Synthesis of 5-substituted ether derivatives of 5-hydroxymethyldeoxyuridine and their a-anomers1

G. L. BUBBAR AND V. S. GUPTA' Department of Plzysiology, Western College of Veterinary Medicine,

University of Saskatchewan, Saskatoon, Saskatchewan Received March 6, 1970

The synthesis and properties of ether derivatives 6a-dof 5-hydroxymethyldeoxyuridine and their corre- sponding a-anomers 7a-d are described. Acid-catalyzed etherification of 5-hydroxymethyluraci1 (1) gave the corresponding 5-alkoxymethyluracils 2a-e in excellent yields. Treatment of compounds 2a-e with trimethylchlorosilane gave bis(trimethylsilyl)pyrimidines 3a-e in high yields which, upon condensa- tion with 3,5-di-(0-p-to1uoyl)-2-deoxy-D-ribofuranosy chloride, furnished an anomeric mixture of substituted nucleosides, p-anomers 4a-e and a-anomers 5(a-e). The nucleosides with p-configuration were the major products in each case and were recovered as crystalline solids in good yields. Deblocking of compounds 4a-d by alcoholysis resulted in the corresponding ether derivatives 6a-d. From mother liquors, two substituted a-anomers, 5a, b were isolated as crystalline solids in small yields. Compounds 7c, d were obtained from mother liquors after hydrolysis by using preparative t.1.c. The structures were confirmed on the basis of p.m.r. studies, U.V. and i.r. absorption spectra, and elemental analysis. The compounds 2a-e and 6a-d did not inhibit thymidylate synthetase at a concentration of 2.5 mM.

Canadian Journal of Chemistry, 48, 3147 (1970)

The hydroxymethylpyrimidine deoxyribonu- corresponding blocked p-anomers 4a-e, and cleotides are unique constituents of viral DNA a-anomers, 5a-e; the p-anomers predominated (1-5); furthermire, 5-substituted pyrimidines have been suggested as possible intermediates during the transformation of thymine to RNA pyrimidines (6). The hydroxymethylation of pyrimidine deoxyribonucleotides is carried out by specific hydroxymethylases; the hydroxy- methyl group originates from the Cl-unit of 5,lO-methylene tetrahydrofolic acid (7, 8). In this commuilication we report the synthesis of 5-substituted ether derivatives of 5-hydroxy-

in all cases.4 After removal of the silyl groups by aqueous ethanol, p-anomers IV (a-e), were isolated from the reaction mixture as crystalline solids in good yields (45-72 %). The F-configura- tion was assigned to 5-alkoxymethyl deoxy- ribonucleosides 4 (a-e) on the basis of a charac- teristic triplet in the p.m.r. spectrum; it exhibited a coupling constant (J,,,,, = J,.,,., of 7.0 + 0.1) c.p.s. for the anomeric proton at 6 6.30-6.35. Treatment of the substituted nucleosides 4a-e

methyldeoxyuridine and their corresponding &- with hot sodium methoxide in methanol or with anomers (Scheme 1). We have prepared these ethanolic sodium hydroxide at room tempera- compounds for the study of the bulk tolerance ture gave the corresponding 5-alkoxymethyl de- of thymidylate synthetase to substituents on the rivatives of deoxyuridine 6a-e in excellent yields 5-position of the pyrimidine ring. (75-85%). In compounds 6a-e the signal of the

The conversion of 5-hydroxymethyluracil (1) anomeric hydrogen (1 '-H) appeared in the form was successfully modified (9) to obtain 5-alkoxy- of a triplet as 6 6.16 with a coupling constant of methyluracils 2a-e in excellent yields (92-97%). J,,,,, = J,,,,., = 6.95 + 0.1 c.p.s. This triplet These ethers 2(a-e) on treatment with trimethyl- due to the anomeric proton (1 '-H) is characteristic chlorosilane in the presence of triethylamine (10) of deoxyribonucleosides having the P-configura- gave the corresponding bis(trimethylsily1) deriva- tion (15, 16).' Signals of other protons appeared tives 3a-e in high yields (70-90 %). The structure

I of silyl ethers 3a-e was confirmed by i.r. and 3Since the trans-rule is not obeyed in the case of spectra' Condensation of 3a-e with 3755i- deoxyr~bonucleosides, both anomers are formed during

(0-p-toluoyl)-2-deoxy-~-ribof~~~~0~~~ chloride the condensation reaction (12). by a modified silyl condensation procedure (1 1) "The silyl condensation method has been observed pre- at 185-195" an anomeric mixture3 of the viously to favor the p-configuration (13).

5Thvmidine. whose O-configuration has been unequiv- oca~l~~establ ished (14); exhibTted a triplet at 6 6.16-(see

'This work was supported by the Medical Research also Lemieux (15) for a detailed discussion of the theor- Council of Canada (Grant MA-31 36). etical basis of the p.m.r. spectra of thymidine and its

'To whom all enquiries should be sent. a-anomer.

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CANADIAN JOURNAL O F CHEMISTRY. VOL. 48, 1970

R1'OPc' ; R0

J < ~ H Z O R '

R"0 3a-e

1 1 0

Rr'0H2C

H N + H ~ ~ R ,

R r t O H 2 d q 7f)-CH20k

OR" 0

4a-e 5a-e

1 1

at the expected positions and the observed et al. (17) for the preparation of 6b." The mixture integration ratios were equal to calculated values. m.p., R, value, U.V. and p.m.r. spectra, and

Further proof of the structural assignment specific rotation of 6c prepared by this procedure and configuration for the compounds 6a-e was

65-Hydroxyrnethyl deoxyuridine was poorly soluble by the in poor yield 6c in the higher alcohols and preparation of compounds

essentially the same procedure as used by Baker 6c-e by this procedure resulted in low yield (<lo%).

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BUBBAR AND GUPTA: DERIVATIVES 0

and the silyl condensation method were iden- tical.

The two substituted a-anomers, 1-[2-de- oxy - 3,5 - di - ( 0 - p - toluoy1)-a-D-ribofuranosyll-5- methoxymethyluracil (5a) and 1 - [2-deoxy-3,5- di- ( 0 - p - toluoy1)- a-D-ribofuranosyl] - 5-ethoxy- methyluracil (5b), were recovered as crystalline solids in small amounts from the mother liquom7 In these compounds, 5a, b, the anomeric hydro- gen (1'-H) exhibited a broad doublet at 6 6.60 and on raising the temperature to 70°, it split into a quartet (coupling constant = J,,,,,, = 3.20 and J,,,,, = 7.25 c.p.s.). After deblocking, 1 -(2- deoxy - a - D- ribofuranosyl) - 5 - propyloxy- methyluracil (7c) and I-(2-deoxy-a-D-ribofuran- osy1)-5-butyloxymethyluracil (7d) were isolated by preparative t.1.c. In compounds 7a-d, the signal of the anomeric hydrogen (I '-H) appeared as a quartet at F 6.48 (coupling constant = J,.,,,, = 3.2 and J,,,,, = 7.8 c.p.s.). The quar- tet due to the anomeric hydrogen (1'-H) is char- acteristic of deoxyribonucleosides having the a-configuration (15, 16).

It is noteworthy that compounds 6a-d and their corresponding a-anomers, 7a-d did not obey Hudson's rules of isorotation. These observations are consistent with the phenomenon previously observed with other pyrimidine deoxyribonucleoside anomers (1 6, 18-20).

The compounds 2a-e and 6a-d failed to inhibit thymidylate synthetase of pig thymus gland at a concentration of 2.5 mM.

Experimental Melting points were determined with a GallenKamp

apparatus and are uncorrected. Specific rotations were measured (1 dm tube) with a Perkin-Elmer model 141 automatic digital readout polarimeter. The p.m.1. spectra were recorded on a Varian Associates A-100 spectrometer. All compounds were dissolved in dimethylsulfoxide (DMSO-d6). The i.r. spectra were measured with a Perkin- Elmer model 421 spectrophotometer. A Unicam spectro- photometer, model SP-800, was used to measure the complete U.V. absorption spectra. Microanalyses were performed by the College of Pharmacy and the Prairie Regional Research Laboratory,Saskatoon, Saskatchewan. Silica gel (HFZ54), was purchased from E. Merck, Darmstadt, Germany. The t.1.c. was carried out on silica gel plates (0.3 mm) at room temperature and spots were detected under U.V. illumination. Paper chromatograms

'The poor yield of a-anomers can be attributed to the high stereospecificity of the silyl condensation method for 0-configuration, vide supra (13).

were run overnight on Whatman No. 1 paper. Solvents used (by volume) were: (A) 11-butanol- glacial acetic acid - water (4:1:5), upper phase; (B) i-propanol- N H 4 0 H - water (7:1:2); (C) CHC1,-MeOH (8:2). All evaporations under reduced pressure were performed on rotary evaporator at 35".

5-HydroxymethyluraciI (1) was prepared by base- catalyzed hydroxymethylation of uracil (9) and 5-hy- droxymethyldeoxyuridine was synthesized by the method of Baker et al. (17).

Ether Derivations of 5-Hydroxyn~ethyl~rvacil (2a-e) The ether derivatives of 1 were synthesized by the

following modification of the procedure of Cline et al. (9). To 100 ml of each alcohol containing 1 ml of con- centrated HCl was added 2 g of 1. The suspension was stirred at room temperature for 15 min, and then kept on a steam bath for 3 4 h. The reaction mixture became homogeneous after 3 0 4 5 min in all instances, and t.1.c. of a sample (90 min or thereafter) using silica gel in solvent C for development showed only a single quenching spot. The reaction mixture was refrigerated and filtered to remove the crystals (yield 85-88%). Mother liquors on concentration gave an additional yield (5-10%) of the pure products. The m.p., total yields of the crystalline material, and Rf x 100 values in solvents A, B, and C, respectively, were: for 5 methoxymethyluracil (Za), 203-204" (lit. (9) 203-204"), 97.7%, 30, 46, 37; 5-ethoxy- methyluracil (26), 212-213" (lit. (9) 212-213"), 97.2%, 54, 61, 68; 5-propyloxymethyluracil (2c), 221-222", 95 %, 77, 78, 73; 5-butyloxymethyluraci1 (24 , 215-217" (lit. (9) 215-217"), 9474, 81, 78, 79; and 5-benzyloxymethyluracil (2e), 205-206" (lit. (16) 202-203"), 94.5%, 81, 84, 88.

Bis(trimetlgvlsilyl) Derivatives of 5-Alkoxytt~etlgvluracil (3a-e)

T o a suspension (12.4 mmoles) of each uracil ether 2a-e in anhydrous benzene (25 ml), was added trimethyl- chlorosilane (4.07 g, 37.5 mM). Triethylamine (3.79 g, 37.5 mM) in benzene (10 ml) was then added dropwise. and the reaction mixture was stirred overnight at room temperature. The precipitated triethylamine hydrochlo- ride was collected by filtration and washed with benzene (3 x 5 ml). The filtrate and washings were combined and the solvent was evaporated under reduced pressure. The viscous oily liquid of each silyl pyrimidine was distilled under high vacuum to yield compounds 3a-e. I n the i.r. spectrum (liquid film) the silyl pyrimidines exhibited absorption bands at 1250 (vs), 835 (s), 753 (vs), Si(CH&; 1037 (vs), Si-0-C; 1087-1092 (s), Si-0-C and C-0-C and 1595 cm-I (s), pyrimidine ring 110, 21). The physical properties and yields of these compounds are summarized in Table 1.

1-[2-Deoxy-3,5-di-(O-p-toluoyl)-~-~-ribofurat1osyl]- 5-akoxyt~~etlgvluracil (4a-e)

Anhydrous 3a-e (5 mM) and 3,5-di-(0-p-toluoy1)-2- deoxy-D-ribofuranosyl chloride (1.94 g, 5 mM) (22) were mixed and introduced into a preheated (to 170") oil bath. After the halogenose melted, it was flushed with a gentle stream of nitrogen, the temperature of the oil bath was rapidty increased, and the reaction mixture was kept at 185-195" for an additional 30 min. The gummy residue

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CANADIAN JOURNAL OF CHEMISTRY. VOL. 48, 1970

TABLE 1

Physical properties and yields of the trimethylsilyl pyrimidines

Boiling point Yield Proton magnetic resonance data* of ether Compound ("C/mm Hg) ( %) linkage 6 (p.p.m.)

Bis(trimethylsily1)-5-methoxy 12010.3 72 5.53 (3H,s,0CH3)? methyluracil (3a)

Bis(trimethylsily1)-5- 120/0.1 88 5.67 (2H,q,-OCHz-), 3.39 (3H,t,-CH3) ethoxymethyluracil (36)

Bis(trimethylsily1)-5- 13310.15 80 5.62 (2H,t,-OCHz-), 3.62 (2H,se,-CH2-), propyloxymethyluraci1 (3c) 3.16 (3H,t,-CH,)

Bis(trimethylsily1)-5- 13710.1 72 5.62 (2H,t,-OCHz-), 3.69 (4H,m,-CH2- butyloxymethyluracil (3d) CHz-), 3.12 (3H,t,-CH,)

Bis(trimethylsily1)-5- 152-15412 87 6.56 (2H,s,-OCHz-); 7.22 (5H,m,5-phenyl benzyloxymethyluracil (3e) protons)$

'Formic acid external. Abbreviations: singlet (s), doublet (d), triplet (t). quartet (q), sextet (se) and multiplet (m). ?In addition thesignals common in compounds 3a-e were at 6 2.57 i 0.02 (9H, s , 2 or 4 (3CH3), 2.61 f 0.02 (9H, s, 2 or 4 (3CHP)3 and

6.60 +- 0.12 (2H, s, 5-CH2-with ring) p.p.m. The s~gnal of the hydrogen (H,) of the pyrimidine ring was located by using TMS (internal) and in 30-e appeared at 6 1.86 f 0.02 p.p.m.

$Located by using TMS (internal).

TABLE 2

Properties of 3,5-disubstituted pyrimidine 2-deoxy-D-ribonucleosides -

Ultraviolet absorption data [aIDz5" E t o ~ (95 %) CHC13 Proton magnetic resonance data of ether linkage

Compound (Concentration)* h,,, (mp) E,,, x lo4 6 (P .P .~ . )

4c -61.4" ' 241.0 3.75 3.20 (2H,q,-OCHz-), I .31 (2H,se,--CHz-), (1 .14) 0.78 (3H,t,-CH3)?

4d -65.0" 240.0 3.75 3.23 (2H,q,-OCHZ-), 1 .l-1.46(4H,m,-CHz- (1.17) CHz-) 0.81 (3H,t,-CH,)?

4e - 68" 242.0 3.20 4.35 (2H,s,-OCHz-), 7.29 (SH,s,phenyl ring (1 .24) at 5-position)?

'Concentration = glml. tTMS internal. $TMS external.

was dissolved in 30 ml of ethanol (95 %), refluxed for 30 min, and filtered hot through charcoal-Celite. The charcoal-Celite mixture was washed with hot ethanol (3 x 20 ml), filtrate and washings were combined, solvent was reduced in volume to 25 rnl, and refrigerated. The crystals were collected by filtration and upon crystallization from ethanol and then methanol, yielded the substituted nucleosides 4a-e as white needles. The total amount of blocked nucleosides formed (based on the amounts of 3a-e used), m.p., and yields of the crystalline material based on total nucleoside formed were: for 1-[2-deoxy-3,5-di-(0-p-toluoy1)-p-D-ribofurano- syll-5-methoxymethyluracil (4a), 70%, 170-172", 45%; 1 - [2-deoxy-3,5-di-(O-p-toluoyl)-~-~-ribofuranosyl]-5- ethoxymethyluracil (46), 73 %, 183-185" (lit. (16), 185-186"), 54%; 1-[2-deoxy-3,5-di-(0-p-toluoy1)-p-D- ribofuranosyl]-5-propyloxymethyluracil (4c), 86%, 187-

188", 68.7 %; I - [2-deoxy~3,5-di-(0-~-toluoyl)-~-~-ribo- furanosyll-5-butyloxymethyluracil (4d), 88 %, 160-1 62" (lit. (16) 162-163"), 60%; and 1-[2-deoxy-3,5-di-(0-p- toluoyl) - !3 - D - ribofuranosyl] - 5 - benzyloxymethyluraci1 (4e), 60.5%, 142-143" (lit. (16) 141-142"), 44.8%. The chromatography (t.1.c.) of the mother liquors after deblocking indicated that the major constituent (ca. 80%) in every case was the p-anomer. However, none of the substituted nucleosides 4a-e could be induced to crystallize even after long standing. The physical prop- erties are summarized in Table 2 and the signals of protons common to 4a-e in the p.m.r. spectra appeared at 6 7.66 (1 H, s, 6-H), 11.40-11.45 (1 H, s, 3-NH), 3.94 f 0.02 (2H, s, 5-CHz-with ring), 6.35 (lH, t, J,.,2. = J,.,,,, = 7.0 c.p.s., 1'-H), 5.62 (lH, m, 3'-H), 4.57(2H, s, 5'-CHz- and 4.47, m, 4'-H overlapped with 5'-CHz-), 2.37-2.60 (2H, m, solvent and 2', 2"-H), 7.87 and 7.95

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BUBBAR AND GUPTA: DERIVATIVES OF

(4H, a , 2 sets, 3-H and 5-H of two phenyl rings), 7.28, 7.38 (4H, d, 2 sets, 2-H and 6-H of two phenyl rings), 2.40 (6H, s, two p-methyl groups of benzene) p.p.m.

Anal. Calcd. for 46, C28H30N208 (522.5): C, 64.35; H, 5.79; N, 5.36. Found: C, 64.20; H, 5.83; N, 5.37.

I-[2-Deoxy-3,5-di-(O-p-toluoyl)-a-~-ribofuranosyl]- 5-methoxymetl~yluracil (5a)

Mother liquors left after removal of the B-anomer, 4a, were combined, dried, and the residue (575 mg) was shaken with warm benzene (3 x 10 ml), filtered, con- centrated until turbid, and left overnight at room tem- perature. The crystalline mass (160 mg; m.p. 140-145") was filtered and recrystallized twice from methanol to give 5a, as white shining needles (85 mg); m.p. 160-161". The physical properties are given in Table 2 and common proton signals for compounds 5a, b in the p.m.r. spectra were, at 6 11.75 (IH, s, 3-NH), 8.29 (lH, s, 6-H), 4.38 (2H, s, 5-CH2-with ring), 6.59 ( lH, d, broad) which, at 70°, split into a quartet, 1'-H), 3.18-3.38 ( lH, m, 2'-H), 2.74-2.88 (IH, m, 2"-H and the solvent), 5.90 (IH, d, 3'-H), 5.37 ( lH, t, 4'-H), 4.79 (2H, d, 5'-CHZ), 7.59, 7.67 (4H, d, 2 sets, two phenyl rings 2-H and 6-H), 8.11, 8.17 (4H, d, 2 sets, two phenyl rings 3-H and 5-H), and at 2.70 (6H, s, two-p-methyl groups attached to benzene rings) p.p.m.

I-/2-Deoxy-3,5-di- (0-p-toI1ioyl) -a-D-ribofura~~osyl]- 5-etl~oxymetlgvl~iracil (56)

The solvent was evaporated from the mother liquors and the residue (830 mg) was dissolved in boiling ether (50 ml). On cooling, the crystals (575 mg; m.p. 112-146") were filtered and twice recrystallized from ether to give 56 as a fine microcrystalline powder (165 mg, m.p. 117-118").

Anal. Calcd. for C28H30N208 (522.5); C, 64.35; H, 5.79; N, 5.36. Found: C, 64.20; H, 5.83; N, 5.39.

Physical properties are summarized in Table 2.

I-(2-Deoxy-~-~-ribofirm1osyl)-5-alkoxyn1etl1yli~racil (6a-d)

300 mg each of compound 4a-d was dissolved in 4.0 ml of NaOH (2 N) containing EtOH (8.0 ml) and the solution was left at room temperature overnight. After dilution with water (3 ml), Dowex 50 (H+; X8; 200400 mesh) was added and the resulting solution at p H 4 5 was stirred for 30 min at room temperature. The resin was filtered, washed with water (3 x 4 ml), and the combined filtrate was evaporated to dryness. The residue was triturated with water (20 ml) and precipitated toluic acid was collected by filtration; mother liquors were freed of residual toluic acid by extraction with ether (2 x 25 ml). The aqueous phase on evaporation gave 6a-d as an amorphous white powder (yield 80-92%). The solvent of crystallization, yields of crystalline material, m.p., and Rr x 100 values in solvents A and B, respectively, were: for I-(2-deoxy-e-D-ribofuranosy1)-5-methoxy- methyluracil (6a), EtOAc-cyclohexane, 45 %, 120-125", 55, 57; I-(2-deoxy-e-D-ribofuranosy1)-5-ethoxymethyl- uracil (6b), EtOAc-MeOH, 54%, 136-137" (lit. (17) 135-136"), 67, 66; I-(2-deoxy-~-~-ribofuranosyl)-5-pro- pyloxymethyluracil (6c), EtOAc-ether, 68%, 127-128", 76, 73; 1-(2-deoxy-e-D-ribofuranosy1)-5-butyloxymethyl-

uracil (6d), EtOAc+zther, 75%, 119-12l0, 83, 79. The physical properties of compounds 6a-d are summarized in Table 3; signals for common protons in the p.rn.r. spectra were, at 6 11.38 (lH, s, 3-NH), 7.92 (IH, s, 6-H), 4.03 (2H, s, 5-CH2-with ring), 6.16 (lH, t, J1,,2. = Jlr,2,. = 6.95 c.P.s., 1'-H), 2.09 (2H, q, 2', 2"-H), 4.23 ( lH, t, 3'-H), 5.21 (lH, d, proton of hydroxyl 3'-CHOH), 3.77 (IH, q, 4'-H), 3.57 (2H, s, (broad), 5'-CH2-), 4.99 ( lH, t, proton of hydroxyl group 5-CH20H). Each mother liquor showed a homogeneous quenching spot on paper chromatograms in solvents A and B and Rc values were the same; the crystalline products were not obtained in significant quantity even on long standing, however.

Direct Synthesis of 6c (17) 5-Hydroxymethyl deoxyuridine (500 mg) was sus-

pended in n-propanol (50 ml) containing concentrated HC1 (0.1 ml). The reaction mixture was stirred at room temperature for 30 min and then kept on a steam bath for 4 h. Solvent was removed 61 vacno, the residue was taken up in MeOH (5 ml) and examined by paper chromatography (solvent A). In addition to 6c (Rr 0.76, major spot), there were two minor quenching spots: Rc 0.34, starting material (15%) and Rr 0.05, probably polymerized material (5%). 'The product did not crys- tallize and was therefore chromatographed on a column (2 x 25 cm) of silica gel packed in benzene. Elution was begun with CHC13 (200 ml) and followed with CHC13-MeOH (95:5, 500 ml); 25 ml fractions were collected and monitored by t.1.c. (solvent C). Fractions containing mostly 6c (tubes no. 15-28) were combined and evaporated in vacrio. The residue (112 mg) after crystallization from ethyl acetate - ether gave 6c as a microcrystalline solid (75 mg). The physical properties of this product were identical with those of 6c obtained by the condensation method. Mother liquors on standing at 4" gave an additional amount (20 mg) of material with a slightly lower m.p. 121-123", but it was chromato- graphically homogeneous.

I-(2-Deoxy-a-~-ribofrIranosy~)-~-i~1et/1oxymet/1y~riraci~ (70)

The reaction mixture, consisting of 300 mg 5a, MeONa (150 mg), and anhydrous MeOH (6.0 ml), was refluxed for 30 min, deionized, and worked up as described under 6a. The residue (150 mg) was twice crystallized from ethylacetate and 7a was recovered as a fine microcrys- talline solid (85 mg), m.p.149-15O0, Rf (A , B), 51, 73; sig- nals common to compounds 7a-d in p.nl.r. were: at 6 11.65 (IH, s, 3-NH), 8.27 ( lH, s, 6-H), 4.42(2H, s, 5-CH2-with ring), 6.47 ( lH, q, J,,,2,. = 3.0 + 0.2 c.p.s. and J1,,2, = 7.5 + 0.1 c.p.s., 1'-H); 2.78-3.06 ( lH, m, partially buried under the solvent, 2'-H), 2.17 and 2.32 (lH, two triplets, 2"-H), 4.53 (2H, m, 3'-H and 4'-H), 5.66 (lH, s (broad), proton of the hydroxyl group 3'-CHOH), 5.18 (lH, t, proton of the hydroxyl group 5'-CH20H), 3.75 (2H, t, overlapped with DOH, 5'-CH2-) p.p.m. Mother liquors on prolonged standing at 4' gave an additional quantity (30 mg) of 7a. This product was chromato- graphically homogeneous but the n1.p. (146-149") was slightly lower. The physical properties and analytical values of compounds 7a-d are given in Table 3.

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TABLE 3

Properties of pyrimidine 2-deoxy-D-ribonucleosides --

Ultraviolet absorption data 0.1 M HCI (pH 1.0) Analyses

ID^^^ Proton magnetic resonance EtOH, 95 % h,,, Emax data of ether linkage Formula Calcd. Found 1:

Compound (Concentration)* (mp) x lo3 6 (P .P .~ . ) (mol. wt.) ( %) ( %) 5 - P

6a +21.7" 206 11.4 3.50(3H,s,0CH3)? C11H16N206 C,48.52; H, C,48.35; H,5.95; N,10.40 (0.01) 264 10.7 (272.26) 5.92; N.10.28 o

6b +21 .go 208 11.8 3.34(2H,q,-OCH2-), 1.10 C12H18N206 C,50.32; H, C,50.25; H,6.28; N,9.75 (0.096) 264 11.3 (3H,t,-CH3)$ (286.17) 6.34; N,9.78

6c +21.7" 208 11.9 3.35 (2H,q,--0CH2-over- B

C13HzoNzOs C,51.99; H, C,52.1; H,6.74; N,9.40 $ (0.012) 264 11.5 lapped with DOH of solvent), (300.32) 671 ; N,9.32

1.32-1.37 (2H,se,-CHj-), o v 0.87 (3H,t,-CH,)? n

6d +19.0° 207 10.7 3.36 (2H,q,-0CH2-over- C14H22N206 C,53.49; H, C,53.20; H,7.15; N,9.05 (0.01) 264 10.3 lapped with DOH of solvent), (314.35) 7.05; N,8.91

1.13-1.61 (4H,m,-CH2-CH2-), I - V)

0.87 (3H,t,) 7a +9.0° 207 10.5 3.61(3H,s,5-0CH3)$

P C1lH16N2o6 C,48.52; H, C,48.75; H,5.80; N,10.50 5

(0.04) 263.5 9.8 (272.26) 5.92; N,10.28 207

< 7b +7.S0 10.9 3.75(2H,q,-OCH2-), 1.48 C12Hl~N206 C,50.32; H, N,9.85 0

(0.02) 263.5 10.4 (3H,t,-CH3) (286.17) 6.34; N,9.78 !-

7c +6.7" 204 9.6 3.58(2H,q,partially buried Cl3H2~N2O6 C,51.99;H, C,52.3;H,6.84;N,9.10 -% (0.015) 263.5 8.5 under DOH,-0CH2-), (300.32) 6.71; N,9.32 -

1.40-1.84 (2H,m,-CH,) w 21

7d +7.75" 208.5 8.6 3.60(2H,q,-OCH,-), C14H22N206 C,53.49; H, N,8.87 0

1.40-1.84 (314.35) 7.05; N,8.91 (4H,m,-CH2-CH2-)

'Concentration = g/ml. tTMS internal. STMS external.

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BUBBAR AND GUPTA: DERIVATIVES OF 5-HYDROXYMETHYLDEOXYURIDINE 3153

I-(2-Deoxy-a-~-ribofuranosyl)-5-ethoxymet~yuraci (76) (Pharmacy College) for microanalysis facilities, and The reaction mixture, consisting of 56 (100 mg), F. M. Loew (Veterinary College) for helpful suggestions

MeOH (1.5 ml), and MeONa (50 mg), was worked up as in the preparation of this manuscript; Mr. T. Mazurek described under 6a. The residue (52 mg) was dissolved for measuring the p.m.r. spectra and Mr. B. Haid for in ethyl acetate and precipitated twice with cyclohexane recording the i.r. spectra. (43 mg); Rc (A,B) 50, 71 (see Table 3 for other physical properties). 1. J. G . FLAKS and S. S. COHEN. J. Biol. Chem. 234, 1-(2-Deoxy-a-~-ribofuranosyl)-5-propyloxymethyluracil 3501 (1959).

( 7 ~ ) 2. R. G . KALLEN, M. SIMON, and J. MARMUR. J. Mol.

Biol. 5, 248 (1963). Mother liquors left after the removal of 4c were dried, 3. S. oKUBO, B. sTRAUSS, and M. sTODOLSKY. Viral.

the white fluffy residue (0.6 g) was suspended in MeOH a, 552 (1964). (15 ml)containingNaOMe(400mg), refluxed for 30min 4. J. J. PENE and J. MARMUR. Fed. Proc. 23, 318 and worked up as described under 6a. The aqueous layer (1964). was concentrated to dryness, the residue was dissolved 5. D. H. ROSCOE and R. G . TUCKER. Biochem. in MeOH (10 ml), spread on silica gel plates (6, 20 x 20 Biophys. Res. Gxnmun. 16, 106 (1964). cm, 0.65 mm) which were developed three times with 6. M. T. ABBorr, R. J. KADNER, and R. M. J.

Biol. Chem. 239, 156 (1964). CHCkMeOH @:I). The respective zones were marked 7. D. H. RoscoE and R. G . TUCKER. Viral. 29, 157 under U.V. illumination and the nucleoside was extracted (1966). with hot methanol. After removal of solvent, the residue 8. A. H. ALEGRIA, F. M. KAHAN and J. MARMUR. (60 mg) was dissolved in EtOAc and precipitated twice Biochem. 7, 3179 (1968). with cyclohexane (49 mg), R,, 49, 74 (solvents A and B). 9. R. E. CLINE, R. M. FINK, and K. FINK. J. Amer.

Chem. Soc. 81, 2521 (1959). 1-(2-Deoxy-a-~-ribofuranosyl)-5-butyloxymethyluracil 10. T. NISHIMURA and I. IWAI. Chem. Pharm. Bull.

(7d) Tokyo, 12, 352 (1964). After recovery of 4d, the mother liquors were dried 11. T. NISHIMURA and B. SCHIMIZU. Agr. Biol. Chem.

in vacuo, and the residue (0.82 g) was hydrolyzed using Tokyo, 28, 224 (1964). MeONa (0.4 g) in anhydrous MeOH (20 ml) and worked 12. B. R. BAKER. The chemistry and biology of ~urines. up as described under 6a. The precipitate (300 mg) was Ciba Churchill, Lond0n.

1957. p. 123. dissolved in MeOH (5 ml) and purified using preparative 13. H. G . STOUT and R. K. ROBINS. J. Org. Chem. 33, t.1.c. (vide supra). The gum remaining after the removal 1219 (1968). of methanol was dissolved in EtOAc and precipitated 14. A. M. MICHELSON and A. R. TODD. J. Chem. Soc. twice with cyclohexane to give a white fluffy powder 816 (1955). (44 mg); R,, 51, 75 in solvents A and B. The analysis and 15. R. U. LEMIEUX. Can. J. Chem. 39, 116 (1961). physical properties are given in Table 3. 16. R. BROSSMER and E. ROHM. Hoppe-Seyler's Zeitsch.

Assay of Thymidylate Synthetase Phys. Chem. 348, 1431 (1967). 17. B. R. BAKER, T. J. SCHWAN, and D. V. SANTI. J.

The direct spectrophotometric assay (23) was used for ~ ~ d . them. 9, 66 (1966). determining the activity of thymidylate synthetase. The 18. J. J. Fox, N. C. YUNG, I. WEMPEN, and M. HOFFER. contents of the reaction mixture and assay conditions J. Amer. Chem. Soc. 83, 4066 (1961). were the same as described by Wahba and Friedkin (23), 19. J. FARKAS, L. KAPLAN, and J. J. FOX. J. Org. except that deoxyuridylate was used at a concentration Chem. 29, 1469 (1964). of 50 pm. For inhibition experiments, the enzyme was 20. R. LEMrEUX and M. H°FFER. Can. J. Chem. 397

pre-incubated with each compound for 10 min and then (Ig6').

other co-factors were added. The reaction was run at 21' L' J' BELLAM'. The infrared 'pectra of molecules. 2nd ed. John Wiley and Sons, Inc., New

room temperature. York, N.Y. 1958. p. 115 and 334. 22. M. HOFFER. Chem. Ber. 93, 277 (1960).

The authors gratefully thank Drs. J. C. MacDonald 23. A. J. WAHBA and M. FRIEDKIN. J. Biol. Chem. (PRL) for allowing use of the polarimeter, J. R. Dimmock 236, PC1 1 (1961).

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