Arylazoamidoximes and related compounds as NO-modulators

Article abstract of DOI:10.1002/ardp.200900060

Three amidinoarylhydrazines 1, three arylazoamidines 2, and nine arylazoamidoximes 3 have been synthesized and investigated for their potential to function as nitric oxide (NO) modulators. In-vitro studies demonstrated that 2 and 3 inhibited platelet aggregation (2c, IC₅₀ = 3 μM) which could also be shown in vivo by inhibition of thrombus formation in arterioles (3a, 22%). Moreover, for all compounds antihypertensive effects were examined in vivo with SHR rats, with 2a being the most potent candidate by lowering blood pressure by 19%. However, no common underlying mechanism of action could be shown. Some of these compounds released HNO non-enzymatically. Incubations with NO synthase isoforms (NOSs) revealed, that compounds 1 to 3 were weak substrates for NOSs but arylazoamidoximes 3 remarkably elevated the NOSs activity in the presence of L-arginine (3h, up to fivefold). In addition, we examined effects on arginase and dimethylarginine dimethylaminohydrolase (DDAH), two further enzymes involved in the complex regulation of NO biosynthesis, to elucidate whether the observed in-vivo effects can be traced back to their modulation. Furthermore, the metabolic fate of arylazoamidoximes 3 was addressed by investigation of a possible N-reductive biotransformation. In summary, novel NO-modulating compound classes are presented, among which arylazoamidoximes 3 are potent activators of NOS isoforms, and arylazoamidines 2 exert in-vivo effects by unknown mechanisms.

Full text of DOI:10.1002/ardp.200900060

Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
A. Schrꢀder et al.
9
Full Paper
Arylazoamidoximes and Related Compounds as NO-
modulators
Alexander Schrꢀder1, Jꢁrke Kotthaus2, Dennis Schade2, Bernd Clement2, and Klaus Rehse1
1 Pharmaceutical Institute, Freie Universitꢁt Berlin, Berlin, Germany
2 Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Kiel, Germany
Three amidinoarylhydrazines 1, three arylazoamidines 2, and nine arylazoamidoximes 3 have
been synthesized and investigated for their potential to function as nitric oxide (NO) modula-
tors. In-vitro studies demonstrated that 2 and 3 inhibited platelet aggregation (2c, IC50 = 3 lM)
which could also be shown in vivo by inhibition of thrombus formation in arterioles (3a, 22%).
Moreover, for all compounds antihypertensive effects were examined in vivo with SHR rats, with
2a being the most potent candidate by lowering blood pressure by 19%. However, no common
underlying mechanism of action could be shown. Some of these compounds released HNO non-
enzymatically. Incubations with NO synthase isoforms (NOSs) revealed, that compounds 1 to 3
were weak substrates for NOSs but arylazoamidoximes 3 remarkably elevated the NOSs activity
in the presence of L-arginine (3h, up to fivefold). In addition, we examined effects on arginase
and dimethylarginine dimethylaminohydrolase (DDAH), two further enzymes involved in the
complex regulation of NO biosynthesis, to elucidate whether the observed in-vivo effects can be
traced back to their modulation. Furthermore, the metabolic fate of arylazoamidoximes 3 was
addressed by investigation of a possible N-reductive biotransformation. In summary, novel NO-
modulating compound classes are presented, among which arylazoamidoximes 3 are potent
activators of NOS isoforms, and arylazoamidines 2 exert in-vivo effects by unknown mechanisms.
Keywords: Antihypertensive / Antiplatelet / Antithrombotic / HNO–NO formation / NOS activation /
Received: March 20, 2009; Accepted: July 7, 2009
DOI 10.1002/ardp.200900060
neuromediator in basic physiological functions, includ-
ing modulation of pain [1, 2]. Accordingly, it is vital that
NO formation is strictly regulated, and it is not surpris-
ing that dysregulation leads or contributes to the devel-
opment of numerous diseases [3]. Insufficient NO avail-
ability is associated with hypertension [4], coronary heart
disease [5], heart failure and myocardial infarction as
well as erectile dysfunction [6]. The predominant
enzymes in this regulation process are NOSs, arginases,
Introduction
Nitric oxide (NO) is endogenously formed from L-arginine
via nitric oxide synthases (NOSs) and is involved in vari-
ous physiological processes, such as regulation of vascu-
lar tone, inhibition of platelet aggregation, and leuko-
cyte adhesion on the endothelial surface [1]. In addition,
it is generated in large quantities by macrophages during
host defence and immunological reactions as well as by
neurons of the central nervous system, where it acts as a
and
dimethylarginine
dimethylaminohydrolases
(DDAHs).
Physiologically, NOSs activity is regulated by the
endogenous, competitive inhibitors asymmetric Nx,Nx-
dimethyl-L-arginine (ADMA) and Nx-monomethyl-L-argi-
nine (NMMA). These compounds, which are released by
proteolysis of various proteins containing methylated
arginine residues, are degraded by DDAHs to L-citrulline
and either dimethylamine or methylamine [7].
Correspondence: Prof. Dr. Klaus Rehse, Pharmaceutical Institute –
Freie Universitꢁt Berlin, Kꢀnigin-Luise-Str. 2 + 4, D-14195 Berlin, Ger-
many.
E-mail: rehiwer@zedat.fu-berlin.de
Fax: +49 30 304-2925
Abbreviations: dimethylarginine dimethylaminohydrolases (DDAHs); ni-
tric oxide (NO); nitric oxide synthases (NOSs); spontaneously hyperten-
sive rat (SHR)
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10
A. Schrꢀder et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
Interestingly, a regulatory mechanism between argi-
nase and NOSs becomes more and more apparent, since
these metabolic pathways compete for their common
substrate L-arginine [8]. Essentially, arginase inhibition
leads to increased NO levels by elevating the substrate
pool for NOSs.
In this paper, we have concentrated on amidinoarylhy-
drazines 1, arylazoamidines 2, and arylazoamidoximes 3,
which were expected to be alternate substrates for NOSs
or, respectively, would release NO by other mechanisms.
Therefore, typical NO-dependent effects were investi-
gated by several methods such as the inhibition of plate-
let aggregation, antithrombotic effects or by blood pres-
sure measurements in SH (spontaneously hypertensive)
rats. Moreover, in this study we examined if the observed
effects depend on NOS activation or were due to a modu-
lation of arginase or DDAH, two other enzymes of the NO
generating system.
Results and discussion
Scheme 1. Synthesis of amidinoarylhydrazines 1, arylazoami-
dines 2, and arylazoamidoximes 3. For details see Experimen-
tal.
Chemistry
The synthesis of type 1–3 compounds was performed
according to modified literature procedures (see Scheme
1). Reaction of hydrazines with S-methylisothiouronium
sulfate yield amidinoarylhydrazines 1 in 60–66% overall
yield [9]. Oxidation with silver oxide delivered arylazo-
amidines 2 in only hardly reproducible manner [10]. Di-
azotation of anilines and reaction with cyanide yield aryl-
azonitriles which were not isolated but converted further
to the arylazoamidoximes 3 with hydroxylamine [11].
In-vivo antithrombotic effects
In the laser thrombosis assay format, compound 3a
showed the best potency (see Table 1). Arylazoamidines 2,
which seemed to be superior in the Born test, were less
effective in this in-vivo study possibly due to low absorp-
tion after oral application. At physiological pH, these
compounds are positively charged which impairs absorp-
tion. Amidoximes are much less basic and therefore
show improved oral bioavailability [13].
Biology
Secondary effects of nitric oxide formation were meas-
ured by the following methods. Two in-vivo test systems
were used to examine the relevance of the in-vitro results
for therapeutical purposes.
In-vivo effects on blood pressure
The blood pressure measurement showed that most com-
pounds reduced blood pressure in SHR rats, among
which the most active candidates were 1a, 1b, 2a, 2c, 3a,
3c, and 3d (see Table 1). Obviously, 4-substituted substan-
ces of all classes (1–3) were particularly potent. However,
In-vitro effects on platelet aggregation
First, stimulation of guanylate cyclase by nitric oxide and
subsequent inhibition of blood platelet aggregation was
determined via the Born test [12]. As can be seen in Table the observed antihypertensive potency of arylazo-
1, two classes of compounds (2, 3) inhibited platelet amidines 2 appeared surprising, because of their lack of
aggregation. Arylazoamidines 2 represent the most
potent compounds with IC50 values between 3 to 30 lM.
Moreover, the results showed that a 4-fluoro-substitution
potency in the laser-thrombosis model which can be
explained by a poor oral bioavailability (see above).
of 2c is superior to a 4-Cl moiety. In addition, by compar- In-vitro NO formation by NOS isoforms
ing 2a and 2b a substitution in position 4 seems to
As compounds 1–3 are structurally similar to N-hydroxy-
improve the potency. This is also underlined by the
guanidines, they were thus examined as alternate sub-
results of type 3 compounds. Within these compounds strates for NOSs or, respectively, as activators of the
3a, 3c, and 3f were almost as effective as 2b, whereas all
other substitution patterns were less favorable.
enzyme (see Table 2). NO formation was measured indi-
rectly by detecting nitrite via the Griess assay.
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Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
Arylazoamidoximes and Related Compounds
11
Table 1. Results of compounds 1a–c, 2a–c and 3a–i in the Born-, laser thrombosis and blood pressure test.
Born-testa)
IC50
(lM)
Laser thrombosis testb)
Inhibition in arterioles (%)
60 mg/kg
Blood pressure testc)
Max. Blood-pressure reduction
after 2 h (%)
Compound
R
30 mg/kg
60 mg/kg
1-Amidino-2-arylhydrazines
1a
1b
1c
4-Cl
3-Cl
4-F
>300
>300
>300
7 € 1
7 € 2
6 € 2
17 € 4
7 € 6
9 € 5
n. m.
n. m.
n. m.
Arylazoamidines
2a
2b
2c
4-Cl
3-Cl
4-F
14
30
3
8 € 1
4 € 1
n. s.
13 € 7
n. s.
11 € 5
19 € 4
n. m.
n. m.
Arylazoamidoximes
3ad)
3b
3cd)
3d
3e
4-Cl
3-Cl
4-F
2-Cl
3-F
2-F
4-CF3
4-phenyl
2,6-Cl
37
160
25
210
170
30
230
210
150
22 € 2
3 € 2
n. s.
7 € 1
9 € 2
10 € 2
6 € 1
9 € 2
7 € 2
10 € 3
n. s.
10 € 3
4 € 3
12 € 2
14 € 6
15 € 14
5 € 3
6 € 5
n. s.
n. s.
8 € 3
n. m.
n. m.
n. m.
n. m.
n. m.
n. m.
3f
3g
3h
3i
a)
Born-test: In-vitro inhibition of platelet aggregation induced with collagen.
laser thrombosis test: In-vivo inhibition of laser-induced thrombosis formation in mesenterial vessels of male Wistar rats after a
single oral administration.
blood-pressure test: Blood-pressure reduction of hypertensive rats, measured at the rat tail after a single oral administration.
described by Bade [30]; n. m. = not measured; n. s. = not significant.
b)
c)
d)
Most arylazoamidoximes 3 enhanced nitrite formation Further studies will be performed to investigate the acti-
in the presence of L-arginine, whereas in the absence of L- vation of NOS isoforms more precisely.
arginine only small amounts of nitrite were detectable.
It is remarkable that 3h is the most potent activator of
The biphenyl derivative 3h showed a 34% release of NOSs in vitro, since it showed low activity in in-vivo mod-
nitrite compared to L-arginine by nNOS. However, in the els. This might be explained by a possible N-reductive bio-
presence of L-arginine the activation of NOSs was much transformation to the respective arylazoamidine which
greater. In particular, this is striking for 3h which acti- is well recognized for N-hydroxyguanidines and amidox-
vates nNOS threefold, eNOS fourfold, and iNOS even five- imes [13, 14]. Ambiguously, all other arylazoamidoximes
fold. In addition, 3a and 3i were also potent activators of 3 reveal in-vivo efficacies but should be metabolically
NOS isoforms. In summary, all type-3 compounds repre- reduced as well.
sent weak substrates for NOSs but potentiate NOSs activ-
ity substantially in the presence of L-arginine, however, it Non-enzymatical release of HNO and NO
has to be noted that the employed Griess-assay format is In order to examine whether the observed in-vivo effects
susceptible to artifacts, which we cannot entirely rule are due to a non-enzymatical release of NO or HNO – both
out. Though, this study may at least provide a clue for the of them are attributed several biological effects [15] – com-
underlying mechanism for the observed in-vivo effects. pounds 1–3 were incubated for 1 h under anaerobic condi-
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12
A. Schrꢀder et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
Table 2. Incubations of compounds 1–3 (1 mM) with NO-synthase isoenzymes (nNOS, eNOS, iNOS) in the absence and presence
of L-arginine (0.5 mM).a)
Compound
R
nNOS
eNOS
iNOS
nNOS + L-arg.
eNOS + L-arg.
iNOS + L-arg.
1-Amidio-2-arylhydrazines
1a
1b
1c
4-Cl
3-Cl
4-F
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
69 € 20
n. m.
34 € 3
22 € 3
n. m.
13 € 2
46 € 4
n. m.
12 € 1
Arylazoamidines
2a
2b
2c
4-Cl
3-Cl
4-F
n. s.
n. s.
n. s.
18 € 2
n. s.
27 € 3
19 € 8
n. s.
11 € 3
67 € 13
n. m.
25 € 1
29 € 2
n. m.
9 € 5
67 € 7
n. m.
31 € 3
Arylazoamidoximes
3a
3b
3c
3d
3e
3f
3g
3h
3i
4-Cl
3-Cl
4-F
2-Cl
3-F
2-F
4-CF3
4-phenyl
2,6-Cl
n. s.
n. s.
n. s.
n. s.
n. s.
n. s.
16 € 3
34 € 3
n. s.
n. s.
n. s.
n. s.
n. s.
332 € 9
103 € 3
140 € 12
107 € 2
100 € 4
93 € 3
n. m.
310 € 7
183 € 5
388 € 25
107 € 2
105 € 10
115 € 3
99 € 4
96 € 6
n. m.
387 € 10
150 € 30
484 € 29
112 € 7
149 € 2
113 € 8
100 € 8
87 € 13
n. m.
10 € 1
17 € 2
n. s.
11 € 7
9 € 2
15 € 2
4 € 3
28 € 11
17 € 7
n. s.
12 € 9
8 € 6
20 € 13
n. s.
507 € 5
193 €15
a)
Nitrite formation (%) compared to a control with 0.5 mM L-arginine as substrate (100%); n. m. = not measured; n. s. = not signifi-
cant.
tions in phosphate buffer in the presence of trifluoroace-
Our studies clearly showed that these compounds do
toxy-iodosobenzene as an oxygen-donor at 378C. Further- not affect activity of arginase and DDAH-1 at concentra-
more, to mimic a CYP450-mediated HNO- or NO-release, a tions of 100 and 500 lM. Thus, the observed in-vivo effects
synthetic FeIII-porphyrine complex (FeIIITPPCl) was added seem to be due to an increase in NOSs activity. Particu-
to the incubation mixture [16, 17]. HNO was indirectly larly 2a was expected to affect one of these enzymes,
detected by measuring its stable decomposition product since it showed significant antihypertensive effects and
N2O by gas chromatography [18], whereas NO could be no modulation of NO, neither direct via NOSs nor non-
detected bychemiluminescence [19].
enzymatically. This phenomenon could have been
Only for the arylazoamidoximes 3 N2O could be explained by an arginase inhibition. Obviously, this com-
detected (3a: 5.7%, 3c: 4.5%) as a result of simple chemical pound reduces blood pressure by a different mode of
degradation. In the presence of FeIIITPPCl the formation action. A possible explanation would be that arylazoami-
of N2O was significantly enhanced (3a: 14.9%, 3c: 19.3%, dines 2 have structural similarities to guanfacine, which
3g: 4.3%). These findings indicate possible mechanisms lowers blood pressure by activating a2-receptors [20].
for a purely chemically and CYP450-mediated release of
Moreover, our investigations revealed that these com-
HNO in vivo which may be responsible for the observed pounds are not converted as alternate substrates, neither
biological effects. In contrast, the formation of NO was by arginase nor DDAH-1.
not significant for all compounds.
Reduction of arylazoamidoximes
Effects on bovine arginase and human DDAH-1
Meanwhile, a reduction of N-hydroxylated guanidines
Exemplary for all arylazoamidines and arylazoamidox- and amidines is well known [13, 14]. Therefore, we exam-
imes, the effects of 2a, 3a, and 3c on bovine liver arginase ined a possible physiological reduction of type-3 com-
and recombinantly expressed human DDAH-1 were pounds. 3a, 3b, and 3c were reduced by human and por-
examined. These enzymes are involved in the complex cine mitochondria, and microsomes purified from liver
regulation of NO bioavailability and might be responsi- and kidney. 3a is most intensely reduced with a maximal
ble for the effects observed in in-vivo studies. An addi- reduction rate of 17.16 nmol/(min mg) by human liver
tional inhibition of arginase would enhance NO forma- microsomes. Reduction rates for 3b and 3c are lower (for
tion, whereas inhibition of hDDAH-1 would impair NO detailed reduction rates see Table 3). This might, in part,
biosynthesis.
explain the differences in the described in-vitro and in-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
Arylazoamidoximes and Related Compounds
13
Table 3. Reduction rates of arylazoamidoximes 3 to the corre-
sponding arylazoamidines 2 by different enzyme sources.
pounds effectively lowered blood pressure in vivo. This
might be caused by a second mode of action such as the
stimulation of a2-receptors, considering their structural
similarity to guanfacine. Any further possible modula-
tion of the NO generating system explaining these effects
was ruled out by investigations with arginase and DDAH.
Specific activity
(nmol/(min mg protein)
3a
3b
3c
4-Cl
3-Cl
4-F
Mitochondria
Human liver
Human kidney
Porcine liver
1.22
3.25
0.81
3.92
0.10
0.12
0.17
0.17
n. s.
n. s.
1.04
1.77
Experimental
Porcine kidney
Chemistry
Microsomes
Human liver
Human kidney
Porcine liver
Porcine kidney
M.p. (uncorr.): Linstrꢀm apparatus (Bꢁhler, Tꢁbingen, Germany).
Elemental analysis: Elementar Vario EL (Elementar, Germany).
IR: Perkin-Elmer 1420 Ratio Recording IR-Spectrophotometer
(Perkin-Elmer, Norwalk, CT, USA). ATI Mattson Genesis Serie
FTIR (Unicam Analytische Systeme, Kassel, Germany). NMR:
Bruker AC 300 and Bruker Avance / DPX 400 (Bruker Bioscience,
USA). EI-MS: CH-7A-Varian MAT (70 eV; Varian, USA) and Kratos
MS 25 RF (80 eV; Kratos Analytical, UK).
17.16
6.17
1.68
1.99
0.38
0.12
0.11
0.12
2.89
n. s.
0.11
0.20
n. s. = not significant
vivo studies. In contrast to arylazoamidines 2, the investi-
gated arylazoamidoximes 3 were potent activators of all
NOS isoforms in vitro, whereas their in-vivo effects on
blood pressure and thrombosis formation were compara-
ble to the arylazoamidines. Probably, arylazoamidoximes
are physiologically reduced which seems to limit their
effects in vivo.
The synthesis of amidinoarylhydrazines 1a and 1c has already
been reported [21]. 3a and 3c were synthesized as described by
Rehse et al. [22]. All other compounds were prepared for the first
time.
Wistar rats for the in-vivo tests were purchased at Tierzucht
Schꢀnwalde, GmbH.
General procedure for the synthesis of 1-amidino-2-
arylhydrazines 1
According to a modified method of Ohuchida et al. [9], 26 mmol
of the aromatic hydrazine N HCl are mixed with equal molaric
amounts of NaOH and S-methylisothiourea-sulfate in an aque-
ous solution at 80–908C and stirred for 4–6 h. After cooling and
removing of solvent the residue is boiled in absolute ethanol
and filtered hot. The filtrate is concentrated in a vacuum and
the crude product recrystallized.
Conclusions
Within our investigations, arylazoamidines 2a, 2c and
arylazoamidoximes 3a, 3c turned out to be the most
potent compounds. Thus, chloro- or fluoro-substitution,
respectively, in position 4, seemed to be most favorable.
These compounds inhibited platelet aggregation and
thrombosis formation and also lowered blood pressure
of SHR rats. We assume that the observed effects were
due to a release of NO either by the compounds them-
selves or by an increase in NOSs activity. Our studies
clearly revealed that they did not liberate NO non-enzy-
matically, but to a small extent the release of HNO was
observed, which might, in part, explain their biological
effects.
Moreover, our studies with NOS isoforms showed that
arylazoamidines 2 and arylazoamidoximes 3 were only
weak alternate substrates for NOSs. However, 3a, 3c, and
3h were capable of enhancing NOSs activity in the pres-
ence of L-arginine up to fivefold. Despite this considerable
activation of NOSs, the in-vivo effects on blood pressure
were comparable to that of 2a and 2c, which seem to be
due to a rapid physiological N-reduction to the corre-
sponding arylazoamidines. In addition, it is striking that
2 did not enhance NOSs activity, whereas these com-
1-Amidino-2-(4-chlorophenyl)-hydrazine-monohydrate
1a [9]
From 3,29 g 4-chlorophenylhydrazine N HCl (18,6 mmol) and 2 g
(10,7 mmol) S-methylisothioureasulfate. Beige crystals (H2O),
m.p.: 2138C, yield: 1.3 g (66%). Anal. C7H11ClN4O (202). IR (KBr) m
[cm–1]: 3465, 3357, 3199, 3118, 3001, 2857, 1881, 1674, 1647,
1589, 1492, 1435, 1260, 1088, 825; 1H-NMR (400 MHz, DMSO-d6) d
[ppm]: 6.76 (d, J = 8.7 Hz, 2H, aromat. 2-H, 6-H), 7.23 (d, J = 8.7 Hz,
2H, aromat. 3-H, 5-H), 7.44 (br. s, 1H, D2O exchange,
ClC6H4NHNH), 7.69 (br. s, 1H, D2O exchange, ClC6H4NH), 8.23 (s,
2H, D2O exchange, -NH2), 9.61 (s, 1H, D2O exchange, =NH); MS (EI,
2108C) m/z (%): 184 (86) [M+9], 167 (44), 142 (18), 132 (27), 125
(100), 111 (21), 99 (18), 90 (25), 43 (37), 36 (53), 28 (11).
1-Amidino-2-(3-chlorophenyl)-hydrazine-monohydrate 1b
From 3.29 g 3-chlorophenylhydrazine N HCl (18.6 mmol) and 2 g
(10.7 mmol) S-methylisothioureasulfate. Beige crystals (ethylace-
tate), m.p.: 1648C, yield: 1.2 g (61%). Anal. C7H9ClN4 (202). IR (KBr)
m [cm–1]: 3435, 3215, 3156, 3054, 1672, 1644, 1598, 1502, 1476,
1415, 685; 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 6.75 (m, 2H, aro-
mat. 2-H, 6-H), 6.89 (d, J = 8.1 Hz, 1H, aromat. 4-H), 7.27 (t, 1H, aro-
mat. 5-H), 8.13 (s, 2H, D2O exchange, NH2), 8.43 (s, 1H, D2O
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A. Schrꢀder et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
exchange, =NH), 10.01 (s, 2H, D2O exchange, NH-NH); MS (EI,
3308C) m/z (%): 184 (100) [M+9], 167 (46), 142 (23), 132 (57), 125
(69), 111 (23), 90 (30), 75 (16), 43 (72), 36 (80).
dized with NaNO2 below 58C. The mixture was adjusted to pH = 8
with Na2CO3. Diethylether was added and the two-phase system
was stirred with 36 mmol KCN until the color difference of the
two phases was stable (ca. 30 min). The ether phase was collected
and stirred with an aqueous solution of 36 mmol hydroxylami-
ne N HCl and 63 mmol NaOH until the color difference of the two
phases was stable (20 to 60 min). The aqueous phase was washed
with diethylether and, thereafter, acidified with glacial acetic
acid. The precipitate was recrystallized.
1-Amidino-2-(4-fluorophenyl)-hydrazine-dihydrate 1c [9]
From 4.9 g 4-fluorophenylhydrazine N HCl (30 mmol) and 4.2 g
(30 mmol) S-methylisothioureasulfate. Beige crystals (THF), m.p.:
1658C, yield: 3.0 g (60%). Anal. C7H13FN4O2 (204.2). IR (KBr) m
[cm–1]: 3443, 3380, 3290, 3200, 3136, 3009, 1668, 1644, 1511,
1212, 829, 631. 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 6.75 (dd, J =
9.0 Hz, J = 6.8 Hz, 2H, aromat. 2-H, 6-H), 7.08 (t, J = 8.8 Hz, 2H, aro-
mat. 3-H, 5-H), 7.44 (br. s, 1H, D2O exchange, FC6H4NHNH), 7.67
(br. s, 1H, D2O exchange, FC6H4NHNH), 8.11 (s, 2H, D2O exchange,
-NH2), 9.64 (s, 1H, D2O exchange, =NH); MS (EI, 808C) m/z (%): 168
(?) [M+9], 126 (100), 110 (71), 95 (17), 83 (74), 57 (39), 43 (33), 36
(82), 28 (51).
3-Chlorophenylazoamidoxime-semihydrate 3b
From 5.1 g 3-chloraniline (40 mmol) and 2.50 g (36 mmol)
hydroxylamine N HCl. Crystals (ethylacetate/n-hexane), m.p.:
1628C, yield: 2,3 g (29%). Anal. C7H7ClN4O N 1/2 H2O (207.61). IR
(KBr) m [cm–1]: 3481, 3359, 3073, 2819, 1681, 1552, 1475, 1451,
1414, 1020, 979, 780; 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 5.91
(s, 2H, NH2, D2O exchange), 7.62 (dd, J = 4.3 Hz, J = 3.3 Hz, 1H, aro-
mat. 4-H), 7.64 (t, 1H, aromat. 5-H), 7.77 (s, 1H, 2-H), 7.80 (dd, 1H,
6-H), 11.35 (br. s, 1H, D2O exchange, NOH); MS (EI, 2008C) m/z (%):
198 (8) [M+9], 168 (56), 111 (73), 75 (31), 59 (100), 44 (23).
General procedure for the synthesis of arylazoamidines 2
According to a modified method of Henrion [10], 10 mmol of the
respective 1-amino-2-arylhydrazine 1 are dissolved in 20 mL of
methanol and suspended with 20 mmol of freshly precipitated
silver oxide for 60 min at room temperature. After filtration aryl-
azoamidines 2 are precipitated from the filtrate as their hydro-
chloride or maleate.
2-Chlorophenylazoamidoxime 3d
From 5.1 g 2-chloraniline (40 mmol) and 2.5g (36 mmol) hydrox-
ylamine N HCl. Crystals (chloroform), m.p.: 1798C, yield: 2.1 g
(26%). Anal. C7H7ClN4O N 1/2 H2O (207.61). IR (KBr) m [cm–1]: 3491,
3382, 3162, 2853, 1662, 1582, 1537, 1463, 1094, 1059, 1014, 766;
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 5.81 (s, 2H, NH2, D2O
exchange), 7.23 (t, 1H, aromat. 4-H), 7,57 (t, 1H, aromat. 5-H), 7.61
(d, 1H, 3-H), 7.83 (d, J = 7.7 Hz, 1H, 6-H), 11.52 (s, 1H, D2O
exchange, NOH); MS (EI, 1758C) m/z (%): 198 (8) [M+9], 168 (63), 139
(13), 111 (85), 75 (45), 59 (100), 44 (29).
4-Chlorophenylazoamidine N HCl 2a
From 1.24 g 1a (6.7 mmol) and 3.11 g (13.44 mmol) silver oxide.
Red crystals, m.p.: 1528C, yield: 0.5 g (34%). Anal. C7H8Cl2N4
(219.1). IR (KBr) m [cm–1]: 3282, 3142, 3089, 1720, 1485, 1447,
1407, 1281, 1165, 1153, 1088, 1014, 922. 1H-NMR (400 MHz,
DMSO-d6) d [ppm]: 7.81 (d, J = 8.7 Hz, 2H, aromat. 3-H, 5-H), 8.00
(d, J = 8.7 Hz, 2H, aromat. 2-H, 6-H), 9.65 (s, 2H, D2O exchange,
+
NH2), 9.78 (s. 2H, D2O exchange, NH2 ); MS (EI, 508C) m/z (%): 183
3-Fluorophenylazoamidoxime-semihydrate 3e
(0) [M+9], 111 (11), 43 (100).
From 4.44 g 2-chloraniline (40 mmol) and 2.5 g (36 mmol)
hydroxylamine N HCl. Crystals (ethylacetate/n-hexan), m.p.:
1768C, yield: 1.84 g (24%). Anal. C7H7FN4O N 1/2 H2O (197.16). IR
(KBr) m [cm–1]: 3484, 3363, 3075, 2825, 1681, 1598, 1475, 1433,
1239, 1021, 981, 792; 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 5.92
(s, 2H, NH2, D2O exchange), 7.41 (dt, J = 8.4 Hz, J = 1.9 Hz, 1H, aro-
mat. 5-H), 7,54 (dd, 1H, aromat. 4-H), 7.64 (dd, J = 8.0 Hz, J = 5.9
Hz, 1H, 6-H), 7.71 (d, J = 8.0 Hz, 1H, 6-H), 11.47 (s, 1H, D2O
exchange, NOH); MS (EI, 2008C) m/z (%): 182 (38) [M+9], 152 (83), 95
(100), 75 (31), 59 (100), 44 (32).
3-Chlorophenylazoamidine-maleate 2b
From 3.0 g 1b (16.2 mmol) and 8.0 g (34.5 mmol) silver oxide.
Beige powder, m.p.: 1318C, yield: 0.1 g (2%). Anal. C11H13ClN4O4
(300.7). IR (KBr) m [cm–1]: 3343, 3072, 1731, 1623, 1573, 1475,
1388, 1361, 1195, 1071, 863; 1H-NMR (400 MHz, DMSO-d6) d
[ppm]: 6.16 (s, 2H, maleat.), 7.85 (t, J = 8.0 Hz, 1H, aromat. 5-H),
7.97 (d, 1H aromat. 4-H), 8.02 (s, 1H, aromat. 2-H), 8.08 (d, J = 8.0
+
Hz, 1H, aromat. 6-H), 9,78 (m, 4H, D2O exchange, NH2, NH2 ); MS
(EI, 908C) m/z (%): 184 (2) [M+9], 111 (13), 99 (14), 75 (10), 72 (96), 43
(100), 55 (31), 27 (49).
2-Fluorophenylazoamidoxime 3f
From 4.44 g 2-fluoraniline (40 mmol) and 2.5g (36 mmol) hydrox-
ylamine N HCl. Yellow crystals (ethylacetate/n-hexane), m.p.:
1768C, yield: 2.20 g (29%). Anal. C7H7FN4O (188). IR (KBr) m [cm–1]:
3487, 3369, 3090, 2850, 1682, 1553, 1484, 1228, 1106, 1020, 763;
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 5.83 (s, 2H, NH2, D2O
exchange), 7.31 (t, J = 7.8 Hz, 1H, aromat. 4-H), 7.48 (t, J = 8.5 Hz,
1H, aromat. 5-H), 7.64 (m, 2H, 3-H, 6-H), 11.44 (s, 1H, D2O
exchange, NOH); MS (EI, 358C) m/z (%): 182 (12) [M+9], 152 (77), 123
(14), 110 (32), 95 (96), 75 (37), 59 (100), 44 (30).
4-Fluorophenylazoamidin-maleate 2c
From 2.97 g 1c (17.7 mmol) and 6.0 g (26.0 mmol) silver oxide.
Yellow powder, m.p.: 1598C, yield: 0.5 g (6%). Anal. C11H11FN4O4
(282.2). IR (KBr) m [cm–1]: 3433, 1720, 1630, 1504, 1367, 1237,
1141, 852, 794; 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 5.95 (s, 2H,
maleinic acid), 7.53 (t, J = 8.6 Hz, 2H, aromat. 3-H, 5-H), 8.01 (dd,
J = 8.5 Hz, J = 5,2 Hz, 2H, aromat. 2-H, 6-H), 9.62 (m, 4H, D2O
+
exchange, NH2, NH2 ), MS (EI, 908C) m/z (%): 166 (?) [M+9], 123 (1),
95 (10), 72 (13), 43 (100).
4-Trifluoromethylphenylazoamidoxime 3g
General procedure for the synthesis of
arylazoamidoximes 3
According to Longo [11], 40 mmol of the respective aromatic
amine was dissolved or suspended in 50 mL of 10% HCl and oxi-
From 6.44 g 4-trifluoraniline (40 mmol) and 2.5 g (36 mmol)
hydroxylamine N HCl. Yellow crystals (ethylacetate/n-hexane),
m.p.: 1528C, yield: 4.15 g (45%). Anal. C8H7F3N4O (232.2). IR (KBr) m
[cm–1]: 3470, 3385, 3086, 2827, 1668, 1542, 1409, 1323, 1176,
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Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
Arylazoamidoximes and Related Compounds
15
1133, 1066, 1011, 850; 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 5.99
(s, 2H, NH2, D2O exchange), 7.97 (dd, J = 8.9, J = 4.3 Hz, 4H, aromat.
2-H, 3-H, 5-H, 6-H), 11.61 (s, 1H, D2O exchange, NOH); MS (EI,
908C) m/z (%): 232 (7) [M+9], 202 (59), 145 (99), 95 (12), 59 (100), 44
(20), 28 (23).
EDTA. Compounds were dissolved in DMSO and diluted to a final
concentration of 1 mM in 1.25% DMSO. Some incubation sam-
ples additionally contained 0.5 mM L-arginine. Reactions were
performed in a final volume of 80 lL and incubated for 30 min
at 378C. Since excessive NADPH can cause low values by reducing
the diazonium cation as well as the azo dye, it is necessary to
remove NADPH using an additional incubation with 20 lL lac-
tate dehydrogenase (20 U/mL) and 1.6 mM sodium pyruvate [26].
After incubation for 20 min at 378C, reactions were terminated
by adding 50 lL of ice-cold acetonitrile and centrifugation
(100006g, 15 min, 48C). 100 lL of supernatants were mixed
with 20 lL of Griess reagent (sulfanilamide 5.4% and N-(1-naph-
thyl)-ethylenediamine 0.9% in 1 N HCl) and the absorption of the
formed azo dye was measured at 543 nm against a blank con-
taining all cofactors diluted in assay buffer.
4-Biphenylazoamidoxime 3h
From 2.5 g 4-biphenylaniline (14.8 mmol) and 0.97 g (14.0 mmol)
hydroxylamine N HCl. Yellow crystals (ethylacetate/n-hexane),
m.p.: 1488C, yield: 1.2 g (13%). Anal. C13H12N4O (240.2). IR (KBr) m
[cm–1]: 3465, 3370, 3064, 2830, 1654, 1599, 1551, 1483, 1405,
1006, 849, 767, 695; 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 5.89 (s,
2H, NH2, D2O exchange), 7.41 (t, J = 7.2 Hz, 1H, aromat. 49-H), 7.51
(t, J = 7.1 Hz, 2H, aromat. 39-H, 59-H), 7.76 (d, 2H, aromat. 29-H, 69-H)
7,92 (m, 4H, aromat. 2-H, 3-H, 5-H, 6-H), 11,24 (s, 1H, D2O
exchange, NOH); MS (EI, 908C) m/z (%): 240 (13) [M+9], 210 (51), 168
(55), 153 (100), 59 (100), 43 (15), 28 (10).
Incubations containing 0.5 mM L-arginine but no test com-
pound were referred to 100%. Specific activities of the applied
enzyme sources were 800 nmol min–1 mg–1 for nNOS, 184 nmol
min–1 mg–1 for eNOS, and >1 nmol min–1 mg–1 for iNOS.
4.1.3.7 2,6-Dichlorophenylazoamidoxime 3i
From 6.48 g 2,6-dichloraniline (40 mmol) and 2.5 g (36 mmol)
hydroxylamine N HCl. Red crystals (ethylacetate/n-hexane), m.p.:
1808C, yield 5.0 g (54%). Anal. C7H6Cl2N4O (232). IR (KBr) m [cm–1]:
3465, 3365, 3078, 2849, 1668, 1559, 1451, 1432, 1347, 1092,
1027, 792; 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 6.01 (s, 2H, NH2,
D2O exchange), 7.41 (t, J = 8.1 Hz, 1H, aromat. 4-H), 7.62 (d, J = 8.1
Hz, 2H, aromat. 3-H, 5-H), 11.54 (s, 1H, D2O exchange, NOH; MS
(EI, 908C) m/z (%): 232 (1) [M+9], 202 (15), 187 (12), 173 (14), 160
(28), 145 (48), 124 (10), 109 (25), 75 (12), 59 (100), 44 (24).
Detection of HNO and NO
Compounds 1–3 were dissolved in phosphate buffer/ethanol
(1:1) (3 lM) and argon was flushed through the solution for 20
min. Trifluoroacetoxy-iodosobenzene and FeIIITPPCl were added
to obtain a final concentration of 1 mM. The mixtures were incu-
bated for 1 h at 378C.
The chromatographical determination of N2O was carried out
with a Perkin-Elmer F22 gas-chromatograph, head-space tech-
nique, using a Porapak Q-column (Sigma-Aldrich, Germany) and
electron capture detector [18]. NO was detected by the chemilu-
minescence-method as published by Schmidt [19], using an
Ansyco NO-analyzer AC 30M (Anysco, Karlsruhe, Germany).
Biology
Platelet aggregation
The Born test was performed with collagen as inducer as
described in the literature [12, 23]. Due to solubility issues, all
compounds were assayed with 0.3% DMSO, which did not affect
platelet aggregation.
Expression and purification of 66His-tagged hDDAH-1
Human DDAH-1 was expressed in Escherichia Coli BL21 and puri-
fied via Ni-NTA-agarose as described previously [27].
Thrombosis model
In-vitro hDDAH-1 assay
The laser thrombosis test was performed as described in detail
in the literature [24]. Test compounds were suspended in a solu-
tion of arabic gum (1%) and orally administered in a concentra-
tion of 60 mg/kg. Control experiments with acetylsalicylic acid
(ASS) showed an inhibition of thrombosis in arterioles of 48 €
10%.
Inhibition rates of hDDAH-1 were determined as described in
detail by Kotthaus et al. [28] with the following alterations: Inhib-
itors were applied in final concentrations of 100 and 500 lM and
L-NNMA at a concentration of 200 lM. Due to solubility issues
assays contained 5% DMSO.
In-vitro arginase assay
Blood pressure measurements
A colorimetric assay was carried out with 0.3 lg of bovine liver
arginase in 150 lL of 50 mM Tris buffer pH = 7.4 containing 100
lM MnCl2 and 7 mM L-arginine. Inhibitors were applied at con-
centrations of 100 and 500 lM. Due to solubility issues, assays
contained 5% DMSO. Samples were incubated in a 96-well micro-
plate at 378C for 30 min, and the reaction was stopped by addi-
tion of 200 lL Colder reagent [29]. Microplates were sealed with
sealing tape, incubated at 958C for 20 min and then read at 526
nm.
The blood pressure model has been performed as described in
detail in the literature [25].
In-vitro NO-synthase assay
NO formation was measured indirectly by determining nitrite
formation via the Griess assay. eNOS and nNOS were obtained
from Prof. Dr. B. Mayer (University Graz, Austria), iNOS was pur-
chased from Alexis Biochemicals (Enzo Life Sciences GmbH, Lꢀr-
rach, Germany). Incubations were performed in assay buffer con-
taining 50 mM triethanolamine pH = 7.5, 0.5 mM NADPH, 5 lM
FMN, 5 lM FAD, and 10 lM H4B. Assay buffer for incubations
with eNOS and nNOS additively required 0.5 mM CaCl2 and 10
lg/mL calmoduline. iNOS buffer additionally contained MgCl2 in
a final concentration of 1 mM and nNOS buffer additively con-
tained 1 mM CHAPS, 10 mM 2-mercaptoethanol, and 0.5 mM
Reduction of arylazoamidoximes
Compounds were dissolved in DMSO and diluted to a final con-
centration of 3 mM in 150 lL of 100 mM phosphate buffer pH =
6.3 containing 3% DMSO. Incubations additively contained 1
mM NADH and reactions were started by the addition of 300 lg
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www.archpharm.com

16
A. Schrꢀder et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 9–16
enzyme source. Incubations were performed for 30 min at 378C
and reactions terminated by adding 150 lL of ice-cold methanol.
Samples were centrifuged (90006g, 5 min) and analyzed by
HPLC-analytics.
[12] G. V. Born, Nature 1962, 194, 927–929.
[13] B. Clement, Drug Met. Rev. 2002, 34, 565–579.
[14] B. Clement, T. Kunze, S. Heberling, Biochem. Biophys. Res.
Commun. 2006, 349, 869–873.
Metabolites were separated on a LiChrospher 60 RP-select B
(46250 mm; 5 lm; Merck Chemicals, Germany) with a RP-select
B 464.0 mm guard column, autosampler Waters 700 Satellite
WISP, Waters 600 Controller, and a Waters 486 absorbance
detector set at 320 nm (Waters Corporation, Milford, MA, USA).
Eluent: 10 mM octylsulfonate/acetonitrile 73:27, pH = 6.3, and
the flow-rate was kept at 1 mL/min. All compounds were sepa-
rated within 25 min.
[15] J. C. Irvine, R. H. Ritchie, J. L. Favaloro, K. L. Andrews, R. E.
Widdop, B. K. Kemp-Harper, Trends Pharmacol. Sci. 2008, 29,
601–608.
[16] H. Volz, T. Barth, Justus Liebigs Ann. Chem. 1989, 171–175.
[17] H.-J. Duchstein, S. Riederer, Arch. Pharm. 1995, 328, 317–
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[18] D. A. Wink, M. Feelisch, in Methods in Nitric Oxide Research
(Eds.: M. Feelisch, J. S. Stamler), John Wiley & Sons, Chi-
chester, New York, Brisbane, Toronto, Singapur 1996, pp.
403–412.
The authors have declared no conflict of interest.
[19] H. H. H. W. Schmidt, Med. Monatsschr. Pharm. 1994, 17,
168–185.
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www.archpharm.com