Identification, synthesis and pharmacological activity of moxonidine metabolites

Article abstract of DOI:10.1016/S0223-5234(01)01293-4

The metabolism of moxonidine, 4-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-6-methoxy-2-methyl-5- pyrimidinamine, LY326869, in rats, mice, dogs, and humans has been examined. At least 17 metabolites were identified or tentatively identified in the different species by HPLC, LC/MS and LC/MS/MS. The identities of seven of the major metabolites have been verified by independent synthesis. The metabolites are generally derived from oxidation and conjugation pathways. Oxidation occurred at the imidazolidine ring as well as the methyl at the 2 position of the pyrimidine ring. All seven metabolites were examined in the spontaneously hypertensive rats (3 mg kg^-1, i.v.) for pressure and heart rate. Only one, 2-hydroxymethyl-4-chloro-5-(imidazolidin-2-ylidenimino)-6-methoxypyrimidine, exerted a short-lasting decrease in blood pressure, albeit attenuated in magnitude compared to moxonidine.

Full text of DOI:10.1016/S0223-5234(01)01293-4

Eur. J. Med. Chem. 37 (2002) 23–34
Original article
Identification, synthesis and pharmacological activity of
moxonidine metabolites
David D. Wirth a,*, Minxia M. He b, Boris A. Czeskis b, Karen M. Zimmerman b,
Ulrike Roettig c, Wolfgang Stenzel c, Mitchell I. Steinberg b
a Lilly Research Laboratories, Eli Lilly and Co., Lafayette, IN 47905, USA
b Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN, USA
c Lilly Forshung GmbH, Hamburg, Germany
Received 20 June 2001; received in revised form 12 November 2001; accepted 14 November 2001
Abstract
The metabolism of moxonidine, 4-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-6-methoxy-2-methyl-5-pyrimidinamine, LY326869,
in rats, mice, dogs, and humans has been examined. At least 17 metabolites were identified or tentatively identified in the different
species by HPLC, LC/MS and LC/MS/MS. The identities of seven of the major metabolites have been verified by independent
synthesis. The metabolites are generally derived from oxidation and conjugation pathways. Oxidation occurred at the imidazo-
lidine ring as well as the methyl at the 2 position of the pyrimidine ring. All seven metabolites were examined in the spontaneously
hypertensive rats (3 mg kg−1, i.v.) for pressure and heart rate. Only one, 2-hydroxymethyl-4-chloro-5-(imidazolidin-2-yliden-
imino)-6-methoxypyrimidine, exerted a short-lasting decrease in blood pressure, albeit attenuated in magnitude compared to
´
moxonidine. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: moxonidine; antihypertensive; metabolites
1. Introduction
sites as compared to clonidine [6,7]. Thus, in pre-clini-
cal studies, the antihypertensive effects of moxonidine
and related imidazolines appear to correlate with its
potency at brainstem imidazoline sites rather than to a2
receptors [8–10].
Moxonidine (4-chloro-N-(4,5-dihydro-1H-imidazol-
2 - yl) - 6 - methoxy - 2 - methyl - 5 - pyrimidinamine, LY-
326869, 1) is a new antihypertensive agent that acts
within the rostroventral lateral medulla to decrease
sympathetic outflow [1,2]. It is marketed throughout
Europe under the trade names Cynt™ and Physiotens®.
Moxonidine is generally well tolerated by hypertensive
patients with fewer adverse reactions such as sedation
and rebound hypertension compared to the first genera-
tion a2 agonist, clonidine [3–5]. The basis for the
greater therapeutic index of moxonidine compared to
clonidine is not clear, but studies suggest that mox-
onidine has greater selectivity in the central nervous
system for specific imidazoline receptive sites over a2
In elderly patients, the clearance of moxonidine is
reduced and the area under the curve of unchanged
drug is greater in comparison to younger patients sug-
gesting the existence of an age-related decrease in
metabolism [11]. However, despite widespread clinical
use of moxonidine, there is little information available
on its metabolic fate or on the activity its metabolites.
In part, this reflects the limited analytical technologies
available when the compound was originally developed.
Data are available demonstrating that the structurally
related imidazoline clonidine is degraded by extensive
first-pass metabolism after oral administration in rab-
bits and that levels of intact drug in the brain correlate
with blood pressure effects [12]. The pharmacological
* Correspondence and reprints.
E-mail address: wirth@lilly.com (D.D. Wirth).
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(01)01293-4

24
D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
activity of clonidine metabolites was also assessed fol-
lowing peripheral or central administration in rabbits,
and only one metabolite, p-hydroxy clonidine, induced
weak decreases in blood pressure by either route. Five
metabolites of clonidine, derived from hydroxylation of
the phenyl ring and oxidation of the imidazoline ring
followed by splitting of the ring were also identified in
dog urine [13].
idine (10). Nitration of 10 with a mixture of nitric and
acetic acids led to 4,6-dihydroxy-2-hydroxymethyl-5-ni-
tropyrimidine (11). Minimal volume of the nitration
solution was used in order to achieve a reasonable yield
of the nitro product. Initial attempts to transform 11
into a dichloropyrimidine using phosphorus oxychlo-
ride, resulted in formation of 2-chloromethyl-4,6-
dichloro-5-nitropyrimidine as
a
main product.
Moxonidine has a relatively short half-life (about
2–3 h), however; it has long duration of effect and is
given only once daily clinically. The differences between
the pharmcokinetics and pharmacodynamics of mo-
xonidine were not clear. To determine whether the long
duration of moxonidine is attributed to the production
of active metabolite(s), we have identified and synthe-
sised the major metabolites of moxonidine and tested
their pharmacological activity.
Protection of the primary hydroxyl as an acetate under
standard conditions using acetic anhydride in the pres-
ence of base in an aprotic solvent was not possible
because of the very low solubility of triol 11 in most
organic solvents. Its reaction with acetic anhydride in
the presence of trimethylsilyl chloride [28,29] also
failed, leaving only unreacted starting material. Acety-
lation was successfully accomplished when 11 was
heated in a mixture of acetic acid and acetyl chloride or
acetic acid and acetic anhydride, following by simple
evaporation of the reaction mixture under vacuum. The
resulting 2-acetoxymethyl-4,6-dihydroxy-5-nitropyrim-
idine (12), was treated with phosphorus oxychloride in
the presence of N,N-diethylaniline to form 2-ace-
toxymethyl-4,6-dichloro-5-nitropyrimidine (13). Reduc-
tion of the nitro group by hydrogenation over Raney
nickel smoothly gave 2-acetoxymethyl-4,6-dichloro-5-
aminopyrimidine (14), without hydrogenolysis of the
aromatic chloride. Coupling of amine 14 with N-
acetylimidazolidin-2-one [30] in the presence of phos-
phorus oxychloride afforded 15. Finally, simultaneous
hydrolysis of the acetyl groups and nucleophilic substi-
tution using sodium methoxide in methanol gave the
desired product 5.
For the synthesis of acid 4, outlined in Fig. 2, the
acetate 13 was reduced with diisobutylaluminum hy-
dride under mild conditions to give 2-hydroxymethyl-
4,6-dichloro-5-nitropyrimidine (16) [31]. Jones oxi-
dation of alcohol 16 followed by the methylation of
the resulting 2-carboxy-4,6-dichloro-5-nitropyrimidine
yielded 2-methoxycarbonyl-4,6-dichloro-5-nitropyrim-
idine (17). Reduction of its nitro group afforded 2-
methoxycarbonyl-4,6-dichloro-5-aminopyrimidine (18),
which was converted into 2-methoxycarbonyl-4-chloro-
5-amino-6-methoxypyrimidine (19), by selective
methanolysis. A low yield of 20 in the next step of the
coupling with N-acetylimidazolidin-2-one may be ex-
plained by the low nucleophilicity of the heteroaromatic
amino group due to the presence of the electron with-
drawing methoxycarbonyl group. Finally, simultaneous
hydrolysis of the ester and amide fragments of 20 with
lithium hydroxide quantitatively gave the target com-
pound 4.
2. Chemistry
The key synthetic approach to moxonidine and its
analogs has been the POCl3-mediated coupling of a
5-aminopyrimidine with a protected cyclic urea such as
N-acetylimidazolidin-2-one, methodology used for the
synthesis of moxonidine itself [14]. This tactic was used
for the synthesis of metabolites derived from oxidation
on the pyrimidine ring while those involving functiona-
lisation of the imidazolidine ring were prepared by
closing an oxidised intermediate to form this five-mem-
bered ring. The structures and key mass spectral frag-
mentations of the metabolites are shown in Table 1.
The simplest route to hydroxy compound 5 would be
direct oxidation of the methyl group of moxonidine
itself. Attempts to use a number of oxidizing agents,
including ceric ammonium nitrate [15–17], chromic
acid [18–20], peroxydisulfate [21,22], and N-bromosuc-
cinimide [23–25] failed. This failure was initially at-
tributed to the sensitivity of the guanidine fragment
toward the oxidation. However, application of the same
methods to the corresponding nitro-compound (4,6-
dichloro-5-nitro-2-methylpyrimidine) was also unsuc-
cessful due to its poor stability under oxidation
conditions. Thus, a total synthesis of 5 was required
and is summarised in Fig. 1.
The key structural feature of the target molecule is
the 2-hydroxymethylpyrimidine fragment, which can be
made by condensation of hydroxyacetamidine with
malonates [26,27]. Hydroxyacetamidine hydrochloride
(9), was prepared by known procedures from
paraformaldehyde and hydrogen cyanide and subse-
quent reaction with ethanolic hydrogen chloride, and
ammonolysis of the resulting imino-ether hydrochloride
[26,27]. Reaction of 9 with diethyl malonate in the
presence of sodium ethoxide in refluxing ethanol
smoothly gave 4,6-dihydroxy-2-hydroxymethylpyrim-
The synthesis of metabolites 2, 3, 6, and 7 is sum-
marised in Fig. 3. Although 5-amino-4-chloro-6-
methoxy-2-methylpyrimidine (22), was previously
known [32,33], its present synthesis represents a signifi-
cant improvement in its yield from the known

D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
25
dichloropyrimidine (21) [34]. Reaction of 22 with an ex-
cess of cyanamide in ethanol provided the metabolite 3.
The synthesis of 6 and 2 was more challenging,
however. Attempted alkylation of 3 with bromoac-
etaldehyde diethyacetal was unsuccessful. Likewise, the
possible condensation of amine 22 with the known
cyanamide derived from aminoacetaldehyde diethyl
acetal [35] failed to produce the desired coupling. Simi-
larly disappointing was the failure of the amine 22 to
react with cyanogen bromide to give the cyanamide, 25
[36].
The successful synthesis of hydroxymoxonidine 6,
is shown in Fig. 3. Condensation of 22 with benzoyl
thiocyanate gave the acyl urea 23 in high yield as
long as the temperature was kept at ambient or
below. The thiourea 24, produced by methanolysis of
23, was unstable even at ambient temperature. The
100 °C temperature recommended for the lead-based
Table 1
Moxonidine and its metabolites

26
D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
The synthesis of the dihydroxylated compound 7, was
effected by the addition of glyoxal to the guanidine 3.
This compound was a mixture of cis and trans diols and
the ratio was somewhat variable and changed as a
function of pH, suggesting an equilibrium with open-
chain forms.
Due to the ready displacement of the chlorine of
moxonidine, the synthesis of metabolite 8 was accom-
plished by simple heating of a mixture of moxonidine,
L
-cysteine, and sodium bicarbonate in water. It was
purified by chromatography on a polystyrene resin.
3. Metabolism studies
The metabolism of moxonidine was first characte-
rised in Fisher 344 rats after a single oral administration
Fig. 1. Synthetic scheme for metabolite 5. Reagents: (a) (i)
CH2(CO2Et)2, EtONa, EtOH, (ii) HCl; (b) HNO3, AcOH; (c) AcOH,
Ac2O; (d) POCl3, PhNEt2; (e) H2, NiꢀRa; (f) POCl3; (g) MeONa,
MeOH.
Fig. 2. Synthesis of metabolite 4. Reagents: (a) DIBAL-H, CH2Cl2;
(b) (i) H2CrO4, (ii) CH2N2; (c) H2, RaꢀNi; (d) MeONa, MeOH; (e)
POCl3; (f) LiOH, THF, MeOH.
desulfurisation reaction [37], of thioureas to cyanamides
caused extensive degradation. The cyanamide 25 was
best prepared at 60 °C and, although it could be iso-
lated and characterised somewhat, it proved beneficial
to condense it directly with commercially-available
aminoacetaldehyde diethylacetal to produce the acetal
26. Careful hydrolysis of this acetal and isolation of the
resultant hydroxymoxonidine 6, under acidic conditions
resulted in a filterable solid which was quite pure.
Czarnik and Leonard developed these conditions for a
similar compound [38]. When subjected to neutral or
alkaline conditions, the hydroxymoxonidine readily de-
hydrated to the imidazole metabolite 2.
Fig. 3. Synthesis of metabolites 2, 3, 6, and 7. Reagents: (a) MeONa,
MeOH; (b) NH4SCN, PhCOCl, C3H6O; (c) MeONa, MeOH; (d)
Pb(OAc)2, H2O; (e) H2NCH2CH(OEt)2, KH2PO4; (f) HCl; (g) KOH;
(h) H2NCN; (i) HCOCHO.

D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
27
Fig. 4. The effect of moxonidine and its metabolites (3 mg kg−1, i.v.) on the change in mean arterial pressure (MAP) in conscious SHR monitored
by telemetry. The numbers in parenthesis indicate replicate animals. Asterisks indicate significantly different from the vehicle group by Dunnett’s
test.
Fig. 5. The effect of moxonidine and its metabolites (3 mg kg−1, i.v.) on the heart rate in conscious SHR monitored by telemetry. The numbers
in parenthesis indicate replicate animals. Asterisks indicate significantly different from the vehicle group by Dunnett’s test.
of 5 mg kg−1 of 14C-moxonidine. Metabolites were
identified in rat urine, plasma and bile samples by
HPLC, LC/MS and LC/MS/MS [39]. Additional
metabolism studies were conducted in CD-1 mice, bea-
gle dogs and healthy volunteers at a single dose of 10,
0.2 mg kg−1 and 1 mg of 14C-moxonidine, respectively.
At least 17 metabolites have been identified or tenta-
tively identified in biological fluids (i.e. urine, bile and
plasma) in different species. The metabolites are gene-
rally derived from oxidation and conjugation pathways.
Oxidation occurred at the imidazolidine ring as well as
the methyl at the two position of the pyrimidine ring.
The most abundant and frequently observed mo-
xonidine metabolites were synthesised for determina-
tion of pharmacological activities. Their structures and
mass spectral information are given in Table 1.
4. Results and discussion
As shown in Fig. 4, of all the compounds studied
only moxonidine and its hydroxymethyl metabolite 5,
lowered mean blood pressure compared to vehicle in
the spontaneously hypertensive rat (SHR).
The effects of moxonidine on mean pressure were
apparent within the first hour and lasted for ca. 12 h.
Only compound 5 showed a decrease in pressure that
lasted 2 h as compared to vehicle. Accompanying the
effect of moxonidine on blood pressure were pro-
nounced falls in heart rate that were most prominent
4 h after dosing as shown in Fig. 5.
Consistent with the shorter effect of 5 on blood
pressure, there was also a significant fall compared to
vehicle in heart rate apparent during the first hour after

28
D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
dosing. Compound 6 also demonstrated a significant
decrease in heart rate 11–18 h after dosing that was not
accompanied by changes in mean blood pressure.
6.1.2. 4,6-Dihydroxy-2-hydroxymethyl-5-
nitropyrimidine (11)
To a stirred mixture of HNO3 (fuming, 4.2 mL) and
AcOH (glacial, 2.1 mL) was added 10 (2.3 g, 16.2
mmol) over the period of 40 min at 10–15 °C. The
reaction mixture was stirred for 5.5 h at r.t. then cooled
to 5 °C and diluted with cold H2O (3 mL). The precip-
itate was collected by filtration, washed with EtOH (5
mL) and Et2O (5 mL), and dried under vacuum to give
2.44 g (80.5%) of 11 as a colorless solid. IR (KBr): 539,
789, 1105, 1297, 1358, 1630, 1674, 2828, 2920, 3402,
3460 cm−1; UV (EtOH): umax (m) 322 (3354), 213 nm
(17626); 1H-NMR (DMSO-d6) l 4.42 (s, 2H); MS (FD):
187 [M+, 100%]. Anal. Calc. for C5H5N3O5: C, 32.10;
H, 2.69; N, 22.46. Found: C, 31.44; H, 2.91; N, 21.83%.
5. Conclusions
Seven metabolites of moxonidine were definitively
identified and prepared by independent synthesis. The
hydroxymethyl metabolite of moxonidine 5, has been
identified in urine of mice, rats, dogs, and humans and
has been shown to possess antihypertensive and brady-
cardic activity in conscious unrestrained hypertensive
rats when administered intravenously at 3 mg kg−1
.
Compound 5 was less active and shorter acting than
moxonidine at this dose; its half-life remains to be
determined, however. Although, this metabolite is less
active and shorter acting than moxonidine, it may
contribute somewhat to the long antihypertensive effect
and the overall clinical profile of moxonidine. No other
identified metabolites to date show similar antihyper-
tensive activity in this model.
6.1.3. 2-Acetoxymethyl-4,6-dihydroxy-5-nitropyrimidine
(12)
A mixture of 11 (2.35 g, 12.56 mmol), AcOH (glacial,
15 mL), and Ac2O (15 mL) was heated at 105 C for 2
h, evaporated under vacuum, and re-evaporated with
C6H5CH3 (three times). The residue was dried under
vacuum to give 2.843 g (99%) of 12 as a yellow solid.
IR (KBr): 545, 787, 1212, 1326, 1354, 1367, 1493, 1650,
1673, 1758, 2939, 3082, 3141 cm−1; UV (EtOH): umax
(m) 327 nm (3365); 1H-NMR (DMSO-d6): l 2.14 (s,
3H), 4.98 (s, 2H); MS (FD): 229 [M+, 100%]. HRMS
Calc. for C7H7N3O6: 229.0335. Found: 229.0339.
6. Experimental
6.1. Chemistry
The NMR spectra were obtained on a Bruker AC300
or a General Electric QE-300 at 300 (1H) and 75 (13C)
MHz. Chemical shifts are reported in parts per million
(ppm) downfield from TMS. The Physical Chemistry
Research Department of Lilly Research Laboratories
provided Microanalytical, IR, UV, and MS data. Flash
chromatography was performed on silica gel 60 (230–
400 mesh). TLC was done on silica gel 60 F254 pre-
coated plates.
6.1.4. 2-Acetoxymethyl-4,6-dichloro-5-nitropyrimidine
(13)
To a mixture of 12 (2.75 g, 12.0 mmol) and phospho-
rus oxychloride (13 mL) was added N,N-diethylaniline
(2.5 mL, 15.7 mmol) dropwise. The reaction mixture
was refluxed for 2 h and evaporated under vacuum.
The residue was diluted with Et2O (15 mL) and poured
onto ice. After stirring for 3 min, the organic layer was
separated, washed with saturated aq. solutions of
NaHCO3 and NaCl, dried over Na2SO4 and evaporated
under vacuum. Flash chromatography of the residue on
silica gel (20% ether in C6H14) gave 2.446 g (77%) of 13
as a white solid. Rf 0.60 (C6H14–EtOAc, 3:2); IR
(KBr): 836, 853, 1218, 1550, 1755 cm−1; UV (EtOH):
6.1.1. 4,6-Dihydroxy-2-hydroxymethylpyrimidine (10)
To a solution of C2H5ONa prepared from Na (1.38
g, 60.0 mmol) and EtOH (29 mL), was added hydroxy-
acetamidine hydrochloride (9) [26] (2.21 g, 20.0 mmol)
and after 5 min ethyl malonate (3.2 g, 20.0 mmol). The
reaction mixture was refluxed for 3 h, cooled to room
temperature (r.t.), diluted with H2O (10 mL) and aci-
dified with concd. HCl (4 mL). The precipitate was
filtered off, washed with H2O (10 mL), EtOH (5 mL)
and Et2O (5 mL), and dried under vacuum to give 2.24
g (79%) of 10 as a light-brown solid. IR (KBr): 527,
1
umax (m) 259 nm (3290); H-NMR (CDCl3): l 2.28 (s,
3H), 5.33 (s, 2H). Anal. Calc. for C7H5Cl2N3O4: C,
31.60; H, 1.89; N, 15.79. Found: C, 31.84; H, 1.85; N,
15.69%.
6.1.5. 2-Acetoxymethyl-4,6-dichloro-5-aminopyrimidine
(14)
1106, 1324, 1571, 1639, 1682, 2910, 3029, 3084 cm−1
;
A mixture of 13 (255 mg, 0.96 mmol) and Raney
nickel (30 mg) in EtOH (25 mL) was hydrogenated in a
Parr apparatus (50 psi of hydrogen) for 5.5 h. The
resulting suspension was filtered through celite and
evaporated under vacuum. Flash chromatography of
the residue on silica gel (40% EtOAc in C6H14) gave 202
1
UV (EtOH): umax (m) 256 (4235), 239 nm (3398); H-
NMR (DMSO-d6) l 4.28 (s, 2H), 5.14 (s, 1H); MS
(FD): 142 [M+, 100%]. Anal. Calc. for C5H6N2O3: C,
42.26; H, 4.26; N, 19.71. Found: C, 41.85; H, 3.96; N,
19.02%.

D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
29
mg (89%) of 14 as a colorless solid. Rf 0.41 (C6H14–
EtOAc, 3:2); IR (KBr): 793, 907, 1249, 1366, 1459,
1513, 1621, 1731, 3332, 3428 cm−1; UV (EtOH): umax
chromatography of the residue on silica gel (from 55 to
60% ethyl ether in C6H14) gave 634 mg (53%) of 16 as
a white solid. Rf 0.31 (C6H14–ethyl ether, 1:1); IR
(KBr): 833, 841, 1056, 1107, 1264, 1309, 1345, 1437,
1519, 1554, 3335, cm−1; UV (EtOH): umax (m) 260 nm
(2713); H-NMR (CDCl3): l 3.00 (s, 1H), 5.00 (s, 2H).
Anal. Calc. for C5H3Cl2N3O3: C, 26.81; H, 1.35; N,
18.76. Found: C, 27.11; H, 1.35; N, 18.72%.
1
(m) 314 (5676), 253 nm (11 629); H-NMR (CDCl3): l
2.21 (s, 3H), 4.55 (s, 2H) 5.16 (s, 2H); MS (FD): 235
[M+, 100%]. HRMS Calc. for C7H7Cl2N3O2: 234.9917.
Found: 234.9920.
1
6.1.6. 2-Acetoxymethyl-4,6-dichloro-5-(1-acetyl-
imidazolidin-2-ylidenimino)-pyrimidine (15)
6.1.9. 2-Methoxycarbonyl-4,6-dichloro-5-
To a solution of 14 (191 mg, 0.81 mmol) in phospho-
rus oxychloride (1 mL) was added N-acetylimidazo-
lidin-2-one [15] (114 mg, 0.89 mmol) in one portion.
The reaction mixture was heated at 105–110 °C (bath)
for 3 h and evaporated under vacuum. The residue was
diluted with ice H2O (2 mL), made alkaline with aq.
NaOH (5N, 2 mL) and extracted with CH2Cl2 (3×5
mL). The extract was washed with saturated aq. solu-
tions of NaHCO3 and NaCl, dried over Na2SO4 and
evaporated under vacuum. Flash chromatography of
the residue on silica gel (from 50 to 60% EtOAc in
C6H14) gave 166 mg (59%) of 15 as a colorless solid. Rf
0.21 (C6H14–EtOAc, 1:1); IR (KBr): 815, 1033, 1074,
1219, 1355, 1378, 1414, 1497, 1657, 1675, 1752, 3254,
nitropyrimidine (17)
To a solution of 16 (625 mg, 2.79 mmol) in C3H6O
(15 mL) at 0–5 °C was added Jones reagent (8N, 2.9
mL) dropwise. The reaction mixture was allowed to
reach r.t. over the period of 2 h and treated with
isopropyl alcohol (2 mL). The resulting suspension was
stirred for 15 min and evaporated under vacuum. The
residue was diluted with H2O (5 mL) and extracted with
Et2O (60 mL). The extract was washed with saturated
aq. NaCl, and the combined water layer was re-ex-
tracted with Et2O. The combined organic extract was
dried over MgSO4 and evaporated under vacuum to
give 2-carboxy-4,6-dichloro-5-nitropyrimidine as
a
white solid, which was dissolved in Et2O (35 mL),
treated with an excess of ethereal diazomethane, and
evaporated under vacuum. Flash chromatography of
the residue on silica gel (45% ethyl ether in C6H14) gave
449 mg (64%) of 17 as a white solid. Rf 0.61 (C6H14–
ethyl ether, 1:1); IR (KBr): 816, 845, 836, 974, 1171,
1211, 1283, 1300, 1330, 1345, 1430, 1521, 1536, 1558,
1
cm−1; UV (EtOH): umax (m) 245 nm (12 118); H-NMR
(CDCl3): l 2.23 (s, 3H), 2.70 (s, 3H), 3.58 (t, J=7.98
Hz, 2H), 4.11 (t, J=7.98 Hz, 2H), 4.74 (s, 1H), 5.19 (s,
2H); HRMS (FAB) Calc. for C12H13Cl2N5O3: 346.0474.
Found: 346.0481.
1
6.1.7. 2-Hydroxymethyl-4-chloro-5-(imidazolidin-2-
ylidenimino)-6-methoxypyrimidine (5)
1746, 3420 cm−1; H-NMR (CDCl3): l 4.14 (s, 3H).
Anal. Calc. for C6H3Cl2N3O4: C, 28.60; H, 1.20; N,
16.67. Found: C, 28.87; H, 1.31; N, 16.56%.
A solution of 15 (87 mg, 0.25 mmol) and CH3ONa
(14 mg, 0.26 mmol) in MeOH (1.2 mL) was refluxed for
3 h. The reaction mixture was evaporated under va-
cuum to one half of the initial volume and cooled to
5 °C. The precipitate was collected by filtration,
washed with H2O (1 mL) and dried under vacuum to
give 32 mg (50%) of 5 as a colorless solid. IR (KBr):
720, 1042, 1091, 1283, 1307, 1376, 1440, 1471, 1533,
1659, 3030, 3225, 3340 cm−1; UV (EtOH): umax (m) 254
6.1.10. 2-Methoxycarbonyl-4,6-dichloro-5-
aminopyrimidine (18)
A mixture of 17 (449 mg, 1.78 mmol) and Raney
nickel (80 mg) in EtOH (40 mL) was hydrogenated in a
Parr apparatus (50 psi of hydrogen) for 3.5 h. The
resulting suspension was filtered through silica gel and
evaporated under vacuum. Flash chromatography of
the residue on silica gel (from 50 to 60% EtOAc in
C6H14) gave 235 mg (59%) of 18 as a light yellow solid.
Rf 0.42 (C6H14–EtOAc, 1:1); IR (KBr): 742, 983, 1181,
1222, 1341, 1365, 1442, 1511, 1606, 1717, 3301, 3461
1
nm (8577); H-NMR (DMSO-d6): l 3.34 (s, 4H), 3.87
(s, 3H), 4.39 (d, J=6.18 Hz, 2H), 5.19 (t, J=6.18 Hz,
1H), 6.26 (s, 2H); HRMS (FAB) Calc. for
C9H12ClN5O2: 258.0758. Found: 258.0759.
cm−1; UV (EtOH): umax (m) 301 nm (12932); H-NMR
1
6.1.8. 2-Hydroxymethyl-4,6-dichloro-5-nitropyrimidine
(16)
(CDCl3): l 4.06 (s, 3H), 5.03 (s, 2H). Anal. Calc. for
C6H5Cl2N3O2: C, 32.46; H, 2.27; N, 18.93. Found: C,
32.55; H, 2.47; N, 18.90%.
To a solution of 13 (1.42 g, 5.34 mmol) in CH2Cl2
(40 mL) at −78 °C was added di-isobutylaluminum
hydride (1M in C6H5CH3, 15 mL, 15.0 mmol) drop-
wise. The reaction mixture was stirred at −78 °C for
15 min, treated with isopropyl alcohol (1.5 mL), diluted
with CH2Cl2 (40 mL), washed with saturated aq. solu-
tions of potassium sodium tartrate and NaCl, dried
over Na2SO4 and evaporated under vacuum. Flash
6.1.11. 2-Methoxycarbonyl-4-chloro-5-amino-6-
methoxypyrimidine (19)
A solution of 18 (390 mg, 1.76 mmol) in MeOH (25
mL) containing CH3ONa (90 mg, 1.67 mmol), was kept
at r.t. for 30 min, diluted with EtOAc, washed with
brine, dried over Na2SO4, and evaporated under

30
D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
vacuum to give 320 mg (82%) of 19 as an amorphous
solid. H-NMR (CDCl3): l 6.4 (br s, 2H), 4.1 (s, 3H),
3.9 (s, 3H). HRMS Calc. for C7H8ClN3O3: 217.0255.
Found: 217.0261.
added and the mixture was cooled to about 30 °C
whereupon it crystallised. After stirring for 1 h at
ambient temperature, the product was collected by
filtration and was rinsed with about 35 mL of C6H14.
The filtrate was distilled at atmospheric pressure until
about 275 mL had been removed. When cooled to
about 40 °C, an oil separated but formed crystals upon
continued stirring. After a stir of one-half hour at
ambient temperature, the slurry was stirred for 1 h in
an ice bath. The second crop was isolated by filtration
and was rinsed with cold C6H14. Both crops were dried
in vacuo overnight at 30 °C. The yield was 26.66 g in
the first crop and 10.42 g in the second crop for a
combined yield of 95%. The purity of the first crop by
isocratic HPLC was 99% (m.p. 58–59 °C) [40,41] and
the second about 95% (m.p. 56–58 °C); they were
1
6.1.12. 2-Methoxycarbonyl-4-chloro-5-(1-acetyl-
imidazolidin-2-ylidenimino)-6-methoxypyrimidine (20)
A mixture of 19 (460 mg, 2.11 mmol), N-acetyl-2-imi-
dazolidone (285 mg, 2.22 mmol), and phosphorous
oxychloride (15 mL) was heated in an oil bath of
110 °C for 2.25 h, cooled, and evaporated under va-
cuum. The residue was stirred with CH2Cl2–ice water,
made strongly basic with NaOH, and extracted well
with 3:1 CH2Cl2–isopropyl alcohol. The organic solu-
tion was washed with brine, dried over Na2SO4, and
evaporated under vacuum. The residue was chro-
matographed on silica gel, eluting with a gradient from
50 to 100% C6H14–EtOAc to give 125 mg (18%) of 20
1
combined and used in the next step. H-NMR: 5.1 (s,
2H), 3.9 (s, 3H), 2.3 (s, 3H). 13C-NMR: 157.55, 153.13,
138.27, 124.52, 54.18, 24.12. MS EI+: 173 (50, M+1),
175 (18), 144 (22), 120 (15), 89 (30), 42 (100); CI+: 174
(100), 176 (30), 202 (12), 138 (22); HRMS found
174.0437, theory for C6H9N3OCl (M+H) is 174.0434.
1
as an amorphous solid. H-NMR (CDCl3) l 7.4 (br s,
1H), 4.1 (t, 2H), 4.0 (s, 3H), 3.9 (s, 3H), 3.5 (t, 2H), 2.6
(s, 3H); MS (FD): 327.2 [M+, 35Cl, 100%], 329.2 [M+,
37Cl, 38%]. HRMS Calc. for C12H14ClN5O4: 327.0736.
Found: 327.0732.
6.1.15. N-[4-Chloro-6-methoxy-2-methylpyrimid-5-yl]-
N%-benzoylthiourea (23)
6.1.13. 2-Carboxy-4-chloro-5-(imidazolidin-2-
ylidenimino)-6-methoxypyrimidine (4)
Ammonium thiocyanate (16.3 g, 0.21 mol) was added
to a 1 L round-bottomed flask fitted with a mechanical
agitator, a nitrogen purge, and a thermocouple. Ace-
tone (222 mL) was added and the mixture stirred until
homogeneous. Benzoyl chloride (24.4 mL, 0.21 mol)
was added and the mixture was stirred for 2 h at
ambient temperature. Aminopyrimidine 22 (37.0 g, 0.21
mol) was added along with 148 mL of C3H6O. After
stirring for 2 h at ambient temperature, 370 mL of
water was added. The mixture was stirred for 1.5 h at
ambient temperature, filtered, and the product was
washed with 150 mL of a 1:1 mixture of C3H6O and
water. The product was dried in vacuo at 50 °C
overnight to give 67.7 g (94%) of an off-white solid.
1H-NMR: 11.9 (s, 1H), 11.8 (s, 1H), 7.9 (d, 2H), 7.6 (m,
1H), 7.5 (t, 2H), 3.9 (s, 3H), 2.6 (s, 3H). 13C-NMR:
181.28, 168.00, 165.49, 165.38, 156.82, 133.15, 133.67,
128.63, 128.35, 116.36, 54.75, 25.08. MS, CI+: 301
(100), 337 (40), 339 (18); ES+: 301 (100), 337 (90), 339
(35); due to its instability, HRMS analysis showed only
the ion from C7H9N4OS corresponding to the loss of
benzamide and HCl, found 197.0492, theory is
197.0497.
To a solution of 20 (120 mg, 0.366 mmol) in THF (20
mL) and MeOH (5 mL) was added LiOH (15 mg, 0.626
mmol). After stirring for 16.75 h the suspension was
treated with aq. HCl (1N, 0.63 mL, 0.63 mmol) and
evaporated under vacuum. The residue was triturated
with THF and filtered to give 110 mg (100%) of 4 as a
hygroscopic solid. MS (FD): 271.2 [M+, 35Cl, 100%],
273.2 [M+, 37Cl, 37%]. HRMS Calc. for C9H10ClN5O3:
271.0474. Found: 271.0475.
6.1.14. 5-Amino-4-chloro-6-methoxy-2-methyl-
pyrimidine (22)
Sodium metal (5.43 g, 0.236 mol) was added in
portions to 265 mL of anhydrous MeOH in a 500 mL
round-bottomed flask fitted with a mechanical agitator,
a nitrogen purge, and a thermocouple. After a homoge-
neous solution was obtained, it was cooled to 20 °C
and 40.0 g (0.225 mol) of 5-amino-4,6-dichloro-2-
methylpyrimidine (21), [34] was added along with a
small MeOH rinse. The mixture was stirred overnight
at ambient temperature, filtered, and concentrated in
vacuo to a residue. Ethyl acetate (200 mL) and water
(65 mL) were added and the phases were separated. The
organic layer was washed with 30 mL of dilute aq.
NaCl and the combined aq. layers were extracted with
30 mL of EtOAc. The combined organic layers were
distilled at atmosphere pressure until the still tempera-
ture reached 93 °C. Hexane, 133 mL, was added and
the mixture was distilled again until the internal tem-
perature was 80 °C. Another 335 mL of C6H14 was
6.1.16. N-(4-Chloro-6-methoxy-2-methylpyrimid-5-yl)-
thiourea (24)
Sodium metal (4.09 g, 0.18 mol) was added in por-
tions to 600 mL of anhydrous MeOH in a 1 L round-
bottomed flask fitted with a mechanical agitator, a
nitrogen purge, and a thermocouple. After a homoge-
neous solution was obtained, it was cooled to 17 °C

D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
31
and 60.0 g (0.18 mol) of compound 23 was added,
along with a rinse of 20 mL of MeOH. The mixture was
stirred for 5 h at this temperature, then at ambient
temperature for one-half hour. It was cooled in an ice
bath and 5% aq. HCl was added until a pH of 7 was
obtained. The resultant slurry was stirred for an hour in
the ice bath then an hour in an ice–C3H6O bath. The
product was collected by filtration, rinsed with 60 mL
of cold 4:1 MeOH–water, and dried in vacuo overnight
at ambient temperature to give 38.5 g (93%) of a white
solid which was 96.4% pure by gradient HPLC. The
sample was stored in the freezer to minimise decompo-
added to the mixture and the resultant slurry was
stirred at ambient temperature for 15 min. The crude
material was isolated by filtration, rinsed with C6H14,
and dried in vacuo at 30 °C for 30 min. Its purity was
85% by the gradient HPLC method. The acetal was
purified by adding the partially dried solid to a clean
flask with 80 mL C6H14 and heating to 60 °C. Ethyl
acetate (40 mL) was added at this temperature and the
heating was removed. After stirring for one-half hour at
ambient temperature, the product was isolated by filtra-
tion, was rinsed with 2:1 C6H14–EtOAc, and was dried
in vacuo at 30 °C. The yield was 6.0 g (34%) of an
off-white solid, m.p. 81–91 °C. The purity by gradient
1
sition. H-NMR: 9.02 (br s, 1H), 7.8 and 7.2 (very br
singlets, 1H ea.), 3.93 (s, 3H), 2.47 (s, 3H). 13C-NMR:
182 (br), 166.46, 165.25 (br), 157.95, 128.5 (3 or 4 lines).
MS ES+: 233 (9, M+1), 235 (3), 197 (100); EI+: 196
(30), 167 (25), 125 (20), 60 (75), 29 (100); due to its
instability, HRMS analysis showed only the ion from
C7H9N4OS corresponding to the loss of HCl, found
197.0497, theory is 197.0497.
HPLC was 96.8%. H-NMR: 5.58 (t, J=7, 1H), 5.2 (s,
1
2H), 4.63 (t, J=7, 1H), 3.82 (s, 3H), 3.6 (m, 2H), 3.5
(m, 2H), 3.15 (t, J=7, 2H), 2.38 (s, 3H), 1.1 (t, J=7,
6H). 13C-NMR: 163.25, 156.77, 152.46, 150.29, 126.86,
100.55, 61.67, 53.79, 43.53, 24.55, 15.29. MS ES+: 332
(58, M+1), 334 (20), 286 (100), 288 (60); HRMS found
332.1493, theory for C13H23N5O3Cl (M+H) is
332.1489.
6.1.17. N-(2,2-Diethoxyethyl)-N%-(4-chloro-6-methoxy-
2-methylpyrimid-5-yl)-guanidine (26)
6.1.18. 4-Chloro-5-[4-hydroxy-imidazolidin-2-
Potassium hydroxide (10.6 g, 85% assay, 0.16 mol)
was added to a 250 mL round-bottomed flask fitted
with a mechanical agitator and a thermocouple. Water
(75 mL) was added and the solution was heated to
55 °C. Lead(II) acetate trihydrate (9.0 g, 0.024 mol)
was added followed by thiourea 24 (5.0 g, 0.022 mol)
and a rinse of 10 mL of water. The slurry was stirred
rapidly at 60 °C for 45 min and cooled to ambient
temperature. Glacial AcOH was added until the pH of
the black slurry was 7. About 3 g of filter aid was
added. The mixture was filtered on a pad of filter aid
and the cake was rinsed with about 10 mL of water
[42]. The aq. solution was washed twice with 100 mL
portions of EtOAc and evaporated in vacuo (bath
temperature of 50 °C) to a weight of 46 g. The purity
of this solution of cyanamide 25 was 98% by gradient
HPLC. The aq. solution of the cyanamide and 40 mL
of MeCN were combined in a clean flask and amino-
acetaldehyde diethyl acetal (6.2 mL, 0.043 mol) was
added followed by sufficient solid KH2PO4 to achieve a
pH of 8.5. The mixture was heated at 60 °C for 3 h,
cooled to ambient temperature, and treated with solid
KOH until the pH was 10. Filter aid was added and the
mixture was filtered to remove polymeric materials. The
phases were separated and the upper layer was evapo-
rated to a weight of 15 g in vacuo. Water (50 mL) and
EtOAc (100 mL) were added and the mixture was
agitated, then separated. The organic layer was concen-
trated in vacuo to a weight of about 14 g. Upon
standing overnight in the freezer, the product crys-
tallised. This slurry was combined with similar slurries
from one and one-half other such reactions (total of
12.5 g of 24 starting material). Hexane (150 mL) was
ylidenimino]-6-methoxy-2-methyl-pyrimidine (6)
Acetal 26 (2.0 g, 0.006 mol) and 21 mL of water were
combined in a flask with a magnetic stirrer and concd.
HCl (5.0 mL, 0.066 mol) was added dropwise at ambi-
ent temperature. After 85 min, the solution was con-
centrated over a period of 6 min in vacuo with a 50 °C
bath, removing 23 g. Acetone (200 mL) was added to
the residue and the resulting mixture stirred rapidly at
ambient temperature for one-half hour and then in an
ice bath for 1 h. The mixture was filtered, and the
product was rinsed with C3H6O and dried in vacuo at
27 °C for 1 h to give 1.21 g (68%) of a white solid. The
purity based on the isocratic HPLC method (but 70:30
aqueous–MeCN) was 98.1%. The NMR spectra show
hindered rotation, i.e. two structures of equal amounts:
1H-NMR: 8.8 (d, J=8, 1H), 8.3 (br s), 5,55 (m, 1H),
3.9 and 3.85 (s, s, 3H), 3.9 (m, 1H), 3.35 (m, 1H), 2.5 (s,
3H). 13C-NMR: 168.56 and 168.43, 168.36 and 167.44,
161.13 and 159.31, 156.76 and 156.61, 111.15, 84.21
and 84.04, 56.26 and 56.08, 50.15 and 49.88, 26.26. MS
ES+: 258 (100, M+1), 260 (38), 242 (95), 240 (92);
HRMS found 258.0748, theory for C9H13N5O2Cl (M+
H) is 258.0758.
6.1.19. N-(4-Chloro-6-methoxy-2-methylpyrimid-5-
yl)-1H-imidazol-2-amine (2)
Acetal 26 (1.0 g, 0.003 mol) and 10 mL of water were
combined in a flask with a magnetic stirrer and concd.
HCl (2.5 mL, 0.033 mol) was added dropwise at am-
bient temperature. After 4 h solid KOH was added
until the pH rose to 12. The mixture was stirred one-
half hour at ambient temperature and filtered. The solid
was rinsed with water and dried in vacuo, at 35 °C for

32
D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
2 days. The yield was 0.43 g (60%) of a pink solid, m.p.
175–178 °C. The purity by HPLC was 98.2%. 1H-
NMR: 6.55 (s, 2H), 3.88 (s, 3H), 2.49 (s, 3H). 13C-
NMR: 164.35, 160.66, 152.65, 145.85, 119.26, 118 (br),
54.48, 24.77. MS EI+: 239 (50, M+1), 241 (18), 204
(100), 189 (19), 174 (29), 56 (65), 42 (90); CI+: 240
(100), 242 (32), 268 (20), 204 (85); ES−: 238 (100), 240
(33), 202 (32), 206 (35); HRMS found 240.0651, theory
for C9H11N5OCl (M+H) is 240.0652.
collected, combined, and concentrated in vacuo to pro-
duce 0.43 g of colorless oil with a purity of 99.3% by
the gradient HPLC method. 1H-NMR (C3H6O-d6):
11.3, 11.2, 10.2, 7.6 (broad d, d, s, s, total 2H), 5.7 and
5.4 (m, m, total 2H), 4.10, 4.02, 4.00 (s, s, s, total 3H),
2.61, 2.60, 2.55 (s, s, s, total 3H). MS ES+: 274 (100,
M+1), 276 (29); HRMS found 274.0709, theory for
C9H13N5O3Cl (M+H) is 274.0707.
6.1.22.
L-S-[5-(Imidazolidin-2-ylidenimino)-6-methoxy-
6.1.20. N-(4-Chloro-6-methoxy-2-methylpyrimid-5-
yl)-guanidine (3)
2-methyl-pyrimidine-4-yl]-cysteine (8)
Moxonidine (1, 2.0 g, 8.3 mmol), NaHCO3 (0.7 g, 1.0
Compound 22 (4.0 g, 0.023 mmol) and 2.0 g
cyanamide (2.0 equiv. Aldrich Chem. Co.) were com-
bined in a 50 mL round-bottomed flask. Ethanol (20
mL) and concd. HCl (2.2 mL, 3 equiv.) were added and
the mixture was heated at 50 °C. An additional 2
equiv. of cyanamide and 2 equiv. concd. HCl were
added at this temperature and at 1.5–2 h intervals until
8 equiv. of cyanamide had been added. The mixture
was stirred overnight and the EtOH was evaporated.
The residue was dissolved in 30 mL water and washed
twice with 20 mL of methylene chloride. The pH of the
aq. layer was raised to 9.5 with solid NaOH. The
resultant slurry was stirred 1 h at ambient temperature
and one-half hour in an ice bath. The product was
isolated by filtration, washed with water, and dried for
2 days at 40 °C to yield 4.22 g (85%). Its purity was
equiv.) and L-cysteine (2.0 g, 2.0 equiv.) were mixed
with 20 mL of water and 10 mL of MeOH in a 50 mL
round-bottomed flask and heated to 55 °C. After 3 h at
this temperature, the mixture was cooled and concen-
trated on the rotary evaporator. It was purified along
with a similar batch from a second reaction at the same
scale on an 800 mL Biotage HP20 column, eluted with
water. Fractions rich in the desired adduct (retention
time 4 min on the isocratic HPLC method) were com-
bined and the water was removed on a rotary evapora-
tor with a bath temperature of 40 °C. The resulting
solid was further dried on the vacuum pump to give 1.5
g of a white foam. Its purity by HPLC was 97.2% with
1.2% of a late-eluting (11 min) degradation product.
1H-NMR (D2O): 4.1 (m, 1H), 3.9 (s, 3H), 3.8 (dd,
J=3, 15 Hz, 1H), 3.6 (s, 4H), 3.5 (dd, J=6, 15 Hz,
1H), 2.4 (s, 3H). 13C-NMR (D2O): 171.71, 167.28,
166.16, 164.09, 158.21, 110.08, 55.20, 54.27, 42.64,
37.15, 24.37. MS direct infusion ES+: 327 (100), 313
(30), 281 (35), 240 (60); HRMS found 327.1244, theory
for C12H19N6O3S (M+H) is 327.1239.
1
98.9% by the isocratic HPLC method. H-NMR: 5.25
(s, 4H), 3.82 (s, 3H), 2.36 (s, 3H). 13C-NMR: 163.56,
156.64, 153.53, 150.36, 126.96, 53.80, 24.58. MS EI+:
215 (20, M+1), 217 (8), 173 (18), 150 (12), 143 (15), 43
(100); CI+: 216 (100, M+1), 218 (30); HRMS found
216.0658, theory for C7H11N5OCl (M+H) is 216.0652.
6.2. Analytical methods
6.1.21. 4-Chloro-5-(3-4-dihydroxyimidazolidin-2-
ylidenimino)-6-methoxy-2-methyl-pyrimidine (7)
6.2.1. Isocratic HPLC
Compound 3 (1.0 g, 4.7 mmol) and 10 mL of water
were combined in a 50 mL round-bottomed flask and
1.0 equiv. of concd. HCl (0.38 mL) was added.
Aqueous glyoxal (40%, 0.96 mL, Aldrich Chem. Co.)
was added and the mixture was heated at 50 °C for 4 h.
After standing for 3 days at ambient temperature, it
was heated for 4 h at 60 °C. This produced a mixture
of the two isomers of the product and 3 in a ratio of
19:74:1.5 by the isocratic HPLC method (retention
times 5.1, 5.4, and 6.6 min, respectively). The water was
removed on the rotary evaporator with a bath tempera-
ture of 50 °C. Acetone, 15 mL, was added and evapo-
rated. Ethyl acetate, 25 mL, was added and the mixture
stirred at ambient temperature for 1 h. The solid was
filtered, rinsed with EtOAc, and dried at 40 °C for a
day to give 1.68 g of a white powder. It was purified by
HPLC on a 25 cm×23 mm Zorbax RX-C8 column
eluted with 92% water (containing 0.08% TFA) and 8%
MeCN at 22 mL min−1. The main fractions were
A Zorbax RX-C8 column, 25 cm×4.6 mm with 5 m
particles, was eluted at 1.0 mL min−1 at ambient
temperature with 75% aq. eluent (20 mmol sodium
pentanesulfonate, H2SO4 to pH 3) and 25% MeCN.
Detection was by UV at 230 nm.
6.2.2. Gradient HPLC
The same column and eluent solutions were used.
Detection was at 255 nm and the program was as
follows, all at 1.00 mL min−1: 75:25 (aq.: CH3CN) for
6 min, ramp linearly to 45:55 at 10 min, hold at 45:55
until 20 min, return to 75:25 at 25 min.
6.2.3. Metabolite identification
6.2.3.1. Sample preparation. Urine samples collected
from rats, mice and dogs were diluted 1:1 with 25 mM
CH3COONH4 buffer (pH 5.0) before analysis. Urine
samples collected from healthy volunteers were

D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
33
concentrated by adding an equal volume of MeCN
followed by evaporation to dryness at ca. 37 °C under
N2. Samples were reconstituted with 25 mM CH3-
COONH4 buffer (pH 5.0) before analysis. The bile
samples collected from bile-duct cannulated rats were
diluted 1:1 with 25 mM CH3COONH4 (pH 5.0) before
analysis. Plasma samples were mixed with 20% of
trichloroacetic acid (TCA) and centrifuged at ca. 3800
rpm for ca. 5 min to sediment precipitated proteins.
The entire volume of the supernatant fraction of the
sample was transferred to a Varian C-18, 200 mg, 3
mL solid phase extraction cartridge, which had been
conditioned with 2 mL of MeOH and equilibrated
with 2 mL of 1% TCA. The cartridge was then rinsed
with 2 mL of 1% TCA and moxonidine-related sub-
were anesthetised with 2% isoflurane (Aerrane,
Anaquest, Madison, WI) for implantation of blood
pressure transmitters (model TA11PA-C40, Data Scien-
ces Int. (DSI), St. Paul, MN). The abdomen was
shaved, scrubbed with Betadine®, and a 4.5-cm ab-
dominal incision was made beginning just caudal to
the approximate location of the kidneys. The abdomi-
nal aorta was isolated and gently cleaned of connec-
tive tissue with a sterile cotton swab. A small spatula
was used to raise a portion of the aorta away from
the vena cava in an area just rostral to the iliac artery
bifurcation. A bulldog clamp was placed caudal to the
left renal artery, and the aorta was punctured rostral
to the common iliac using a 21-gauge needle (bent at
a 45° angle with the bevel down). A fluid-filled
catheter (0.7 mm OD, 8 cm in length) attached to the
hermetically sealed transmitter was inserted and ad-
vanced to the bulldog clamp using the bent needle as
a guide. The area was dried with a cotton swab and
tissue adhesive applied at the entry point while the
clamp was removed. The entry point was further
sealed using tissue adhesive and a cellulose fiber patch
(Vetland, 3M Co). The body of the transmitter was
sutured to the muscles of the inner abdominal wall
using non-absorbable 4-0 silk, the muscle layers were
approximated with sterile 3-0 silk, and the final inci-
sion closed with sterile metal wound clips. All animals
were administered 10,000 units of penicillin intramus-
cularly (Ambi-Pen®, Butler), housed individually in
shoe box cages with food and water ad libitum, and
permitted to recover for at least 1 week before study.
Digitised pressure signals were acquired for 30 s every
10 min using DSI Dataquest IV 2.0 software. Mean
arterial pressure was calculated as the arithmetic mean
of the waveform sampled at a frequency of 500 Hz.
Heart rate was obtained from the pressure readings.
The 6 hourly values were averaged to obtain a mean
blood pressure over a 1 h interval. Pressure and heart
rate values in the drug groups were compared to the
vehicle group using Dunnett’s test for multiple com-
parisons. Statistical comparisons were performed with
JMP software (SAS, Cary, NC) with values of PB0.05
considered significant.
stances were eluted with
2
mL of 25 mM
CH3COONH4 (pH 5.0)–MeCN (50:50). The effluent
was evaporated to dryness at ca. 37 °C under N2.
Samples were reconstituted with 25 mM CH3-
COONH4 buffer (pH 5.0) before analysis.
6.2.3.2. LC/MS/MS analysis. Separation was per-
formed using a C-18 Supelcosil LC-ABZ column (25
cm×4.6 mm, 5 m) at a flow rate of 1 mL min−1
(Waters 600MS pump). The flow was split to allow
250 mL to flow into the electrospray source and 750
mL to a Ramona 5-LS radiochemical detector with a
solid flow cell, which provided simultaneously detec-
tion of radioactivity and total ion chromatogram. A
mobile phase gradient of (A) 25 mM CH3COONH4
(pH 5) and (B) MeCN was programmed as follows:
started with 100% A, changed to A–B at 95:5 from 0
to 20 min, held at 95:5 from 20 to 25 min, changed to
100% A from 25 to 26 min. The gradient was changed
slightly to optimise the separation of metabolites in
each matrix. The characterisation of the metabolites
was performed on a Finnigan TSQ 700 Mass Spec-
trometer. The samples were introduced using atmo-
spheric pressure ionisation (API) with electrospray.
Samples were analyzed in the positive ion mode using
a spray voltage of +4500 V, capillary heater temper-
ature of 270 °C, sheath gas of 80 psi (N2) and an
auxiliary gas flow (N2) of 30 mL min−1. For full scan
analysis, the mass spectrometer was scanned from 150
to 600 amu in 1 s. The MS/MS analysis was per-
formed utilizing collision energy of −30 eV and colli-
sion gas pressure of 1.5 mTorr of argon.
6.3.2. Animal studies
Animals were briefly sedated with 2% isoflurane.
Test compounds, moxonidine, or vehicle (water) were
administered via the tail vein in a total volume of
0.3–0.4 mL. Test solutions were prepared by adding
ca. 50 mL of 1 N HCL to the test compound and
slowly diluting with water to the appropriate concen-
tration. Rats were returned to telemetry cages and
their blood pressure was monitored for the subsequent
24 h. Monitoring was initiated immediately except
that the values for 15 min prior and subsequent to
dosing were omitted.
6.3. Pharmacology
6.3.1. Telemetry implantation
Spontaneously hypertensive rats (Tac:N(SHR)fBR)
were obtained from Taconic Farms (Germantown,
NY) at 14–16 weeks of age (295–325 gm) and housed
under a 12-h light–dark cycle (lights on from 06:00 to
18:00 h). Following a 1-week acclimation period, rats

34
D.D. Wirth et al. / European Journal of Medicinal Chemistry 37 (2002) 23–34
[18] V. Askam, R.H.L. Deeks, J. Chem. Soc. Sect. C (1969) 1935–
Acknowledgements
1936.
[19] H. Maehr, J.M. Smallheer, J. Org. Chem. 46 (1981) 1752–1755.
[20] R. Rangarajan, E.J. Eisenbraun, J. Org. Chem. 50 (1985) 2435–
2438.
[21] A. Belli, C. Giordano, A. Citterio, Synthesis (1980) 477–499.
[22] A.D. Cort, L. Mandolini, S. Panaioli, Synth. Commun. 18
(1988) 613–616.
The authors gratefully acknowledge technical assis-
tance from Nicholas Bach, Steve Gregg, Gary Thomas,
Thomas J. Lindsay, and Trent L. Abraham. We also
are indebted to Lisa Shipley and Bill Wheeler for
helpful discussions and suggestions.
[23] H.J. Dauben, L.L. McCoy, J. Am. Chem. Soc. 81 (1959) 4863–
4872.
[24] A. Sudalai, G.S.K. Rao, Indian J. Chem. 28B (1989) 520–521.
[25] R.J. Hambalek, J. Just, Synth. Commun. 23 (1993) 209–215.
[26] G.E. McCasland, D.S. Tarbell, J. Am. Chem. Soc. 68 (1946)
2393–2395.
[27] A.E. Frissen, A.T.M. Marcelis, D.G. Buurman, C.A.M. Poll-
mann, H.C. van der Plas, Tetrahedron 45 (1989) 5611–5620.
[28] N.C. Barua, R.P. Sharma, J.N. Baruah, Tetrahedron Lett. 24
(1983) 1189–1190.
References
[1] P. Ernsberger, H. Elliott, H.-J. Weiman, A. Raap, M.A. Haxhiu,
E. Hofferber, A. Loew-Kroeger, J.L. Reid, H.-J. Mest, Cardio-
vasc. Drug Rev. 11 (1993) 411–431.
[2] B. Szabo, R. Urban, Arzneim.-Forsch./Drug Res. 47 (1997)
1009–1015.
[3] V. Pla¨nitz, J. Clin. Pharmacol. 27 (1987) 46–51.
[4] J.-P. Ollivier, V. Bouchet, Am. J. Cardiol. 70 (1992) 27C–36C.
[5] J. Webster, H. Koch, J. Cardiovasc. Pharmacol. 27 (1996) S49–
S54.
[6] P. Ernsberger, T. Damon, L. Graff, S. Scha¨fer, M. Christen, J.
Pharmacol. Exp. Ther. 264 (1993) 172–182.
[7] P. Ernsberger, Rev. Contemp. Pharmacother. 9 (1998) 441–462.
[8] P. Ernsberger, M. Graves, L.M. Graff, N. Zakieh, P. Nguyen,
L.A. Collins, K.L. Westbrooks, G.G. Johnson, Ann. N.Y. Acad.
Sci. 763 (1995) 22–42.
[29] D.S. Matteson, E.C. Beedle, A.A. Kandil, J. Org. Chem. 52
(1987) 5034–5036.
[30] J.G. Roberts, J. Chem. Soc. (1964) 176–178.
[31] Attempts to hydrolyze 5 with NaOH or LiOH led to the
substitution of the chloride, whereas treatment with LiAlH4
caused partial reduction of the aromatic nitro group.
[32] M.P. Nemeryuk, A.L. Sedov, I. Krepelka, Y. Benes, T.S.
Safonova, Chem. Het. Compds. (1983) 1113–1115.
[33] R. Urban, O. Schinder, Helv. Chim. Acta 41 (1958) 1806–1816.
[34] A. Albert, D.J. Brown, H.C.S. Wood, J. Chem. Soc. Pt. 4 (1954)
3832–3839.
[35] F. Loftus, US Patent 4,497,949, issued Feb. 5, 1985.
[36] R. Niwa, H. Kamada, E. Shitara, J. Horiuchi, N. Kibushi, T.
Kato, Chem. Pharm. Bull. 44 (1996) 2314–2317.
[37] F. Kurzer, Org. Synthesis 31 (1951) 19–20.
[38] A.W. Czarnik, N.J. Leonard, J. Org. Chem. 45 (1980) 3514–
3517.
[39] M. He, T.L. Abraham, T.J. Lindsay, S.H. Chay, B.A. Czeskis,
L.A. Shipley, Drug Metab. Dispos. 28 (2000) 446–459.
[40] Nemeryuk et al. report the m.p. as 56–58 °C [17].
[41] Urban and Schinder report the m.p. as 60–61 °C [17].
[42] A mixture of the cyanamide and KOAc was isolated from this
mixture by evaporation of the water. It gave the following
spectra. 1H-NMR: 3.8 (s, 3H), 2.26 (s, 3H). 13C-NMR: 161.82,
148.80, 142.32, 129.47, 123.69, 53.44, 23.97. MS ES+: 199 (70,
M+1), 201 (28), 123 (100); HRMS found 199.0378, C7H8N4OCl
theory is 199.0387.
[9] P. Ernsberger, J.E. Friedman, R.J. Koletsky, J. Hypertens. 15
(Suppl. 1) (1997) S9–S23.
[10] J.J. Buccafusco, C.A. Lapp, K.L. Westbrooks, P. Ernsberger, J.
Pharmacol. Exp. Ther. 273 (1995) 1162–1171.
[11] R. Theodor, H.-J. Weimann, M. Mu¨ller, H. Mosberg, K.
Michaelis, Eur. J. Clin. Res. 8 (1996) 63–74.
[12] W. Hoefke, S. Darda, W. Gaida, Arzneim.-Forsch. 35 (1985)
401–405.
[13] S. Darda, H.J. Forster, H. Stahle, Arzneim.-Forsch. 28 (1978)
255–259.
[14] Stenzel W., Fleck W., Cohnen E., Armah B., US Patent
4,323,570, issued Apr. 6, 1982.
[15] W.S. Trahanovsky, B.L. Young, J. Org. Chem. 31 (1966) 2033–
2035.
[16] T.L. Ho, Synthesis (1973) 347–354.
[17] T. Thyrann, D.A. Lightner, Tetrahedron Lett. 36 (1995) 4345–
4348.