the three compounds on apomDrE)hine-iriduced emesi:, in dogs was not observed with doses up to 10 rng/kg (sc), while chlorpromazine u a s able to b1oc.k tlie emesis completely with 1.5 mg, kg. I n summary, :Lmong the ten compound\ t csted, 2 with favorable pharmacological properties zeems to bc
promising niititu.sivc agent in that it shon s approximately equal untitussive effect t o codeine \tit11 slightl! le+ toxicit). Hcre also thc rcil~of piperidirio groril) i t 1 m:tnifeqtatiori of antitussive :tr(ivity K:L> i1lul;tmt (,ti Tlii:, subqtmcc i. ~ i o \ viindergoing c1inic:d trial. LL- , i i i iiiltitussivc agtnt :L
Synthesis of 5-Mercaptouridiiie1~'
Reaction of ~-ace~\ilmercapto-2,4-bis(trimethylsiloxy)pyriniidirie (1) N ith (niiorneiic) 2,3,j-lri-O-beii/c)?.I-i,ribofuranosyl chloride led to an anomeric mixture of the blocked nucleo4de. Reaction of I with 2,3,5-tri-O-pariisoyl-o-ribofuranosyl chloride resulted, instead of coupling with the blocked ribosyl group, in p-anisoylatioii of the NI position of the pyrimidine. Stereoselective synthesis of >-me1 captouridine (MVR) was achieved by fusion in vacuo of I with 2,3,5-tri-0-p-chlorobenzoyl-or-~-ribofurarios~l bromide (prepared from the ariomericall> pure p-1-p-nitrobenxoate), followed by removal of the blocking groups. N U R showed inhibitory activity on preliminary testing in bacterial and tissue culture assayf ERC.iPTOURIDINE
Journal of Medicinal Cheniistry, 1970, 5'01. 13, S o . 4 711
a t 110". A homogeneous melt was obtained within a few min which then gradually solidified. After 30 min the resulting solid was dissolved in C6Hs(600 ml) and H20 (10 ml) was added. A4fter 1 hr, the CsHs solution containing some ppt was concentrated to dryness in vacuo, and the residue was dried by repeated addition of C6Hs (2 X 1.50 ml) followed by evaporation. The residual solid was treated with boiling CsH6 ( 2 1.) and some insoluble substance was removed by filtration. This was identical with an authentic sample of S-acetyl-5-mercaptouracil by mp (251-252'), mixture melting point, and ir spectrum. Recovery of unreacted pyrimidine varied between 30-50x based on the starting matmerial,silyl pyrimidine 1. The CsHe filtrate was concentrat,ed and, after 24 hr, a crystallized product V was collected; mp 186-189"; yield 3.8-5.3 g (80% based on the amount, of reacted silyl pyrimidine). Two recryst,allizations from C6H6yielded an analytically pure sample, mp 192-194'. The nmr spectrum (DXSO-d6) contained only 3 singlets, a t 6 2.4 (3 H, COCH3), 3.9 (3 H, OCHII, and 8.2 ppni (1 H, C-6), in addition to aromatic protons showing a typical para splitting pat'tern at 6 7.0-8.0 ppm (4 H ) . All resonance peaks attributable to the protons of the ribose moiety were tot,ally absent,. d n a l . for X I - p anisoyl-S-acet,yl-5-mercaptouracil (V) (C14HlZX2SO:): C, H, N, S. I n a control experiment, the syrupy 2,3,5-tri-O-p-anisoyl-nribofuranosyl chloride (IV) was prepared as described above and transferred into a microdist,illation apparatus. On attempted vacuum distillation, no distillable mat,erial was obt,ained. hdded Experimental Section authentic p-anisoyl chloride could be fully recovered by distilling it out from the mixture a t 108" (1.5 mm). All melting points were taken on a Mel-Temp apparatus and Methyl 2,3,5-Tri-0-p-chlorobenzoyl-~-o-ribofuranoside 0'11). are uncorrected. I r spectra (KBr) were recorded on a Perkin-To a solution of D-ribose (15.0 g, 0.1 mol) in dry MeOH( 250 Elmer Infracord or Beckman IR8. Nmr spectra were recorded ml) was added 1 ml concd HzSO4, and the mixture was stirred a t on a Varian Model 8-60 spectrophotometer in CDCL, unless 5" for 15 hr. Dry CsHJ (50 ml) was then added, and the solution otherwise indicated, with T M S or t-BuOH as an internal standard. was concentrated in vacuo to yield a syrup (anomeric methyl Uv spectra were obtained on a Beckman D B recording spectroriboside, VI).2a Addition of dry C S H ~ N ( 2 X 40 ml) and evaporaphotometer. Optical rotations were measured in a d m tube using tion was repeated two more t,imes. The residue was then disa Perkin-Elmer Model 141 automatic polarimeter a t 589 mp. solved in 70 ml of dry CLH5X,and the solution was cooled in an Elemental analyses22 were performed by Galbraith Laboratories, ice bath, while p-C1C&COCI (70 g, 0.5 mol) was added gradually. Knoxville, Tenn. Anomeric S-Acetyl-N1-(2,3,5-tri-0-benzoyl-~-ribofuranosyl)-5-After standing overnight', H20 (200 ml) and CH.f.212 (250 ml) were added to the partially solidified reaction mixture, and after commercaptouraci1s.-To 2,3,5-tri-0-benzoyl-~-ribofuranosyl chloplete dissolut,ion, two layers were separated. The CH2C12 layer ride (syrup) prepared from 7.56 g (15 mmol) of 2,3,5-tri-Owas washed (H20,ice-cold 3.0 N H,SO,, H20 saturated NaHCO,), benzoyl-1-0-acetyl-p-n-ribofuranoseaccording to Kissman, et al., l 8 dried (MgSO,), and evaporated in vacuo. The residual white wm added 5-acetylmercapto-2,4-bis(trimethylsiloxy)pyrimidine~ solid was crystallized from EtOH to give VI1 (22.96 g, 40Yc), (I, 5.17 g, 15.7 mmol), and the mixture was mechanically stirred mp 102-104". -4small sample for analysis was twice recrystalin vucuo a t 155" (oil bath) for 40 min. The result,ing homogeneous lized from EtOH to give needles, mp 106-108"; [ol]'OD +89.3" melt was dissolved in CsHs (300 ml), and 3 ml of H 2 0 was added. (c 5.23, CHC13).24$rial. (C2?H2,CL08)C, H, C1. After standing for 1 hr, the solution containing some ppt w&s Anomeric 2,3,5-Tri-O-p-chlorobenzoyl-~-ribofuranose (VIII) evaporated in vacuo and the residue was dried by azeotropic evapand the CorrespondingEthyl 6-Glycoside (IX).-To a solution of oration with C6Hs (2 X 150 ml). VI1 (2.90 g, 0.005 mol) in CH2C12 (20 ml) was added a solution of The residue wm t,hen dissolved in hot CCl, and some insol 30Oj, HBr in glacial AcOH (16 ml), and the mixture was stirred material was removed by filtration. This was shown to be Sfor 1 hr. More AcOH (10 ml) was then added, and the soln was acet~yl-5-mercaptouraci18 by mp (251-252'), ir spectrum, and kept below 10" while H 2 0 (10 ml) was gradually added. The mixture melting point with an aut,hentic sample. The amount of two-phase mixture was stirred for an additional 40 min and then recovered pyrimidine varied between 25 and 35y0 based on the poured into ice-water (100 ml) and CHzC12 (100 ml). The silyl pyrimidine I used. From the CCl, solution, after standing layers were separat,ed, and the CH2C12 layer was washed several in the cold for 24 hr, the anomeric blocked nucleoside I11 deposited times with WaHC03 solut,ion, then dried (1jfgS04). Evaporat,ion in the form of somewhat sticky crystals (3.2 g, 50-607, based on reacted silyl pyrimidine). Rfelt,ing range 80-130°, which did not of the CH2C12 in vucuo yielded a syrup which was crystallized from cyclohexane, then recrystallized from petsroleum ether, to change on repeated attempts of recrystallization from various give the anomeric mixture, VIII, (0.25 g, 8.5%); mp 73-82'. solvents, nor did the ratio (2: 1) of the two nmr peaks a t 6 2.30 The ir spectrum showed absorption for free (C-1) OH of the and 2.36 ppm (SCOCH,) change appreciably on recrystallization. ribofuranose at, 3500 cm-l, and C=O for the acyl blocking groups However, the ratio of these two nmr peaks varied somewhat ~O H,~ )C1. at 1735 cm-'. Anal. ( C Z B H I S C ~C, from run to run. The substance gave a single uv-absorbing spot on tlc (silica gel H F ) with several eluent systems. An attempt to crystallize t,he originally obtained syrup (crude Reaction of the Silylpyrimidine I with 2,3,5-Tri-O-p-anisoyI-n- VIII) from EtOH result,ed in the isolation of the corresponding ribofuranosyl Chloride (LV).-Methyl 2,3,5-tri-O-p-anisoyl-p-n- ethyl @-glycoside ( I X ) in 55yGyield. This was purified by repeated recryst,allizat,ion from EtOH and finally from n-hexane; ribofuranoside, prepared according to the method of Haga and 0~ (c 1.9; CHCl,). The nmr spectrum nip 104-107"; [ a l Z +8.5.j0 Ness," (16.98 g, 30 mmol) was dissolved in ice-cold anhydrous showed a doublet for the anomeric proton at 6 3.88 ppm ( J l , 2 = Et20 (320 ml) which had been saturated with dry HC1 at L O ' ' 5.0 H z ) . ~4~n d . (CzsH23CLOs) C, H, C1. and the solution was kept a t -10" for 1 week. The EtzO was 2,3,5-Tri-0-p-chlorobenzoyl-1 -P-O-p-nitrobenzoyl-~-riboevaporated in vucuo, two 100-ml portions of dry Et20 were added and again evaporated, to give a syrupy residue of the blocked furanose (X).-The crude, syrupy anomeric 2,3,5-p-chlorobenzoyl-n-ribofuranose (VIII) obtained above was redissolved in dry halogenose IV. To t,his was added the silyl pyrimidine I (9.90 g, 30 mmol), and the mixture was stirred vigorously and heated in vacuo CHzClZ (15 ml). To this was added a solution of p-nitrobenzoyl chloride (1.86 g, 0.01 mol) in a mixt,ure of dry CH2CL (20 ml) and dry CSHSK (1.5 ml) while t,he react>ion flask was kept in (20) A . Bloch, hI. H. Fleysher, R. Thedford, R. F. Maue, and R. H. ice-water bath. After 4-5 hr stirring, chips of ice were added
captouridine (XIV) which, however, contained approximately 5% of the oxidized form, bis-(5-uridinyl) disulfide (XV) . On recrystallization, the disulfide content increased, due to air oxidation. The uv spectrum of XIV at neutral or alkaline p H shows the characteristic absorption peak of the 5-thiolate ion at Amax 335 mp which disappears on oxidation to the disulfide, but reappears upon the addition of dithiothreitol (DTT), due to reduction to the thioL8 Thus, the relative amount of the disulfide XV in the sample can be estimated on the basis of the absorbancy at 335 mp in the presence and absence of DTT. I n the preliminary biological testing, 5-mercaptouridine showed significant inhibitory activity in the Streptococcus fueculis assay system20 (Ijo2 X Ai!)21 but was less active against leukemia L1210 cells in culture (Ijo 4 X Further studies, relating to the activity spectrum and the mode of action of this analog are in progress.
Hall. J . .Wed. Chem., 9, 886 (1966). (21) .I. Bloch, personal communication. ( 2 2 ) Where analyses are indicated only by symbols of t h e elements, analytical results obtained for those elements were within =k0.4% of t h e theoretical values.
(23) R. Barker and €1. G. Fletcher, Jr., J . 070. Chem.. 26, 4605 (1Y61). (24) Assignment of anomeric configuration tentative, based on analogies of preparation and comparative optical rotation of similar glycosides.'3 17
