Synthesis of 2-ethylthio-6-(3-hydroxy-1,2-O- isopropylidenepropyl)pteridin-4(3H)-one

Article abstract of DOI:10.3987/COM-00-8881

A strategy has been described for the synthesis of 2-ethylthio-6-(3- hydroxy-1,2-O-isopropylidenepropyl)pteridin-4(3H)-one, which can be used as a useful intermediate for the conversion of neopterin to biopterin.

Full text of DOI:10.3987/COM-00-8881

HETEROCYCLES, Vol.53, No.7, 2000, p.1551 - 1557, Received, 23rd February, 2000
SYNTHESIS OF 2-ETHYLTHIO-6-(3-HYDROXY-1,2-O-
ISOPROPYLIDENEPROPYL)PTERIDIN-4(3H)-ONE
Yonghan Kang,*1 Seungjin Kim,1 Youngchan Myoung,2 and Daejin Baek3
1Department of Chemistry, Hanyang University, Ansan, Kyunggi-do 425-791,
Korea, 2Department of Chemical Science and Technology, Agency for Technology
3
and Standards, MOCIE, Kwacheon, Kyunggi-do, 427-010, Korea, Department of
Chemistry, Hanseo University, Seosan, Chungnam 356-820, Korea
Abstract - A strategy has been described for the synthesis of 2-ethylthio-6-(3-
hydroxy-1,2-O-isopropylidenepropyl)pteridin-4(3H)-one, which can be used as a
useful intermediate for the conversion of neopterin to biopterin.
Pterin derivatives are natural materials biosynthesized from guanosinetriphosphate (GTP) in biological
systems and can be isolated in all living things. Biopterin is reported as 6-(D-erythro-1,2-
dihydroxypropyl)pterin isolated from human urine.1 Its biosynthetic pathways have drawn attention since
they play an important role in amino acid hydroxylation as a tetrahydroform. The atypical phenylketouria
disease is caused by a defect in the biosynthetic process for tetrahydrobiopterin.2,3 Neopterin has been
identified as 6-(D-erythro-1,2,3-trihydroxypropyl)pterin in the larva of bee, royal jelly4,5 and human
urine.6 Wachter and his coworkers report that the urine of tumor patients had a higher fluorescent
composition than normal and this was linked to a neopterin.7 It is assumed that neopterin is produced in
large amounts specifically from macrophages upon stimulation with interferon-gamma.8 Recently, the
study of the relation between cancer and neopterin and its application to cures for cancer or viral diseases
have been the focus of many studies.
O
OH OH
O
OH OH
H
N
N
CH3
H
N
N
CH2OH
N
N
H
H
H
H
H2N
N
H2N
N
Neopterin
Biopterin
Neopterin is synthesized by the Viscontini reaction of 2,5,6-triaminopyrimidin-4(3H)-one (1) with D-
arabinose phenylhydrazone, with more than 80% yield,9 biopterin was obtained from the reaction of 1

with 5-deoxy-L-arabinose phenylhydrazone in 40% yield.10 In general, a high yield of sugar hydrazone
was produced from the simple reaction of sugar with phenylhydrazine, however, 5-deoxy-L-arabinose
phenylhydrazone was obtained through several steps.
Neopterin and biopterin are formed by the respective addition of 1,2,3-trihydroxypropyl group and 1,2-
dihydroxypropyl group to the 6-position of the pterin. Therefore, the conversion of the terminal
hydroxymethy group to the methyl group in neopterin is strongly sought to improve the synthetic process
of biopterin. The selective protection to the 1,2-dihydroxyl groups, the secondary alcohols, is necessary
for the conversion of neopterin into biopterin. There were a few attempts to protect the two secondary
hydroxyl groups of the neopterin derivatives.11-13 However, the trials failed because the primary 3-OH in
the 1,2,3-trihydroxypropyl group has a higher chemical reactivity than the 1,2-dihydroxyl groups.
In general, pterin [2-aminopteridin-4(3H)-one] derivatives are insoluble in water or organic solvents. To
induce to selective protecting group at the 1,2-dihydroxyl group of secondary alcohol in neopterin, a 2-
ethylthiopterin derivative related to neopterin was synthesized. 2-Alkylthiopteridin-4(3H)-one provided
better solubility than ordinary pterins and could be converted easily to the corresponding pterins by
replacement reaction of 2-alkylthio group with amine nucleophiles.14
6-Amino-2-ethylthiopyrimidin-4(3H)-one (3) was prepared from the reaction of 6-amino-2(1H)-thio-
pyrimidin-4(3H)-one (2)15 with diethyl sulfate in KOH(aq) in 70% yield. The treatment of 3 with sodium
nitrite in acidic medium provided 6-amino-2-ethylthio-5-nitrosopyrimidin-4(3H)-one (4) as a dark blue
powder in 82% yield. We obtained 5,6-diamino-2-ethylthiopyrimidin-4(3H)-one (5), from the reduction
of nitrosopyrimidine (4) with hydrosulfite as a colorless needle in 60 % yield (Scheme 1).
Scheme 1
O
N
O
O
O
H
S
H
H
NO
H
NH2
NH2
c
a
b
N
N
N
N
NH2
CH3CH2S
N
NH2
CH3CH2S
N
NH2
CH3CH2S
N
H
2
3
4
5
a: 10% KOH(aq), (CH3CH2O)2SO2 b: i) NaNO2 / H2O ii) AcOH c: Na2S2O4, 60-70 oC
5,6-Diamino-2-ethylthiopyrimidin-4(3H)-one (5) was reacted with the D-arabinose phenylhydrazone (6),
which was obtained from the reaction of D-arabinose with phenylhydrazine in acidic medium by a
method similar to the reported procedure,16 and the diastereomeric mixture of 8-ethylthio-3,4,4a(R,S),5,10,
10a(S,R)-hexahydro-3,4-dihydroxy-2H-pyrano[3,2-g]pteridin-6(7H)-one (7) was separated (Scheme 2).
The 1H NMR spectrum of 7 showed the presence of two D2O exchangable N10-H peaks17 at 7.74 (J = 4.8

Hz) and 7.24 (J = 4.0 Hz) ppm, which were not found in the compound (8). Diastereomeric mixture (7)
was converted to 2-ethylthio-6-(D-erythro-1,2,3-trihydroxypropyl)pteridin-4(3H)-one (8) by handling
with O2 in a pH 10 buffer solution in 64% yield. In the 1H NMR spectrum of 8, the aromatic proton at C7
was identified at 8.70 ppm. Without separation of the diastereomeric mixture, 7 was reacted with 2,2-
1
dimethoxypropane in the presence of p-toluenesulfonic acid as a catalyst, and the H NMR spectrum of
the isolated product (9) showed the isopropylidene group (1.25 and 1.43 ppm) and N10-H peaks (δ 7.43
and 7.81 ppm). Compound (9), therefore, was expected to be 8-ethylthio-3,4,4a(R,S),5,10,10a(S,R)-
hexahydro-3,4-O-isopropylidene-2H-pyrano[3,2-g]pteridin-6(7H)-one (Scheme 2).
Scheme 2
N
NH Ph
NH NH Ph
O
O
N
H
N
OH
H
H
H
H
OH
H
5
a
N
OH
H
H
HO
HO
OH
H
OH
H
OH
CH3CH2S
N
HOH2C
HOH2C
6
O
N
OH
OH
O
N
H
N
OH
O
H
H
H
N
N
H
OH
N
OH
b
N
CH3CH2S
CH3CH2S
N
H
8
7
c
O
N
O
O
O
N
H
N
O
O
H
H
H
N
N
H
O
b
N
N
CH2OH
CH3CH2S
CH3CH2S
N
H
10
9
a
aq b
N
: 50% CH3OH( ), 5 HCl, HSCH2CH2OH, N2, reflux, 1 h : O2, pH 10, rt
c: 2,2-dimethoxypropane, p-TsOH, DMF, rt, 16 h
The oxidation of compound (9) was carried out by a procedure similar to the preparation of 8. The
obtained product (10) was characterized as 2-ethylthio-6-(D-erythro-3-hydroxy-1,2-O-isopropylidene-
propyl)pteridin-4(3H)-one by the 1H NMR spectrum and elemental analysis.

EXPERIMENTAL
General Procedures. All chemicals used were purchased from commercial sources with an analytical
grade. The solvents were purified by distillation and the other reagents were used without further
1
purification. H-NMR spectra were measured at 300 MHz using a Varian Unity Plus 300 spectrometer.
The chemical shift values are reported as δ downfield from TMS as an internal standard. Melting points
were determined on a Büchi 530 melting point apparatus and are uncorrected. Elemental analyses were
performed by Fisons EA 1108.
6-Amino-2-ethylthiopyrimidin-4(3H)-one (3)
To a well-stirred solution of 6-amino-2(1H)-thiopyrimidin-4(3H)-one (2)15 (25 g, 0.17 mol) in 10%
aqueous KOH solution (100 mL), diethyl sulfate (25 mL, 0.17 mol) at ambient temperature was added
dropwise. After being stirred for 2 h at ambient temperature, the precipitated solids were filtered off,
washed with water and ethanol, and recrystallized from ethanol to give 3 as a colorless needle (20.5 g,
70%): mp 205-206 oC; 1H NMR (DMSO-d6) 1.25 (3H, t, J = 7.14 Hz, -SCH2-CH3), 3.02 (2H, q, J = 7.32
Hz, -SCH2-CH3), 4.88 (1H, s, C5-H), 6.41 (2H, br s, C6-NH2), 11.22 (1H, br s, N3-H); Anal. Calcd for
C6H9N3OS: C, 42.09; H, 5.30; N, 24.54. Found C, 41.72; H. 5.29; N, 24.41.
6-Amino-2-ethylthio-5-nitrosopyrimidin-4(3H)-one (4)
Compound (3) (20 g, 0.12 mol) and NaNO2 (5.1 g, 0.12 mol) were suspended in water (350 mL) and
treated with acetic acid (16 mL). The reaction mixture was stirred at ambient temperature for 8 h. The
precipitated solids were filtered off and washed with water and ethanol, and recrystallized from water to
give 4 as a dark blue powder (19.7 g, 82%): mp 198 oC (decomp); 1H NMR (DMSO-d6) 1.29 (3H, t, J =
7.14 Hz, -SCH2-CH3), 3.15 (2H, q, J = 7.32 Hz, -SCH2-CH3), 9.05 and 11. 24 (2H, 2 s, C6-NH2), 12.67
(1H, br s, N3-H); Anal. Calcd for C6H8N4O2S: C, 35.99; H, 4.03; N, 27.98. Found C, 35.76; H, 4.33; N,
27.71.
5,6-Diamino-2-ethylthiopyrimidin-4(3H)-one (5)
o
To a suspension of compound (4) (10 g, 0.05 mol) in water (400 mL) at 60-70 C, Na2S2O4 (10 g, 0.06
mol) was added until a clear solution was obtained. The resulting solution was cooled in an ice bath. The
precipitated solids were filtered off and washed with water and ethanol, and recrystallized from water to
o
1
give 5 as a colorless needle (5.66 g, 60%): mp 172 C (decomp); H NMR (DMSO-d6) 1.23 (3H, t, J =
6.39 Hz, -SCH2-CH3), 3.01 (2H, q, J = 7.30 Hz, -SCH2-CH3), 5.6 (2H, br s, C5-NH2), 6.82 (2H, br s, C6-
NH2), 11.02 (1H, br s, N3-H); Anal. Calcd for C6H10N4OS: C, 38.70; H, 5.41; N, 30.08. Found C, 38.60;

H, 5.49; N, 29.79.
8-Ethylthio-3,4,4a(R,S),5,10,10a(S,R)-hexahydro-3,4-dihydroxy-2H-pyrano[3,2-g]pteridin-6(7H)-
one (7).
To a suspension of compound (5) (3.72 g, 0.02 mol) in aqueous methanol (50%, 360 mL) stirred under
nitrogen, D-arabinose phenylhydrazone (6)16 (6.5 g, 0.03 mol), 5 N HCl (6 mL), and mercaptoethanol
(1 mL) were added successively, and the resulting mixture was heated at reflux for 1 h and then cooled to
ambient temperature. The precipitated solids were filtered off and washed with water and ethanol to give
the diastereomeric mixture (7) as a pale yellow solid (3.81 g, 63%), which was used in the following
reaction without further purification and separation: 1H NMR (DMSO-d6) 3.30~1.24 (C4-H, C3-H, C2-H,
and -SCH2CH3 of major and minor isomers), 3.06 (s, 4a-H of minor isomer), 3.10 (s, 4a-H of major
isomer), 4.21 (br s, N5-H of minor isomer), 4.26 (d, J = 4.27 Hz, 3-OH of minor isomer, D2O
exchangable), 4.40 (br s, N5-H of major isomer), 4.52 (s, 10a-H of minor isomer), 4.62 (d, J = 4.51 Hz, 3-
OH of major isomer, D2O exchangable), 4.79 (d, J = 4.16 Hz, 4-OH of major isomer, D2O exchangable),
4.81 (s, 10a-H of major isomer), 5.41 (d, J = 4.36 Hz, 4-OH of major isomer), 7.74 (d, J = 4.8 Hz, N10-H
of major isomer), 7.24 (d, J = 4.0 Hz, N10-H of minor isomer), 12.11 (br s, N7-H).
2-Ethylthio-6-(D-erythro-1,2,3-trihydroxypropyl)pteridin-4(3H)-one (8)
A suspension of diastereomixture (7) (5.26 g, 17.5 mmol) in buffer solution (pH 10, 300 mL) was
protected from light with aluminum foil and stirred under oxygen atmosphere until the starting material
completely disappeared. The resulting solution was filtered, and then the pH of the filtrate was adjusted to
6 by addition of ambelite IR 120(H+). After the resin was filtered out, the filtrate was evaporated in vacuo
to dryness. Recrystallization of the residual solid from a mixture of methanol/acetone (50:600, v/v)
afforded 8 as a pale yellow powder (3.3 g, 64%): mp 162 oC (decomp); 1H NMR (DMSO-d6) 1.28 (3H, t,
J = 7.32 Hz, S-CH2CH3), 3.05 (2H, q, J = 7.32 Hz, S-CH2CH3), 3.42(2H, m, C3′-H2), 3.76 (2H, m, C2′-H
and C3′-OH), 4.68 (1H, d, J = 5.67 Hz, C1′-H), 4.82 (1H, s, C2′-OH, D2O exchangable), 5.62 (1H, s, C1′-
OH, D2O exchangable), 8.70 (1H, s, C7-H), 12.4 (1H, br s, N3-H, D2O exchangable); Anal. Calcd for
C11H14N4O4S: C, 44.29; H, 4.73; N, 18.78. Found C, 43.98; H, 4.97; N, 18.51.
8-Ethylthio-3,4,4a(R,S),5,10,10a(S,R)-hexahydro-3,4-O-isopropylidene-2H-pyrano[3,2-g]pteridin-
6(7H)-one (9)
To a suspension of compound (7) (3.72 g, 0.02 mol) in dry DMF (150 mL), 2,2-dimethoxypropane (6.4
mL, 0.05 mol) and p-toluenesulfonic acid monohydrate (0.76 g, 4 mmol) were added, and the resulting
mixture was stirred at ambient temperature for 16 h. The reaction mixture was treated with a small

amount of sodium carbonate powder. After the insoluble solids were filtered off, the filtrate was
evaporated in high vacuo to dryness. Dichloromethane and water were then added to the residual solid,
and the organic layer was washed with water twice. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column chromatography over silica gel eluting with
dichloromethane: methanol (35:1, v/v) to give 9 (4.4 g, 74%) as a pale yellow solid, which was used in
the following reaction without further separation: 1H NMR (DMSO-d6) 1.28 and 1.43 (6H, 2 s, 2 x CH3 of
isopropylidene), 1.25~3.27 (C4-H, C3-H, C2-H, and -SCH2CH3 of major and minor isomers), 3.06 (s, 4a-
H of minor isomer), 3.10 (s, 4a-H of major isomer), 4.45 (br s, N5-H of minor isomer), 4.78 (s, 10a-H of
minor isomer), 4.81 (br s, N5-H of major isomer), 5.42 (s,10a-H of major isomer), 7.81 (br s, N10-H of
major isomer), 7.43 (br s, N10-H of minor isomer), 11.95 (br s, N7-H).
2-Ethylthio-6-(D-erythro-3-hydroxy-1,2-O-isopropylidenepropyl)pteridin-4(3H)-one (10)
Following the procedure for prepararing 8 from 7, the product (10) was obtained from 9 in 68% yield: mp
110 oC (decomp); 1H NMR: (DMSO-d6) δ 1.27 (3H, t, J = 7.34 Hz, -S-CH2CH3), 1.41 and 1.59 (2 s, CH3
x 2 in isopropylidene), 3.00 (4H, m, S-CH2CH3 and C3′-2H), 4.47 (1H, m, C2′-H), 4.68 (1H, br s, C3′ OH,
D2O exchangable), 5.36 (1H, d, J = 7.32 Hz, C1′-H), 8.60 (1H, s, C7-H), 12.6 (1H, br s, N3-H, D2O
exchangable); Anal. Calcd for C14H18N4O4S: C, 49.69; H, 5.36; N, 16.56. Found C, 49.36; H, 5.61; N,
16.41.
ACKNOWLEDGMENT
The author wishes to acknowledge the financial support of Hanyang University, Korea, made in the
program year of 1999.
REFERENCES
1. S. Kaufmann, in ‘Unconjugated Pterins in Neurobiology’, ed. by W. Lovenberg and R. Levine,
Taylor & Francis, London, 1987, p. 1.
2. S. Kaufmann, N. Holtzman, S. Milstein, I. J. Buther, and A. Krumholtz, New Engl. J. Med., 1975,
293, 785.
3. T. Nagatsu, T. Yamaguchi, T. Kato, T. Sugimoto, S. Matsuura, M. Akino, I. Nagatsu, R. Iizuka, and
H. Naraoyashi, Clin. Chim. Acta, 1981, 109, 305.
4. H. Rembold and L. Buschmann, Ann., 1963, 662, 72.
5. H. Rembold and L. Buschmann, Chem. Ber., 1963, 96, 1406.
6. A. Sakuri and M. Goto, J. Biochem. (Tokyo), 1967, 61, 142.

7. H. Wachter, K. Grassmayer, W. Gutter, A. Hausen, and G. Sallaberger, Wien, Klin. Wochenschr.,
1972, 84, 586.
8. L. Leohirun, P. Thuvasethakul, T. Pholcharoen, and S. Lapanant, Pteridines, 1993, 4, 67.
9. M. Viscontini, R. Provenzale, S. Ohlgart, and J. Mallevialle, Helv. Chim. Acta, 1970, 53, 1202.
10. M. Viscontini, in ‘Biochemical and Clinical Aspects of Pteridines’, ed. by H. Wachter, H. Ch.
Curtius, and W. Pfleiderer, Walter de Gruyter, Berlin, 1984, Vol. 3, p. 19.
11. A. Kaiser and H. P. Wessel, Helv. Chim. Acta, 1987, 70, 766.
12. Y. Kang, R. Soyka, W. Hutzenlaub, M. Wiesenfeldt, W. Leskopf, and W. Pfleiderer, in ‘Chemistry
and Biology of Pteridines 1986’, ed. by B. Cooper and V. Whitehead, Walter de Gruyter, Berlin,
1986, p. 31.
13. R. Soyka and W. Pfleiderer, in ‘Biochemical and Clinical Aspects of Pteridines’, ed by H. Wachter,
H. Ch. Curtius, and W. Pfleiderer, Walter de Gruyter, Berlin, 1985, Vol. 4, p. 33.
14. T. Sugimoto, S. Matsuura, and T. Nagatsu, Bull. Chem. Soc. Japan, 1980, 53, 2344.
15. W. Traub, Ann., 1904, 331, 64.
16. T. Sugimoto and S. Matsuura, Bull. Chem. Soc. Japan, 1975, 48, 3767.
17. R. Soyka, W. Pfleiderer, and R. Prewo, Helv. Chim. Acta, 1990, 73, 808.