Hydroxyurea analogues as kinetic and mechanistic probes of the nitric oxide producing reactions of hydroxyurea and oxyhemoglobin

Article abstract of DOI:10.1021/jm0301538

Derivatives of N-hydroxyurea that contain an N-hydroxy group react with oxyhemoglobin to form methemoglobin and variable amounts of nitrite/nitrate. Compounds with an unsubstituted -NHOH group produce the most nitrite/nitrate, which provides evidence for nitric oxide formation. The rate of reaction of these N-hydroxyurea derivatives with oxyhemoglobin correlates well with that compound's oxidation potential. Aromatic N-hydroxyureas react 25-80-fold faster with oxyhemoglobin than with N-hydroxyurea, suggesting other N-hydroxyurea analogues may be superior nitric oxide donors. Electron paramagnetic resonance spectroscopy shows that the formation of a low-spin methemoglobin - hydroxyurea complex is critical for iron nitrosyl hemoglobin formation. These results show that iron nitrosyl hemoglobin formation from the reaction of hydroxyureas and hemoglobin requires an unsubstituted -NHOH group and that the nitrogen atom of the non-N-hydroxy group must contain at least a single hydrogen atom. These results should guide the development of new hydroxyurea-based nitric oxide donors and sickle cell disease therapies.

Full text of DOI:10.1021/jm0301538

3748
J . Med. Chem. 2003, 46, 3748-3753
Hyd r oxyu r ea An a logu es As Kin etic a n d Mech a n istic P r obes of th e Nitr ic Oxid e
P r od u cin g Rea ction s of Hyd r oxyu r ea a n d Oxyh em oglobin
J inming Huang,† Zhou Zou,† Daniel B. Kim-Shapiro,‡ Samir K. Ballas, and S. Bruce King*,†
Departments of Chemistry and Physics, Wake Forest University, Winston Salem, North Carolina 27109, and
The Cardeza Foundation, Department of Medicine, J efferson Medical College, Philadelphia, Pennsylvania 19107
Received April 4, 2003
Derivatives of N-hydroxyurea that contain an N-hydroxy group react with oxyhemoglobin to
form methemoglobin and variable amounts of nitrite/nitrate. Compounds with an unsubstituted
-NHOH group produce the most nitrite/nitrate, which provides evidence for nitric oxide
formation. The rate of reaction of these N-hydroxyurea derivatives with oxyhemoglobin
correlates well with that compound’s oxidation potential. Aromatic N-hydroxyureas react 25-
80-fold faster with oxyhemoglobin than with N-hydroxyurea, suggesting other N-hydroxyurea
analogues may be superior nitric oxide donors. Electron paramagnetic resonance spectroscopy
shows that the formation of a low-spin methemoglobin-hydroxyurea complex is critical for
iron nitrosyl hemoglobin formation. These results show that iron nitrosyl hemoglobin formation
from the reaction of hydroxyureas and hemoglobin requires an unsubstituted -NHOH group
and that the nitrogen atom of the non-N-hydroxy group must contain at least a single hydrogen
atom. These results should guide the development of new hydroxyurea-based nitric oxide donors
and sickle cell disease therapies.
In tr od u ction
The in vitro reaction of hydroxyurea with normal
oxyhemoglobin (oxyHb) produces methemoglobin (metHb)
as the major protein product and HbNO as a minor
product along with nitrate and nitrite.13 Cyanide trap-
ping studies indicate that the formation of HbNO from
this reaction proceeds through the intermediacy of
metHb.13 EPR studies show the formation of the nitrox-
ide radical (2, Scheme 1), which has been proposed to
decompose to NO,14 during the reaction of hydroxyurea
and oxyHb.15 Direct incubation of hydroxyurea with
metHb initially produces an EPR detectable low spin
hydroxyurea-metHb complex that ultimately forms
HbNO.13 Scheme 1 depicts a general model for the
reactions of hydroxyurea and oxy and metHb with
subsequent HbNO formation.13 For oxyHb, hydrogen
atom abstraction from hydroxyurea by the oxygen bound
to the iron of oxyHb produces metHb, hydrogen perox-
ide, and the nitroxide radical (2, first reaction, Scheme
1). Hydroxyurea coordinates to the ferric iron of metHb
and inner sphere electron transfer produces deoxyHb
and the nitroxide radical (2) and deoxyHb (second
reaction, Scheme 1). Nitric oxide formed from the
decomposition of 2 rapidly combines with deoxyHb to
give HbNO or oxidizes remaining oxyHb to metHb and
nitrate (third reaction, Scheme 1). These results and
model find precedence in the known conversion of oxyHb
to deoxyHb through the intermediacy of metHb by many
organic reductants including phenylhydroxylamine.16
Hydroxyurea (1) is a recently approved drug for
treating sickle cell disease,1,2 a condition that affects
about one out of six hundred people of African descent
born in the United States.3 Hydroxyurea benefits sickle
cell patients by increasing the level of fetal hemoglobin
(HbF) a genetically distinct hemoglobin that inhibits
polymerization of deoxygenated sickle cell hemoglobin
(HbS).1,2,4 In addition, hydroxyurea may benefit sickle
cell patients by influencing red cell-endothelial cell
interactions, the rheological properties of red cells, and
through myelosuppressive actions.4 Hydroxyurea also
acts as a source of nitric oxide (NO), a biologically
important messenger molecule involved in the mainte-
nance of normal blood pressure and flow that has drawn
considerable interest as a sickle cell disease therapy.5-7
Electron paramagnetic resonance (EPR) studies dem-
onstrate the in vivo formation of iron nitrosyl hemoglo-
bin (HbNO) in the blood of both rats and humans upon
the administration of hydroxyurea.8,9 Sickle cell patients
on hydroxyurea therapy show increased plasma and red
cell nitrite, nitrate, HbNO, and cyclic guanylate mono-
phosphate (cGMP) levels.10,11 These results clearly as-
sociate hydroxyurea therapy with NO generation, which
could potentially benefit sickle cell patients in a number
of ways.6,7 Recent in vitro work reveals that hydroxy-
urea and two other mechanistically different NO donors
increase γ-globin gene expression and HbF levels in
human erythroid progenitor cells by a soluble guanylate
cyclase (sGC) dependent pathway providing further
evidence for a role of NO in hydroxyurea therapy.12
Scheme 1 indicates that ease of oxidation of hydroxy-
urea and its ability to bind the ferric iron of metHb
should play important roles in the formation of NO and
HbNO from its reaction with oxyHb. Using a combina-
tion of electrochemical, kinetic, and spectroscopic mea-
surements and a group of synthetic hydroxyurea de-
rivatives (3-8) (Chart 1), we have investigated how
structure influences these factors in relationship to a
compound’s ability to produce NO and HbNO. These
* To whom correspondence should be addressed. Department of
Chemistry, Wake Forest University, Winston Salem, NC 27109,
phone: 336-758-5774, FAX: 336-758-4656, email: kingsb@wfu.edu.
† Department of Chemistry, Wake Forest University.
‡ Departments of Physics, Wake Forest University.
J efferson Medical College.
10.1021/jm0301538 CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/18/2003

Hydroxyurea Analogues As Kinetic Probes
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3749
Sch em e 1
F igu r e 1. Absorption scanning kinetics of the reaction of
oxyHbA (70 µM) and 50 mM N′-phenyl N-hydroxyurea (5) in
0.1 M sodium phosphate buffer (pH 7.3) at room temperature
(25 °C) under an atmosphere of air. Scans taken every 5 min
total for 3 h.
Ch a r t 1
Ta ble 1. Apparent Rate Constants for the Reactions of
Hydroxyurea Derivatives (50 mM) with OxyHb (x ( SD, n ) 3),
Chemical Potentials and pKa’s of Hydroxyurea (1) and
Derivatives (4-8)
derivative apparent rate constant (min-1
)
Ep2/2 (mV)a
pKa
1
4
5
6
7
8
7.54 × 10-4 ( 2.16 × 10-5
3.93 × 10-2 ( 1.68 × 10-3
6.24 × 10-2 ( 6.18 × 10-3
8.56 × 10-3 ( 1.87 × 10-4
2.26 × 10-3 ( 8.16 × 10-5
1.94 × 10-3 ( 5.50 × 10-4b
948.0
588.0
433.0
693.0
705.0
560.0
10.65
9.95
10.30
11.00
10.80
10.20
a
b
Half-peak potential vs Ag/AgCl. 5 mM.
hydroxyurea derivatives (50 mM). Such analysis gives
a rate constant for the reaction of 1 with oxyHb similar
to that previously reported.17,18 Similar to 1, the rate
constants of reactions of oxyHbS and the various hy-
droxyurea derivatives remain similar to the rate con-
stants of the corresponding reaction with oxyHb (data
not shown).17,18 Due to poor solubility, the reaction of
N′-4-methoxyphenyl N-hydroxyurea (8) with oxyHb was
performed at a lower concentration (5 mM).
studies show that the structure of the hydroxyurea
derivative directly controls both the ability of the
compound to generate NO and HbNO and the rate of
NO release. Such information may ultimately be useful
for the design and preparation of hydroxyurea deriva-
tives with improved NO releasing profiles.
Resu lts
Table 1 summarizes the chemical potential (Ep/2, half
peak potential) of each of the hydroxyurea derivatives
(1, 4-8) as determined by cyclic voltammetry (CV).
Consistent with an earlier report, hydroxyurea and its
derivatives (4-8) demonstrate irreversible redox be-
havior and the measured value for hydroxyurea com-
pares well to a previously reported value.19 Table 1
shows that the compounds with the lowest chemical
potentials (easiest to oxidize) react faster with oxyHb
than compounds with higher chemical potentials. Hy-
droxyurea (1) reacts the slowest and is the most difficult
to oxidize of all of the hydroxyureas examined, while
the aromatic hydroxyureas (5 and 8) oxidize the easiest
and react the fastest (adjusted for the concentration of
8). Titration measurements provide the pKa’s of each of
the hydroxyurea derivatives (1, 4-8, Table 1). These
experiments show that the first pKa for hydroxyurea is
10.65, which compares well with a previously reported
pKa of 10.60 for hydroxyurea.20,21 The first pKa’s of
hydroxyurea and its derivatives (4-8) all fall between
9.95 and 11.00, indicating that these compounds pre-
Absorption measurements indicate the formation of
mainly metHb from the reaction of normal oxyHb with
an excess of 4-8 (Figure 1). Figure 1 shows a repre-
sentative scanning kinetic spectra from the reaction of
oxyHb and 5 where the increasing absorbance at 500
and 635 nm and the decreasing absorbance at 540 and
577 nm indicate the conversion of oxyHb to metHb. The
reactions of oxyHb and 5 and 6 occur appreciably faster
than the reaction of oxyHb and 1 and demonstrate
increased levels of turbidity and protein precipitation.
In general, these results parallel previous results from
absorption studies that show that 1 reacts with oxyHb
to predominately form metHb.13 Analogue 3 does not
react with oxyHb and 4 converts oxyHb to metHb as
previously demonstrated.13
The various hydroxyurea derivatives react with oxy-
Hb at different rates and singular value decomposition
(SVD) and global analysis of the change in absorption
over time provide apparent rate constants for the
reactions. Table 1 summarizes the apparent rate con-
stants for the reactions between oxyHb and the various

3750 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Huang et al.
Ta ble 2. Nitrite and Nitrate Production from the Reactions of
OxyHb with Hydroxyurea Derivatives (50 mM, n ) 3)
derivative NO2- (µM) NO3- (µM) HbNO MetHb complex
1
3
4
5
6
7
9.4 ( 0.6
0
6.3 ( 0.5
7.3 ( 1.1
2.4 ( 0.8
6.1 ( 0.5
25.1 ( 0.5
0
7.5 ( 1.1
26.1 ( 2.3
3.5 ( 1.3
18.5 ( 1.2
yes
no
no
yes
yes
no
yes
no
yes
yes
yes
no
F igu r e 3. EPR spectra for the reactions of hydroxyurea
derivatives with methemoglobin. Methemoglobin solutions
(1-2 mM in heme) were treated with hydroxyurea derivatives
(50 mM) in 0.1 M sodium phosphate buffer (pH 7.3) and
allowed to react for 5 min or 12 h at room temperature under
air. (A) EPR spectrum of the reaction of metHbA and 5 after
5 min, (B) EPR spectrum of the reaction of metHbA and 5 after
12 h, (C) EPR spectrum of the reaction of metHbA and 7 after
5 min, (D) EPR spectrum of the reaction of metHbA and 7 after
12 h. EPR spectra were taken on a Bruker ER 200D spec-
trometer using identical conditions described in Figure 2. For
all spectra: X ) high spin metHbA, O ) low spin metHbA
hydroxyurea derivative complex, and Y ) HbNO.
F igu r e 2. EPR spectra for the reactions of hydroxyurea
derivatives with oxyhemoglobin. Hemoglobin solutions (1-2
mM in heme) were treated with hydroxyurea derivatives (50
mM) in 0.1 M sodium phosphate buffer (pH 7.3) and allowed
to react for 12 h at room temperature. These reaction solutions
were transferred to an EPR tube and frozen in liquid nitrogen
(77 K). EPR spectra were taken on a Bruker ER 200D
spectrometer using 8.5 mW microwave power, 5.0 G modula-
tion amplitude, and 9.32 GHz microwave frequency. (A) The
reaction of oxyHbA and 1. (B) The reaction of oxyHbA and 3.
(C) The reaction of oxyHbA and 4. (D) The reaction of oxyHbA
and 5. (E) The reaction of oxyHbA and 6. (F) The reaction of
oxyHbA and 7. For all spectra: X ) high spin metHbA, O )
low spin metHbA hydroxyurea complex, and Y ) iron nitrosyl
hemoglobin (HbNO).
of hydroxyurea and oxyHb produces an EPR spectrum
characterized by resonances at g ) 2.01, corresponding
to HbNO, g ) 6 for high-spin metHb and g ) 2.52, 2.24,
and 1.86 for a low-spin metHb-hydroxyurea complex
(Figure 2A).13 Consistent with the absorbance experi-
ments, 3 does not react with oxyHb (Figure 2B).13 The
reaction of N-methyl N-hydroxyurea (4) with oxyHb only
produces metHb species and no HbNO (Figure 2C).13
The reactions of oxyHb with derivatives 5 and 6 produce
spectra nearly identical to the reaction of hydroxyurea,
which indicate the formation of HbNO, high-spin metHb,
and a low-spin metHb-hydroxyurea complex (Figure
2D,E). N′,N′-diethyl N-hydroxyurea (7) only produces
high-spin metHbA; no low-spin metHbA-hydroxyurea
complex or HbNO form during this reaction (Figure 2F).
The poor water solubility of 8 limited our ability to
perform EPR measurements, which require higher
concentrations of both protein and hydroxyurea ana-
logue. Table 2 also summarizes HbNO and metHb
formation from the reactions of these compounds with
oxyHb.
Further EPR measurements show the ability of the
hydroxyurea derivatives to form a low-spin complex
with metHb. Previous work shows the rapid formation
of a low-spin metHb-hydroxyurea complex upon addi-
tion of either hydroxyurea or derivative 4 to metHb.13
The hydroxyurea and metHb mixture produces HbNO
over time, while the reaction with 4 fails to form any
HbNO.13 Similar to hydroxyurea, the addition of deriva-
tives 5 and 6 to metHb rapidly produces a low-spin
metHb-hydroxyurea complex (within 5 min) and HbNO
over 12 h (Figure 3A,B). The reaction of 5 and metHb
actually produces some HbNO within 5 min (Figure 3A).
The addition of N′,N′-diethyl N-hydroxyurea (7) to
metHb does not form any low spin-metHb complex or
HbNO even after 12 h (Figure 3C,D).
dominately exist in the un-ionized form at pH 7.3 (used
for both kinetic and CV measurements).
Chemiluminescence NO analysis following reaction
mixture reduction with a potassium iodide/glacial acetic
acid solution or a refluxing vanadium (III) chloride/HCl
solution provides a method for the measurement of the
stable oxidation decomposition products of NO, nitrite,
and nitrate (through subtraction). Table 2 summarizes
the production of nitrite and nitrate from the reactions
of the derivatives (3-7) with oxyHb in comparison with
hydroxyurea. The reactions of oxyHb with all the
derivatives except 3 produce both nitrite and nitrate,
and no large differences exist in reactions in which HbS
is used in place of Hb (data not shown). Except for
derivatives 4 and 6, the reactions with oxyHb produce
much more nitrate than nitrite, and the amount of
nitrate formed increases with hydroxyurea concentra-
tion.13
EPR experiments reveal the formation of HbNO and
metHb from the reaction of hydroxyurea and its deriva-
tives with oxyHb (1-2 mM). Figure 2 shows the EPR
spectra from the frozen (77 K) reaction mixtures of
oxyHb and the hydroxyurea derivatives. The reaction

Hydroxyurea Analogues As Kinetic Probes
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3751
Discu ssion
only hydroxyureas (1, 5, 6) capable of forming a low-
spin metHb-hydroxyurea complex ultimately produce
HbNO (Figure 3, Table 2). These results suggest elec-
tron transfer within this complex reduces the iron and
generates the corresponding nitroxide radical (2 for
hydroxyurea, Scheme 1, second reaction). Nitric oxide
formation from this process within the heme pocket
followed by rapid binding to the ferrous iron may
eliminate competing reactions of NO with oxygen or
oxyHb and explain HbNO formation only from hydroxy-
ureas capable of forming a low-spin metHb complex
(Scheme 1, reactions 2 and 3).25 Direct addition of these
hydroxyureas (1, 5, 6) to metHb also forms HbNO with
the reactions of 5 and 6 with metHb producing HbNO
within 5 min (Figure 3).13 While these results clearly
show the involvement of a low-spin metHb-hydroxy-
urea complex in the formation of HbNO; the precise
details of electron transfer and NO release from nitrox-
ide radicals, such as 2, remain to be defined thus
limiting mechanistic interpretations. In addition, the
kinetics of HbNO formation during the reaction of these
hydroxyureas and metHb remain to be fully explored.
These results provide further support for Scheme 1
as a mechanistic pathway of NO and HbNO formation
from the reaction of hydroxyurea and oxyHb and
identify structural features important for both NO
release and HbNO formation. As the hydroxyurea
derivatives (4-8) each contain an N-hydroxy group,
Scheme 1 predicts that oxyHb will oxidize these com-
pounds in a similar fashion as hydroxyurea and other
organic reductants.13,16 Absorption spectroscopy experi-
ments show that oxyHb oxidizes each of these deriva-
tives with the formation of metHb. The inability of
O-methyl hydroxyurea (3) to react with oxyHb further
supports Scheme 1.
Absorption spectroscopy provides kinetic information
regarding these reactions, and cyclic voltammetry re-
veals a direct relationship between the rate of these
reactions and the ease of oxidation of the hydroxyurea
derivatives. Compounds with the lowest oxidation po-
tentials (most easily oxidized) react the fastest with
oxyHb (Table 1). These results also support hydrogen
atom abstraction as the first step in the reaction
between hydroxyurea and oxyHb (Scheme 1, first reac-
tion). The structure of the hydroxyurea influences the
rate of reaction with oxyHb, and in general the addition
of any alkyl or aryl group to either nitrogen atom of
hydroxyurea increases the rate (Table 1). Specifically,
the greatest rate increases occur with the addition of a
methyl group to the N-hydroxy nitrogen (4) or the
addition of aromatic groups to the other (non-N-hydroxy
nitrogen, 5 and 8, adjusted for the difference in concen-
tration, Table 1). The rate increases observed with these
derivatives most likely reflect the ability of these groups
to stabilize the initially formed nitroxide radical (analo-
gous to 2) from the hydrogen atom abstraction of the
parent hydroxyurea (Scheme 1, reaction 1). These
results demonstrate the potential of these substituted
hydroxyureas as superior NO donors compared to the
parent (1).
The reactions of hydroxyurea and the derivatives (4-
7) with oxyHb also produce nitrite and nitrate providing
evidence for NO release (Table 2). The formation of NO
from the proposed decomposition of nitroxide radicals,
such as 2, provides a mechanism for the formation of
these species.14 Recent support for NO production from
the direct decomposition of 2 comes from work that
shows the formation of NO and formamide from the Cu-
(II) oxidation of hydroxyurea.22 Reaction of hydroxyurea-
derived NO with deoxyHb would produce HbNO, with
oxyHb would produce metHb and nitrate, and with
oxygen would yield nitrite.23,24 Gas chromatographic
headspace measurements fail to identify the formation
of nitrous oxide in these reactions arguing against the
involvement of nitroxyl (HNO) in these reactions. The
diminished amount of nitrite and nitrate formed in the
reaction of 6 may be related to the significant protein
precipitation observed in this reaction, which could limit
nitrite/nitrate formation. The lack of nitrite/nitrate
formation in the reaction of 3 with oxyHb further
indicates the inability of this compound to undergo
reaction.
EPR spectroscopy shows that 4 forms a low spin
complex with metHb but that neither the reaction of 4
with oxyHb or metHb forms HbNO.13 The failure to
detect HbNO from these reactions indicates that 4
cannot efficiently transfer NO to the heme, and the lack
of NO transfer in these reactions again most likely
relates to the fact that formation of NO from 4 requires
C-N bond cleavage that may not be kinetically compat-
ible with HbNO formation.
Absorption spectroscopy shows that derivative 7
reacts with oxyHb to give metHb, but EPR experiments
reveal that no HbNO forms during these reactions even
though 7 contains an unsubstituted hydroxylamine
group and produces nitrite/nitrate. A closer examination
of the EPR spectrum for the reaction of 7 and oxyHb
(Figure 2F) provides some understanding as to the lack
of HbNO formation. This spectrum shows a resonance
for high-spin metHb and no resonances for HbNO or a
low-spin metHb complex. Figure 3 also shows that the
mixture of 7 and metHb does not produce a metHb-
hydroxyurea complex. As the formation of such a
complex represents an important step in the proposed
mechanism of HbNO formation from hydroxyurea, the
failure of 7 to form such a complex and reduce the ferric
iron would explain the lack of HbNO production in these
reactions and support Scheme 1. Absorption spectros-
copy of the reaction of metHb and 7 under an atmos-
phere of carbon monoxide fails to show the formation
of any HbCO confirming the inability of 7 to reduce the
iron atom.
The inability to form a metHb complex must be
related to the structural differences between 1, 4-7. The
compounds that form complexes with metHb (1, 4-6)
each have at least a single hydrogen atom on the non-
hydroxy nitrogen of the hydroxyurea group. These
compounds must be capable of arranging themselves in
the heme pocket in a manner that allows iron coordina-
tion. However, in 7 both of the hydrogen atoms of this
nitrogen are replaced with alkyl groups, and this added
substitution apparently cannot be sterically tolerated
and prevents the association of the compound with the
iron. These results indicate that HbNO formation from
While these results show the ability of hydroxyureas
with an N-hydroxy group to produce NO during their
reaction with oxyHb, EPR measurements indicate that

3752 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Huang et al.
temperature. These reaction solutions were transferred to an
EPR tube and frozen in liquid nitrogen (77K) and EPR spectra
were taken as previously described.13
hydroxyureas require at least one hydrogen substituent
on the non-hydroxy nitrogen of the urea group and
further refine the structural requirements of HbNO
formation.
Nitr ite a n d Nitr a te An a lysis. Nitrite and nitrate were
measured with a Sievers 280 Nitric Oxide Analyzer chemilu-
minescence detector as reported earlier.13
The described reactivity of hydroxyurea and its
analogues with oxyHb could have important implica-
tions in sickle cell disease therapy. Sickle cell patients
demonstrate elevated levels of ferrous cell-free (outside
of the red cell yet within the plasma) hemoglobin that
rapidly scavenges NO thus reducing its bioavailability.26
The ability of hydroxyurea to react with oxyHb to form
metHb, which reacts slowly with NO,27 and to produce
NO that rapidly reacts with both oxy and deoxyHb to
give metHb and HbNO could be an important mecha-
nism of action of hydroxyurea in addition to its ability
to stimulate HbF synthesis. The conversion of ferrous
cell-free hemoglobin to cell-free metHb or cell-free
HbNO through reactions with hydroxyurea or hydroxy-
urea-derived NO would allow endogenously produced
NO to assume its normal function.
p Ka Deter m in a tion of Hyd r oxyu r ea Der iva tives. The
pKa’s of the hydroxyurea derivatives were determined by
titration as previously described.20
Electr och em istr y of Hyd r oxyu r ea Der iva tives. Cyclic
voltammetry (CV) was carried out in a three-electrode mini-
cell (2-3 mL) containing a glassy carbon-working electrode
(diameter 1.5 mm), a platinum flag counter electrode, and a
reference electrode (saturated Ag/AgCl) at a scan rate of 100
mV/s. Potentials were reported against the saturated Ag/AgCl
electrode. Samples were typically 2.5 mM in 0.01 M phosphate
buffer (pH 7.3) with 0.2 M NaClO4 as the supporting electro-
lyte. Solutions were purged with argon prior to use and kept
under argon during the experiment. The glassy carbon-
working electrode was polished with 1.0, 0.3, and 0.05 µm
R-alumina (Buehler Corp.) and rinsed with deionized water.
This cleaning treatment was repeated using 0.05 µm R-alu-
mina for polishing between runs. Data were collected using a
Pine AFRDE4 (Pine Instrument Company, Grove City, PA)
with bi-potentiostat/waveform generator.
Exp er im en ta l Section
Hydroxyurea was purchased from Aldrich Chemical CO
(Milwaukee, WI). All other chemicals were of the highest
analytical grade commercially available.
P r ep a r a tion of Hem oglobin . HbS and normal adult
hemoglobin (Hb) were prepared and stored at -80 °C as
previously described.13 Hemoglobin concentrations are ex-
pressed in terms of heme and were determined using previ-
ously reported extinction coefficients.28
Syn th esis of Hyd r oxyu r ea Der iva tives. O-Methyl N-
hydroxyurea (3), N-methyl-N-hydroxyurea (4), N′-phenyl N-
hydroxyurea (5), and N′-n-butyl N-hydroxyurea (6) were
synthesized as previously described.13,29,30
Ack n ow led gm en t. The authors wish to thank Dr.
Howard Shields for assistance with EPR experiments
and Drs. Ronald E. Noftle and J ian Dai for assistance
with cyclic voltammetry experiments. This work was
supported by the National Institutes of Health (HL62198,
S.B.K.). The NMR spectrometers used in this work were
purchased with partial support from NSF (CHE-
9708077) and the North Carolina Biotechnology Center
(9703-IDG-1007).
Syn th esis of N′,N′-Dieth yl N-Hyd r oxyu r ea (7). N,N-
diethylcarbamoyl chloride (3 g, 22.1 mmol, 2.8 mL) was added
dropwise to a solution of hydroxylamine hydrochloride (1.8 g,
25.9 mmol) and potassium carbonate (2.5 g, 23.6 mmol) in
ethyl acetate (15 mL) and distilled water (0.25 mL) at 0 °C
with stirring. This solution was allowed to stand at room
temperature overnight. After evaporation of the solvents, the
crude product was purified by flash chromatography (ethyl
acetate) to afford 7 as a white powder (1.7 g, 58%): mp 64-
66 °C; 1H NMR (300 MHz, CD3SOCD3) δ 8.76 (s, 1H), 7.87 (s,
1H), 3.15 (q, 4H, J ) 7 Hz), 1.00 (t, 6H, J ) 7 Hz); 13C NMR
(75 MHz, CD3SOCD3) δ 159.8, 40.3, 13.9. Anal. (C5H12N2O2)
C, H, N.
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Syn th esis of N′-4-Meth oxyp h en yl N-Hyd r oxyu r ea (8).
4-Methoxyphenylisocyanate was substituted for phenylisocy-
anate in the previously described preparation of 5:29 mp 158-
1
162 °C; H NMR (300 MHz, CD3SOCD3) δ 8.86 (s, 1H), 8.74
(s, 1H), 8.61 (s, 1H), 7.54 (d, 2H, J ) 9 Hz), 6.88 (d, 2H, J )
9 Hz), 3.75 (s, 3H); 13C NMR (75 M Hz, CD3SOCD3) δ 163.3,
159.1, 136.8, 125.3, 118.1, 59.6. Anal. (C8H10N2O3) C, H, N.
P r ep a r a tion of Hyd r oxyu r ea Der iva tives Solu tion s.
Solutions of hydroxyurea derivatives in sodium phosphate
buffer (0.1 M, pH 7.3) were prepared fresh daily.
Absor p tion Sp ectr oscop y. Hemoglobin solutions (70 µM,
in heme) in 0.1 M sodium phosphate buffer (pH 7.3) were
treated with hydroxyurea derivatives (50 mM) unless other-
wise indicated. Absorption measurements were made every 5
min for 12 h or unless otherwise noted on a Cary 100 Bio UV-
Visible spectrophotometer at 25 °C. Kinetic data was analyzed
with Specfit (Spectrum Software Associates, Boston, MA) using
singular value decomposition (SVD) and global analysis.17,18
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with hemoglobin were obtained in triplicate and averaged.
Electr on P a r a m a gn etic Sp ectr oscop y. Hemoglobin so-
lutions (1-2 mM in heme) were treated with hydroxyurea
derivatives (50 mM) and allowed to react for 12 h at room

Hydroxyurea Analogues As Kinetic Probes
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3753
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