Synthesis and biological evaluation of oxadiazole derivatives as inhibitors of soluble guanylyl cyclase

Article abstract of DOI:10.1016/j.bmc.2009.12.027

Soluble guanylyl cyclase (sGC) is an ubiquitously expressed enzyme that generates the second messenger cGMP and hence, leads to a number of physiological responses including vasodilation, inhibition of platelet aggregation and neurotransmission. Whilst ma

Full text of DOI:10.1016/j.bmc.2009.12.027

Bioorganic & Medicinal Chemistry 18 (2010) 1288–1296
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of oxadiazole derivatives as inhibitors
of soluble guanylyl cyclase
Margarete von Wantoch Rekowski a, Anastasia Pyriochou b, Nektarios Papapetropoulos b, Anne Stößel a,
a,
Andreas Papapetropoulos b, , Athanassios Giannis
*
*
a Department of Chemistry and Mineralogy, Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, D-04103 Leipzig, Germany
b Department of Pharmacy, Laboratory of Molecular Pharmacology, University of Patras, GR-26504 Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Soluble guanylyl cyclase (sGC) is an ubiquitously expressed enzyme that generates the second messenger
cGMP and hence, leads to a number of physiological responses including vasodilation, inhibition of plate-
let aggregation and neurotransmission. Whilst many activating and stimulating modulators of sGC were
identified and studied in recent years, only two selective inhibitors are known: ODQ and NS 2028. Fur-
thermore, a synthetic approach to these inhibitors has not been reported yet. Herein, we describe a novel
and efficient synthesis of these inhibitors, as well as the preparation of three different classes of NS 2028
analogues. Biological evaluation of this library using rat aortic smooth muscle cells revealed four new
compounds with good to moderate sGC inhibitory activity. Our experiments underline the major impor-
tance of the oxadiazole ring in ODQ and NS 2028 for the efficiency of this class of inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 23 October 2009
Revised 2 December 2009
Accepted 8 December 2009
Available online 23 December 2009
Keywords:
Soluble guanylyl cyclase
Inhibitor
Oxadiazole synthesis
1. Introduction
and leucocyte adhesion through the vessel wall by modulating
the activity of phosphodiesterases, cGMP-dependent protein ki-
Endogenously produced nitric oxide participates in a number of
cell communication and signal transduction cascades by activation
of soluble guanylyl cyclases (sGC). These in turn, generate the sec-
ond messenger cGMP which can influence physiological processes,
as vasodilation or immune response. The NO–sGC–cGMP pathway
plays an important role in cell life and has attracted much interest
in medicinal research. Hence, several details concerning the mode
of action of sGCs and their downstream effects have been collected.
It is widely known that sGCs are heterodimeric proteins, consisting
nases and cGMP-gated ion channels.10,11
Dysfunction of the NO–sGC–cGMP pathway demonstrably in-
duces an array of cardiovascular, immunological or neurodegenera-
tive diseases. Several NO donors are currently in clinical use,
although they are limited due to unspecific effects of NO with other
biomolecules, lacking efficiency because of insufficient metabolism
and long-term resistances development. Hence, the trend goes to
novel sGC activators that do not release NO, and do not require the
presence of heme. Such compounds are being tested in pulmonary
hypertension and peripheral arterial occlusive disease.12–19
In contrast to sGC activators only two specific inhibitors are
known. The first generation of sGC inhibitors included methylene
blue and LY83583. However, later it was shown that these agents
do not act as sGC inhibitors, but rather block NO-stimulated cGMP
formation through the generation of superoxide anions that react
with NO and inactivate it. In the mid-to-late 1990s, two chemically
closely related sGC inhibitors became available, ODQ and NS 2028
(Fig. 1).
of a bigger
a
and a smaller b subunit. Two isoforms of each subunit
1b1 type
have been discovered in different mammalian cells. The
a
has been found to be the most abundant variant of sGCs.1–5 The b
subunit provides the histidine moiety His105, an essential element
for the interaction with NO. By coordination of His105 to a pros-
thetic heme group a complex is formed that serves as NO receptor.
It is assumed that NO binding induces the oxidation of ferrous
heme to its ferric form, leading to the cleavage of its axial bond
with His105 and ultimately, to heme pivoting. This in turn, causes
a conformational change that is transmitted to the C-terminus of
the enzyme harbouring the catalytic site to increase conversion
of GTP to cGMP. The activation of sGC by NO increases the rate
of cGMP formation several hundred-fold.6–9 Once formed, cGMP
promotes smooth muscle relaxation, inhibits platelet aggregation
O
O
O
O
N
N
Br
N
N
N
O
ODQ
NS 2028
* Corresponding authors. Fax: +30 2610 969337 (A.P.); +49 341 9736599 (A.G.)
E-mail addresses: apapapet@upatras.gr (A. Papapetropoulos), giannis@uni-leip
zig.de (A. Giannis).
Figure 1. Chemical structures of selective inhibitors ODQ and NS 2028.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.027

M. von Wantoch Rekowski et al. / Bioorg. Med. Chem. 18 (2010) 1288–1296
1289
These compounds have big advantages over the first generation
O
O
H
N
of NO inhibitors, as they do not produce superoxide anions, and do
not inhibit basal sGC activity. Moreover, they are selective for sGC,
since they do not reduce particulate GC activity. Both ODQ and NS
2028 are thought to act as selective heme oxidants, rendering sGC
insensitive to NO. These agents are valuable tools in unravelling
the role of sGC in physiology and disease. Besides their utility in
basic science studies, sGC inhibitors could become useful in treat-
ing hypotension, such as that seen during septic shock.20–23
Although ODQ and NS 2028 are known for approximately
15 years, their synthesis has not been reported yet. Moreover, the
pharmacophore structure of these compounds has not been dis-
closed in detail. Some analogues of ODQ and NS 2028 have been
prepared which were modified at the benzoxazine part. Thus, the
molecules contained polar substituents at the phenyl ring or had
a benzoxathine core. However, the oxadiazole element was not
changed.23 The aim of the present study is to establish a straight-
forward and efficient synthesis of ODQ and NS 2028 and further-
more to prepare analogues that provide information on the parts
of the ODQ/NS 2028 scaffold that are crucial for sGC inhibitory
activity.
N
N
N
Cl
N OH
N
a
b
N
ODQ
N
6
5
O
O
H
Br
NH2
N
Br
NO2
OH
Br
N
Br
N
X
c
d
b
OH
O
O
8
7
9: X = O
NS 2028
e
f
10: X = S
11: X = NOH
Scheme 2. Synthesis of ODQ and NS 2028. Reagents and conditions: (a)
H2NOHꢀHCl, Na2CO3, DMSO, rt, 24 h (40%); (b) CDI, THF, reflux, 24 h (79% for
ODQ; 95% for NS 2028); (c) SnCl2ꢀ2H2O, concd HCl, MeOH, 0 °C?rt, 4.5 h (quant.);
(d) i—NaHCO3, H2O, 4-methyl-2-pentanone; ii—chloroacetylchloride, 0 °C?reflux,
4 h (86%); (e) Lawesson reagent, THF, rt, 24 h (95%); (f) H2NOHꢀHCl, Et3N, EtOH, rt,
24 h (78%).
It is worth to point out that the use of 4-methyl-2-pentanone as
solvent is important for a satisfactory yield.26
Lactam 9 was converted into NS 2028 in three steps according
to the described retrosynthesis (Scheme 1). This procedure was
performed in analogous way for all following oxadiazole com-
pounds and is herein discussed using the example of NS 2028
(Scheme 2). At first, lactam 9 (respectively, 12 and 13 in Scheme
3, as well as 26 in Scheme 4) was thionated with Lawesson reagent
by stirring for 24 h in THF at rt27 yielding thiolactam 10 in 95%
yield (respectively, 70–86%). For compound 23 (Scheme 4) a mod-
ified method was used, heating the reaction mixture in toluene at
70 °C increased the yield by 25% and decreased reaction time to
5 h.28 The following nucleophilic substitution with hydroxylamine
hydrochloride under basic conditions provided oxime 11 (respec-
tively, 16, 17 as well as 24, 28) without any problems.24
In case of the desired analogue 21 the oxadiazole ring is substi-
tuted by a triazole ring (Scheme 3). Therefore, thiolactam 10 was
treated with 8 equiv hydrazine monohydrate for 12 h at rt.29 In
that way, hydrazone 20 was obtained in 58% yield.
The last step of the oxadiazole and triazole synthesis was the
carbonylative cyclisation. For this purpose oxime 11 (respectively,
16, 17 and 24, 28 as well as hydrazone 20) was treated with CDI in
refluxing THF.25 The oxadiazole compound NS 2028 (respectively,
18, 19 and 25, 29) was prepared in five steps in an overall yield
of 61%.
Analogues 18 and 19 were obtained by Suzuki coupling of lac-
tam 9 with corresponding boronic acids (Scheme 3). We first in-
tended to do a coupling as last step of the synthesis, which
means with NS 2028, but that strategy was unsuccessful, since
2. Results and discussion
2.1. Chemistry
We first planned to establish a straightforward and efficient
synthesis of the known inhibitors ODQ and NS 2028. In addition,
we were interested in the preparation of a small library of inhibi-
tors belonging to different substance classes in order to study
structure–activity relationships. Hence, we intended to compare
the inhibitory activity of compounds lacking the common feature
in both ODQ/NS 2028 inhibitors with others that maintain this
structure element but differ in their general scaffold.
A general retrosynthetic analysis of the oxadiazole ring is de-
picted in Scheme 1. Most of the desired compounds were synthe-
sized in this manner. In this scheme all compounds are shown in
a simplified manner for a better visualisation.
We planned to generate oxadiazole ring 1 from oxime 2 by
insertion of CO and the following ring closure. The hydroxylimine
functionality in 2 would arise from nucleophilic substitution of
thiolactam 3 that in turn, would be generated by thionation of lac-
tam species 4 with Lawesson reagent. The appropriate lactams
would be bought from commercial sources or synthesized
beforehand.
In the case of ODQ the synthesis of the oxadiazole ring proved to
be easier, since oxime 6 was readily prepared in one step from
commercially available 2-chloroquinoxaline 5 (Scheme 2). The
nucleophilic substitution was performed with hydroxylamine
hydrochloride under basic conditions and resulting oxime 6 was
converted to ODQ by carbonylation with CDI24 followed by cyclisa-
tion. Thus, ODQ was prepared in two steps with an overall yield of
32%.
O
O
H
N
H
N
R1
X
R1
N
N
12, 14, 16, 18:
R1 = C H p
Br
O
b
a
d
OMe
5
6
O
O
O
18, 19
13, 15, 17, 19:
The synthesis of NS 2028 started with the reduction of 4-bro-
mo-2-nitrophenol 7 with tin(II)chloride dihydrate in concd hydro-
chloric acid.25 Ring closure with chloroacetyl chloride under basic
conditions gave rise to 6-bromo-2H-1,4-benzoxazin-3(4H)-one 9.
9
R1 = C6H5mCF3
12, 13: X = O
b
14, 15: X = S
c
16, 17: X = NOH
O
H
NH
Br
N
X
N
Br
N
d
O
O
21
10: X = S
20: X = NNH2
e
O
H
N
H
N
H
N
O
O
N OH
S
Scheme 3. Synthesis of compounds 18, 19 and 21. Reagents and conditions: (a)
approp. boronic acid, Pd(OAc)2, PPh3, Na2CO3, 1,4-dioxane/water, reflux, 12 h (96%
for 12 and 99% for 13); (b) Lawesson reagent, THF, rt, 24 h (70–86%); c) H2NOHꢀHCl,
Et3N, EtOH, rt, 24 h (78%); (d) CDI, THF, reflux, 24 h (57–69%); (e) N2H4ꢀH2O, EtOH,
rt, 12 h (58%).
N
N
4
2
3
1
Scheme 1. Retrosynthetic analysis of the oxadiazole element.

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O
able to interact better with the enzyme than the methoxy group.
O
H
N
H
N
X
NH
The difference in activity could also be associated with the opposed
electronic properties of these functional groups: the trifluorome-
thane residue has a strong electron-withdrawing effect, whereas1
the methoxy group is a good electron-donator. The additional H-
bridge donor in compound 29 has also a positive effect on inhibi-
tory activity, showing similar efficiency as ODQ. For compound
25 the expansion of the oxazine ring to a benzodiazepinone ele-
ment results in a molecule that even at the highest concentration
employed was a poor sGC inhibitor (although a trend to decrease
cGMP is seen, this does not reach statistical significance at
N
O
d
a
O
N
N
O
O
O
25
22: X = O
23: X = S
24: X = NOH
b
c
R2
O
O
N
O
H
H
N
R1
N
O
N
R1
O
N
N
N
X
N
d
f
O
O
29
O
10 lM). Probably, the additional pyrrolo element and the changed
spatial configuration of the seven-membered ring lead to this
result.
31: R1 = H, R2 = H
32: R1 = Br, R2 = H
33: R1 = H, R2 = Cl
30: R1 = H, R2 = H
9: R1 = Br, R2 = H
26: X = O
27: X = S
e
c
28: X = NOH
Substitution of the oxadiazole ring by a quinazoline element
generally implicates loss of activity. An exception is compound
33 that is effective in blocking SNP-induced cGMP production
when used at high concentrations. An explanation for this observa-
tion may be the chloride atom in position 9 of 33 which presum-
ably interacts with the active site of the enzyme and thus blocks
it. Surprisingly, compound 21 showed no inhibitory activity on
sGCs. It differs from the inhibitor NS 2028 only by substitution of
the oxygen atom in the oxadiazole ring by a nitrogen atom. The
inactivity of triazole 21 may occur due to the inability of the NH
moiety to serve as H-bridge acceptor. Alternatively, it is possible
that the oxadiazole derivatives act by covalently binding to the en-
zyme and blocking its activity; a similar mode of action has been
shown to occur in carbamates induced inhibition of acetylcholines-
terase.31 Consequently, the exchange of the oxadiazole structure
by a less reactive triazole would lead to a loss activity. In order
to obtain support for this hypothesis we incubated human recom-
binant sGC with NS 2028 and after, proteolytic processing of the
sample we subjected it to mass spectrometric analysis. In these
experiments, we could not detect any peptide fragment of sGC that
is covalently bound to NS 2028 (data not shown); we were, thus,
unable to prove our original hypothesis that NS 2028 inhibits
sGC after covalent binding to the enzyme.
Scheme 4. Synthesis of compounds 25, 29 and 31, 32 and 33. Reagents and
conditions: (a) -proline, DMF, reflux, 4 h (quant.); (b) Lawesson reagent, toluene,
70 °C, 5 h (86%); (c) H2NOHꢀHCl, Et3N, EtOH, rt, 24 h (76% for both oximes); (d) CDI,
THF, reflux, 24 h (84% for 25 and 79% for 29); (e) Lawesson reagent, THF, rt, 24 h
(84%); (f) POCl3, DCE, rt, 15 min, then approp. anthranilic acid, DCE, Et3N, 0 °C?rt,
1 h, then reflux 1 h (88% for 31, 67% for 32 and 48% for 33).
L
the oxadiazole ring decomposed to oxime 9 by treatment with pal-
ladium. Therefore the coupling was done in an earlier step, with
intermediate lactam 9. Hence, we performed the Suzuki reaction
in a two phase system (dioxane/water, 3:1, v/v) with palla-
dium(II)acetate as catalytic species, triphenylphosphine as ligand
and sodium carbonate as base. As a result we obtained lactams
12 and 13, which were converted into the oxadiazoles 18 and 19
as described above in 27% and 29% overall yield.
Since benzodiazepines are privileged structures, we intended to
investigate the influence of this scaffold on inhibitory activity.
Hence, lactam 22 was synthesized by condensation of isatoic anhy-
dride with L
-proline28, which was subsequently converted into
oxadiazole 25 by above procedure in an overall yield of 55%
(Scheme 4). Furthermore, in an analogous way commercially avail-
able 2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one 26 led to compound
29 in 40% overall yield. This compound contains an additional H-
bridge donor possibly leading to a stronger interaction with the en-
zyme. As last substance class, we prepared three differently substi-
tuted quinazolinbenzoxazine derivatives 31, 32 and 33, lacking the
oxadiazole structure element (Scheme 4). These compounds were
prepared by one pot synthesis according to a procedure of Sastry30
from either commercially available 2H-1,4-benzoxazin-3(4H)-one
30 or lactam 9 in an overall yield of 48–88%.
3. Conclusions
We describe a straightforward and efficient synthesis of the
known inhibitors ODQ and NS 2028, as well as a series of different
classes of analogues. The biological activity was evaluated in vitro
with RASM cells. Thereby two novel inhibitors with good activity
have been found, compounds 18 and 19, as well as inhibitors 29
and 33 that showed to be more moderate. Furthermore, the results
of our studies revealed new structure–activity relationships: the
crucial role of the oxadiazole ring for inhibitory activity. In partic-
ular, compound 21 shows that the exchange of one atom leads to
the loss of activity, as it comprises a triazole instead of the oxadi-
azole element. Considering the chemical reactivity of carbamides
in comparison to carbamates this observation may be a reference
to a different mode of inhibition.
In subsequent biological assays all these compounds were
tested on their inhibitory activity against sGC.
2.2. Biological evaluation
The inhibitory activity of the synthesized compounds was eval-
uated in vitro using rat aortic smooth muscle cells. cGMP levels in
resting cultures were low and increased by more than 100-fold
after stimulation with SNP. Pre-treatment of cells with ODQ
dose-dependently inhibited SNP-stimulated cGMP formation, with
the lower ODQ concentration reducing by 85%; the highest ODQ
concentration almost abolished NO-stimulated cGMP formation.
From all experiments the most significant results are presented
in Figure 2. The benzodiazepine 25, quinazolinbenzoxazine deriva-
tives 31 and 32 as well as triazole 21 do not exhibit an inhibitory
effect on sGC, whilst oxadiazoles 18, 19 and 29 as well as quin-
azolinbenzoxazine 33 are good to moderate inhibitors.
4. Experimental
4.1. Chemistry
All reagents were commercially obtained from Acros, Sigma–Al-
drich, Merck and Fluka and used without further purification un-
less otherwise stated. Anhydrous solvents were transferred via
oven-dried syringe or cannula. Flasks were flame-dried under vac-
uum and cooled under a constant stream of argon. Melting points
were measured with a NAGEMA K8-micro hot stage and are uncor-
rected. 1H, 13C and 19F NMR spectra were recorded on Varian
Although compound 19 features an extended scaffold, it is still
as effective and potent as ODQ. In contrast, compound 18 has low-
er potency, as it only partially inhibits SNP-induced cGMP forma-
tion at 0.1 lV. Presumably, the trifluoromethane moiety of 19 is

M. von Wantoch Rekowski et al. / Bioorg. Med. Chem. 18 (2010) 1288–1296
1291
Figure 2. Effect of selected NS 2028/ODQ derivatives on cGMP accumulation. RASM cells were treated with ODQ or the newly synthesized compounds at the indicated
concentration for 20 min. Cells were then stimulated with 10 V SNP for 15 min, in the presence of the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX; 1 mM),
prior to extraction of cGMP with HCl. Values are means SEM, n = 11–12 wells; p < 0.05 versus SNP.
l
*
Gemini 200 (200 MHz for 1H NMR, 50 MHz for 13C NMR, 188 MHz
for 19F NMR), 300 (300 MHz for 1H NMR, 75 MHz for 13C NMR,
228 MHz for 19F NMR) and 400 (400 MHz for 1H NMR, 100 MHz
for 13C NMR, 367 MHz for 19F NMR). The chemical shifts are re-
ported relative to the residual solvent peak, which was used as
an internal reference (chemical shifts in d values, J in Hz). HRMS
were obtained on a Bruker Daltonics APEX II for ESI and on a Mas-
slab Manchester VG 12-250 for EI. Optical rotations were measured
with half automatical polarimeter Polartronic D (Schmidt + Hae-
nsch) and are uncorrected. Reactions involving moisture-sensitive
reactants were performed in flame-dried glassware under an
atmosphere of argon, reactants being added via syringe. Flash col-
umn chromatography was performed on silica gel (Acros 60 A,
0.035–0.070 mm) and analytical TLC on pre-coated silica gel plates
(Merck 60 F254, 0.25 mm).
Thereby fawn crystals precipitated, which were filtered, washed
with water and dried in vacuo to yield pure 9 (8.43 g, 36.96 mmol,
86%) as fawn crystals. Mp 221–223 °C. 1H NMR (300 MHz, DMSO-
d6)32: d = 4.57 (s, 2H, 2-CH2); 6.89 (d, 1H, 3J = 8.8 Hz, arom. 8-CH);
7.0 (d, 1H, 4J = 2.2 Hz, arom. 5-CH); 7.05 (dd, 1H, 4J = 2.2 Hz,
3J = 8.8 Hz, arom. 7-CH); 10.78 (br, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): d = 66.6 (2-CH2); 113.2 (arom. 6-CBr); 118.0 (arom. 8-
CH and arom. 5-CH); 125.3 (arom. 7-CH); 129.0 (arom. 4a-CNH);
142.6 (arom. 8a-CO); 164.6 (3-C@O). HRMS-ESI: m/z [M+H]+ calcd
for C8H6NO2BrH: 227.96547; found: 227.96546; [2M+H]+ calcd for
C16H12N2O4Br2H: 454.92366; found: 454.92376.
4.2. General procedure for the preparation of 2H-1,4-
benzoxazin-3(4H)-one derivatives (12) and (13) via Suzuki
reaction
4.1.1. 2-Amino-4-bromophenol (8)
6-Bromo-2H-1,4-benzoxazin-3(4H)-one 9 and the appropriate
boronic acid (1 equiv) were suspended in 1,4-dioxane (3 mL/
100 mg) under argon atmosphere. Afterwards water (1 mL/
100 mg), 2 M Na2CO3 solution (0.44 mL/100 mg), Pd(OAc)2
(0.02 equiv) and triphenylphosphine (0.04 equiv) were added.
The reaction mixture was refluxed for 12 h and afterwards ex-
tracted three times with ethyl acetate and the combined organic
phases were washed with satd NaHCO3 solution and brine. After
drying over Na2SO4 the solvent was removed in vacuo and the
crude product was purified by column chromatography.
A solution of tin(II)chloride dihydrate (41.4 g, 183.7 mmol) and
88 mL concd hydrochloric acid in 160 mL methanol was cooled to
0 °C and 4-bromo-2-nitrophenol 7 (8.0 g, 36.7 mmol) was added in
one portion. The mixture was stirred for 4.5 h at room tempera-
ture, whereas the yellow solution turned colourless. Afterwards
the solution was diluted with ethyl acetate and neutralized with
satd NaHCO3 solution (to pH 7). Thereby, a white solid precipi-
tated, which was filtered and washed with ethyl acetate. The or-
ganic phase was separated and the water phase was extracted
three times with ethyl acetate. The combined organic phases were
dried over Na2SO4 and the solvent was removed in vacuo to yield
pure 8 (6.9 g, 6.74 mmol; quantitative) as a fawn solid. Mp: 130–
132 °C; 1H NMR (300 MHz, DMSO-d6): d = 4.76 (br, 2H, NH2);
6.47 (dd, 1H, 4J = 2.3 Hz, 3J = 8.2 Hz, arom. 5-CH); 6.54 (d, 1H,
3J = 8.2 Hz, arom. 6-CH); 6.69 (d, 1H, 4J = 2.3 Hz, arom. 3-CH);
9.23 (br, 1H, OH). 13C NMR (100 MHz, DMSO-d6): d = 110.6 (arom.
4-CBr); 115.6 (arom. 3-CH); 116.0 (arom. 6-CH); 118.1 (arom. 5-
Cq); 138.8 (arom. 2-CNH2); 143.3 (arom. 1-COH). HRMS-ESI: m/z
[M+H]ꢁ calcd for C6H5NOBr: 185.95600; found: 185.95563.
4.2.1. 6-(4-Methoxyphenyl)-2H-1,4-benzoxazin-3(4H)-one (12)
According to the general procedure this compound was pre-
pared from 6-bromo-2H-1,4-benzoxazin-3(4H)-one
9 (200 mg,
0.88 mmol) and 4-methoxy boronic acid (133 mg, 0.88 mmol)
to give pure 12 as a colourless solid (216 mg, 0.85 mmol, 97%).
Mp 205 °C. 1H NMR (400 MHz, DMSO-d6): d = 3.77 (s, 3H,
OCH3); 4.57 (s, 2H, 2-CH2); 6.96–7.0 (m, 3H, 2ꢂ arom. 30-CH
and arom. 8-CH); 7.08 (d, 1H, 4J = 2.0 Hz, arom. 5-CH); 7.12
(dd, 1H, 4J = 2.0 Hz, 3J = 8.3 Hz, arom. 7-CH); 7.44–7.46 (dd, 2H,
3
4J = 2.0 Hz, J = 6.8 Hz, 2ꢂ arom. 20-CH); 10.73 (br, 1H, NH). 13C
4.1.2. 6-Bromo-2H-1,4-benzoxazin-3(4H)-one (9)
NMR (100 MHz, DMSO-d6): d = 55.1 (OCH3); 66.8 (2-CH2); 113.5
(arom. 8-CH); 114.4 (2C, 2ꢂ arom. 30-CH); 116.5 (arom. 5-CH);
120.9 (arom. 7-CH); 127.3 (2C, 2ꢂ arom. 20-CH); 127.6 (arom.
10-Cq); 132.0 (arom. 4a-CNH); 134.4 (arom. 6-Cq); 142.4 (arom.
8a-CO); 158.7 (arom. 40-C(OCH3)); 164.9 (3-C@O). HRMS-ESI:
m/z [M+H]+ calcd for C15H13NO2H: 256.09682; found:
To
a
suspension of 2-amino-4-bromophenol
8
(8.00 g,
42.55 mmol) in 25 mL 4-methyl-2-pentanone were added NaHCO3
(8.54 g, 101.68 mmol) and 25 mL H2O. The suspension was cooled
to 0 °C and chloroacetylchloride (3.89 mL, 48.93 mmol) was added
dropwise. After refluxing for 4 h the mixture was cooled to 0 °C.

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M. von Wantoch Rekowski et al. / Bioorg. Med. Chem. 18 (2010) 1288–1296
256.09686; [2M+H]+ calcd for C30H26N2O4H: 511.18636; found:
511.18675.
(3-C@S). HRMS-ESI: m/z [M+H]+ calcd for C8H6NOBrSH:
243.94262; found: 243.94257.
4.3.2. 6-(4-Methoxyphenyl)-2H-1,4-benzoxazine-3(4H)-thione
(14)
4.2.2. 6-[3-(Trifluoromethyl)phenyl]-2H-1,4-benzoxazin-3(4H)-
one (13)
According to the general procedure this compound was pre-
pared from 6-(4-methoxyphenyl)-2H-1,4-benzoxazine-3(4H)-one
12 (200 mg, 0.78 mmol) to yield pure 14 (162 mg, 0.6 mmol;
77%) as a yellowish solid. Mp 175 °C. 1H NMR (400 MHz, DMSO-
d6): d = 4.84 (s, 3H, OCH3); 4.84 (s, 2H, 2-CH2); 7.0–7.04 (m, 3H,
2ꢂ arom. 30-CH and arom. 8-CH); 7.24–7.27 (m, 2H, arom. 5-CH
According to the general procedure this compound was pre-
pared from 6-bromo-2H-1,4-benzoxazin-3(4H)-one
9 (300 mg,
1.32 mmol) and 3-trifluoromethylphenyl boronic acid (250 mg,
1.32 mmol) to yield pure 13 (381 mg, 1.3 mmol, 99%) as a colour-
less solid. Mp 227 °C. 1H NMR (400 MHz, DMSO-d6): d = 4.61 (s,
2H, 2-CH2); 7.04 (d, 1H, 3J = 8.4 Hz, arom. 8-CH); 7.19 (d, 1H,
4J = 2.2 Hz, arom. 5-CH); 7.28 (dd, 1H, 4J = 2.2 Hz, 3J = 8.4 Hz, arom.
7-CH); 7.67 (m, 2H, arom. 40-CH and arom. 50-CH); 7.81 (s, 1H,
arom. 20-CH); 7.85 (m, 1H, arom. 60-CH); 10.74 (br, 1H, NH). APT
(100 MHz, DMSO-d6): d = 66.8 (2-CH2); 114.2 (arom. CH); 116.8
(arom. CH); 121.8 (arom. CH); 122.5 (q, 3J = 4 Hz, arom. CH);
123.7 (q, 3J = 4 Hz, arom. CH); 124.2 (q, 1J = 273 Hz, CF3); 127.8
3
and arom. 7-CH); 7.46–7.48 (d, 2H, J = 8.7 Hz, 2ꢂ arom. 20-CH);
12.77 (br, 1H, NH). 13C NMR (75 MHz, DMSO-d6): d = 55.2
(OCH3); 72.3 (2-CH2); 114.0 (arom. 8-CH); 114.4 (2C, arom. 30-
CH); 116.7 (arom. 5-CH); 123.0 (arom. 7-CH); 126.6 (arom. 10-
Cq); 127.4 (2C, arom. 20-CH); 131.7 (arom. 4a-CNH); 134.5 (arom.
6-Cq); 144.0 (arom. 8a-CO); 158.8 (arom. 40-C(OCH3)); 190.8 (3-
C@S). HRMS-ESI: m/z [M+H]+ calcd for C15H13NO2SH: 272.07398;
found: 272.07404.
2
(arom. Cq); 129.8 (q, J = 32 Hz, arom. 30-Cq); 130.1 (arom. CH);
130.3 (arom. CH); 132.9 (arom. Cq); 140.6 (arom. Cq); 143.5 (arom.
Cq); 164.7 (3-C@O). 19F NMR (282 MHz, DMSO-d6): d = ꢁ61.55
(CF3). HRMS-ESI: m/z [M+H]+ calcd for C15H10NO2F3H: 294.07364;
found: 294.07373; [2M+H]+ calcd for C30H20N2O4F6H: 587.14000;
found: 587.13979.
4.3.3. 6-[3-(Trifluoromethyl)phenyl]-2H-1,4-benzoxazine-
3(4H)-thione (15)
According to the general procedure this compound was pre-
pared from 6-[3-(trifluoromethyl)phenyl]-2H-1,4-benzoxazine-
3(4H)-one 13 (300 mg, 1.02 mmol) to yield pure 15 (222 mg,
0.72 mmol; 70%) as a yellowish solid. Mp 185–187 °C. 1H NMR
(400 MHz, DMSO-d6): d = 4.88 (s, 2H, 2-CH2); 7.09 (d, 1H,
3J = 8.3 Hz, arom. 8-CH); 7.38 (d, 1H, 4J = 2.2 Hz, arom. 5-CH);
7.41 (dd, 1H, 4J = 2.2 Hz, 3J = 8.3 Hz, arom. 7-CH); 7.68–7.7 (m,
2H, arom. 40-CH and arom. 50-CH); 7.83 (s, 1H, arom. 20-CH);
7.84–7.87 (m, 1H, arom. 60-CH); 12.77 (br, 1H, NH). APT
(100 MHz, DMSO-d6): d = 72.3 (2-CH2); 114.8 (arom. CH); 117.0
(arom. CH); 122.7 (q, 3J = 4 Hz, arom. CH); 123.9 (2C, 2ꢂ arom.
CH); 124.1 (q, 1J = 273 Hz, CF3); 126.8 (arom. Cq); 129.8 (q,
2J = 32 Hz, 30-Cq); 130.2 (arom. CH); 130.4 (arom. CH); 133.0 (arom.
Cq); 140.3 (arom. Cq); 145.2 (arom. Cq); 190.8 (3-C@S). 19F NMR
(188 MHz, DMSO-d6): d = ꢁ61.76 (CF3). HRMS-ESI: m/z [M+H]+
calcd for C15H10NO2F3SH: 310.05080; found: 310.05088.
4.2.3. (11aS)-2,3-Dihydro-1H-pyrrolo[2,1-
c][1,4]benzodiazepine-5,11(10H,11aH)-dione (22)
A suspension of isatoic anhydride (2.0 g, 12.26 mmol) and L-
proline (1.41 g, 12.26 mmol) in 11 mL DMF was heated to 160 °C
for 4 h. The solvent was removed in vacuo and the crude product
was purified by column chromatography (dichloromethane/ethyl
acetate 2:1 v/v). Benzodiazepine 22 (2.65 g, 12.26 mmol; quantita-
tive) was gained as fawn crystals. Mp 215 °C. ½a D23
ꢃ
+493 (c 1.03,
MeOH). 1H NMR (400 MHz, CDCl3): d = 1.97–2.08 (m, 3H, 2-CH2,
1-CHaHb); 2.72–2.80 (m, 1H, 1-CHaHb); 3.56–3.63 (m, 1H, 3-
NCHaHb); 3.77–3.82 (m, 1H, 3-NCHaHb); 4.07 (d, 1H, 3J = 6.2 Hz,
11a-NCH); 7.04 (d, 1H, 3J = 8.1 Hz, arom. CH); 7.23–7.27 (m, 1H,
arom. CH); 7.46 (m, 1H, arom. CH); 7.99 (dd, 1H, 4J = 1.6 Hz,
3J = 7.9 Hz, arom. 6-CH); 8.92 (br, 1H, NH). APT (100 MHz, CDCl3):
d = 23.6 (2-CH2); 26.4 (1-CH2); 47.4 (3-NCH2); 56.8 (11a-NCH);
121.2 (arom. CH); 125.2 (arom. CH); 127.3 (arom. 5a-Cq); 131.3
(arom. CH); 132.6 (arom. CH); 135.5 (arom. 9a-CNH); 165.6 (5-
C@O); 171.52 (11-C@O). HRMS-ESI: m/z [M+H]+ calcd for
C12H12N2O2H: 217.09715; found: 217.09719; [2M+H]+ calcd for
C24H24N4O4H: 433.18703; found: 433.18697.
4.3.4. (11aS)-11-Thioxo-1,2,3,10,11,11a-hexahydro-5H-
pyrrolo[2,1-c][1,4]ben-zodiazepin-5-one (23)
To a suspension of 2,3-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodi-
azepine-5,11(10H,11aH)-dione 22 (1.0 g, 4.62 mmol) in 100 mL
toluene were added Lawesson reagent (0.94 g, 2.31 mmol). The
yellow suspension was heated to 70 °C for 5 h. After this time a yel-
low solid has precipitated which was recrystallized from ethanol to
yield pure 23 (923 mg, 3.97 mmol; 86%) as yellow crystals. Mp
4.3. General procedure for the preparation of thiolactams (10),
(14), (15), (23) and (27)
253–254 °C. ½a 2D3
ꢃ
+873 (c 1.026, DMSO). 1H NMR (200 MHz,
DMSO-d6): d = 1.84–2.07 (m, 3H, 2-CH2, 1-CHaHb); 2.54 (m, 1H,
1-CHaHb); 3.4–3.5 (m, 2H, 3-NCH2); 4.24 (d, 1H, 3J = 6.0 Hz, 11a-
NCH); 7.23–7.34 (m, 2H, 2ꢂ arom. CH); 7.50–7.58 (m, 1H, arom.
CH); 7.8 (dd, 1H, 4J = 1.7 Hz, 3J = 7.7 Hz, arom. 6-CH); 12.43 (br,
1H, NH). APT (100 MHz, DMSO-d6): d = 22.7 (2-CH2); 29.0 (1-
CH2); 46.8 (3-NCH2); 59.8 (11a-NCH); 121.8 (arom. CH); 125.6
(arom. CH); 127.7 (arom. 5a-Cq); 130.2 (arom. CH); 132.2 (arom.
CH); 136.4 (arom. 9a-CNH); 164.2 (5-C@O); 202.0 (11-C@S).
HRMS-ESI: m/z [M+Na]+ calcd for C12H12N2OSNa: 255.05625;
found: 255.05642; [2M+Na]+ calcd for C24H24N4O2S2Na:
487.12329; found: 487.12355.
To a solution of the appropriate benzoxazine or benzodiazepine
derivative in abs. THF (5 mL/mmol) was added Lawesson reagent
(0.5 equiv). The yellow suspension was stirred for 12 h, in which
a yellowish precipitate has formed. Subsequently, the solvent
was removed in vacuo and the crude product was purified by col-
umn chromatography.
4.3.1. 6-Bromo-2H-1,4-benzoxazine-3(4H)-thione (10)
According to the general procedure this compound was pre-
pared from 6-bromo-2H-1,4-benzoxazine-3(4H)-one
9 (8.40 g,
36.84 mmol) to yield pure 10 (8.56 g (35.07 mmol, 95%) as a yel-
lowish solid. Mp 207 °C. 1H NMR (400 MHz, DMSO-d6): d = 4.84
(s, 2H, 2-CH2); 6.95 (d, 1H, 3J = 7.8 Hz, arom. 8-CH); 7.16–7.2
(m, 2H, arom. 7-CH and arom. 5-CH); 12.77 (br, 1H, NH). 13C
NMR (100 MHz, DMSO-d6): d = 72.2 (2-CH2); 113.2 (arom. 6-
CBr); 118.3 (arom. 8-CH); 118.6 (arom. 5-CH); 127.4 (arom. 7-
CH); 127.6 (arom. 4a-CNH); 144.3 (arom. 8a-CO); 191.2
4.3.5. 2H-Pyrido[3,2-b][1,4]oxazine-3(4H)-thione (27)
The preparation was done according to the general procedure
starting from 2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one 26 (1.0 g,
6.66 mmol). The yellow precipitate of 27 (933.7 mg, 5.62 mmol;
84%) was filtered, washed with cold n-hexane and dried in vacuo.
Mp >239 °C (dec).33 1H NMR (200 MHz, DMSO-d6): d = 4.9 (s, 2H,

M. von Wantoch Rekowski et al. / Bioorg. Med. Chem. 18 (2010) 1288–1296
1293
2-CH2); 7.08 (dd, 1H, 3J = 4.9 Hz, 3J = 7.9 Hz, arom. 7-CH); 7.4 (dd,
1H, 4J = 1.2 Hz, 3J = 7.9 Hz, arom. 8-CH); 7.97 (dd, 1H, 4J = 1.2 Hz,
3J = 4.9 Hz, arom. 6-CH); 13.13 (br, 1H, NH). 13C NMR (50 MHz,
DMSO-d6): d = 72.3 (2-CH2); 120.9 (arom. 7-CH); 123.6 (arom. 8-
CH); 140.1 (arom. 8a-CO); 141 (arom. 4a-C@N(NH)); 141.1 (arom.
6-CH); 193.6 (3-C@S). HRMS-EI: m/z [M]+ calcd for C7H6N2OS:
166.02009; found: 166.01802.
160.1 (arom. 40-C(OCH3)). HRMS-ESI: m/z [M+H]+ calcd for
C15H14N2O3H: 271.10772; found: 271.10790.
4.4.3. 6-[3-(Trifluoromethyl)phenyl]-2H-1,4-benzoxazin-3(4H)-
one-oxime (17)
According to the general procedure this compound was pre-
pared from 6-[3-(trifluoromethyl)phenyl]-2H-1,4-benzoxazine-
3(4H)-thione 15 (200 mg, 0.65 mmol) to yield 17 (148 mg,
4.3.6. 6-Bromo-3-hydrazono-3,4-dihydro-2H-1,4-benzoxazine
(20)
0.48 mmol, 74%) as a
colourless solid. Mp 121 °C. 1H NMR
(400 MHz, DMSO-d6): d = 4.52 (s, 2H, 2-CH2); 6.96 (d, 1H,
To a solution of 6-bromo-2H-1,4-benzoxazine-3(4H)-thione 10
(214.8 mg, 0.88 mmol) in 4 mL ethanol were added hydrazine
monohydrate (352.4 mg, 7.04 mmol). The yellow suspension was
stirred at room temperature for 12 h. After removal of the solvent
in vacuo the crude product was washed with ethyl acetate to give
20 (123.6 mg, 0.51 mmol; 58%) as colourless solid. Mp 261 °C. 1H
NMR (200 MHz, DMSO-d6): d = 4.7 (s, 2H, 2-CH2); 6.83 (d, 1H,
3J = 8.1 Hz, arom. 8-CH); 6.95 (dd, 1H, 4J = 2.2 Hz, 3J = 8.1 Hz, arom.
7-CH); 7.39 (d, 1H, 4J = 2.2 Hz, arom. 5-CH); 9.89 (br, 1H, NH). 13C
NMR (50 MHz, DMSO-d6): d = 65.5 (2-CH2); 113.7 (arom. 6-CBr);
118.0 (arom. CH); 118.1 (arom. CH); 123.3 (arom. CH); 130.2
(arom. 4a-CNH); 143.6 (arom. 8a-CO); 145.7 (3-C@N). HRMS-ESI:
m/z [M+H]+ calcd for C8H8N3OBrH: 241.99253; found: 241.98122.
3J = 8.3 Hz, arom. 8-CH); 7.12 (dd, 1H, 4J = 2.1 Hz, 3J = 8.3 Hz arom.
4
7-CH); 7.48 (d, 1H, J = 2.1 Hz, arom. 5-CH); 7.66 (m, 2H, arom. 40-
CH and arom. 50-CH); 7.8 (s, 1H, arom. 20-CH); 7.83–7.85 (m, 1H,
arom. 60-CH); 9.42 (br, 1H, OH); 9.92 (br, 1H, NH). APT (100 MHz,
DMSO-d6): d = 63.7 (2-CH2); 113.8 (arom. CH); 116.8 (arom. CH);
118.9 (arom. CH); 122.4 (q, 3J = 4 Hz, arom. CH); 123.5 (q,
3J = 4 Hz, arom. CH); 124.2 (q, 1J = 273 Hz, CF3); 129.4 (arom. Cq);
2
129.7 (q, J = 33 Hz, arom. 30-Cq); 130.0 (arom. CH); 130.2 (arom.
CH); 132.9 (arom. Cq); 141.0 (arom. Cq); 141.4 (arom. Cq); 144.4
(arom. 3-C@N). 19F NMR (376 MHz, DMSO-d6): d = ꢁ61.55 (CF3).
HRMS-ESI: m/z [M+H]+ calcd for C15H11N2O2F3H: 309.08454;
found: 309.08452.
4.4.4. (11aS)-2,3-Dihydro-1H-pyrrolo[2,1-c][1,4]benzodiaze-
pine-5,11(10H,11aH)-dione-11-oxime (24)
4.4. General procedure for the preparation of oximes (11), (16),
(17), (24) and (28)
According to the general procedure this compound was pre-
pared from (11aS)-11-thioxo-1,2,3,10,11,11a-hexahydro-5H-pyr-
rolo[2,1-c][1,4]benzodiazepin-5-one 23 (500 mg, 2.15 mmol) to
yield 24 (380 mg, 1.64 mmol, 76%) as colourless crystals. Mp
The appropriate thiolactam was dissolved in ethanol (2 mL/
mmol) and hydroxylamine hydrochloride (2 equiv) was added.
After cooling to 0 °C triethyl amine (2 equiv) was added. The light
yellow suspension was stirred at room temperature for 24 h,
whereupon the mixture decolourised and H2S was released. The
solvent was removed in vacuo, the residue taken with dichloro-
methane and washed three times with water. The organic phase
was dried with Na2SO4 and the solvent removed in vacuo. The
crude product was purified by column chromatography.
132 °C. ½a 2D3
ꢃ
+427 (c, 1.026 in MeOH). 1H NMR (400 MHz, DMSO-
d6): d = 1.84–2.0 (m, 3H, 2-CH2, 1-CHaHb); 2.55–2.59 (m, 1H, 1-
CHaHb); 3.47–3.6 (m, 2H, 3-NCH2); 4.29–4.31 (dd, 1H, 4J = 2.3 Hz,
3J = 7.8 Hz, 11a-NCH); 7.0–7.08 (m, 1H, arom. CH); 7.23–7.25 (m,
1H, arom. CH); 7.34–7.39 (m, 1H, arom. CH); 7.66 (dd, 1H,
4J = 1.6 Hz, 3J = 7.8 Hz, arom. 6-CH); 8.69 (br, 1H, OH); 10.03 (br,
1H, NH). APT (100 MHz, DMSO-d6): d = 23.0 (2-CH2); 25.5 (1-
CH2); 46.8 (3-NCH2); 54.0 (11a-NCH); 121.2 (arom. CH); 121.7
(arom. CH); 125.1 (arom. 5a-Cq); 130.4 (arom. CH); 131.9 (arom.
CH); 138.2 (arom. 9a-CNH); 149.0 (11-C@N); 165.2 (5-C@O).
HRMS-ESI: m/z [M+Na]+ calcd for C12H13N3O2Na: 254.09000;
found: 254.09013; [2M+Na]+ calcd for C24H26N6O4Na: 485.19077;
found: 485.19087.
4.4.1. 6-Bromo-2H-1,4-benzoxazin-3(4H)-one-oxime (11)
According to the general procedure this compound was pre-
pared
from
6-bromo-2H-1,4-benzoxazine-3(4H)-thione
10
(8.50 g, 34.82 mmol) to yield 11 (6.57 g, 27.02 mmol, 78%) as a col-
ourless solid. Mp 196–198 °C. 1H NMR (300 MHz, DMSO-d6)
d = 4.47 (s, 2H, CH2), 6.79 (d, 1H, 3J = 9.0 Hz, arom. 8-CH), 6.86
(dd, 1H, 3J = 9.6 Hz, 4J = 2.4 Hz, arom. 7-CH), 7.22 (d, 1H,
4J = 2.1 Hz, arom. 5-CH), 9.56 (s, 1H, OH), 10.01 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6): d = 63.3 (2-CH2); 13.6 (arom. 6-CBr);
117.5 (arom. 8-CH); 118.1 (arom. 5-CH); 122.5 (arom. 7-CH);
130.6 (arom. 4a-CNH); 140.9 (arom. 8a-CO); 143.4 (3-C@N).
HRMS-ESI: m/z [M+H]+ calcd for C8H7N2OBrH: 242.97691; found:
242.97633.
4.4.5. 2H-Pyrido[3,2-b][1,4]oxazin-3(4H)-one-oxime (28)
According to the general procedure this compound was prepared
from 2H-Pyrido[3,2-b][1,4]oxazine-3(4H)-thione 27 (550 mg,
3.31 mmol) to yield 28 (400 mg, 2.42 mmol, 76%) as a colourless
powder. Mp 253 °C. 1H NMR (200 MHz, DMSO-d6): d = 4.56 (s, 2H,
2-CH2); 6.83 (dd, 1H, 3J = 4.9 Hz, 3J = 7.9 Hz, arom. 7-CH); 7.24 (dd,
1H, 4J = 1.4 Hz, 3J = 7.9 Hz, arom. 8-CH); 7.83 (dd, 1H, 4J = 1.4 Hz,
3J = 4.9 Hz, arom. 6-CH); 9.59 (br, 1H, OH); 10.15 (br, 1H, NH). 13C
NMR (50 MHz, DMSO-d6): d = 63.5 (2-CH2); 116.9 (arom. 7-CH);
122.9 (arom. 8-CH); 139.9 (arom. 8a-CO); 141.0 (arom. 6-CH);
141.1 (arom. 4a-C@N(NH)); 142.6 (3-C@N). HRMS-EI: m/z [M]+ calcd
for C7H7N3O2: 165.05386; found: 165.05375.
4.4.2. 6-(4-Methoxyphenyl)-2H-1,4-benzoxazin-3(4H)-one-
oxime (16)
According to the general procedure this compound was prepared
from 6-(4-methoxyphenyl)-2H-1,4-benzoxazine-3(4H)-thione 14
(100 mg, 0.37 mmol) to yield 16 (77 mg, 0.29 mmol, 77%) as colour-
less needles. Mp 207–208 °C. 1H NMR (300 MHz, acetone-d6):
d = 3.82 (s, 3H, OCH3); 4.52 (s, 2H, 2-CH2); 6.92 (d, 1H, 3J = 8.3 Hz,
arom. 8-CH); 6.99 (m, 2H, 2ꢂ arom. 30-CH); 7.05 (dd, 1H,
4J = 2.2 Hz, 3J = 8.3 Hz, arom. 7-CH); 7.4 (d, 1H, 4J = 2.2 Hz, arom. 5-
4.4.6. Quinoxalin-2(1H)-one oxime (6)
To a solution of 2-chloroquinoxaline 5 (1 g, 6.08 mmol) in 4 mL
dimethyl sulfoxide were added hydroxylamine hydrochloride
(0.84 g, 12.16 mmol) and anhydrous sodium carbonate (0.71 g,
6.69 mmol). The mixture was stirred at room temperature for
24 h. Afterwards the solution was diluted with water and extracted
several times with dichloromethane. The combined organic phases
were washed with brine and dried over MgSO4. After concentration
the solution was stored in the refrigerator over night. The yellow
precipitate of 6 (388 mg, 2.4 mmol; 40%) was collected by filtration
3
CH); 7.50–7.53 (dd, 4J = 1.9 Hz, J = 6.8 Hz, 2H, 2ꢂ arom. 20-CH);
8.57 (br, 1H, OH); 9.08 (br, 1H, NH). APT (100 MHz, acetone-d6):
d = 55.6 (OCH3); 64.9 (2-CH2); 114.2 (arom. 30-CH); 114.3 (arom.
30-CH); 115.1 (arom. 8-CH); 117.6 (arom. 5-CH); 119.8 (arom. 7-
CH); 128.4 (2C, 2ꢂ arom. 20-CH); 130.0 (arom. 10-Cq); 134.0 (arom.
4a-CNH); 136.3 (arom. 6-Cq); 143.2 (arom. 8a-CO); 144.8 (3-C@N);

1294
M. von Wantoch Rekowski et al. / Bioorg. Med. Chem. 18 (2010) 1288–1296
and dried in vacuo. Mp 183 °C (dec). 1H NMR (400 MHz, DMSO-d6)
d = 6.88–6.92 (m, 1H, arom. 8-CH), 7.18–7.20 (m, 2H, arom. 6-CH,
arom. 7-CH), 7.34 (d, 1H, 3J = 7.2 Hz, arom. 5-CH), 7.80 (d, 1H,
4J = 2.4 Hz, arom. 3-CH), 10.14 (br, 1H, OH), 10.41 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6): d = 114.4 (arom. CH); 120.9 (arom.
CH); 128.0 (arom. CH); 129.4 (arom. CH); 132.5 (arom. Cq); 133.0
(arom. Cq); 142.4 (N@CH); 150.1 (C@N(NH)). HRMS-EI: m/z [M]+
calcd for C8H7N3O 161. 05892; found: 161.05903.
CH); 128.2 (2C, 2ꢂ arom. 20-CH); 131.9 (arom. 10-Cq); 137.3 (arom.
8-Cq); 143.6 (arom. 5a-CO); 151.0 (3a-C@N(N)); 154.3 (arom.
1-C@O); 159.7 (arom. 40-C(OCH3)). IR (neat) 1769, 1634, 1498 cm
ꢁ1. HRMS-ESI: m/z [M+Na]+ calcd for C16H12N2O4Na: 319.06948;
found: 319.06900; [2M+Na]+ calcd for C32H24N4O8Na: 615.14918;
found: 615.14858.
4.5.4 8-[3-(Trifluoromethyl)phenyl]-4H-[1,2,4]oxadiazolo[3,4-c]-
benzoxazin-1-one (19)
4.5. General procedure for the preparation of triazole (21) and
oxadiazoles ODQ, NS 2028, (18), (19), (25) and (29)
According to the general procedure this compound was pre-
pared from 6-[3-(trifluoromethyl)phenyl]-2H-1,4-benzoxazin-
3(4H)-one-oxime 17 (100 mg, 0.32 mmol) to yield 19 (52 mg,
0.16 mmol, 69% concerning conversion) as colourless needles. Mp
163 °C. 1H NMR (300 MHz, CDCl3): d = 5.16 (s, 2H, 4-CH2); 7.22
(d, 1H, 3J = 8.4 Hz, arom. 6-CH); 7.47 (dd, 1H, 4J = 2.1 Hz,
3J = 8.4 Hz, arom. 7-CH); 7.54–7.66 (m, 2H, arom. 40-CH and arom.
50-CH); 7.74–7.78 (m, 2H, arom. 20-CH and arom. 60-CH); 8.32 (d,
1H, 4J = 2.1 Hz, arom. 9-CH). APT (75 MHz, CDCl3): d = 60.4 (4-
CH2); 115.2 (arom. 9-CH); 118.6 (arom. 6-CH); 121.7 (arom. 9a-
In an argon atmosphere the appropriate oxime or hydrazone
was dissolved in abs. THF (4 mL/mmol) and subsequently treated
with 1,10-carbonyl diimidazole (1.1 equiv). The reaction mixture
was refluxed for 24 h. Subsequently, the solvent was removed in
vacuo, the residue taken with dichloromethane and washed three
times with water. The organic phase was dried over Na2SO4 and
the solvent was removed in vacuo. The crude product was purified
by column chromatography.
3
CN); 123.9 (q, J = 4 Hz, arom. 20-CH); 124.2 (q, 1J = 273 Hz, CF3);
124.7 (q, 3J = 4 Hz, arom. 40-CH); 126.7 (arom. 7-CH); 129.7 (arom.
2
50-CH); 130.5 (arom. 60-CH); 131.6 (q, J = 33 Hz, arom. 30-Cq);
4.5.1. 8-Bromo-2,4-dihydro-1H-[1,2,4]triazolo[3,4-c][1,4]-
benzoxazin-1-one (21)
136.1 (arom. 8-Cq); 140.2 (arom. 10-Cq); 144.6 (arom. 5a-CO);
150.7 (3a-C@N(N)); 154.2 (1-C@O). 19F NMR (282 MHz, CDCl3):
d = ꢁ63.03 (CF3). IR (neat) 1777, 1634, 1488 cmꢁ1. HRMS-ESI: m/z
[M+Na]+ calcd for C16H9N2O3F3Na: 357.04574; found: 357.04590;
[2M+Na]+ calcd for C32H18N4O6F6Na: 691.10227; found:
691.10284.
According to the general procedure this compound was pre-
pared from 6-bromo-3-hydrazono-3,4-dihydro-2H-1,4-benzox-
azine 20 (174 mg, 0.72 mmol) to yield 21 (86.1 mg, 0.32 mmol,
63%) as colourless needles. Mp 232 °C. 1H NMR (300 MHz, DMSO-
d6): d = 5.14 (s, 2H, 4-CH2); 7.08 (d, 1H, 3J = 8.2 Hz, arom. 6-CH);
7.34 (dd, 1H, 4J = 2.3 Hz, 3J = 8.2 Hz, arom. 7-CH); 8.2 (d, 1H,
4J = 2.3 Hz, arom. 9-CH); 12.17 (br, 1H, NH). 13C NMR (75 MHz,
DMSO-d6): d = 61.3 (4-CH2); 113.6 (8-CBr); 118.3 (arom. 9-CH);
119.4 (arom. 6-CH); 124.3 (arom. 9a-CN); 128.7 (arom. 7-CH);
138.3 (3a-C@N(N)); 144.1 (arom. 5a-CO); 151.3 (1-C@O). IR (neat)
4.5.5. (13aS)-11,12,13,13a-Tetrahydro-3H,9H-[1,2,4]oxadiazolo-
[4,3-a]pyrrolo [2,1-c]-[1,4]-benzodiazepine-3,9-dione (25)
According to the general procedure this compound was pre-
pared from (11aS)-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiaze-
pine-5,11(10H,11aH)-dione-11-oxime 24 (231 mg, 1 mmol) to
yield 25 (164 mg, 0.64 mmol, 84%) as a colourless solid. Mp
2923, 1647, 1462 cmꢁ1
.
HRMS-ESI: m/z [M+H]+ calcd for
C9H6N3O2BrH: 267.97162; found: 267.97179; [2M+H]+ calcd for
C18H12N6O4BrH: 534.93595; found: 534.93596.
176 °C. ½a 2D3
ꢃ
+158 (c 1.098, CHCl3). 1H NMR (400 MHz, DMSO-d6):
d = 2.04–2.13 (m, 2H, 12-CH2); 2.27–2.32 (m, 1H, 13-CHaHb);
2.74–2.78 (m, 1H, 13-CHaHb); 3.63–3.68 (m, 1H, 11-NCHaHb);
3.80–3.86 (m, 1H, 11-NCHaHb); 4.54–4.57 (dd, 1H, 4J = 3.0 Hz,
3J = 8.4 Hz, 13a-NCH); 7.43–7.46 (m, 1H, arom. 7-CH); 7.56–7.61
(m, 1H, arom. 6-CH); 7.74 (d, 1H, 3J = 8.0 Hz, arom. 5-CH); 7.94–
7.96 (dd, 1H, 4J = 1.3 Hz, 3J = 7.7 Hz, arom. 8-CH). 13C NMR
(100 MHz, DMSO-d6): d = 23.5 (12-CH2); 25.7 (13-CH2); 47.9 (11-
NCH2); 51.3 (13a-NCH); 122.5 (arom. 5-CH); 128.7 (arom. 8a-Cq);
128.8 (arom. 7-CH); 128.8 (arom. 4a-CN); 132.2 (arom. 8-CH);
132.8 (arom. 6-CH); 156.4 (3-C@O); 158.2 (13b-C@N(N)); 164.2
(6-C@O). IR (neat) 1770, 1620 cmꢁ1. HRMS-ESI: m/z [M+H]+ calcd
for C13H11N3O3H: 258.08787; found: 258.08759; [2M+H]+ calcd
for C26H22N6O6H: 515.16791; found: 515.16709.
4.5.2. 8-Bromo-4H-[1,2,4]oxadiazolo[3,4-c]benzoxazin-1-one
(NS 2028)
According to the general procedure this compound was pre-
pared from 6-bromo-2H-1,4-benzoxazin-3(4H)-one-oxime 11
(4.00 g, 16.45 mmol) to yield NS 2028 (4.21 g, 15.65 mmol, 95%)
as a colourless solid. Mp 163–164 °C. 1H NMR (200 MHz, DMSO-
d6): d = 5.36 (s, 2H, 4-CH2), 7.18 (d, 1H, 3J = 8.8 Hz, arom. 6-CH),
7.47 (dd, 1H, 3J = 8.8 Hz, 4J = 2.2 Hz, arom. 7-CH), 8.02 (d, 1H,
4J = 2.2 Hz, arom. 9-CH). 13C NMR (75 MHz, DMSO-d6): d = 59.9
(4-CH2), 113.8 (arom. 8-CBr), 117.9 (arom. 9-CH), 119.7 (arom. 6-
CH), 122.5 (arom. 9a-C-N), 129.9 (arom. 7-CH), 144.0 (arom. 5a-
C-O), 151.1 (3a-C@N(N)), 153 (1-C@O). IR (neat) 1782, 1627,
1493 cmꢁ1
.
HRMS-ESI: m/z [M+Na]+ calcd for C9H4N2O3Na:
290.93758; found: 290.93772; [2M+Na]+ calcd for C18H8N4O6Na:
558.88593; found: 558.88599.
4.5.6. 6H-[1,2,4]Oxadiazolo[4,3-d]pyrido[3,2-b][1,4]oxazin-9-
one (29)
According to the general procedure this compound was pre-
pared from 2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one-oxime 28
(310 mg, 1.88 mmol) to yield 29 (281 mg, 1.47 mmol, 79%) as col-
ourless crystals. Mp 215 °C. 1H NMR (300 MHz, DMSO-d6): d = 5.34
(s, 2H, 6-CH2); 7.34 (dd, 1H, 4J = 4.7 Hz, 3J = 8.2 Hz, arom. 3-CH);
7.62 (dd, 1H, 4J = 1.5 Hz, 3J = 8.2 Hz, arom. 4-CH); 8.15 (dd, 1H,
4J = 1.5 Hz, 3J = 4.7 Hz, arom. 2-CH). APT (75 MHz, DMSO-d6):
d = 60.7 (6-CH2); 124.1 (arom. 3-CH); 126.3 (arom. 4-CH); 136.5
(arom. 10a-CN); 141.9 (arom. 4a-CO); 142.3 (arom. 2-CH); 152.5
4.5.3. 8-(4-Methoxyphenyl)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]-
benzoxazin-1-one (18)
According to the general procedure this compound was pre-
pared from 6-(4-methoxyphenyl)-2H-1,4-benzoxazin-3(4H)-one-
oxime 16 (70 mg, 0.26 mmol) to yield 18 (54 mg, 0.2 mmol, 57%
concerning conversion) as colourless needles. Mp 209 °C. 1H NMR
(400 MHz, CDCl3): d = 3.86 (s, 3H, OCH3); 5.12 (s, 2H, 4-CH2);
3
6.98 (dd, 2H, 4J = 1.9 Hz, J = 6.8 Hz, 2ꢂ arom. 30-CH); 7.16 (d, 1H,
3J = 8.0 Hz, arom. 6-CH); 7.41 (dd, 1H, 4J = 2.0 Hz, 3J = 8.0 Hz, arom.
(9-C@O); 153.0 (6a-C@N(N)). IR (neat) 1775, 1633, 1450 cmꢁ1
.
3
7-CH); 7.51 (dd, 2H, 4J = 1.9 Hz, J = 6.8 Hz, 2ꢂ arom. 20-CH); 8.27
HRMS-ESI: m/z [M+Na]+ calcd for C8H5N3O3Na: 214.02231; found:
214.02246; [2M+Na]+ calcd for C16H10N6O6Na: 405.05540; found:
405.05579.
(d, 1H, 4J = 2.0 Hz, arom. 9-CH). APT (100 MHz, CDCl3): d = 55.5
(OCH3); 60.3 (4-CH2); 114.5 (2C, 2ꢂ arom. 30-CH); 114.6 (arom.
9-CH); 118.3 (arom. 6-CH); 121.5 (arom. 9a-CN); 126.0 (arom. 7-

M. von Wantoch Rekowski et al. / Bioorg. Med. Chem. 18 (2010) 1288–1296
1295
4.5.7. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)
According to the general procedure this compound was pre-
pared from quinoxalin-2(1H)-one oxime 6 (40 mg, 0.25 mmol) to
yield ODQ (45 mg, 0.24 mmol, 97%) as fawn crystals. Mp 163–
164 °C. 1H NMR (300 MHz, CDCl3): d = 7.55 (td, 1H, 4J = 1.2 Hz,
3J = 7.8 Hz, arom. 7-CH2); 7.66 (td, 1H, 4J = 1.5 Hz, 3J = 7.8 Hz, arom.
8-CH2); 7.92 (dd, 1H, 4J = 1.2 Hz, 3J = 7.8 Hz, arom. 6-CH); 8.54 (dd,
1H, 4J = 1.2 Hz, 3J = 8.4 Hz, arom. 9-CH); 8.68 (s, 1H, 4-CH). 13C NMR
(75 MHz, CDCl3): d = 114.8 (arom. CH); 124.5 (arom. Cq); 127.8
(arom. CH); 130.6 (arom. CH); 131.9 (arom. CH); 134.8 (arom.
Cq); 141.0 (4-CH); 146.7 (C@N(N)); 154.3 (C@O). IR (neat) 1780,
130.8 (arom. 3-CH); 135.1 (arom. 9-CH); 145.8 (arom. 7a-CN);
148.1 (arom. 6a-C@N(N)); 148.2 (arom. 4a-CO); 159.8 (12-C@O).
IR (neat) 1702, 1629, 1473, 1341 cmꢁ1. HRMS-ESI: m/z [M+Na]+
calcd for C15H9N2O2BrNa: 350.97396; found: 350.97387;
[2M+Na]+ calcd for C30H18N4O4Br2Na: 678.95870; found:
678.95885.
4.6.3. 9-Chloro-12-oxo-6,12-dihydroquinazolino[2,3-c][1,4]-
benzoxazine (33)
According to the general procedure this compound was pre-
pared from 2H-1,4-benzoxazin-3(4H)-one 30 (149.2 mg, 1.0 mmol)
and 4-chloroanthranilic acid (171.6 mg, 1.0 mmol) to yield 33
(136 mg, 0.48 mmol, 48%) as orange needles. Mp 168–170 °C. 1H
NMR (400 MHz, CDCl3): d = 4.95 (s, 2H, 6-CH2); 7.13–7.18 (m, 2H,
arom. 2-CH and arom. 4-CH); 7.23–7.27 (m, 1H, arom. 3-CH);
7.46 (dd, 1H, 4J = 2.4 Hz, 3J = 8.6 Hz, arom. 10-CH); 7.65 (d, 1H,
4J = 2.4 Hz, arom. 8-CH); 8.27 (d, 1H, 3J = 8.6 Hz, arom. 11-CH);
8.54 (d, 1H, 3J = 8.0 Hz, arom. 1-CH). APT (75 MHz, CDCl3):
d = 69.3 (6-CH2); 117.8 (arom. 4-CH); 120.9 (arom. 11a-Cq); 122.9
(arom. 2-CH); 123.4 (arom. 1-CH); 125.0 (arom. 13a-CN); 126.8
(arom. 8-CH); 128.2 (arom. 3-CH and arom. 10-CH); 129.2 (arom.
11-CH); 141.1 (arom. 9-Cq); 147.1 (arom. 7a-C@N); 149.2 (arom.
4a-CO); 150.3 (arom. 6a-C@N(N)); 159.3 (12-C@O). IR (neat)
.
1769, 1573, 1462 cmꢁ1 HRMS-ESI: m/z [M+Na]+ calcd for
C9H5N3O2Na: 210.02740; found: 210.02732; [2M+Na]+ calcd for
C18H10N6O4Na: 397.06557; found: 397.06584.
4.6. General procedure for the preparation of 12-
oxoquinazolino-benzoxazines (31), (32) and (33)30
A suspension of the appropriate 2H-1,4-benzoxazin-3(4H)-one
derivative and phosphoryl chloride (1.5 eq) in dichloroethane
(1 mL/mmol) was stirred for 15 min at room temperature. Subse-
quently triethyl amine (1.5 equiv) and anthranilic acid (1 equiv)
were added dropwise at 0 °C. The resulting suspension was stirred
for one hour at room temperature and then it was refluxed for an-
other hour. After dilution of the mixture with dichloroethane and
cold water the organic phase was separated and washed with
10% solution of sodium carbonate and water. The organic phase
was dried over Na2SO4 and filtered, the solvent was removed in va-
cuo yielding the crude product which was recrystallized from
ethanol.
1687, 1595, 1349 cmꢁ1
.
HRMS-ESI: m/z [M+H]+ calcd for
C15H9N2O2ClH: 285.04253; found: 285.04257; [2M+H]+ calcd for
C30H18N4O4Cl2H: 569.07779; found: 569.07719.
4.7. Biology
Cell culture media and serum were obtained from Life Technol-
ogies Gibco–BRL (Paisley, UK). Penicillin and streptomycin from
Applichem (Darmstadt, Germany). All cell culture plasticware
4.6.1. 12-Oxo-6,12-dihydroquinazolino[2,3-c][1,4]benzoxazine
(31)
was purchased from Corning-Costar Inc. (Corning, NY); L-NAME
According to the general procedure this compound was pre-
pared from 2H-1,4-benzoxazin-3(4H)-one 30 (1.49 g, 10 mmol)
and anthranilic acid (1.37 g, 10 mmol) to yield 31 (2.19 g,
8.75 mmol, 88%) as yellowish needles. Mp 123 °C. 1H NMR
(400 MHz, CDCl3): d = 4.98 (s, 2H, 6-CH2); 7.13–7.18 (m, 2H, arom.
2-CH and arom. 4-CH); 7.23–7.27 (m, 1H, arom. 3-CH); 7.52 (m,
1H, arom. 10-CH); 7.66 (dd, 4J = 0.8 Hz, 3J = 8.0 Hz, 1H, arom. 8-
CH); 7.77 (m, 1H, arom. 9-CH); 8.37 (dd, 1H, 4J = 1.2 Hz,
3J = 8.0 Hz, arom. 11-CH); 8.59 (dd, 1H, 4J = 1.6 Hz, 3J = 8.4 Hz,
arom. 1-CH). APT (100 MHz, CDCl3): d = 69.5 (6-CH2); 117.8 (arom.
4-CH); 122.5 (arom. 11a-Cq); 122.9 (arom. 2-CH); 123.5 (arom. 1-
CH); 125.2 (arom. 13a-CN); 127.1 (arom. 8-CH); 127.6 (arom. 10-
CH); 127.7 (arom. 11-CH); 128.0 (arom. 3-CH); 134.9 (arom. 9-
CH); 146.1 (arom. 7a-C@N); 149.0 (arom. 6a-C@N(N)); 149.3
(arom. 4a-CO); 160.0 (12-C@O). IR (neat) 1689, 1625, 1490,
and isobutyl-methylxanthine (IBMX), were purchased from Sig-
ma–Aldrich (St. Louis, MO). The cGMP EIA kit was purchased from
Assay Designs (Ann Arbor, MI).
4.7.1. Rat aortic smooth cell culture
Rat aortic smooth muscle cells (RASMC) were grown on 100-
mm dishes in high glucose DMEM supplemented with 10% foetal
calf serum, 50 U/mL penicillin and 50 lg/mL streptomycin. RASMC
between passages 3 and 4 were used for all experiments.
4.7.2. cGMP immunoassay
Confluent RASMC were initially treated with isobutyl-methyl-
xanthine (IBMX; 1 mM, 5 min) to inhibit phosphodiesterase activ-
ity and were then exposed to the tested molecules (0, 1 or 10
for 20 min in the presence of IBMX. At the end of the incubation
period cells were treated with SNP (10 V) for 15 min. Media were
then aspirated, and 200 l of 0.1 N HCl were added to each well to
lM)
1352 cmꢁ1
.
HRMS-ESI: m/z [M+Na]+ calcd for C15H10N2O2Na:
l
273.06345; found: 273.06368; [2M+Na]+ calcd for C30H20N4O4Na:
523.13768; found: 523.13720.
l
extract cGMP. After 30 min, HCl extracts were collected and centri-
fuged at 600g for 10 min to remove debris. The supernatants were
directly analysed for cGMP by enzyme immunoassay.
4.6.2. 2-Bromo-12-oxo-6,12-dihydroquinazolino[2,3-c][1,4]-
benzoxazine (32)
According to the general procedure this compound was pre-
Acknowledgment
pared from 6-bromo-2H-1,4-benzoxazin-3(4H)-one
9 (228 mg,
1.0 mmol) and anthranilic acid (137 mg, 1.0 mmol) to yield 32
(221 mg, 0.67 mmol, 67%) as orange needles. Mp 149–151 °C. 1H
NMR (400 MHz, CDCl3): d = 4.97 (s, 2H, 6-CH2); 7.02 (d, 1H,
3J = 8.8 Hz, arom. 4-CH); 7.35 (dd, 1H, 4J = 2.4 Hz, 3J = 8.8 Hz, arom.
3-CH); 7.54 (m, 1H, arom. 10-CH); 7.66 (d, 1H, 3J = 8.0 Hz arom. 8-
CH); 7.77 (m, 1H, arom. 9-CH); 8.35 (dd, 1H, 4J = 1.4 Hz, 3J = 7.8 Hz,
arom. 11-CH); 8.80 (d, 1H, 4J = 2.4 Hz, arom. 1-CH). APT (100 MHz,
CDCl3): d = 69.3 (6-CH2); 115.0 (arom. 2-CBr); 119.1 (arom. 4-CH);
122.2 (arom. 11a-Cq); 126.1 (arom. 13a-CN); 126.3 (arom. 1-CH);
127.2 (arom. 8-CH); 127.7 (arom. 11-CH); 127.9 (arom. 10-CH);
We gratefully acknowledge Dr. Lothar Hennig for recording
NMR spectra and the University of Leipzig for financial support.
References and notes
1. Murad, F. Angew. Chem. Int. Ed. 1999, 38, 1856.
2. Madhusoodanan, K. S.; Murad, F. Neurochem. Res. 2007, 32, 681.
3. Murad, F. N. Eng. J. Med. 2006, 355, 2003.
4. Evgenov, O. V.; Pacher, P.; Schmidt, P. M.; Haskó, G.; Schmidt, H. H. H. W.;
Stasch, J.-P. Nat. Rev. 2006, 5, 755.
5. Pyriochou, A.; Papapetropoulos, A. Cell. Signal. 2005, 17, 407.

1296
M. von Wantoch Rekowski et al. / Bioorg. Med. Chem. 18 (2010) 1288–1296
6. Wedel, B.; Humbert, P.; Harteneck, C.; Foerster, J.; Malkewitz, J.; Böhme, E.;
Schultz, G.; Koesling, D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2592.
7. Zhao, Y.; Schelvis, J. P. M.; Babcock, G. T.; Marletta, M. A. Biochemistry 1998, 37,
4502.
H.; Stahl, E.; Straub, A.; Wunder, F.; Stasch, J.-P. Chem. Med. Chem. 2009, 4,
853–865.
20. Garthwaite, J.; Southam, E.; Boulton, C. L.; Nielsen, E. B.; Schmidt, K.; Mayer, B.
Mol. Pharmacol. 1995, 48, 184–188.
8. Bellamy, T. C.; Garthwaite, J. Mol. Cell. Biochem. 2002, 230, 165.
9. Ballou, D. P.; Zhao, Y.; Brandish, P. E.; Marletta, M. A. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 12097–12101.
10. Biel, M.; Sautter, A.; Ludwig, A.; Hofmann, F.; Zong, X. Naunyn Schmiedebergs
Arch. Pharmacol. 1998, 358, 140–144.
11. Hanafy, K. A.; Krumenacker, J. S.; Murad, F. Med. Sci. Monit. 2001, 7, 801–819.
12. Ko, F.-N.; Wu, C.-C.; Kuo, S.-C.; Lee, F.-Y.; Teng, C.-M. Blood 1994, 84,
4226–4233.
13. Mülsch, A.; Bauersachs, J.; Schäfer, A.; Stasch, J.-P.; Kast, R.; Busse, R. Br.
J. Pharmacol. 1997, 120, 681–689.
14. Stasch, J.-P.; Schmidt, P. M.; Nedvetsky, P. I.; Nedvetskaya, T. Y.; Kumar, A.;
Meurer, S.; Deile, M.; Taye, A.; Knorr, A.; Lapp, H.; Müller, H.; Turgay, Y.;
Rothkegel, C.; Tersteegen, A.; Kemp-Harper, B.; Müller-Esterl, W.; Schmidt, H.
H. H. W. J. Clin. Invest. 2006, 116, 2552–2561.
15. Straub, A.; Benet-Buckholz, J.; Fröde, R.; Kern, A.; Kohlsdorfer, C.; Schmitt, P.;
Schwarz, T.; Siefert, H.-M.; Stasch, J.-P. Bioorg. Med. Chem. 2002, 10, 1711–1717.
16. Stasch, J.-P.; Alonso-Alija, C.; Apeler, H.; Dembowsky, K.; Feurer, A.; Minuth, T.;
Perzborn, E.; Schramm, M.; Straub, A. Br. J. Pharmacol. 2002, 135, 333–343.
17. Stasch, J.-P.; Dembowsky, K.; Perzborn, E.; Stahl, E.; Schramm, M. Br. J.
Pharmacol. 2002, 135, 344–355.
18. Stasch, J.-P.; Schmidt, P.; Alonso-Aija, C.; Apeler, H.; Dembowsky, K.;
Haerter, M.; Heil, M.; Minuth, T.; Perzborn, E.; Pleiss, U.; Schramm, M.;
Schroeder, W.; Schröder, H.; Stahl, E.; Steinke, W.; Wunder, F. Br. J.
Pharmacol. 2002, 136, 773–783.
19. Mittendorf, J.; Weigand, S.; Alonso-Alija, C.; Bischoff, E.; Feurer, A.; Gerisch, M.;
Kern, A.; Knorr, A.; Lang, D.; Muenter, K.; Radtke, M.; Schirok, H.; Schlemmer, K.
21. Feelisch, M.; Kotsonis, P.; Siebe, J.; Clement, B.; Schmidt, H. H. H. W. Mol.
Pharmacol. 1999, 56, 243–253.
22. Zhao, Y.; Brandish, P. E.; DiValentin, M.; Schelvis, J. P.; Babcock, G. T.; Marletta,
M. A. Biochemistry 2000, 39, 10848–10854.
23. Olesen, S.-P.; Drejer, J.; Axelsson, O.; Moldt, P.; Bang, L.; Nielsen-Kudsk, J. E.;
Busse, R.; Mülsch, A. Br. J. Pharmacol. 1998, 123, 299–309.
24. Bartsch, H.; Erker, T.; Neubauer, G. Monatsh. Chem. 1989, 120, 81–84.
25. Nugiel, D. A.; Jacobs, K.; Cornelius, L.; Chang, C.-H.; Jadhav, P. K.; Holler, E. R.;
Klabe, R. M.; Bacheler, L. T.; Cordova, B.; Garber, S.; Reid, C.; Logue, K. A.; Gorey-
Feret, L. J.; Lam, G. N.; Erickson-Viitanen, S.; Seitz, S. P. J. Med. Chem. 1997, 40,
1465–1474.
26. Shridhar, D. R.; Jogibhukta, M.; Krishnan, V. S. H. OPPI Briefs 1982, 14,
195–197.
27. Bartsch, H.; Erker, T.; Neubauer, G. Monatsh. Chem. 1988, 119, 1439–1444.
28. Kamal, A.; Howard, P. W.; Reddy, N.; Reddy, P.; Thurston, D. E. Tetrahedron
1997, 53, 3223–3230.
29. Gillard, A.; Foloppe, M.; Rault, S. J. Heterocycl. Chem. 1997, 34, 445.
30. Sastry, C. V. R.; Rao, K. S.; Krishnan, V. S. H.; Rastogi, K.; Jain, M. L. Synthesis
1988, 4, 336–337.
31. Palmer, T. In Goodman and Gilman’s The Pharmacological Basis of Therapeutics;
Brunton, L. L., Ed.; McGraw-Hill: New York, 2006; pp 203–204.
32. Powell, N. A.; Ciske, F. L.; Cai, C.; Holsworth, D. D.; Mennen, K.; Van Huis, C. A.;
Jalaie, M.; Day, J.; Mastronardi, M.; McConnell, P.; Mochalkin, I.; Zhang, E.;
Ryan, M. J.; Bryant, J.; Collard, W.; Ferreira, S.; Gu, C.; Collins, R.; Edmunds, J. J.
Bioorg. Med. Chem. 2007, 15, 5912–5949.
33. Clauson-Kaas, N.; Lei, J.; Heide, H. Acta Chem. Scand. 1971, 25, 3135–3143.