadeiiiries aiid 9-u-arttbinofiir:Iiiosyl-2-fluoroadeniries from 3,6-dichloropiiririe is de ihed. The c>-toiosicity of these compoiinds, and also o f ;r;'-deoxy-2-fliioz.oadeuoaine and F)-8-o-x~lof~iranosyl-~-fl~ior(iadeiiirie, t o a riiiinbet of HEp-9 (.ell lines i n mli lire has been determined. The data permit certain conclii4oiis coiiceriiiiig the probable metabolism and niech:mimi of action of these nucleosides.
'2-B'luoro:tdeiiosirie2is readily analiolizec13-5 hut not c:it~Jiolizcd"-" in cells in culturr or in uico. I t is highly highly toxic to ant1 hiis hroailspcctrum mtibncterial activity.1i,12I t has a sl-nergistic vffect on the antimicrobial action of acti~iobolin'~ aiid is ;LII iiihibitor of blood-platelet aggregation. l4 The liroatl a l i d high-level tiiologic activity of 2-fluoroadcnosine has iiiatle tlu: study of other nucleosidcls of 2-fluoroadeiiino tlclsirahle. The preparation of 2-ami1io-2'-tlroxyade1iosi1ic (8-6) iii 1.7:; over-all yield and its a anoiiier in 1.3,'; over-all yieltl froni 2-amino-6-chloropurine by the conveiitioiliil c:hloromcrcuri procedure has been rcportetl.15 hi :ti1 (+fortto improve the yields of both anomers of 6 aiicl t o ol)t:Lin analytical samples of thtsc conipouiids, thtbir preparatioii from the chloronierruri derivative o f 2-1~eiix~~1iiido-N-~ieiizoyladeiiiii~~~ aiitl :3,~-di-C)-(p-ciilorol~eiizoyl)-2-tlcox\--n-erythro-pe1ito~ur~~1ios~l rhloriilcl; wis iiivcstigated m i d found t o givr 4 :is a i i npproxiinatcly 1: 1 mixture of a and moiiiers ill a total yitld of 36c/;, (Schenie I ) . Trcatineiit of 4 with S u O J l c i i i t tic usual niaiiiier resulted i i i ticcoriipoaitioii of tiiv I iuc*lcositle,whercns treatniei i t v i t li inct,haiiolic S H :It .' i rcmovcd only t.ht I,-clilorol)eiizo?-l groups. Tht. iijiportd by fiiiidi. f r ~ i ntlir S(iutlirin IZehealrli I i i h t i t i i t t . , I'oundatior~, arid t h e C'aiiwr Chemotherapj, National Serviw i'rntrr, National Canwr Institritr. Natioii:rl I n i t i n i t w of Ht.iilth, i P ) J . .4. Montgomery atid K. H r w w i i , .I. . I mer. ( ' h e m . Suc., 79, 4 K. Hariseii. J . B i o l . Cheni., 242, d I. I,. Briinrtt, Jr.. ibid., 242, 1 , Y. I). S:inipoii, and 31. 1,. llrlorli. .Ivc/I, ier, ibid., 102. 77 (IWA). .-L ntimicrobial .Iyen18 C h e m o i t ~ e r a p l i .1 W!,.
NH. I
p C I C , , H 4 C O O C a
p-ClC,H,COO 4.R=SHHi
+
H
O
C
W
+
HO 6
5 , R = I'(
",I
Inik. Bioc/iemisir!/, 4, l 2 X J (I!J(i:). ril)
S. 1;rFderikwii. .Ir.ch. B i o c h r m . R i o p h ! / s . , 113, :i83 (1966).
i 101
13. E. S k i i i ~ ~ J. x , -4. AIoiitgo~ner>.I. I2000 20
3IeJ\lPR
FAIFAR
1 1-2 1
>2000 > 2000 2.4
12 1 1 1 3'-Deoxyadenosine 2-Fluoro-9-p-~-xylofuraii~~syladenine 9-p-D-S ylofuranosyladenine S-p-D-;Zrahinofuranosyl-2-fluoroadenine (p-16e) 9-P-D-Arabinofuranosyladenine 9-a-u-Airabinofuranosyl-2-fluoroadenine (a-16e) 3s 9-a-D-Arat)inofuranosyladenine 37 a The concentration required to inhibit the growth of cells, as measured by clone counts, to 5OCi of untreated controls.
> 10
* See ref 35.
tallization from MeOH gave a sample of pure 01-6: nip 230": [ a ] 2 7 D $73.9 i 0.7" (c 0.75, H 2 0 ), ;, ,A [mp ( E X pH 1-2T2 (10.6), 291 (9.8);pH 7, 13-256 (9.2), 279 (10.2) [lit.l"[a]21u 4-58.8 i 1.0" (H20);, , ,A [mp ( e X pH 7, 14-216 (25.0), 256 (8.9), 280 (9.9); mp 167"l. A n d . ( C I O H ~ c, ~ SH~, S ~ .) Only a single tlc-homogeneous crop of the p anomer (24 mg) was obtained from the EtOH-NeOH crystallization solution. Evaporation of the combined filtrates from the EtOH-1IeOH fractionation t o dryness gave a residue which was dissolved in Ha0 (3 ml). Fractionation of the aqueous solution resulted in the isolation of additional homogeneous p r o k c t in the same Experirnrental Section3' anomeric ratio as the EtOH-NeOH crystallization. 2-Fluoroadenine (7).-2-.4mino-9-( 2-~eosy-aJ~-u-~rythro-pent o2-Benzamido-N-benzoyl-9-[3,5-di-O-(p-chlorobenzoyl)-2-defuranosy1)adenine (6, 270 mg, 1 mmole) was disjolved in 4 (I).---Chloromeroxy- a , p- D-erythro-pentof~ranasyl ladenine HBFa (30 ml a t On),the solution was coo!ed to --ISo, and Sa?jOr curi-2-henzamido-S-henzoyladenine16 (36.6 g of 48" chloro(100 mg, 1.7 mmoles) was added in small portions to the stirred mercuri-(=elite mixture) was azeotropically dried in CsHs (600 ml) and allowed to react a-ith 3,5-di-O-(p-chlorobenzoy1)-2-deoxy-~- reaction mixture. After the addition was complete (15 min), the mixture was stirred for 15 min at -lo", cooled to -3O", and erythro-pentofuranosyl chloride17 (12.8 g, 30 mmoles) under neutralized ( p H 6-7) with 50'; S a O H . After evaporation to reflux. =\fter the usual work-up requiring filtration, evaporation, dryness in v a c q the residue was triturated with EtOH (four and extraction (307; aqueous K I ) of a CHC13 solution of the 10-ml portions). Evaporation of the EtOH filtrate to dryness reaction residue, the crude product was obtained as a glass, the gave the crude product which was identified by tlc (5: 1 CHCI3pmr spectrum of which indicated it was approximately a 1 : l JIeOH) as a mixture of 2-fluoroadenine and 2-deosyriboje. mixture of a- and p-4. .in EtOH (100 ml) solution of the crude 9-( 3,5-Di-O-acetyl-2-deoxy-a- and -8-D-erylh7.o-pentofuranosyl)product was diluted x i t h E t 2 0until a filterable solid precipitated. 2,6-dichloropurine ( a - and p-Sa).--A mixture of 2,fi-dichloroCollection of the solid gave the purified product which was washed purine (10 g, 53 mmoles) and 1,3,5-tri-O-acetyl-2-deoxy-~and dried: yield 2.7 g (12( c,), nip 140-145". Tlc (99: 1 CHCl3erythro-pentofuranose (14 g, 54 mmoles) was fused, with stirring, JIeOH) showed trace impurities. in G ~ L (10 O mm) a t 130-140" for 15 min. The reaction melt was -inadditional 6 g (26(,,) of less pure nucleoside was obtained as dissolved in C ~ H Gthe , unreacted 2,6-dichloropurine that precipia glass by CHC1, extraction of the reaction mixture insoluble tated (2.2 g ) was removed by filtration, and the filtrate was solids. evaporated t o dryness in vacuo. The residue was dissolved in 2-Amino-9-(2-deoxy-a- and -p-u-eryfhro-pentofuranosy1)adenine (6).--h solution of 2,6-dibenzamido-9-[3,5-di-O-(p- EtzO, the solution was seeded with a-Sa, and the crystals that formed were collected after 1 hr a t room temperature, washed, chlorobenzoy1)-u-erythro-pentofuranosyllpurine(4, 4.7 g, 6.2 and dried in vacuo; yield 4.83 g (23"; a-sa): mp 125'; [a]*51u mmoles) in l\IeOH-SH3 (150 ml, saturated at 5") was heated at $4.5 + 1.5' ( e 0.97, CHC13) [lit.24mp 123.5-124.5', [a12711 100" for 6 hr. The reaction solution was evaporated to dryness i0.4" ( e 1.13, lIeOH)] ; 6 [ppm (CDCl,)] 2.00 and 2.14 (CH3 of in vacuo and the residue, after trituration with CHC13, was acetyls), 2.82 m (two 2'-H), 4.25 m (two 5'-H), 4.63 m (4'-H), crystallized by trituration with EtOH (20-30 ml). The mixture 5.33 m (3'-H), 6.55 (q, J l ' 2 t = 6.5 Hz, .J1'2/' = 2.6 Hz, 1'-H), of crystalline anomers was collected by filtration, dried (500 mg, 8.37 (8-H). The filtrate and washings of a-9a were combined, 315>;))and recrystallized from 1:l EtOH-1IeOH (40 ml). Crysthe solution was evaporated to dryness, and the residue was tallization was allowed to take place slowly at room temperature dissolved in EtOH (100 ml). After treatment with Sorit, the and the crops were analyzed by tlc (5:l CHCl,-^\IeOH). The solution m s seeded with crystalline a,P-9a and refrigerated overcrops rontaining the slower traveling a anomer were combined night. The crystals that formed were collected and recrystallized (180 nip) and recrystallized from hIeOH-HaO. A second recrysfrom EtOH (100 ml); yield 6.4 g (315; la:lp-9a), mp 114-116", [ a I 2 j0 ~ i 1.0" (c 0.9, CHC1,). The pmr spectrum of a solution (36) J. J. Brink and G. A . LePage, Can. J . Biochem., 43, 1 ( 1 ' 3 6 5 ) . ( S i ) Silic.iR-TLC-T (;\Iallinckrodt) was used for column and thin layer of these crj-stals indicated that they are a 1 : l a:D mixture. (1-mm thick) chromatographic purifications. Silica gel H (Brinkmann) was The EtOH filtrates from the isolation of crystalline l a : lp-Sa used for thin layer (0.25-mm thick) analyses (tlc). Chromatographic homowere combined and evaporated to dryness in z'aeuo. Trituration geneity was established for all compounds using the solvent systems indicated. of the residue with C6Hs (25 ml) precipitated additional unreacted Spots were detected with either ultraviolet light (256 mfi) after spraying the 2,6-dichloropurine which wits removed by filtration. The filtrate plates n i t h Ultraphor (WT. high11 concentrated) (BASF Colors & Chemicals, was conventrated in z"acz/o and absorbed on a silica gel column Itic., Cliarlotte, N. C ~ . )or heat charring after SKIM? ing with ammoniuni (2.8 X 85 cm, packed and equilibrated (18 hr) with CsH,). The The ultiaviolpt spectra were deteimined i ~ 0i . 1 N HCl, 0.1 N NaOH, and p H i column was eluted with 9 : l CHCls-EtOhc and the fractions buffer with a Cary Model 14 spectrophotometer; the infrared spectra were determined in pressed KBr disks u i t h a Perkin-Elmer Model 521 spectrowere analyzed l)y tlc (Et20). Fractions containing essentially photometer. T h e pmr spectra were determined in the solvents specified with homogeneous &9a were coinlined and evaporated t o dryness a Varian .%-BOA spectrometer using tetramethvlsilane as an internal reference. in vacuo. l ~ e c r y s t a l l i ~ ~oft i the ~ ~ iresidue i ~ ~ from EtOH gave pure
creased cytotoxicity may be due to the fact that the fluorine a t C-2 prevents deamination of the nucleoside, since it is known that 2-fluoroadenosine is not deaminated8 but am-adenine is.36 The ct anomers of 2-fluoro2'-deoxyadenosine and 9-/3-~-arabinofuranosy~-2-fluoroadenine are much lrss cytotoxic, the ED,o values being 6.5 a:id 38 pnioles I., rmpectively.
llelting points, unless otherwise noted, were determined on a Kofler-Heizbank and are corrected. (38) T. Ziminski and E. Boiowski, J . Ciimmafoyr., 23, 480 (1966).
(39) Seed crystals weie ohtailled f l o m a previous r u n by thin l a y ~ chromar tography (Et20).
AIay 1969
?rTCCLEOSIDES OF 2-FLUOROADENISE
503
12.5 mmoles) was hydrogenated in the presence of 5y0 Pd-C (500 mg) as described for p-12a. Aft'er removal of the catalyst and concentration of the EtOH filtrate, the a-12a precipitated and was collected by filtration, washed, and dried; yield 4 g (91%), mp l55', tlc ( 9 : l CHCI3-MeOH). A CHCL solution (50 ml) of 9-(3,5-di-O-acetyl-2-deoxy-a-~ribofuranosyl-2-aminoadenine (a-l2a, 3.5 g, 11 mmoles) was cooled to -20" and diluted with stirring with 48y0 HBFd (80 ml). The resulting mixture was treated with KaSO2 ( 2 g, 30 mmoles, in 2 ml of H20) as described for p-16b. The neutral reaction emulsion was centrifuged and the reaction products were identified by tlc (Et0.k) and isolated as described (see p-16a) giving the following results: isoguanine (42$;,), 2-fluoroadenine ( l o % ) , 9-( 3,5-di-O-acetyl-2-deoxy-a-~-ribofuranosy1)-2, 6-difluoropurine (35;,),and ~ 1 6 (lo?& a mp 183"). Pure a-16a was isolated by tlc ( E t O h c ) followed by C6H6 recrystallization; yield 7 5 ; mp 182"; i-34.4 ZIZ 0.8" ( e 0.99, CHCI,): A,, [mp ( e X l O P ) ] , pH 1-262 (13.4), 267 (sh); pH 7, 13-261 (14.8), 267 (sh);,,,B (em-') 3320, 3170 ( S H , CH), 1750 (C=O). 1680, 1645, 1610, 1580 ( S H , C=C, C = S ) , 1230, 1100, 1060, 1040 (COC); 6 [ppm (CDCl,)] 2.00 and 2.12 (CHI of acetyls), 2.80 (two 2'-H), 4.25 (two 5'-H), 4.59 (4'-H), 5.27 (3'-H), 6.22 broad (SH,), 6.40 (9, .1112/ = 6.5 Hz, . I l f 2 t t = 3.2 Hz, l'-H), 7.33 (benzene), 8.00 (8-H). ilnal. ( C ~ ~ H ~ , F S , O ~ . ' / I ? C ~ H ~ ) C, H, S . The 9-( 3,5-di-0-acetyl-2-deoxy-(~-~-erythro-pentofuranosy~)-2,6difluoropurine was purified by thin layer chromatography (1: 1 CHCl3-EtOAc) and its identity was confirmed by mass spectroscopy. 3',5'-Di-0-acetyl-2-fluoro-2'-deoxyadenosine(@-16a).-3',5'-Di-O-acetyl-2-amino-2'-deoxyadenosine @-12a, 3.6 g, 10.4 mmoles) was dissolved with stirring in 48y0 HBFd (50 ml a t -10") and the solution was cooled t o -20". A solution of SaS02 (1.2 g, 17 mmoles, in 2 ml of H2O) was added dropwise, and the reaction mixture was stirred a t -20 t o -10" for 20 min. After dilution with CHCL (50 ml), the mixture was cooled to -30' and neutralized ( p H 5-6) with 50L.cKaOH. The resulting emulsion was centrifuged to separate the insoluble gel from the liquid layers, which were each washed with more solvent (50 ml). The aqueous layers were discarded and the combined CHC13 solution was set aside. The gel was triturated with EtOH-Et20 until a filterable solid was obtained, and, after the addition of more Et20, the solid was collected, triturated with HzO, dried in vacuo, and identified by spectral analysis as isoguanine: yield 48%. The EtOH9-(2,3,5-Tr~-6-acety~-p-~-arabinofuranosy~)-2-chloroaden~ne Et20 and H20 filtrates were combined and evaporated to dryness (8-14c).--A solut,ion of 9-p-~-arabinofuranosy1-2-chloroadenine** in vacuo, and the residue was triturated with CHC1,. The insolu(p-l4e, 1.1 g, 35 mmoles) in pyridine (14 ml) and .4c~o(10 ml) ble solid m-as collected and identified by spectral analyses as was stirred a t room temperatrire for 2 hr before it was evaporated 2-fluoroadenine, yield 175,. The filtrate was combined with the to dryness in vacuo. A solution of the residue in E t O H was CHCl3 solution that had been set aside, and the resulting solution evaporated to dryness before the residue was crystallized from was washed with H20, dried (MgSOr), and evaporated to dryness EtOH. The crystals were collected in several crops and recrystalin uacuo. The residue was triturated with CsH.5 and the insoluble lized from EtOH giving p-14c: yield 1.1 g (71%), mp 160"; solid was collected and recrystallized from CHCl3 (40 ml); yield tlc (19: 1 C H C I I - R ~ O H ) shorn-ed only trace impurities and 536 mg ( 1 4 . 5 5 ) ; mp 208"; tlc (EtOAc); 6 [ppm (CDCI,)] 2.07 indicated the material was suitable for use as a n intermediate; and 2.11 (CH3 of acetyls), 2.73 m (two 2'-H), 4.35 m (two 5'-H and 4'-H), 5.39 (3'-H), 6.03 broad ( S H Y ) ,6.30 (t, J 1 y . 2=~ 7.0 ~ , . ,A (mp), pH 1, 7-263, pH 13-264.5; 8, (em-') 3360, 3310, 3260,3175 (NH, C H ) , 3000,2940 (CH), 1750, 1735 (C=O), =t0.2 Hz, peak width 13.5 Hz, 1'-H), 7.91 (8-H). 1650 (NH,), 1590, 1570 (C=C, C=N), 1250-1210, 1110, 1060, Tlc ( E t O h c ) indicated the benzene filtrate from the isolation of 1040 (COC); 6 [ppm (CDCl,)] 1.94 and 2.15 d (CHI of acetyls), p-16a contained a small amount of 9-(3,5-di-O-acetyl-2-deosy4.37 m (two 5'-H and 4'-H), 5.04 m (3'-H and 2'-H), 6.37 broad ~-~-ribofuranosyl)-2,6-difluoropurine in addition to the sugar (NHz), 6.53 (d, J I ~=z4.4 ~ Hz, 1'-H), 7.98 (8-H). residues. 2-Amin0-6-cliloro-9-cu-n-arabinofuranosylpurine(a-l5e).-A 9-( 2,3,5-Tri-O-benzyI-p-~ -arabinofuranosyI)-2-fluoroadenine solution of 9-( 2,3,5-tri-~-acetyl-a-~-arabinofuranosyl)-6-chIoro-2-(p-lGb).--.HBFd (48c;) (150 ml) was added to a solution ( 5 " )of fluoropurine (8, 3.5 g, 8 mmoles) in EtOH-XH3 (200 ml, saturated 2-amino- 9- ( 2 , 3 , 5 - t r i - 0 - benzyl-6-D-arabinofuranosy1)adenine a t 5") was refrigerated for 3 days before it was evaporated to (p-l2b, 4.6 g, 8.4 mmoles) in CHCl, (60 ml). The resulting misdryness. The residue was dissolved in H20 and extracted with ture was cooled t o -10" before a solution of SaXOy (1.7 g, CHCL. The crystals that precipitated from the H,O solution on 25 mmoles) in 4 ml of H20 was added dropwise with stirring. concentration were collected in several crops giving the crude -4fter the addition was complete, the mixture was stirred an product, which was recrystallized from H,O; yield 860 mg (345;); additional 40 min at -10 to -5' before it was diluted with CHC1, mp 190" dec (RIel-Temp); tlc (3:l CHC13-hIeOH) showed only (75 ml), cooled t o -20", and neutralized ( p H 5-6) with 50'; trace contaminants and indicated the product suitable for use as S a O H . The CHC1, layer was separated, washed with HsO, an intermediate;, , ,A (mp), pH 1-218,246,310; pH 7, 13-216, dried (MgSOd), and evaporated to dryness in vacuo. The residue, 247, 307;,,,B (cm-') 3465, 3370, 3300, 3180, 3080 (OH, KH), primarily a mixture of p-16b and 9-(2,3,5-tri-O-benz~-l-p-~2940, 2920, 2840 (CH), 1630 (KH), 1615, 1555, 1510 (C=C, arabinofuranosyl)-2,6-difluoropurine,identified by tlc ( 1: 1 C=X), 1050, 1020 (COC). CHCl,-EtOAc), was dissolved in absolute EtOH, and the solution 9-( 3,5-D~-~-acety~-~-deoxy-~-~-r~bofuranosyl)-2-fluoroaden~ne was saturated a t 5' with dry KH3. After refrigeration overnight, (a-lGa).--A solution of 9-(3,5-di-0-acetyl-2-deoxy-a-~-ribo-the reaction solution was evaporated to dryness in vacuo. The furanosyl)-2,6-dichloropurine (a-ga, 4.8 g, 12.5 mmoles) and residue was dissolved in CHC1, (25 ml), and the solution was KaX3 (1.6 g, 25 mmoles, in 7 ml of H2O) in EtOH (100 ml) was filtered through dry Celite to remove inorganic salts. The refluxed for 1.25 hr. After removal of inorganic salts by treatment filtrate was adsorbed on a silica gel column (2.6 X 35 cm, packed with C6&, an E t O H solution (250 ml) of the resulting a - l l a (5 g, and equilibrated with CHC1,). The column was eluted with 85 em, packed and equilibrated (18 h r ) with CeHs). T h e column was eluted wit,h CHC1, (750 ml) before the eluent was changed to 1 9 : l CHCl3-€v1eOH. The fractions were analyzed by tlc (19: 1 CHC13-bIeOH) and those fractions containing homogeneous a-12c were combined and evaporated to dryness in vacuo; yield 3.06 g (45%); 6 [ppm (CDCL)] 2.08 and 2.11 (CHI of acetyls), 4.33 m (two 5'-H), 4.62 t (4'-H), 5.03 and 5.85 broad (NH,), 5.32 m (3'-H), 6.06 m (2'-H and 1'-H), 7.68 (8-H). 2-Amino-9-a-~-arabinofuranosyIadenine (a-12e). A.-A soIrition of Xa?;, (260 mg, 4 mmoles) in HzO (1.5 ml) was added to 9(2,3,5-tri-O-benzyl-a-~-arabinofuranosyl)-2,6-dichloropurine ( aSb, 1.2 g, 2 mmoles) in EtOH (30 ml) and the resulting reaction soliit,ion was reflrixed for 1 hr. -4fter mmoval of inorganic salts by filtration, the filtrate was evaporated to dryness in L'UCUO. The residue was dissolved in absolute EtOH (150 ml) and 5% Pd-C (200 mg) was added. The resulting reaction solution was hydrogenated a t atmospheric pressure. After removal of the catalyst by filtration, the filtrate was evaporated to dryness and the residue was dried in vacuo. The resulting crude a-l2b was suspended with st,irririg in liqriid "3. Xa was added in small portions until a blue color pe ted for 5 min. The reaction was quenched with the addition of KH4C1, and the NH3 was removed in a stream of dry N2. After trituration with C&, the residue was dissolved in H 2 0 (20 ml), and the solution was acidified with AcOH and filtered. The filtrate was concentrated in vacuo, and the crystals that formed were collected by filtration and dried in DUCZLO giving a 49Tc yield of product which showed a major spot' [tlc (3: 1 CHC13-RIeOH)I identical with that prepared by method B (see below). Recrystallization of this product from EtOH-HZO (4: 1 ) followed by two recrystallizations from E t O H gave pure a-12e: yield 90 mg (167,); mp 213'; [ ( U ] ~ O D +42.3 i 0.4" ( e 1.1, H20);Xmax [mp ( e X pH 1-253 (11.2), 292 (9.9); pH 7-256 (9.2), 280 (9.9); pH 13-256 (8.9), 279 (9.8); ,,,B (cm-l) 3360, 3320, 3300, 3200-3100 (OH, NH), 2940-2900 (CH), 1660, 1615, 1600 (SH, C=C, C=K), 1090, 10.50, 1010 (COC). i l n a l . (C1,Hl4K6O4) C, H, N. B.--A solution of 2-amino-6-chloro-9-~-~-arab~nofnranosylpurine ( ~ - 1 5 e500 , mg, 1.6 mmoles) in EtOH-NH3 (40 ml saturated at 5') was heated in a Parr bomb a t 100' for 18 hr. The residue from evaporation of the solution in vacuo was crystallized from H20 (2.5 ml) with Norit, treatment. The crvstals were collected by filtration and dried in vacuo; yield 370 mg (80%); AmSa (mpj, p H 1-253, 292; pH 7, 13-256, 278.
