Investigation of the polarographic properties and potential carcinogenity of some hydroxyurea derivatives by DC polarography

Article abstract of DOI:10.1135/cccc19960656

Polarographic reduction was studied for a series of 7 urea derivatives and the results were used to assess their potential carcinogenity. The polarographic reduction was examined in absolutely anhydrous dimethylformamide by DC polarography. In the conditions applied, the majority of the compounds was reduced within a single two-electron step, only biuret and its formyl derivative were reduced in two one-electron steps. The potential carcinogenity of the substances was assessed based on the tg a value of the slope of dependence of the polarographic wave height on the concentration of α-lipoic acid added as a test substance. For hydroxyurea, which is the only substance in this series for which a carcinogenic activity has been demonstrated, the tg a parameter attained a value of 0.290. Still higher values were obtained for the formyl derivatives - formylbiuret (0.362) and 2-carbamoyl-1-formylguanidine (0.510). So high tg a values warn of a significant potential carcinogenity. The other substances studied exhibited considerably lower tg α values, indicating that their potential carcinogenity will be low.

Full text of DOI:10.1135/cccc19960656

656
Novotny, Vachalkova, Piskala:
INVESTIGATION OF THE POLAROGRAPHIC PROPERTIES AND
POTENTIAL CARCINOGENITY OF SOME HYDROXYUREA
DERIVATIVES BY DC POLAROGRAPHY
a
a
b
Ladislav NOVOTNY , Anna VACHALKOVA and Alois PISKALA
a Cancer Research Institute,
Slovak Academy of Sciences, 812 32 Bratislava, Slovak Republic
b Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic
Received August 29, 1995
Accepted November 19, 1995
Polarographic reduction was studied for a series of 7 urea derivatives and the results were used to
assess their potential carcinogenity. The polarographic reduction was examined in absolutely anhy-
drous dimethylformamide by DC polarography. In the conditions applied, the majority of the com-
pounds was reduced within a single two-electron step, only biuret and its formyl derivative were
reduced in two one-electron steps. The potential carcinogenity of the substances was assessed based
on the tg α value of the slope of dependence of the polarographic wave height on the concentration
of α-lipoic acid added as a test substance. For hydroxyurea, which is the only substance in this series
for which a carcinogenic activity has been demonstrated, the tg α parameter attained a value of
0.290. Still higher values were obtained for the formyl derivatives – formylbiuret (0.362) and 2-car-
bamoyl-1-formylguanidine (0.510). So high tg α values warn of a significant potential carcinogenity.
The other substances studied exhibited considerably lower tg α values, indicating that their potential
carcinogenity will be low.
Key words: Urea derivatives; DC polarography; Carcinogenity.
Hydroxyurea (2) is a derivative of urea (1) exhibiting antineoplastic effects, also with
respect to neoplasms of human origin1–3. Its routine clinical use, however, is limited to
chronic myeloid leukemia4. In cells, hydroxyurea blocks the conversion of ribonucleo-
tides to deoxyribonucleotides, a process which is catalyzed by the enzyme ribonucleo-
tide-reductase4.
In addition to the antineoplastic aspect, hydroxyurea and its derivatives are of interest
because they form parts of the structures of considerably more complex biologically
active molecules of synthetic 1,3,5-triazine (5-azapyrimidine) analogues of natural
components of nucleic acids5.
Since the polarographic reduction of urea derivatives in the series selected has not
been studied extensively as yet, we examined their basic polarographic characteristics
in an aprotic medium by DC polarography. Their potential carcinogenity was assessed
based on their polarographic reduction in the presence of α-lipoic acid6–9
.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Polarography of Hydroxyurea Derivatives
657
EXPERIMENTAL
Melting temperatures were determined on a Kofler stage and are not corrected. Optical rotatory
power was measured on a Perkin–Elmer 141 MCA polarimeter at 22 °C. The purity of the substances
was checked by thin layer chromatography, elemental analysis, melting temperature measurement,
and spectroscopically. UV spectra were scanned on a Beckman DU-65 spectrophotometer.
Urea and hydroxyurea were commercial chemicals obtained from Aldrich-Chemie (Steinheim,
Germany). Biuret, formylbiuret and 2-carbamoylguanidine were prepared by published procedures
(Table I). Dimethylformamide (DMF), which served as the solvent in all polarographic measure-
ments, and tetrabutylammonium perchlorate (TBAP) were products of Fluka (Switzerland). DMF was
purified by double vacuum distillation10 prior to use. The water content of DMF, which was checked
periodically by Karl Fischer dead-stop titrations, never exceeded 0.1%. α-Lipoic acid (D,L-6,8-thioctic
acid) was obtained from Koch-Light Laboratories (Colnbrook, U.K.).
Polarographic measurements were performed on a PA4 polarographic analyzer equipped with a
two-line recorder (Laboratorni pristroje, Prague, Czech Republic). The three-electrode connection
was applied, using a dropping mercury electrode as the indicator electrode (drop time 3 s, flow rate
2.27 mg s–1), a saturated calomel reference electrode (SCE), and an OH 9377 standard platinum com-
pensation electrode (Radelkis, Hungary). Polarographic reduction of the compounds was conducted in
a thermostatted vessel at 25 °C under a stream of dry nitrogen. The analyte concentration was
invariably 0.5 mmol l–1, the concentration of TBAP was 150 mmol l–1. In experiments involving
α-lipoic acid, the acid-to-analyte molar ratio was varied over the range of 0.1 : 1 to 2.4 : 1. The
reversibility or irreversibility of the reduction steps was studied by the switched curve method11. The
number of electrons involved in the reduction was obtained by logarithmic analysis of the polaro-
graphic curves as the log id/(il – id) vs E plots (id is the diffusion current at voltage E, il is the limiting
current).
2-Carbamoyl-1-formylguanidine (6)
2-Carbamoyl-1-formylguanidine was obtained as a minor fraction (8%) from the mother liquors after
hydrogenolysis of 1-(2,3,5-tri-O-benzyl-β-D-arabinosyl)-5-azacytosine using palladium on activated
carbon as the catalyst. The known 1-β-D-arabinufuranosyl-5-azacytosine12 (ara-AC) is the major product
H N CO NH R
2
R
X
1-4
H
1
2
3
4
5
6
7
8
OH
NH
2
H N CO
N
C
CO NH
2
2
NH R
CO NH CHO
5, 6
H
NH
2
CHO
H
HCOO
X
HN CO HN C
HOCH
2
O
OH
CH COO
3
NH
2
R
OH
7, 8
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

658
Novotny, Vachalkova, Piskala:
of this reaction, conducted in the presence of hydrogen chloride in alcohol. Compound 6 apparently
emerges from the hydrolytic splitting of arabinose from 2-(β-D-arabinofuranosylaminocarbonyl)-1-
formylguanidine, which is an intermediate product in the hydrolysis of ara-AC (ref.13). This kind of
intermediate was first detected during the hydrolysis of 5-azacytidine14. Compound 6 failed then to
be prepared because the direct formylation of amidinourea led, with a simultaneous cyclization, to
5-azacytosine. Compound 6: M.p. 214–215 °C (methanol), UV spectrum (water): 234 (4.25). For
C3H6N4O2 (130.1) calculated: 27.70% C, 4.65% H, 43.06% N; found: 27.37% C, 4.82% H, 42.81% N.
2-Deoxy-D-erythropentofuranosylaminocarbonylguanidinium Formate (7)
This compound was prepared by hydrolysis of 2′-deoxy-5-azacytidine15 with an aqueous solution of
ammonia at a concentration of 1 mol l–1, conducted as with 5-azacytidine14. This reaction was ac-
companied by partial anomerization, so that the product was a mixture of the α and β anomers. Com-
pound 7: m.p. 152–154 °C (decomp.) (ethanol), [α]D +36.2° (c 0.50, water) (α : β = 1 : 1), UV
spectrum: 221 (3.85) at pH 7.0, 223 (4.38) at pH 11.0. For C8H16N4O6 (264.2) calculated: 36.36% C,
6.10% H, 21.20% N; found: 36.32 C, 6.07% H, 21.42% N.
D-Ribofuranosylaminocarbonylguanidinium Acetate (8)
This compound was obtained by filtering an aqueous solution of β-D-ribofuranosylaminocarbonylgua-
nidinium picrate14 over a Dowex-1 column in the acetate cycle, evaporating the eluate, and crystal-
lizing the residue from ethanol. This operation was accompanied by anomerization, so that the
product was also a mixture of the α and β anomers. Compound 8: m.p. 135–137 °C (decomp.) (ethanol),
[α]D +92.4° (c 0.15, water) (α : β = 9 : 1), UV spectrum 222 (3.84) at pH 7.0, 224 (4.30) at pH 11.0.
For C9H18N4O7 (294.3) calculated: 36.73% C, 6.16% H, 19.04 N; found: 36.78% C, 6.21% H,
19.30% N.
RESULTS AND DISCUSSION
Except for urea (1), all compounds listed in Table I are polarographically active. Urea
was not reduced in the experimental setup used, with DMF as the solvent. This com-
pound, containing two amino groups in a molecule, gave no polarographic wave. On
the other hand, hydroxyurea (2), where one of the hydrogen atoms of the amino group
is replaced by a hydroxy group, gave a two-electron diffusion wave, which, however,
was ill-developed due to the very negative half-wave potential (Table I). This is con-
sistent with the reducibility data of amino compounds, which do not reduce at all16 (e.g.
urea) or reduce very reluctantly (cf. hydroxyurea). If the hydrogen and hydroxy group
(–HOH) in the hydroxyurea molecule are replaced by an amidine group =C(NH2)2, the
reducibility of the product, viz. 2-carbamoylguanidine (5), is facilitated and its half-
wave potential is nearly 900 mV more positive than that of hydroxyurea (Table I).
Reduction of 5 is a one-step two-electron process and the polarographic wave is of
diffusion-controlled and irreversible nature.
A substantially different polarographic behaviour was observed for 2-carbamoyl-1-
formylguanidine (6). The substitution by a formyl group brings about appearance of an
adsorption wave with a half-wave potential E1/2 = –0.130 V and of a polarographic
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Polarography of Hydroxyurea Derivatives
659
maximum of the 1st kind at –1.950 V vs SCE. The adsorption nature of the former was
proved based on the dependence of its current on the mercury column height and also
based on the fact that its height was independent of analyte concentration.
2-Deoxy-D-erythropentofuranosylaminocarbonylguanidinium formate (7) and D-ribo-
furanosylaminocarbonylguanidinium acetate (8) in anhydrous DMF are reduced in
single two-electron reversible steps with values of E1/2 = –2.120 and –2.270 V vs SCE,
respectively.
Biuret (3) and formylbiuret (4) are the only two compounds in the series that are
reduced in two well-defined steps. The first step is a diffusion-controlled one-electron
reversible process, the other, also diffusion-controlled and one-electron step, is an irre-
versible process. Logarithmic analysis gave a slope of 58 mV for both compounds,
corresponding to a charge number of 1 (ref.17).
Potential carcinogenity of the compounds was examined based on their reduction in
anhydrous DMF in the presence of α-lipoic acid6–9. The diffusion-controlled wave
height increased linearly with increasing concentration of α-lipoic acid (Fig. 1) for all
substances except 7. The slope (tg α) of the corresponding plot (Fig. 2) is a suitable
quantity for assessing the potential carcinogenity of the substances at a pre-screening
level. As a rule of thumb6–9, substances whose tg α values do not exceed 0.100 are
harmless as carcinogens, substances whose tg α values lies within the region of 0.100–0.200
TABLE I
Half-wave potentials E1/2 and the parameter tg α of the urea derivatives
E
1/2, V
Compound
tg α
Ref.
–
(SCE)
Urea (1)
–
not
determined
Hydroxyurea (2)
Biuret (3)
–2.760
0.290
0.100
–
–2.040
–2.350
13
Formylbiuret (4)
–2.100
–2.395
0.362
22
2-Carbamoylguanidine (5)
–1.880
0.060
0.510
23
2-Carbamoyl-1-formylguanidine (6)
–0.130
–1.950
this work
2-Deoxy-D-erythropentofuranosylaminocarbonylguanidinium
formate (7)
–2.120
0.000
0.150
this work
this work
D-Ribofuranosylaminocarbonylguanidinium acetate (8)
–2.270
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

660
Novotny, Vachalkova, Piskala:
possess a marginal potential carcinogenity, and substances with tg α values in excess of
0.200 warn of potential carcinogenity; the higher this parameter, the higher the probability
of a carcinogenic activity of the substance. In the series studied (Table I), tg α values
lower than 0.100 (no carcinogenity) were found for compounds 3, 5, and 7, and a tg α
value between 0.100 and 0.200 (marginal carcinogenity) was observed for compound 8.
Values higher than 0.200 were measured for hydroxyurea (2, 0.29), formylbiuret (4,
0.362) and 2-carbamoyl-1-formylguanidine (6, 0.510). From among the three com-
pounds, carcinogenic activity has only been investigated for hydroxyurea which, ad-
ministrated to laboratory mice in doses of 100 mg/kg body weight, induced
hepatocarcinomas in 45% animals18. Hydroxyurea is also the first known inhibitor of
the enzyme ribonucleotidereductase and exhibits an antineoplastic activity18. Its clinical
application field is not very wide but it is used in the prevention of complications of
intracerebral leukostasis in patients with acute myelogenic leukemia where the blastic
cell counts in peripheral blood are increased significantly19,20
.
In addition to hydroxyurea, its two formyl derivatives – formylbiuret (4) and 2-carba-
moyl-1-formylguanidine (6) – also exhibit high tg α values. The formylamidino deriva-
tive is reduced more readily that formylbiuret (Table I). The reduction mechanism of
formylbiuret is identical with that of biuret. The same applies to the 2-carbamoylgua-
nidine (5) / 2-carbamoyl-1-formylguanidine (6) pair, only the difference in the E1/2
values is somewhat higher (70 mV). It is noteworthy that the introduction of the formyl
group into the parent molecule brings about a significant increase in the tg α value, viz.
from 0.100 to 0.362 for the 3–4 pair and from 0.060 to 0.510 for the 5–6 pair, which is
–1.970
–1.970
–1.970
–1.970
I
1
1 µA
2
3
4
–0.130
–0.130
–0.130
–0.130
0.10
0.10 0.10 0.10
E, V
FIG. 1
Effect of α-lipoic acid on the polarographic reduction of 2-carbamoyl-1-formylguanidine (c = 0.5
mmol l–1) in anhydrous DMF. Concentration of α-lipoic acid cla (mmol l–1): 1 0, 2 0.8, 3 1.6, 4 2.4
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Polarography of Hydroxyurea Derivatives
661
a shift from non-carcinogens to potential carcinogens. The data suggest that formyl-
biuret (4) and, in particular, 2-carbamoyl-1-formylguanidine (6) will exhibit carcino-
genic effects.
The situation is different with 2-deoxy-D-erythropentofuranosylaminocarbonylgua-
nidinium formate (7) and D-ribofuranosylaminocarbonylguanidinium acetate (8). In-
volving sugar components in their molecules, these compounds are closely related to
nucleosides and serve as intermediates in the synthesis of nucleosides. Compound 7 –
the only one in the series examined – exhibits a tg α value of 0.000, that is, its polaro-
graphic wave height is unaffected by the presence of α-lipoic acid, in which respect
this substance resembles the non-carcinogenic polyaromatic compounds: naphthalene,
anthracene, and pyrene6. For compound 8 the tg α value is 0.150, which suggests that
in the environment, this compound will pose no appreciable carcinogenic risk.
The higher tg α values of the formyl derivatives 4 and 6 as compared to the parent
compounds 3 and 5, respectively, may be explained in terms of their formylation acti-
vity, capable of formylating the –SH, –OH, and –NH2 groups in enzymes. This capa-
bility is appreciably higher for 6 than for 4 due to the considerably higher rate of
hydrolytic splitting of the formyl group from formylamidinoureas than from formyl-
biurets13,14. Since sugar derivatives of formylamidinourea have been proved to be inter-
mediates in the hydrolysis14 of the less carcinogenic nucleosides of 5-azacytosine, it is
conceivable that such substances contribute to cause of carcinogenity of 5-azacytosine
nucleosides. Amidinoureas formed by further hydrolysis of the formylamidinoureas do
not seem to be carcinogenic. On the other hand, formylbiuret, which is the intermediate in
the hydrolysis of 5-azauracil to biuret13, is slightly less carcinogenic than 5-azauracil,
17.0
I , µA
d
16.0
15.0
14.0
13.0
FIG. 2
12.0
Dependence of the 2-carbamoyl-1-formyl-
guanidine reduction wave hight on the con-
centration of α-lipoic acid
0
0.2
0.4
0.6
c
0.8
1.0
4
–1
. 10 , mol l
la
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

662
Novotny, Vachalkova, Piskala:
perhaps due to the ability of the latter to form rather stable covalent adducts13 – an
ability that 5-azacytosine derivatives are lacking21.
In conclusion, the results of the polarographic examination suggest that the carci-
nogenic activity of the compounds 2, 5, 7, and 8 will be low, whereas the compounds
3, 4, and 6 are potential carcinogens whose carcinogenity and mutagenity should be
additionally tested by procedures recommended by WHO.
The authors (L. N., A. V.) acknowledge financial support of this project from the Slovak Grant Agency
(Grant No. 1331/95).
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