Synthesis of 1-Hydroxyalkyl-3-Substituted Ureas and Thioureas, Substrates for Alcohol Dehydrogenase

Article abstract of DOI:10.1023/A:1011385405611

A series of 1-(2-hydroxyethyl)- and 1-(3-hydroxyethyl)-3-substituted ureas and thioureas were synthesized. 1-(3-Hydroxyethyl)-3-acylthioureas were shown to be specific substrates for alcohol dehydrogenase in vitro.

Full text of DOI:10.1023/A:1011385405611

Russian Journal of Bioorganic Chemistry, Vol. 27, No. 3, 2001, pp. 191–194. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 3, 2001, pp. 217–220.
Original Russian Text Copyright © 2001 by Leonov, Shcherbakov, Devichensky, Kryukova, Vorontsov, Kuznetsov, Kryukov.
Synthesis of 1-Hydroxyalkyl-3-Substituted Ureas and Thioureas,
Substrates for Alcohol Dehydrogenase
1
O. N. Leonov*, V. M. Shcherbakov*, V. M. Devichensky*, L. Yu. Kryukova**,
E. A. Vorontsov**, S. L. Kuznetsov**, and L. N. Kryukov**
*Institute of Biological Medicine, ul. Akademika Oparina 4, Moscow, 117815 Russia
**Center of Medical, Biological, and Ecological Problems, Russian Academy of Natural Sciences,
Simferopolskii bulvar 8, Moscow, 113149 Russia
Received November 1, 2000; in final form, January 11, 2001
Abstract—A series of 1-(2-hydroxyethyl)- and 1-(3-hydroxyethyl)-3-substituted ureas and thioureas were syn-
thesized. 1-(3-Hydroxyethyl)-3-acylthioureas were shown to be specific substrates for alcohol dehydrogenase
in vitro.
Key words: alcohol dehydrogenase, substrates; thiourea, 1,3-disubstituted; urea, 1,3-disubstituted
1
INTRODUCTION
derivatives (I)–(III) to hydroxypropylurea derivatives
(IV)–(XII) and from ureas (I)–(VII) to thioureas
(VIII)–(X) and, further, to acylthioureas (XI) and
(XII). The properties of 1-(3-hydroxypropyl)-3-(4-flu-
orobenzoyl)thiourea (XII) are of particular importance.
Thus, our results suggest that a further search for
highly specific ADH substrates in the series of 1-(3-
hydroxypropyl)-3-acylthioureas is promising, and
these compounds might be used for the design of new
medicines with the corresponding activity profile.
Despite the medical achievements in the treatment
of chronic alcoholism, the assortment of medicines for
its correction is limited [1, 2]. Therefore, the search for
the compounds affecting the ethanol biotransformation
remains topical.
In this work, 1-hydroxyalkyl-3-substituted ureas
and thioureas, which contain a primary hydroxyl group
and structurally resemble the drug disulfiram
(teturamin) [2, 3], became the subjects under study.
2
EXPERIMENTAL
RESULTS AND DISCUSSION
The following reagents were used: 2-aminoethanol,
3-aminopropanol, benzoyl chloride, and 4-fluoroben-
zoyl chloride from Merck (Germany); n-butyl, tert-
butyl, 3,4-dichlorophenyl, and phenyl isocyanates and
ethyl, 4-fluorophenyl, and 2,5-xylyl isothiocyanates
from Fluka (Switzerland). Other reagents and solvents
were of domestic production.
Ureas (I)–(VII) and thioureas (VIII)–(X) were
obtained by interaction of the corresponding
iso(thio)cyanate with 2-aminoethanol and/or 3-amino-
propanol as shown in Scheme 1. The synthesis of 1-(3-
hydroxypropyl)-3-acylthioureas (XI) and (XII) is
described in Scheme 2.
The kinetic parameters of the oxidation reaction of
(I)–(XII) with a typical isoform of ADH from human
liver were determined in experiments in vitro [4]. The
ratio of the maximal oxidation rate (relative to ethanol,
Vrel) to the Michaelis constant (Km) was chosen as a
parameter that, at low concentrations of the compounds
tested, can characterize the substrate properties of the
compounds and appeared to reflect the in vivo situation
[5] (see table).
Melting points were determined on a Mettler FP62
device (Switzerland).
1H NMR spectra were obtained on a Bruker WD-
80SY (Germany) spectrometer at the working fre-
quency of 80 MHz in DMSO-d6 (Fluka, Switzerland).
Chemical shifts of protons were measured relative to
the residual signal of solvent (δ 2.49 ppm) and are
given in δ scale.
For the quantitative elemental analysis, a Carlo Erba
1106 CHN analyzer (Italy) was used.
Spectrophotometric measurements were performed
on a Hitachi 557 instrument (Japan).
1-(2-Hydroxyethyl)-3-n-butylurea (I). n-Butyl
isocyanate (3.50 g, 35.3 mmol) was added at vigorous
stirring to a solution of 2-aminoethanol (2.16 g,
One can see from the table that the compounds
under study tend to increase their substrate specificities
to ADH when proceeding from hydroxyethylurea
1
To whom correspondence should be addressed; phone/fax: +7
(095) 482-2091; e-mail: doctor.bio@mtu-net.ru.
2
Abbreviations: ADH, alcohol dehydrogenase from human liver
+
(alcohol:NAD oxidoreductase, EC 1.1.1.1).
1068-1620/01/2703-0191$25.00 © 2001 MAIK “Nauka /Interperiodica”

192
LEONOV et al.
X
+
R N C X H2N (CH2)n OH
R NHCNH (CH2)n OH
(I)–(X)
R
X
n
R
X
n
n-C4H9
t-C4H9
C6H5
O
O
O
O
O
2
2
2
3
3
(I)
C6H5
O
O
S
3
3
3
3
3
(VI)
(VII)
(VIII)
(IX)
(X)
(II)
(III)
(IV)
(V)
3,4-Cl2C6H3
C2H5
n-C4H9
t-C4H9
4-FC6H4
S
2,5-(CH3)2C6H3
S
Scheme 1.
35.4 mmol) in dry ether (15 ml). The reaction mixture
was stirred for 1 h, and urea (I) was filtered and recrys-
tallized from 95% ethanol; yield 5.12 g (91%); mp 66–
67°ë (ethanol); 1H NMR: 0.90 (3 H, t, CH3), 1.40 (2 H,
m, CH2), 1.65 (4 H, m, 2 CH2), and 3.40 (4 H, m,
ëç2N + ëç2é). Found, %: C 52.54, H 10.17, N 17.19.
Calculated for C7H16N2O2, %: C 52.48, H 10.07, N
17.48.
1-(2-Hydroxypropyl)-3-n-butylurea (IV) was
similarly obtained from n-butyl isocyanate (47.0 g,
47.4 mmol) and 3-aminopropanol (3.56 g, 47.4 mmol);
1
yield 7.76 g (94%); mp 66–67°ë (ethanol); H NMR:
0.90 (3 H, t, CH3), 1.40 (2 H, m, CH2), 1.65 (4 H, m, 2
CH2), and 3.15 and 3.60 (6 H, m, 2 ëç2N + ëç2é).
Found, %: C 55.21, H 10.32, N 15.96. Calculated for
C8H18N2O2, %: C 55.15, H 10.41, N 16.08.
1-(2-Hydroxypropyl)-3-tert-butylurea (V) was
similarly obtained from tert-butyl isocyanate (2.00 g,
20.2 mmol) and 3-aminopropanol (0.80 g, 10.7 mmol);
amorphous; yield 2.31 g (78%); 1H NMR: 1.30 (9 H, s,
3 CH3), 1.65 (2 H, m, CH2), and 3.15 and 3.60 (4 H, 2t,
CH2N + CH2O). Found, %: C 55.31, H 10.25, N 15.84.
Calculated for C8H18N2O2, %: C 55.15, H 10.41, N
16.08.
1-(2-Hydroxyethyl)-3-tert-butylurea (II) was sim-
ilarly obtained from tert-butyl isocyanate (1.68 g,
16.9 mmol) and 2-aminoethanol (1.03 g, 16.9 mmol);
amorphous; yield 2.50 g (93%); 1H NMR: 1.30 (9 H, s,
3 CH3), 3.15, and 3.65 (4 H, 2 m, CH2N and CH2O).
Found, %: C 52.61, H 9.89, N 17.25. Calculated for
C7H16N2O2, %: C 52.48, H 10.07, N 17.48.
1-(2-Hydroxyethyl)-3-phenylurea (III) was simi-
larly obtained from phenyl isocyanate (8.50 g,
71.4 mmol) and 2-aminoethanol (4.36 g, 71.4 mmol);
1-(3-Hydroxypropyl)-3-phenylurea (VI) was sim-
ilarly obtained from phenyl isocyanate (1.59 g,
13.2 mmol) and 3-aminopropanol (1.00 g, 13.3 mmol);
yield 2.13 g (82%); mp 113–114°C (ethanol); 1H NMR:
1.74 (2 H, m, CH2), 3.30 and 3.62 (4 H, 2m, CH2N +
CH2O), and 7.20 (5 H, m, ë6ç5). Found, %: C 62.03, H
7.12, N 14.28. Calculated for C10H14N2O2, %: C 61.84,
H 7.27, N 14.42.
1
yield 11.92 g (93%); mp 123°ë (ethanol); H NMR:
3.60 (4 H, m, CH2N and CH2O) and 7.20 (5 H, m
C6H5). Found, %: C 60.16, H 6.47, N 15.35. Calculated
for C9H12N2é2, %: C 59.99, H 6.71, N 15.55.
1-(3-Hydroxypropyl)-3-(3,4-dichlorophenyl)urea
(VII) was similarly obtained from 3,4-dichlorophenyl
isocyanate (2.00 g, 10.6 mmol) and 3-aminopropanol
(0.80 g, 10.7 mmol); yield 1.65 g (59%); 1H NMR: 1.75
(2 H, m, CH2), 3.25 and 3.65 (4 H, 2m, CH2N + CH2O),
and 7.50 (3 H, m, C6H3). Found, %: C 45.93, H 4.51, N
10.44. Calculated for C10H12Cl2N2O2, %: C 45.65, H
4.60, N 10.65.
O
O
ZNCS
H2N–(CH2)3–OH
R'–C–Cl
R'–C–N=C=S
O
S
R'–C–NHCNH–(CH2 )3–OH
R'
Z
1-(3-Hydroxypropyl)-3-ethylthiourea (VIII) was
similarly obtained from ethyl isothiocyanate (2.00 g,
23.0 mmol) and 3-aminopropanol (1.78 g, 23.7 mmol);
C6H5
NH4
Na
(XI)
(XII)
4-FC6H4
1
yield 3.21 g (86%); mp 86–87°ë (ethanol); H NMR:
1.15 (3 H, t, CH3), 1.75 (2 H, m, CH2), and 3.55 (4 H,
m, CH2N + CH2O). Found, %: C 44.51, H 8.80, N
Scheme 2.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001

SYNTHESIS OF 1-HYDROXYALKYL-3-SUBSTITUTED UREAS
193
Kinetic parameters of the oxidation reaction of 1,3-disubstituted (thio)ureas catalyzed by ADH
Concentration range,
Compound
Vrel
Km, mM
Vrel/Km
mM
(I)
0.04–0.6
0.04–0.6
0.1–1.4
0.04–0.6
0.5–2.4
0.05–0.3
0.1–1.6
0.4–4.1
0.04–0.4
0.1–0.5
0.1–0.5
0.1–1.6
0.08
0.07
0.11
0.12
0.19
0.16
0.23
0.18
0.23
0.18
0.79
2.71
0.21
0.27
0.46
0.16
1.18
0.10
0.52
1.37
0.07
0.20
0.21
0.54
0.38
0.26
0.24
0.75
0.16
1.60
0.44
0.13
3.30
0.90
3.76
5.00
(II)
(III)
(IV)
(V)
(VI)
(VII)
(VIII)
(IX)
(X)
(XI)
(XII)
17.67. Calculated for C6H14N2OS, %: C 44.41, H 8.70, (3.12 g, 38.5 mmol), 4-fluorobenzoyl chloride (6.10 g,
38.5 mmol) and 3-aminopropanol (2.90 g, 38.6 mmol);
yield 4.31 g (44%); mp 85–86°ë (acetone–water); H
NMR: 1.90 (2 H, m, CH2), 3.80 (4 H, m, CH2N + CH2O),
and 7.50 (4 H, m, C6H4). Found, %: C 51.13, H 5.08, N
10.54. Calculated for ë11ç13FN2O2S, %: C 51.55, H
5.11, N 10.93.
N 17.26.
1
1-(3-Hydroxypropyl)-3-(4-fluorophenyl)thiourea
(IX) was similarly obtained from 4-fluorophenyl
isothiocyanate (2.50 g, 16.3 mmol) and 3-aminopro-
panol (1.22 g, 16.2 mmol); yield 3.20 g (86%); mp
1
112–113°ë (ethanol); H NMR: 1.80 (2 H, m, CH2),
3.80 (4 H, m, CH2N + CH2), and 7.15 (4 H, m, C6H4).
Found, %: C 52.90, H 5.63, N 12.13. Calculated for
C10H13FN2OS, %: C 52.61, H 5.74, N 12.27.
Substrate properties of (I)–(XII) toward ADH.
Alcohol dehydrogenase was isolated from a homoge-
nate of the human liver as described in [6]. The homo-
genate was centrifuged at 30000 g for 10 min, and the
supernatant was once more centrifuged at 105000 g for
120 min and lyophilized. The residue was dissolved in
20 mM Tris-HCl buffer, pH 8.2, and applied onto a col-
umn (2.3 × 50 cm) packed with DEAE cellulose equil-
ibrated in the same buffer. The column was eluted with
a linear gradient of Tris-HCl (pH 7.4) from 0 to 1 M,
and the fraction containing ADH (monitoring at
280 nm and by the dehydrogenase activity, eluted
within the concentration range 0.46–0.52 M) was col-
lected and lyophilized up to the final protein concentra-
tion of 1–2 mg/ml. The preparation was stored at
−80°ë.
1-(3-Hydroxypropyl)-3-(2,5-xylyl)thiourea (X) was
similarly obtained from 2,5-xylyl isothiocyanate (1.09 g,
6.68 mmol) and 3-aminopropanol (0.50 g, 6.66 mmol);
yield 1.07 g (67%); mp 97°ë (ethanol); 1H NMR: 1.75
(2 H, m, CH2), 2.20 and 2.30 (6 H, 2 s, 2 CH3), 4.60
(4 H, m, CH2N + CH2O), and 7.05 (3 H, m, C6H3).
Found, %: C 60.29, H 7.67, N 11.49. Calculated for
C12H18N2OS, %: C 60.47, H 7.61, N 11.75.
1-(3-Hydroxypropyl)-3-benzoylthiourea
(XI).
Benzoyl chloride (14.06 g, 100 mmol) was added to a
suspension of ammonium rhodanide (7.61 g, 100 mmol)
in hot anhydrous acetone (25 ml). The mixture was
refluxed for 5 min, cooled to room temperature, and fil-
tered. The filtrate was added dropwise to a solution of
3-aminopropanol (7.51 g, 100 mmol) in acetone
The ADH activity was spectrophotometrically deter-
(25 ml); the mixture was mixed for 1 h and then poured mined according to the increase in the optical density at
into 10 vols of water. The precipitate of (XI) was sepa-
rated and recrystallized from aqueous acetone to give
(XI); yield 17.60 g (74%); mp 83–84°ë (acetone–
340 nm. The reaction was initiated by addition of etha-
nol or one of (I)–(XII), dissolved in DMSO to final con-
centration given in the table, to a solution (3 ml) con-
taining 0.1 mg/ml ADH and saturating concentration of
NAD+ (3 × 10–3 M) in 50 mM pyrophosphate buffer
(pH 7.4). To calculate the amount of NADH formed, the
molar extinction coefficient ε = 6210 å–1 cm–1 was
used. The values of Km and Vrel were determined graph-
1
water); H NMR: 1.90 (2 H, m, CH2), 3.70 (4 H, m,
CH2N + CH2O), and 7.70 (5 H, m, ë6ç5). Found, %: C
56.00, H 6.23, N 11.43. Calculated for C11H14N2O2S,
%: C 55.44, H 5.92, N 11.76.
1-(3-Hydroxypropyl)-3-(4-fluorobenzoyl)thiourea
(XII) was similarly obtained from sodium thiocyanate ically in the Lineweaver–Burk coordinates [7].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001

194
LEONOV et al.
3. Mashkovskii, M.D., Lekarstva XX veka (Medicines of
REFERENCES
XXI Century), Moscow: Novaya Volna, 1998, p. 108.
1. Registr lekarstvennykh sredstv Rossii, Entsiklopediya
lekarstv, 8 izd., (Russian Register of Medicinal Prepara-
tions, Encyclopedia of Drugs, 8th ed.), Krylov, Yu.F.,
Ed., Moscow: RLS-2001, 2000, p. 1237.
4. Fon Warburg, J.P., Papenberg, J., and Aebi, H., Can.
J. Biochem., 1965, vol. 43, pp. 889–895.
5. Entin, G.M., Lechenie alkogolizma (Treatment of Alco-
holism), Moscow: Meditsyna, 1990.
6. Yin, S.J., Bosron, W.S., Magnes, L.J., et al., Biochemis-
try, 1984, vol. 23, pp. 5847–5853.
2. Mashkovskii, M.D., Lekarstvennye sredstva, 12 izd.
(Medicinal Preparations, 12th ed.), vol. 2, Moscow:
Meditsyna, 1993, p. 239.
7. Lineweaver, H. and Burk, D., J. Am. Chem. Soc., 1934,
vol. 56, pp. 658–660.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001