Investigations on synthesis and structure elucidation of novel [1,2,4]triazolo[1,2-a]pyridazine-1-thiones and their inhibitory activity against inducible nitric oxide synthase

Article abstract of DOI:10.1016/j.bmc.2013.05.064

The inducible nitric oxide synthase (iNOS) is a target of great research interest due to its importance in a number of diseases, for example, septic shock and inflammatory lung diseases. A variety of 3-substituted [1,2,4]triazolo[1,2-a]pyridazine derivati

Full text of DOI:10.1016/j.bmc.2013.05.064

Bioorganic & Medicinal Chemistry 21 (2013) 5518–5531
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigations on synthesis and structure elucidation of novel
[1,2,4]triazolo[1,2-a]pyridazine-1-thiones and their inhibitory
activity against inducible nitric oxide synthase q
Ulrike Schulz, Antje Grossmann, Manja Witetschek, Christian Lemmerhirt, Marcus Polzin, Beate Haertel,
⇑
Heike Wanka, Olaf Morgenstern
Institute of Pharmacy, Ernst Moritz Arndt University Greifswald, Friedrich-Ludwig-Jahn-Straße 17, 17487 Greifswald, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The inducible nitric oxide synthase (iNOS) is a target of great research interest due to its importance in a
number of diseases, for example, septic shock and inflammatory lung diseases. A variety of 3-substituted
[1,2,4]triazolo[1,2-a]pyridazine derivatives was synthesized by ring closure with hexahydropyridazine-
1-carbothioamide by using aliphatic and aromatic aldehydes. The activity of the new substances was
tested on the insulin-secreting rat insulinoma cell line RINm5F. iNOS was expressed through exposure
Received 24 March 2013
Revised 26 May 2013
Accepted 28 May 2013
Available online 12 June 2013
to interleukin-1b (IL-1b) and interferon-c (IFN-c). A number of the investigated compounds were more
Keywords:
active than the reference inhibitor aminoguanidine (AG). Structure–activity relationships showed that
a phenyl substituent in position 3 is apparently essential for inhibition.
Ó 2013 Elsevier Ltd. All rights reserved.
Hexahydropyridazine-1-carbothioamide
[1,2,4]Triazolo[1,2-a]pyridazine-1-thiones
Inducible nitric oxide synthase
RINm5F
1. Introduction
a rather small amount of NO. These two isoforms are of basic
physiological importance. On the other hand, the iNOS is a part
In the 1980s was discovered that the endothelium-derived
relaxing factor (EDRF) is actually nitric oxide (NO).1 Since then
an understanding of its role in physiology has dramatically in-
creased. This radical substance is involved in numerous processes,
from cardiovascular regulation to neuronal signaling and immune
response.2–6
NO is synthesized by a family of enzymes called nitric oxide
synthases. These enzymes are homodimers possessing a bidomain
structure. The oxygenase domain contains binding sites for the
cofactors haem, tetrahydrobiopterine (BH4) and the substrate
of the primary immune defense. It is expressed by the influence
of proinflammatory cytokines or bacterial lipopolysaccharides
(LPS), Ca2+/calmodulin-independent and creates a high nitric oxide
concentration.5,10,11
Although nitric oxide is needed for physiological processes, an
excess can be fatal. In fact overproduction of NO is discussed in
relation to the pathogenesis of many diseases, for example, septic
shock,12,13 multiple sclerosis/Alzheimer’s disease6,14 and COPD/
asthma.15,16 In most of the cases the inducible isoform seems to
be responsible for the massive NO production that leads to patho-
logical changes.
L-arginine. The reductase domain located on the other side of the
enzyme reveals binding regions for cofactors FAD, FMN and
NADPH. These domains are linked by a calmodulin recognition
site.7–9 There are three isoforms known: endothelial (eNOS,
NOS3), neuronal (nNOS, NOS1) and inducible nitric oxide synthase
As the number of diseases connected with an overexpression of
iNOS has increased, so too has the number of inhibitors, however,
to date there is no drug available to treat these clinical conditions.
Potency and especially selectivity are central problems of the drug
design because it is obvious that an exclusive inhibition of iNOS is
required to maintain the physiological function of eNOS and
nNOS. Although an inhibition of iNOS mediated NO production
is also possible through pteridine analogues that block the cofac-
tor tetrahydrobiopterine17,18 as well as by substances that hinder
the dimerization of iNOS19,20, most of the reported iNOS inhibitors
interact with the arginine binding site of the oxygenase domain.
Such inhibitors can be divided into two groups: amino acid-based
and non-amino acid-based substances.21 Amino acid-based
(iNOS, NOS2). They all generate NO by oxidation of
L
-arginine to
x
L-citrulline via N -hydroxy-L-arginine but have different
localization, responsibilities and regulation. eNOS and nNOS are
constitutively expressed, Ca2+/calmodulin-regulated and produce
q
N,N-Coupled heterobicycles from cyclic hydrazine derivatives. Part 14. Part 13:
Morgenstern, O.; Wanka, H.; Röser, I.; Steveling, A.; Kuttler, B. Bioorg. Med. Chem.
2004, 12, 1071.
⇑
Corresponding author. Tel.: +49 3834864838.
E-mail address: omorgens@uni-greifswald.de (O. Morgenstern).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.064

U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
5519
inhibitors, as L-NIL, L L
-NIO22 or -NNA,23 usually show IC50 values
2. Results and discussion
2.1. Synthesis
in the low micromolar range but have poor selectivities.10,21
Exceptions are GW273629 and GW274150, which show high
selectivity.24–26 Non-amino acid-based inhibitors also mimic
L-
arginine but with a wider structural variety. There are guanidines,
for example, aminoguanidine, which was discovered as one of the
first iNOS inhibitors. Due to the high concentrations required for
inhibition, severe side effects in vivo (mouse) and the fact that
selectivity is reduced with increasing concentrations aminoguani-
dine is a poor drug candidate.27–29 Isothioureas such as S-ethyl-
isothiourea and AMT showed very good activities in the lower
nanomolar range but were at most only partially selective.30,31
One of the most potent iNOS inhibitors, ONO-1714, is a represen-
tative of the amidine derivatives, but also showed only low selec-
tivity.32,33 On the other hand, another amidine, 1400W, was
identified as a highly selective and very potent inhibitor.34 In
the non-amino acid-based group also are spirocyclic analogues35
and 2-aminopyridines.36–38 Some of the newer studies investi-
gated sulfur and nitrogen containing heterocyclic structures not
unlike those described here.39,40
Here we report on a series of novel [1,2,4]triazolo[1,2-a]pyrida-
zine-1-thiones as promising iNOS inhibitors, whose hitherto un-
known substructure might also be interesting with regards to
other bioactivities; the 1,2,4-triazolo partial structure has been
investigated in respect of antimicrobial, antifungal41,42 and analge-
sic properties43 as well as HSP9044 and TACE inhibition.45 Some
time ago our group published the synthesis and biological activity
of semicyclic aminoguanidines including their carbothioamide
synthetic precursors.46 Unfortunately, the iNOS inhibitory ability
of both of these substance classes was not improved compared
to the lead structure.
To obtain the 3-substituted heterocycles 2, the starting com-
pound 1a was reacted with various aliphatic and aromatic alde-
hydes (Scheme 1). Aliphatic and araliphatic reagents gave the
compounds 2b,c (Scheme 3) and 2d,e (Scheme 4). The carbothioa-
mide 1a was found to be easily accessible from hexahydropyrid-
azine hydrochloride 4 and potassium thiocyanate.46
However, 2a (Scheme 2) was not directly accessible via this
route.
2.1.1. 3-Methyl and 3-ethyl derivatives 2a,b
The first attempts to obtain the 3-methyl derivative 2a by acid-
catalyzed reaction of 1a with acetaldehyde or acetaldehyde di-
methyl acetal applying conventional synthesic methods were just
as unsuccessful as by using a microwave-assisted synthesis of this
compound with acetaldehyde diethyl acetal. Regarding these re-
sults it was investigated whether 2a could be synthesized by react-
ing 1a with acetic acid to give 3a followed by hydrogenation
(Scheme 2). However, all attempts to obtain 3a by this route failed.
Therefore, the synthesis of 3a was attempted by intramolecular
cyclocondensation of 1b. To get to this intermediate stage, hexa-
hydropyridazine hydrochloride 417 was reacted with acetyl isothi-
ocyanate. Nevertheless, even with modification of the reaction
conditions, compound 5b, the 2-acetyl derivative of 1b, was always
isolated. However, when 5b was heated in methanol the com-
pound rapidly cyclized to 3a. This reaction is similar to the desired
formation of
a
2-thioxo-2,3-dihydro-quinazoline-4(1H)-one
derivative.61
For this reason our attention focused on the related bicyclic
derivatives since these substances possess a more rigid molecular
skeletal structure, which could result in a higher efficacy and
selectivity. The ring closure with C1 reactants, such as carbonyl
compounds, offered an excellent preparative access to a variety
of 3-substituted [1,2,4]triazolo[1,2-a]pyridazine derivatives. First,
the synthesis of the hitherto unknown [1,2,4]triazolo[1,2-a]pyrida-
zine-1-thiones 2 (Scheme 1) and their iNOS inhibitory ability was
investigated. The known representatives of this heterocyclic
system possess sp2-hybridized carbon atoms in the triazol ring
such as [1,2,4]triazolo[1,2-a]pyridazin-1,3-diones,47,48-1,3-dithi-
ones,47,49 1-oxo-47,50 and 1-arylimino-51,52 [1,2,4]triazolo[1,2-a]
pyridazin-3-thiones, and 3-oxo-[1,2,4]triazolo[1,2-a]pyridazin-1-
carboxylic acids (and their esters), respectively.53,54 In contrast,
only very few derivatives with only one sp3-hybridized carbon
atom in this part of the condensed heterocyclic system, as in the
title compounds, have been described previously.55,56 With two
exceptions57,58 the hitherto known [1,2,4]triazolo[1,2-a]pyrida-
zines without substituents at the pyridazine ring are always
substituted at the N(2) atom.
The treatment of 1a with acetic anhydride provided the precur-
sor 5a in excellent yield. This seemed to offer an additional facile
method to obtain 3a. However, all attempts failed to cyclize 5a uti-
lizing the same reaction conditions as for 5b. It seems likely that
the formation of 3a from 5b is favored because the nascent acetic
acid is removed immediately from the reaction by formation of
methyl acetate. The reduction of 3a to 2a succeeded by using so-
dium borohydride under mild conditions (Scheme 2).
Heating of 1a with propionaldehyde provided the 2-ethyl deriv-
ative 2b, which was isolated after chromatographic separation of
the resulting product mixture, but only in poor yield (Scheme 3).
NH·HCl
NH
a
b
S
S
O
4
N
NH2
N
NH
N
H
CH3
NH
b
S
e
N
N
1a
1b
NH
At first, the substitution pattern in the 3-position was varied in
order to gain insight into structure–activity relationships. The iNOS
inhibitory potential was investigated by using a cell culture assay
based on the ability to express iNOS in rat insulinoma cells by addi-
tion of proinflammatory cytokines.59,60
CH3
CH3
g
HN
c
2a
O
N
N
S
O
NH2
f
N
N
S
O
d
d
CH3
S
5b
N
N
CH3
N
5a
S
S
NH
CH3
N
NH2
3a
a
N
N
NH
Scheme 2. (a) KSCN, melt;46 (b) CH3CO-NCS, (C2H5)3N, CH2Cl2, room temperature,
30 min; (c) (CH3CO)2O, room temperature, 3 weeks; (d) CH3OH, H+, reflux, 24 h; (e)
CH3CHO or CH3CH(OCH3)2, H+, different conditions; (g) CH3COOH, 130 °C, 12 h; (f)
NaBH4, C3H7OH, room temperature, 24 h.
1a
2
R
Scheme 1. (a) Different aldehydes; different reaction conditions.

5520
U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
Table 1
S
3-Substituted [1,2,4]triazolo[1,2-a]pyridazine-1-thiones 2
N
NH2
NH
1a
a
b
c
2
R
Reaction time [min]
(Yield [%])
Reaction time [min]
(Yield [%])
S
S
S
conventionala
microwave assistedb
N
N
N
N
N
N
H
NH
NH
f
C6H5
75 (76)
30 (74)
25 (68)
25 (54)
85 (71)
15 (70)
110 (53)
20 (69)
20 (73)
20 (84)
15 (57)
15 (64)
15 (53)
75 (35)
15 (74)
20 (70)
3 (63)
NH
g
h
i
j
k
l
m
n
o
p
q
r
2-CH3–C6H4
3-CH3–C6H4
4-CH3–C6H4
2-Cl–C6H4
3-Cl–C6H4
4-Cl–C6H4
2-NO2–C6H4
3-NO2–C6H4
4-NO2–C6H4
2-OH–C6H4
3-OH–C6H4
4-OH–C6H4
4-CH3O–C6H4
3-(C6H5O)–C6H4
4-[(CH3)2N]–C6H4
C2H5
2b
2c
1c
Scheme 3. (a) C2H5–CHO, H+, CH2Cl2, reflux, 9 h; (b) C6H5–CH2–CHO, H+, 125–130 °C,
4.5 h; (c) C6H5–CH2–CHO, CH2Cl2, 60 °C, 5 min.
6 (50)
6 (64)
S
NH2
N
3 (77)
3 (69)
3 (84)
NH
s
t
u
1a
a
b
a
Heating the educts at 150–155 °C (2l: 130–135 °C; 2u: 100–105 °C).
Additional data are given in the experimental part.
b
S
S
c
N
N
N
N
N
NH
in good yields, usually after a general work-up method, by using
a mixture of 1a and the corresponding substituted benzaldehyde
(excess of 10%; exception: methylbenzaldehydes 50%) in the pres-
ence of catalytic amounts of p-toluenesulfonic acid monohydrate.
The reaction mixture was heated to 150–155 °C (Scheme 1). In
most cases the reaction of 1a with the aldehydes was already com-
pleted within a period of 15–30 min. However, benzaldehyde (2f),
o- and p-chlorobenzaldehyde (2j,l), and p-methoxybenzaldehyde
(2s) reacted significantly slower than the other aldehydes. To avoid
undesirable side reactions, the synthesis of p-chlorophenyl and p-
dimethylamino derivative was done at lower temperatures
(Table 1).
Compound 1d showed a higher reactivity towards benzalde-
hyde than did 1a; thus, the 2,3-diphenyl-2,3,5,6,7,8-hexahydro-
1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thione 6 was already formed
by reacting the mixture at room temperature in good yield
(Scheme 5). Especially with the 3-arylsubstituted [1,2,4]triazol-
o[1,2-a]-pyridazine-1-thione derivatives 2, whose preparation
took an extended period of time by conventional synthesis, the
use of microwave technology was advantageous.
2d,e
3b,c
R
R
R
2d, 3b
2e, 3c
H
NO2
Scheme 4. (a) Compound 2d: C6H5–CH@CH–CHO, CH2Cl2, room temperature, 15 h;
Compound 2e: 2-O2N–C6H5–CH@CH–CHO, H+, 100–105 °C, 20 min; (b) Compound
3b: C6H5–CH@CH–COOH, 3c:2-NO2–C6H5–CH@CH–COOH, H+,180 °C, melt; (c) from
1a via 2d,e–3b: C6H5–CH@CH–CHO, H+, room temperature, 2 h; 3c: 2-O2N–C6H5–
CH@CH–CHO, H+, 125–130 °C, 20 min.
2.1.2. 3-Araliphatic substituted compounds 2c–e
Phenyl acetaldehyde reacted with 1a under mild conditions
without proton catalysis to give exclusively the N-styryl carbo-
thioamide 1c. In contrast, the desired 2c was formed in the proton
catalyzed reaction (Scheme 3).
Reaction of 1a and cinnamaldehyde in dichloromethane under
mild conditions yielded the 3-styryl derivative 2d, but without sol-
vent its oxidation product 3b was isolated instead (Scheme 4). The
latter was also formed by prolonged heating (4 h) at 125–130 °C
but in much lower yield.
In all the cases studied microwave methods, very significant
reduction in reaction times (Table 1) and in some cases (2r,s,t)
an improvement in yields were observed. An exception was the
reaction with p-dimethylaminobenzaldehyde, which under similar
conditions resulted in decomposition with strong gas generation. A
product was not isolable.
The reaction with 20-nitrocinnamaldehyde proceeded much fas-
ter and yielded 3c as the main product, 2e as a byproduct, respec-
tively. The ease of such oxidation process has already been
observed with 1,2,4-triazolidine-3-thiones and utilized for tar-
geted synthesis by other authors.62 Compound 2e was formed
within a short time and in good yield at 100–105 °C (Scheme 4).
However, all attempts to obtain 3b or 3c failed by directly melting
(180 °C) 1a with the corresponding cinnamic acids.
Again, oxidation of the resulting 3-aryl derivatives 2 led to the
formation of the 2,3-didehydro compounds 3, but this was much
S
S
a
N
N
N
H
N
NH
N
2.1.3. 3-Aryl derivatives 2f–u
1d
6
In contrast to the observations described above, the cyclization
reaction with aromatic aldehydes proceeded rapidly and without
significant problems. Compounds 2f–u (Table 1) were obtained
Scheme 5. (a) C6H5–CHO, room temperature, 2 weeks.

U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
5521
S
aromatic ring at C(3). Figure 1 exemplary shows the geometry of
compound 2f.
S
N
N
a
N
N
NH
N
For none of the compounds was the expected signal splitting
observed for the proton at N(2) because the protons at the thioam-
ide N(2) and C(3) occupy an angle of nearly 90° to each other
(Fig. 1). The signal position influenced by the aromatic ring and
the signal splitting of the proton at C(3) of the compounds 2f–u
[R = (un)substituted) aryl] differed from those of the spectra of
2a–e. For the C(3) proton, coupling with an adjacent hydrogen
atom of the aliphatic or araliphatic 3-substituent was observed,
and vice versa the corresponding doublets due to the adjacent pro-
ton(s) of the respective 3-substituent.
2f,l
3d,e
R
R
R
2f, 3d
2l, 3e
H
Cl
Scheme 6. (a) Compound 3d: atmospheric O2; refluxing CH3OH or CDCl3 at room
temperature, beginning after 2 h; 3e: atmospheric O2, DMSO, room temperature,
several days (not definable, see experimental data).
It was intended to investigate the conformation of the ethenyl-
ene part of the compounds 2d and 2e but the 1H NMR provided no
evidence for the coexistence of isomers because only the expected
number of signals was observed. The high coupling constants be-
less pronounced than with 2d,e. In some cases, the corresponding
3 could be isolated. Considerable amounts of 3d could be sepa-
rated after recrystallization of 2f from acetone. It precipitated also
within few hours from a solution of 2f in deuterochloroform. Sim-
ilarly, 3e was formed when a solution of 2l in DMSO was allowed
to stand for several days (Scheme 6). Worth mentioning is that
only the solutions of 2 were sensitive to oxidation. The solids
were demonstrably stable, which was proven by NMR. Most likely
all compounds of type 2 are sensitive to oxidation in solution as
there was precipitation from DMSO stock solutions (3 are obvi-
ously even worse soluble than 2) observed for all biologically
tested samples after several days of storage. Only the yields dif-
fered and only for the above mentioned compounds 3 were the
structures proven.
tween the C(a)H and the C(b)H of the ethenylene moiety with both
compounds indicated the sole presence of the E-isomer.
The electron impact mass spectra (EI-MS) of the 3-arylsubsti-
tuted compounds 2f–u supported the structural assignment and
showed nearly always the same fragmentation pattern of the
molecular ions, which appeared intensive. The fragmentation
behavior resembled that reported for 2-thioxo-quinazoline-4-
ones.63 The first fragmentation step was usually the extrusion of
[ÅCH2NS] from the five-membered ring of the molecular ion. More-
over, the loss of the phenyl moiety together with its substituents
was always observed. Except for 2u, the fragmentation of the
molecular ions led to the formation of the hexahydropyridazine
cation [C4H9N2+].
2.2. Structural investigations
For the verification of the assigned structures of the obtained
compounds of type 2, the exclusion of alternative monocyclic or
bicyclic structures was of particular interest. In particular, pro-
ton and carbon NMR spectroscopy complemented by related 2D
methods provided conclusive information. One of the important
criteria for the existence of the assigned bicyclic thione structure
2 is the presence of a proton at the N(2) in a typical thioamide
range of 6 to 9 ppm, as well as the absence of a proton at the
N(4) [corresponding to the N(2)H of the starting compound 1a,
there at about 3.35 ppm]17 in the proton NMR spectra (solvent:
deuterochloroform). In the 13C NMR spectra the related signals
for the tertiary C(3) atoms were found between 75 and 82 ppm
and for the C(1)@S within the expected range of 175–177 ppm.
The NMR data of all characterized 2 were found to be consistent
with the known values for the respective partial structures. For all
3-arylsubstituted [1,2,4]triazolo[1,2-a]-pyridazine-1-thiones the
magnetic non-equivalence of the chemically equivalent protons
at C(8)H2 and C(5)H2 is characteristic. The different signal positions
of these geminal protons result from the spatial arrangement of the
None of the analyzed spectra (NMR, IR, MS) of the compounds 2
obtained from 1a showed any indications for the existence of alter-
native [1,3,4]thiadiazolo[3,4-a]pyridazine-1-imines of type 2-A.
Thus, the IR spectra gave no evidence of C@N stretching vibration
bands, which would be expected for structure 2-A. In accordance
with the carbon NMR data of structurally related 1,2,4-triazoline-
3-thiones and isomeric 1,3,4-thiazolidine-2-imines64 the spectra
of the title compounds showed the expected carbon signals of
the C@S group and did not indicate the presence of an alternative
C@N structure. The carbon signals for C@N would have been ex-
pected at higher field (140–165 ppm).Moreover, the independent
route to 2a through reduction of 3a and, vice versa, the observed
formation of the didehydro compounds 3 under mild oxidizing
conditions supports the proposed structure of 2. In contrast to
the compounds 2, in the 1H NMR spectra of the didehydro ana-
logues 3 the presence of the C(3)@N(2) double bond is evident
by absence of N(2)H and C(3)H proton signals. Consequently, due
to the absence of a C(3) proton at the compounds 3a–c, no coupling
with protons of the 3-substituents are observed.
Figure 1. Spatial conditions at compound 2f.

5522
U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
Table 2
In the carbon NMR spectra of the compounds 3 the signals for
IC50 values of selected compounds 2
the related C(3) atoms, which in contrast to that of the analog 2
are sp2-hybridized, are strongly shifted to low field (>100 ppm),
additionally supporting structure 3. Moreover, the described mag-
netic non-equivalence of the proton on C(8)H2 and C(5)H2 did not
appear in the 1H NMR spectra of 3.
Compound
IC50 [l
M]a
AG
2d
2e
2g
2i
132.01 14.96
29.89 3.72
9.81 2.12
60.26 20.64
53.23 13.78
62.97 13.14
57.59 17.94
24.16 2.74
16.20 2.90
13.20 3.94
70.03 8.36
40.19 5.07
101.08 18.37
2.3. Biological results
2j
2l
2m
2n
2o
2p
2t
The screening for iNOS inhibitory effects of the title compounds
was performed by using the insulin-secreting rat insulinoma cell
line RINm5F.65,66 Human interleukin-1b (IL-1b) and rat inter-
feron-c (IFN-c
) were added to induce NO production.59,60 This test
system is often used (with variations especially concerning the
cytokine concentrations) to investigate the influence of substances
on the cytokine mediated iNOS expression and has its origin in dia-
betes research.67–70 The choice of this assay was made with respect
to our former investigations, which studied the prevention of NO
mediated b-cell damage as a possible approach to treatment of
autoimmune insulin-dependent type-1 diabetes.46 At that time
the very similar RIN5AH cell line was used for these studies. In
preparation to the screening experiments, the optimization of the
NO production of the RIN5F cells was investigated by varying cyto-
kine concentrations and incubation time pre and post cytokine
addition (data not shown).
2u
a
n = 6 for compounds 2; n = 7 for AG; mean SD.
than AG, the potency was quantified by IC50 value. All compounds
listed in Table 1 and substances 2a,c–e were tested. Because of very
poor yields, the iNOS inhibitory effect of compound 2b could not be
investigated. Due to the observations of chemical instability, the
DMSO stock solutions were freshly made before every run.
Considering the solubility of the majority of the test com-
pounds, 39 and 78 lM were chosen as the test concentration (giv-
ing ca. 20% inhibition for the reference inhibitor AG). Additionally,
there is still a large fraction of uninhibited NO production with
either 39 or 78 lM AG, thus these concentrations serve to compare
the possible inhibitory effect of our newly designed substances rel-
ative to AG.
The efficacy of the substances was determined by the inhibitory
potency on iNOS in comparison to aminoguanidine (AG). Possible
cytotoxicity was also tested. For selected compounds, that inhibited
the NO production in both test concentrations significantly stronger
Figure 2. Concentration-dependent inhibition of iNOS through aminoguanidine and thiones 2 (A). NO production was determined by using griess reaction. Cell viability was
tested on substance treated RINm5F cells using MTT assay (B). Both data sets are displayed as percent of positive control (RIN cells treated with IL and IFN but without
substance). Aminoguanidine (AG) as the reference inhibitor was tested in every concentration as well. Mean SD; n = 5–8 (>30 for AG, as control on every plate).

U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
5523
Figure 3. 3D structures of compounds 2d, 2f, 2c (in order of decreasing activity towards iNOS from left to right).
None of the compounds shown in Figure 2 had a noticeable
cytotoxic effect on RINm5F cells. The aliphatic or araliphatic
substituted compounds 2a and 2c showed nearly no effect on NO
production whereas all other tested substances had at least some
impact on the nitrite level or were even more active than AG.
Twelve substances were selected for IC50 determination together
with AG, again for comparison (Table 2).
Thus, it can be postulated that a substance of type 2 with an
inhibitory activity towards iNOS is characterized by a phenyl ring
in position 3. A spacer group between heterocyclic and phenyl ring
is useful. Additionally, a nitro substitution on the phenyl ring is
also advantageous given the observation that these derivatives
were the most effective inhibitors.
The p-dimethylaminophenyl substituted derivative 2u showed
a slightly lower IC50 value than AG whereas most of the other com-
pound showed ca. two- to three-fold better activity compared to
AG. The nitro containing 2e, 2m, 2n and 2o were the most potent
3. Conclusions
A variety of 3-substituted [1,2,4]triazolo[1,2-a]pyridazine-1-
thiones has been successfully prepared and tested as inhibitors
of inducible nitric oxide. A number of the tested compounds
compounds tested with an IC50 value of 10 to 24 lM. In fact, the
inhibitory efficiency increased from o- over m- to p-substitution.
Compound 2e, which has an ethenylene spacer between the phe-
nyl ring and the bicyclic base structure, showed the best results
and possessed an IC50 value that was about tenfold lower than that
of AG.
showed activity and the IC50 value of 9.8 2.1 lM for the most ac-
tive phenylethenyl derivative 2d demonstrates the usefulness of
this substance class as iNOS inhibitors. Although much more po-
tent substances have been found in other classes of compounds,
the high ligand efficiency and the novelty of this class offers excel-
lent chances for development. Of course, further investigations
concerning the selectivity of the substances towards the constitu-
tively expressed eNOS and nNOS are still required. The presented
biological investigations are to be understood as a proof of con-
cept for the adequacy of this new substance class for iNOS
inhibition.
The oxidized products 3 were not yet tested by themselves be-
cause they were obtained only occasionally and incidentally.
However, some observations, made during the biological tests,
indicate an inactivity of these substances. Although the DMSO
stock solutions for the each screening run were freshly prepared,
they were stored to monitor for instability in solution. For almost
every compound in group 2, a precipitate was observed after sev-
eral days as mentioned above (period of time to first signs varied
from 7 days for, e.g., 2d and 2e to several weeks). Thus the activ-
ity of the aged stocked solutions were retested after dissolving
the precipitate; and indeed the IC50 values for the aged solutions
were greater than the freshly prepared ones. For example, after
only 2 weeks of storage the IC50 of 2e increased so much that it
could no longer be determined. For 2l comparable results were
observed, and for this compound the formation of the oxidation
product 3e could be confirmed. Therefore, it can be assumed that
the 3 representing the didehydro products of the related 2 show
little or no biologically activity towards the iNOS under compara-
ble conditions.
Resulting from the primary and secondary screening, the fol-
lowing structure activity relationships were concurred: firstly, a
phenyl moiety in position 3 appears important for an inhibitory ef-
fect on iNOS but not all aromatic structures are active, as in the
case of 2c with a benzyl group. The higher conformational restric-
tion of the sp2 hybridized ethenylene spacer of 2e in comparison to
the methylene group of 2c appears advantageous. The methylene
group allows the phenyl ring in 2e to only to rotate around the axis
and not to alter its position relative to the heterocycle as it is pos-
sible in 2c. The same applies to 2f, which is slightly more active
than 2c. The additional activity of 2e could be explained with the
insertion of a spacer (Fig. 3).
4. Experimental
4.1. General
The reported melting temperatures were determined on a Ko-
fler–Boëtius apparatus type PHMK 81/3035 (VEB Wägetechnik
Rapido) and are uncorrected. Elemental analyses were done with
the 2400 CHN Elemental Analyzer (Perkin–Elmer). For the
microwave assisted syntheses the Discover LabMate with the Intel-
liVent™ Pressure Control System, circular, single-mode-self-
tuning-system, frequency 2.45 GHz, and CEM’s SynergyT software
(CEM Kamp-Lintfort) was used. The spectra were recorded with
the following instruments and conditions: IR spectra: FT-IR 1600
(Perkin–Elmer), transmission technique (KBr pressed disks) and
for the compounds 2a, 5a, 3a,d,e only IR 200 FT-IR (Thermo Elec-
tron Corporation Nicolet), ATR technique (diamond).; 1H, 1H, 1H
COSY, DEPT-135, 13C NMR spectra: AVANCE DPX 200 and for com-
pounds 2a, 5a, 3a,d,e only the above named and additionally
HSQC-und HMBC spectra: FT-NMR-Spektrometer Avance III™ 400
(both spectrometers from Bruker Analytische Messtechnik GmbH);
temperature 25 °C, solvents used are specified in the data of the re-
lated compound; internal standard tetramethylsilane; chemical

5524
U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
shift d in ppm. Mass spectra: EI for 2f, g, h, i, j, k, m, n, o, p, q, r, s, u;
6: M 40 AMD (Intectra GmbH), electron impact, energy 70 eV,
(with the exception of the molecular ion peaks normally only
peaks >10% are listed); ESI for 2a, e, t, 5a, 3d, e: Shimadzu High Per-
formance Liquid Chromatograph/Mass Spectrometer LCMS-IT-TOF
with the system characterized as following: solvent delivery mod-
ule LC-20AD Prominence, autosampler SIL-20AC HT Prominence,
column oven CTO-20A Prominence, system controller CBM-20A
Prominence, UV/vis photodiode array detector SPD-M20A Promi-
nence, evaporative light scattering detector ESD-LT II, spectromet-
ric detector RF-10A XL, LCMS-IT-TOF workstation, software LCMS
solution Version 3.41; column ChromolithÒ SpeedRod RP-18 end-
capped, 50 mm; mobile phases (m. ph.) given in the data of the
compounds:
4.2. Chemistry
4.2.1. Correction
Unfortunately, the signal assignment in the 1H NMR of the hex-
ahydropyridazine hydrochloride 4 (starting substance for the syn-
thesis of the hexahydropyridazin-1-carbothioamides 1, Scheme 2)
in a previous publication17 was partly incorrect. With a new and
more powerful NMR instrument the detection of a very broad sin-
glet at 5.87 ppm was found for this substance. Further investiga-
tions revealed that this signal should be assigned to the NH. The
signal at 10.26 ppm corresponds to the two protons on the quater-
nary nitrogen.
Correction of the signal assignment in 1HNMR: 1H NMR (DMSO-d6,
ppm): d = 1.67 (s, 4H, –C(4)H2– and -C(5)H2–); 2.99 (s, 4H, –
C(3)H2– and –C(6)H2–); 5.87 (very broad and flat singlet, 1H,
NH); 10.26 (very broad and flat singlet, 2H, NH2+).
m. ph. VI: acetonitrile/water 7:3 (0.1% HCOOH in H2O) (v/v),
flow rate 0.2 mL minꢀ1 (compound 2t);
m. ph. VII: methanol/water 1:1 (0.1% HCOOH in H2O) (v/v), flow
4.2.1.1.
Hexahydropyridazine-1-carbothioamides
1
and
rate: 0.4 mL min-1 (compound 2a);
5. 4.2.1.1.1. Hexahydropyridazine-1-carbothioamides 1a, 1d. The
syntheses and the experimental data were described before.17
4.2.1.1.2. N-(2-Phenylethenyl)-1,2,3,4,5,6-hexahydropyridazine-1-
carbothioamide 1c. A mixture of hexahydropyridazine-1-carbo-
thioamide 1a (0.7 mmol, 0.102 g)) and phenylacetaldehyde
(0.7 mmol, 0.841 g) in dichloromethane (3 mL) was warmed at
60 °C for 5 min. After the solvent was removed, the resulting resi-
due was taken up in a minimum of acetone. The product crystal-
lized when the solution was evaporated off. The formed crystals
were collected, washed with a little methanol and dried in vacuo.
Yield 17%. Colorless crystals. Mp. 136–141 °C (acetone, mp of the
hemihydrate).
m. ph. XI: methanol/water 6:4 (0.1% HCOOH) (v/v), flow rate:
0.4 mL minꢀ1 (compounds 2e, 5a).
ESI for compounds 2f, 3a: Bruker Daltonics MicroOTOF-LC (ESI-
TOF); external calibration (Agilent ESI TuneMix); mobile phase
(m. ph. VIII): gradient propan-2-ol/water 20% Ba’ 50% B; flow rate:
0.3 mL minꢀ1; column Zorbax RP-C18; 2.1 ꢁ 30 mm; 3.5
lm. The
gas chromatographic investigations were done for compounds 2c,
f, l with a FISON Instruments GC 8065 coupled with a mass spec-
trometric detector MD 800 (70 eV); column: Macherey–Nagel
MN FS Hydrodex b-PM (25 m ꢁ 0.25 mm); column system pres-
sure: 10 psi (70 kP); column temperature 220 °C. For TLC alumi-
num foil covered with silica gel 60 F254 (Merck) was utilized as
stationary phase. The running distance for the front of the mobile
phase was 6.5 cm. The following mixtures were used as mobile
phases:
IR (KBr, cmꢀ1):
m
¼ 1168 (C@S); 1435, 1445 (–CH–); 1474
~
(S@C–N–); 1582, 1645 (–CH@CH–); 2854, 2924, 2953, 2964 (–
CH–); 3052, 3075, 3134, 3254 (–NH–). 1H NMR (CDCl3, ppm):
d = 1.58–1.93 (m, 4H, C(4)H2/C(5)H2); 3.20–3.47 (m, 3H, C(3)H/
C(6)H2); 5.22 (d, 1H, 3J = 14 Hz, C(3)H); 5.63 (d, 1H, 3J = 16 Hz; –
CH@CH-ar); 6.09 (br s, 1H, –NH–); 6.45 (d, 1H; 3J = 14 Hz, –
CH@CH-ar); 6.81 (br s, 1H, –NH–); 7.13–7.32 (m, 5H, arom. ring).
13C NMR (CDCl3, ppm): d = 21.43 (C5); 23.75 (C4); 42.30 (C3);
52.28 (C6); 108.64 (–CH@CH-ar); 125.01 (2C, arom. C2 u. C6);
125.96 (arom. C4); 128.73 (2C, arom. C3 u. C5); 132.94 (C1 ar);
136.39 (–CH@CH-ar); 181.55 (C(1)@S). Elemental analysis
[C13H17N3S. 0.5 H2O (256.4), %]: Calcd C, 60.91; H, 7.08; N, 16.39.
m. ph. I: n-hexane/ethyl acetate 1:1 (v/v);
m. ph. II: n-hexane/ethyl acetate 2:8 (v/v);
m. ph. III: dichloromethane/acetone 1:1 (v/v);
m. ph. IX: cyclohexane/ethyl acetate 1:1 (v/v);
m. ph. XIII: water/acetic acid/n-propanol 1:2:8 (v/v)
Each mobile phase (m. ph.) used is specified in the data of the
related compound. The substances were detected with UV radia-
tion (k = 254 nm) or munier spray reagent71 and iodine azide re-
agent by awe.72
The HPLC investigations were done under the following
conditions:
System: LaChrom (Merck Hitachi) consisting of pump L-7100,
autosampler L-7200, column thermostat L-7350, solvent degasser
L-7612, interface D-7000, and diode array detector L-7450
(k = 220 or 240 nm);
Stationary phase: RP columns (given in the HPLC data of the
compounds); the dead time t0 was determined with uracil; each
mobile phase (m. ph.) is given in the data for the related
compound:
Found C, 60.64; H, 5.90; N, 14.85.
4.2.1.1.3. 2-Acetyl-1,2,3,4,5,6-hexahydropyridazine-1-carbothioa-
mide 5a. A solution of hexahydropyridazin-1-carbothioamide
1a (1 mmol, 0.145 g) in acetic anhydride (2 mL) was stored at room
temperature for 3 weeks. The solution was evaporated to dryness
and the crystals formed were collected and washed with a little
ethanol. Yield 70%. Colorless needles. Mp 196–200 °C (methanol).
~
TLC (m. ph. III): Rf = 0.71. IR (cmꢀ1):
m
¼ 1159 (C@S); 1457;
1627 (NH2); 1658 (C@O); 2943, 2971, 2998 (CH2, CH3); 3158;
3264, 3306 (NH2). 1H NMR (CDCl3, ppm): d = 1.73 (m, 3H, C(4)H2
u. C(5)H); 1.88 (m, 1H, C(5)H); 2.81 (dt, 1H, 3J = 4 Hz, 3J = 12 Hz,
C(3)H); 3.01 (dt, 1H, 3J = 4 Hz, 3J = 12 Hz, C(6)H); 4.48 (d, 1H,
3J = 16 Hz, C(3)H); 5.41 (d, 1H, 3J = 16 Hz, C(6)H); 6.50 (br s, 1H,
NH); 6.79 (br s, 1H, NH). 13C NMR (CDCl3, ppm): d = 20.5 (C3);
22.9 (C4); 23.0 (C5); 43.1 (C3); 49.9 (C6); 173,6 (C@O); 182.8
(C@S). HRMS [(ESI) m/z]: calcd for C7H13N3OS+H+: 188.0852; found
[M+H+]: 188.0857. Elemental analysis [C7H13N3OS (187.3),%]:
Calcd C, 44.90; H, 7.00; N, 22.44. Found: C, 44.89; H, 6.90; N, 22.85.
N-[(2-Acetyl-1,2,3,4,5,6-hexahydropyridazine-1-yl)thiocarbonyl]
ethanamide 5b. Method 1–from 4 and acetyl isothiocyanate: To a
mixture of dichloromethane (10 mL), hexahydropyridazine-hydro-
chloride 4 (2 mmol, 0.245 g), and triethylamine (2 mmol, 0.202 g) a
m. ph. IV: acetonitrile/0.02 M KH2PO4 (pH 2.85) 1:1 (v/v);
m. ph. V: acetonitrile/0.02 M KH2PO4 (pH 2.85) 3:7 (v/v);
m. ph. X: acetonitrile/water 4:6 (v/v);
m. ph. XII: acetonitrile/0.02 M KH2PO4 (pH 2.85) 4:6 (v/v)
The values for the dead time t0 and the retention times tR are
given in minutes.

U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
5525
solution of acetyl isothiocyanate (2 mmol, 0.202 g) in dichloro-
methane (5 mL) was added dropwise within 10 min with continu-
ous stirring. The mixture was stirred further 20 min. Subsequently,
the solvent was removed, the residue was crystallized from ace-
tone, and dried. Yield: 45%. Colorless crystals. Mp 139–143 °C
(acetone).
NMR (DMSO-d6, ppm): d = 0.85 (m, 3H, (–CH3)); 1.61 (m, 7H,
C(6)H2 u. C(7)H2 u. C(5)H2 u. C(8)H); 2.84 (m; 2H C(3)-CH2–);
4.16 (m, 2H, C(3)H u. C(8)H); 8.92 (s; 1H; –NH). 13C NMR
(DMSO-d6, ppm): d = 8.33 (C(3)-CH2-CH3); 23.12 (C6); 23.19 (C7);
26.02 (C(3)-CH2–); 44.02 (C8); 53.11 (C5); 78.59 (C3); 175.77
(C(1)@S). HRMS (ESI) m/z (Intens. 100): 369.185336 [2M+H]+. Ele-
mental analysis [C8H15N3S (185.3), %]: Calcd C, 51.86; H, 8.16; N,
22.68; S, 17.30. Found: C, 52.20; H, 8.21; N, 22.83; S, 17.42.
4.2.1.2.3. 3-Phenylmethyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazol-
o[1,2-a]-pyridazine-1-thione 2c. Hexahydropyridazine-1-carbo-
thioamide 1a (1 mmol, 0.145 g), phenylacetaldehyde (1 mmol,
0.120 g), and p-toluenesulfonic acid monohydrate (0.033 mmol,
0.006 g) were completely mixed. Subsequently, the mixture was
heated between 125 and 130 °C for 4, 5 h. The cooled melt was ta-
ken up in hot acetone and the product crystallized by careful re-
moval of the solvent. The resulting crystals were filtered off,
washed twice with a little methanol and dried in vacuo. Yield:
46%. Colorless crystals. Mp 133.5–137.5 °C (acetone).
TLC (m. ph. I): Rf = 0.34. HPLC (m. ph. XII): RP-select B, t0 = 1.95;
~
tR = 2.97; k0 = 0.52. IR (KBr, cmꢀ1):
m
¼ 1186 (C@S); 1523 (NH);
1647 (NH-COCH3), 1724 (N(2)-CO-CH3); 2496, 2623, 2856, 2946,
2978, 3017 (CH2, CH3); 3179 (NH). 1H NMR (CDCl3, ppm):
d = 1.76 (m, 4H, C(4)H2 u. C(5)H2); 2.04 (s, 3H, N(2)-COCH3); 2.64
(s, 3H, CS-NH-CO-CH3); 2.79 (br s, 1H, C(3)H); 3.95 (m, 1H,
C(6)H); 4.53 (d, 1H, 3J = 7 Hz, C(3)H); 5.54 (d, 1H, 3J = 14 Hz,
C(6)H); 8.92 (s, 1H, NH). 13C NMR (CDCl3, ppm): d = 20.84 (–N(2)-
CO-CH3); 22.79 (C4); 22.98 (–CS-NH-CO-CH3); 26.55 (C5); 44.86
(C3); 49.20 (C6); 172.45 (C@O); 173.88 (C@O); 178.12 (C@S). MS
(70 eV, 200 °C) m/z (%): 228.8 (22) [M+]; 186.8 (2) [M+–H2C@C@O];
128.0 (33) [M+–CH3CO-N@C@S]; 127.0 (10); 111.1 (12); 86.0 (31);
85.0 (96) [C4H9N2+]; 70.9 (26); 57.4 (17); 56.4 (31); 43.1 (100)
[CH3CO+]; 30.1 (42); 28.0 (18). Elemental analysis [C9H15N3O2S
(229.3), %]: Calcd C, 47.14; H, 6.59; N, 18.33. Found: C, 47.07; H,
6.61; N, 18.08.
TLC (m. ph. I): Rf = 0.72. IR (KBr, cmꢀ1):
m
¼ 1180 (C@S); 1434,
~
1457 (–CH–);1481 (S@C–N–); 2846, 2943 (–CH–); 3180 (–NH–).
1H NMR (CDCl3, ppm): d = 1.61–1.87 (m, 4H, C(6)H2/C(7)H2);
2.73–3,00 (m, 4H, –C(a)H2-/C(8)H2); 3.20 (br t, 1H, C(5)H); 4.38–
Method 2–from hexahydropyridazine-1-carbothioamide 1a and
acetic anhydride: A mixture of hexahydropyridazine-1-carbothioa-
mide 1a (1 mmol, 0.145 g) and acetic anhydride (1 mL) was re-
fluxed for 10 min. Subsequently, methanol (2 mL) was added and
then the solvent removed in vacuo. The remaining crystals were
washed with ethanol and dried. Yield: 10%.
4.49 (m, 2H, C(3)H/C(5)H); 6.00 (s, 1H, –NH–); 7.19–7.38 (m, 5H,
arom.). 13C NMR (CDCl3, ppm): d = 23.47 and 23.54 (2C, C6 + C7);
40.40 (–CH2–); 44.99 (C8); 53.85 (C5); 79.07 (C3); 127.25 (arom.
C4); 128.91 (2C, arom. C2 + C6); 129.46 (2C, arom. C3 + C5);
135.29 (arom. C1); 176.96 (C@S). GC/MS (220 °C): m/z (%): 247.5
(12) [M+]; 173.2 (13) [M+ꢀCH2NS]; 156.0 (17) [M+ꢀC6H5]; 148.1
(10) [M+ꢂꢀC4H9N2]; 85.3 (100) [C4H9N2+]; 77.2 (14). Elemental
analysis [C13H17N3S (247.4), %]: Calcd C, 63.12; H, 6.93; N, 16.99.
Found: C, 61.32; H, 6.61; N, 16.71.
4.2.1.2. 2,3,5,6,7,8-Hexahydro-1H-[1,2,4]triazolo-[1,2-a]pyrida-
zine-1-thiones 2. 4.2.1.2.1. 3-Methyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]-triazolo-[1,2-a]-pyridazine-1-thione 2a. A mixture of pro-
pan-2-ol (8 mL), 3-methyl-5,6,7,8-tetrahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione 3a (2 mmol, 0.338 g), and sodium tetra-
hydroborate (10 mmol, 0.757 g) was stirred at room temperature
for 24 h. Subsequently, water (10 mL) was added and the stirring
was continued until the batch became clear. The solution was then
extracted three times with dichloromethane (12 mL), the organic
phase was dried with sodium sulphate and evaporated to dryness.
In order to remove the non-reacted compound 3a, the resulting
residue was purified by column chromatography (Ø 1.5 cm, silica
gel H Merck, mobile phase ethyl acetate). Yield: 17%. Colorless
crystals. Mp 118–123 °C (ethyl acetate).
4.2.1.2.4.
3-(2-Phenylethenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2d. A solution of hexahydro-
pyridazine-1-carbothioamide 1a (0.5 mmol, 0.073 g) and of cinna-
maldehyde (0.5 mmol, 0.066 g) in dichloromethane (2.5 mL) were
allowed to stand at room temperature for 15 h. Subsequently, the
solvent was removed and the resulting residue was treated with
a little acetone. The resulting solid was recrystallized with a min-
imum of methanol and the formed crystals were collected, washed
with a little methanol, and dried in vacuo. By concentration of the
methanolic mother liquor additional amounts of compound 2d
were obtained. Yield: 49%. Yellowish crystals. Mp 144–146 °C
(methanol).
TLC (m. ph. III): Rf = 0.79. IR (cmꢀ1):
m
¼ 1184 (C@S); 1486 (NH);
TLC (m. ph. I): Rf = 0.62. IR (KBr, cmꢀ1):
m
¼ 1183 (C@S); 1433,
~
~
2849, 2940 u. 2972 (CH); 3175 (NH). 1H NMR (CDCl3, ppm):
d = 1.33 (d, 3J = 6 Hz, 3H, C(3)CH3); 1.61 (m, 1H, C(7)H); 1.70–
1.83 (m, 3H, C(6)H2 u. C(7)H); 2.66–2.72 (m, 1H, C(5)H); 2.87–
2.90 (m, 1H, C(5)H); 3.11 (br s, 1H, C(8)H); 4.39 (br s, 2H,
C(3)H + C(8)H); 6.83 (br s, 1H, N(2)H). 13C NMR (CDCl3, ppm):
d = 19.7 (C(3)CH3); 23.4 (C6); 23.4 (C7); 45.1 (C8); 53.5 (C5); 74.6
(C3); 176.1 (C(1)@S). HRMS [(ESI) m/z]: Calcd for [C7H13N3S+H]+:
172.0903. Found for [M+H]+: 172.0900.
1452 (–CH–); 1477 (S@C–N–); 1657 (C@C); 2849, 2924, 2946 (–
CH–); 3133 (–NH–). 1H NMR (CDCl3, ppm): d = 1.57–1.84 (m, 4H,
C(6)H2/C(7)H2); 2.63–2.73 (m, 1H, C(5)H); 2.99–3.04 (m, 1H,
C(5)H); 3.19 (br s, 1H, C(8)H); 4.36 (br d, 1H, 3J = 8 Hz, C(8)H);
4.80 (d, 1H, JC(3)H-C( )H = 8 Hz, C(3)H); 6.16 (dd, 1H, JC(
a)H-
)H@C(b)H-ar); 6.39 (s, 1H, –
NH–); 6.68 (d, 1H, JC(b)H-C( )H = 16 Hz, –C( )H@C(b)H-C6H5);
3
3
a
3
C(3)H = 8 Hz, JC( )H-C(b)H = 16 Hz, –C(
a
a
3
a
a
7.29–7.39 (m, 5H, arom.). 13C NMR (CDCl3, ppm): d = 23.20 and
23.24 (2C, C6 + C7); 45.52 (C8); 52.67 (C5); 80.00 (C3); 123.54 (–
CH@CH-ar); 127.01 (arom. C4); 128.74 (2 C, arom. C3 + C5);
128.80 (2C, arom. C2 + C6); 135.26 (arom. C1); 135.91 (–CH@CH-
ar); 176.87 (C@S). Elemental analysis [C14H17N3S (259.4), %]: Calcd
C, 64.83; H, 6.61; N, 16.20. Found: C, 64.25; H, 6.73; N, 15.82.
4.2.1.2.5. 3-[2-(2-Nitrophenyl)ethenyl]-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]-triazolo[1,2-a]pyridazine-1-thione 2e. Variant 1–prepara-
tion as the main product: Hexahydropyridazine-1-carbothioamide
1a (0.3 mmol, 0.044 g), 20-nitrocinnamaldehyde (0.33 mmol,
0.058 g), and p-toluenesulfonic acid monohydrate (0.03 mmol,
0.006 g) were intensively powdered together. Then the mixture
was heated to between 100 °C and 105 °C for 20 min. After the
mixture was cooled, the solidified product was suspended in hot
acetone. The crystals that formed were collected by suction
4.2.1.2.2.
3-Ethyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-
a]pyrid-azine-1-thione 2b. A mixture of hexahydropyridazine-1-
carbothioamide 1a (1 mmol, 0.145 g), dichloromethane (5 mL),
freshly distilled propionaldehyde (2 mmol, 0.116 g), and p-tolu-
enesulfonic acid monohydrate (0.05 mmol, 0.010 g) was refluxed
for 9 h. After complete evaporation the residue was dried in vacuo.
Then a column chromatographic purification was done (mobile
phase petroleum ether/ethyl acetate 2:1 (v/v), column length
10 cm, £ = 1 cm) and the isolated crystals were dried in vacuo.
Yield 15%. Colorless crystals. Mp 115–118 °C [petroleum ether/
ethyl acetate 2:1 (v/v)].
TLC (m. ph. II): Rf = 0.70. HPLC (m. ph. V): RP-select B,
~
t0 = 2.11 min; tR = 6.48 min; k0 = 2.07. IR (KBr, cmꢀ1):
m
¼ 1184
(C@S); 2832, 2854, 2926, 2946 (–CH2–, CH3–); 3170 (–NH–). 1H

5526
U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
filtration, washed with a little methanol and dried in vacuo. Yield:
from the general method by using 1.5 mmol o-methylbenzalde-
61%. Yellow-brown crystals. Mp 163–166.5 °C (acetone).
hyde (0.180 g). Reaction time 30 min. Yield 74%. Colorless crystals.
TLC (m. ph. I): Rf = 0.45. IR (KBr, cmꢀ1):
m
¼ 1187 (C@S); 1433,
Mp 201–206 °C (acetone).
~
TLC (m. ph. I): Rf = 0.69. IR (KBr, cmꢀ1):
¼ 1185 (C@S); 1452 (–
~
m
1453 (–CH–); 1491 (S@C–N–); 1521 (–NO2); 1606 (C@C); 2850,
2949 (–CH–); 3183 (–NH–). 1H NMR (CDCl3, ppm): d = 1.66–1.89
(m, 4H, C(6)H2/C(7)H2); 2.70–2.82 (m, 1H, C(5)H); 3.02–3.19
CH3); 1478 (S@C–N–); 2933, 2951 (–CH3); 3175 (–NH–). 1H NMR
(DMSO-d6, ppm): d = 1.34–1.49 and 1.57–1.72 (m + m, 4H, C(6)H2/
C(7)H2); 2.34 (s, 3H, CH3); 2.50 (m, 1H, C(5)H ? beneath solvent sig-
nal); 2.61–3.02 (m, 2H, C(5)H/C(8)H); 4.18–4.23 (m, 1H, C(8)H); 5.48
(s, 1H, C(3)H); 7.18–7.30 (m, 4H, arom.); 9.32 (s, 1H, –NH–). 13C NMR
(DMSO-d6, ppm): d = 19.16 (arom. CH3); 23.56 and 23.72 (2C, C6 u.
C7); 44.79 (C8); 53.05 (C5); 76.58 (C3); 126.22 and 126.78 (2C, arom.
C3 u. C4), 128.95 (arom. C6); 131.05 (arom. C5); 135.86 (arom. C1);
137.06 (arom. C2); 177.27 (C@S). MS (70 eV, 205 °C): m/z (%): 247.1
(100) [M+]; 187.0 (12) [M+–CH2NS]; 162.0 (11) [M+-C4H9N2]; 156.0
(25) [M+–C6H4–CH3]; 118.0 (10); 91.0 (11); 85.1 (98) [C4H9N2+];
77.3 (10); 56.3 (23); 41.0 (12); 30.1 (35); 28.1 (10). Elemental anal-
ysis [C13H17N3S (247.3), %]: Calcd C, 63.12; H, 6.93; N, 16.99. Found:
C, 62.36; H, 6.81; N, 17.02.
(m, 2H, C(5)H/C(8)H); 4.43 (br d, 1H, 3J = 16 Hz, C(8)H); 4.85 (d,
3
1H, JC(3)H-C( )H = 8 Hz, C(3)H); 6.12 (s, 1H, –NH); 6.13 (dd, 1H,
a
3JC( )H-C(3)H = 8 Hz, JC( )H-C(b)H = 16 Hz, –C(
a)H@C(b)H-ar); 7.22 (d,
3
a
a
3
1H, JC(b)H-C( )H = 16 Hz, –C(
a)H@C(b)H-ar); 7.43–7.52 (m, 1H,
a
arom. C(4)H); 7.60 (m, 2H, arom. C(5)H + C(6)H); 8.00 (d, 1H,
3J = 6 Hz, arom. C(3)H). 13C NMR (CDCl3, ppm): d = 23.18 and
23.37 (2 C, C6 u. C7); 45.53 (C8); 52.93 (C5); 79.37 (C3); 124.80
(2 C, –CH@CH-ar, arom. C3); 128.91 (arom. C4); 129.09 (arom.
C6); 129.24 (arom. C5); 131.26 (arom. C1); 131.33 (–CH@CH-ar);
133.46 (arom. C2); 176.68 (C@S)). HRMS [(ESI) m/z]: Calcd for
[C14H16N4O2S+H]+: 305,1067. Found for [M+H]+: 305,1072.
Elemental analysis [C14H16N4O2S (304.4), %]: Calcd C, 55.25; H,
5.30; N, 18.41. Found: C, 55.25; H, 5.26; N, 18.38.
4.2.1.2.9.
3-(3-Methylphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
Variant 2–isolation as by-product: Hexahydropyridazine-1-car-
bothioamide 1a (0.5 mmol, 0.073 g), 20-nitrocinnamaldehyde
(0.55 mmol, 0.097 g), and p-toluenesulfonic acid monohydrate
(0.035 mmol, 0.007 g) were intensively powdered together. Then
the mixture was heated to between 125 and 130 °C for 20 min.
After the mixture was cooled, the solidified product was suspended
in hot acetone. The formed crystals (compound 3c) were collected
by suction filtration and washed with a little methanol. The filtrate
was evaporated to dryness and the residue was taken up in a small
volume of absolute ethanol. The crystallization was initiated by
friction with a glass stick. Yield 8%. Yellow-brown crystals. Mp
158–160 °C (ethanol).
4.2.1.2.6. 3-Arylsubstituted 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triaz-
olo-[1,2-a]pyridazine-1-thiones 2f–u. Preparation by heating of 1a
with benzaldehydes in a melt–general procedure: A well prepared
mixture of hexahydropyridazine-1-carbothioamide 1a (1 mmol,
0.145 g), the appropriate aromatic aldehyde (1.1 mmol), and p-tol-
uenesulfonic acid monohydrate (0.025 mmol, 0.005 g) was heated
between 150 and 155 °C, whereby the course of the reaction was
observed using TLC. After the starting compound 1a was com-
pletely reacted the cooled down melt was recrystallized from ace-
tone. The formed crystals were collected, washed with a little
methanol and dried in vacuo.
triazolo-[1,2-a]pyridazine-1-thione 2h. The synthesis differed
from the general method by using 1.5 mmol m-methylbenzalde-
hyde (0.180 g). Reaction time 25 min. Yield 68%. Colorless crystals.
Mp 175–177 °C (acetone).
TLC (m. ph. I): Rf = 0.66. IR (KBr, cmꢀ1):
m
¼ 1183 (C@S); 1453 (–
~
CH3); 1474 (S@C–N–); 2930, 2946 (–CH3); 3168 (–NH–). 1H NMR
(DMSO-d6, ppm): d = 1.38–1.50 and 1.58–1.76 (m + m, 4H,
C(6)H2/C(7)H2); 2.31 (s, 3H, CH3); 2.49–2.63 (m, 1H, C(8)H ? be-
neath solvent signal); 2.83–2.98 (m, 2H, C(5)H2); 4.18 (d, 1H,
3J = 14 Hz, C(8)H); 5.18 (s, 1H, C(3)H); 7.16–7.32 (m, 4H, arom.);
9.31 (s, 1H, –NH–). 13C NMR (DMSO-d6, ppm): d = 19.16 (arom.
CH3); 23.56 and 23.72 (2C, C6 + C7); 44.79 (C8); 53.05 (C5);
76.58 (C3); 126.22 (arom. C6); 126.78 (arom. C4); 128.95 (arom.
C5); 131.05 (arom. C6); 135.86 (arom. C1); 137.06 (arom. C3);
177.27 (C@S). MS (70 eV, 180 °C): m/z (%): 247.1 (100) [M+];
ꢂ
187.0 (20) [M+–CH2NS]; 161.9 (12) [M+-C4H9N2 ]; 156.0 (20) [M+–
C6H4-CH3]; 117.9 (16); 91.0 (10); 85.0 (82) [C4H9N2+]; 56.4 (16);
41.0 (10); 30.0 (26). Elemental analysis [C13H17N3S (247.3), %]:
Calcd C, 63.12; H, 6.93; N, 16.99. Found: C, 62.97; H, 7.09; N, 17.05.
4.2.1.2.10. 3-(4-Methylphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2i. The synthesis differed from
the general method by using 1.5 mmol p-methylbenzaldehyde
(0.180 g). Reaction time 25 min. Yield 54%. Colorless crystals. Mp
160.5–164 °C (acetone).
In this manner the following compounds were obtained:
4.2.1.2.7. 3-Phenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-
a]pyridazine-1-thione 2f. With benzaldehyde (0.117 g). Reaction
time 75 min. Yield: 76%. Colorless, bright crystals. Mp 152.5–
155 °C (acetone).
TLC (m. ph. I): Rf = 0.68. IR (KBr, cmꢀ1):
m
¼ 1181 (C@S); 1438 (–
~
CH3); 1469 (S@C–N–); 2926, 2958 (–CH3); 3164 (NH–). 1H NMR
(DMSO-d6, ppm): d = 1.38–1.72 (m, 4H, C(6)H2/C(7)H2); 2.30 (s,
3H, CH3); 2.49–2.61 (m, 1H, C(5)H ? beneath solvent signal);
2.78–3.03 (m, 2H, C(5)H); 4.16 (br d, 1H, 3J = 12 Hz, C(8)H); 5.17
(s, 1H, C(3)H); 7.19 (d, 2H, 3J = 8 Hz, arom. C(3)H + C(8)H); 7.28
(d, 2H, 3J = 8 Hz, arom. C(2)H + C(6)H); 9.30 (s, 1H, –NH–). 13C
NMR (DMSO-d6, ppm): d = 21.27 (C7); 23.50 (C6); 40.32 (arom.
CH3); 45.11 (C8); 52.71 (C5); 79.38 (C3); 127.66 (2C, arom.
C2 + C6); 129.37 (2C, arom. C3 + C5); 135.32 (arom. C1); 138.73
(arom. C4); 176.99 (C@S). MS (70 eV, 160 °C): m/z (%): 247.0
TLC (m. ph. I): Rf = 0.64. HPLC (m. ph. IV): RP-Select B, t0 = 1.98;
~
tR = 4.78; k0 = 1.41. IR (KBr, cmꢀ1):
m
¼ 1184 (C@S); 1429, 1458
(–CH3); 1482 (S@C–N–); 2808, 2850, 2919, 2949, 2977 (–CH3);
3040 (aryl-H); 3185 (–NH–). 1H NMR (CDCl3, ppm): d = 1.56–1.79
(m, 4H; C(6)H2/C(7)H2); 2.59–2.71 (m, 1H, C(5)H); 2.94 (m, 1H,
C(5)H); 3.22 (br s, 1H, C(8)H); 4.36 (d, 1H, 3J = 12 Hz, C(8)H);
5.15 (s, 1H, C(3)H); 6.45 (s, 1H, –NH–); 7.36–7.46 (m, 5H, arom.).
13C NMR (CDCl3, ppm): d = 23.21 and 23.23 (2C, C6 + C7); 45.53
(C8); 52.91 (C5); 80.86 (C3); 127.53 (arom. C4); 128.76 (2C, arom.
C2 + C6); 129.80 (2 C, arom. C3 u. C5); 136.26 (arom. C1); 176.60
(C@S). GC/MS (220 °C): m/z (%): 233.2 (31) [M+]; 173.2 (13) [M+–
ꢂ
(100) [M+]; 187.0 (20) [M+–CH2NS]; 161.9 (15) [M+-C4H9N2 ];
156.0 (12) [M+–C6H4-CH3]; 117.9 (13); 104.5 (12); 91.0 (12); 85.0
(96) [C4H9N2+]; 56.3 (20); 41.0 (12); 30.0 (33). Elemental analysis
[C13H17N3S (247.3), %]: Calcd C, 63.12; H, 6.93; N, 16.99. Found:
C, 62.18; H, 6.75; N, 17.13.
ꢂ
CH2NS]; 156.0 (17) [M+–C6H5]; 148.1 (10) [M+-C4H9N2 ]; 85.3
(100) [C4H9N2+]; 77.2 (14). MS (70 eV, 25 °C) m/z (%): 232.8 (76)
[M+]; 184.9 (98); 169.9 (100); 155.9 (10) [M+–C6H5]; 84.9 (88)
[C4H9N2+]; 70.8 (15); 27.9 (37). Elemental analysis [C12H15N3S
(233.3), %]: Calcd C, 61.77; H, 6.48; N, 18.01. Found: C, 61.61; H,
6.23; N, 17.93.
4.2.1.2.11.
3-(2-Chlorophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2j. The synthesis differed from
the general method by using 1.5 mmol o-chlorobenzaldehyde
(0.211 g). Reaction time 85 min. Yield 71%. Light beige crystals.
Mp 215–217 °C (acetone).
TLC (m. ph. I): Rf = 0.78. IR (KBr, cmꢀ1):
m
¼ 756 (Ar-Cl); 1081
~
4.2.1.2.8.
3-(2-Methylphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2g. The synthesis differed
(–Cl); 1177 (C@S); 1475 (S@C–N–); 2931, 2952 (–CH–), 3161

U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
5527
(–NH–). 1H NMR (CDCl3, ppm): d = 1.66–1.95 (m, 4H, C(6)H2/
C(7)H2); 2.87–3.16 (m, 2H, C(5)H/C(8)H); 3.24 (br t, 1H, C(5)H);
4.49 (d, 1H, 3J = 12 Hz, C(8)H); 5.64 (s, 1H, C(3)H); 6.48 (s, 1H, –
NH–); 7.28–7.42 (m, 3H, arom. C(4)H/C(5)H/ C(6)H); 7.55–7.61
(m, 1H, arom. C(3)H). 13C NMR (CDCl3, ppm): d = 23.28 and 23.63
(2 C, C6 + C7); 45.12 (C8); 53.88 (C5); 77.68 (C3); 127.44 (arom.
C5); 128.07 (arom. C4); 129.82 (arom. C3); 130.38 (arom. C6);
132.94 (arom. C1); 134.55 (arom. C2); 175.97 (C(1)@S). MS
(70 eV, 230 °C): m/z (%): 266.8 (84) [M+]; 231.9 (17) [M+-Cl];
206.8 (14) [M+–CH2NS]; 155.9 (37) [M+–C6H4N-Cl]; 124.9 (11);
101.8 (11); 85.0 (88) [C4H9N2+]; 56.3 (22); 55.3 (11); 41.0 (15);
32.0 (21); 30.1 (28); 28.0 (100). Elemental analysis [C12H14ClN3S
(267.8), %]: Calcd C, 53.82; H, 5.27; N, 15.76. Found: C, 53.49; H,
5.51; N, 15.83.
(38) [M+]; 275.8 (22); 155.9 (12) [M+–C6H4-NO2]; 134.9 (26);
85.0 (25) [C4H9N2+]; 77.3 (11); 41.0 (11); 28.0 (30). Elemental anal-
ysis [C12H14N4O2S (278.3), %]: Calcd C, 51.78; H, 5.07; N, 20.13.
Found: C, 51.50; H, 5.27; N, 20.09.
4.2.1.2.15.
3-(3-Nitrophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo[1,2-a]pyridazine-1-thione 2n. With m-nitrobenzaldehyde
(0.166 g). Reaction time 20 min. Yield 73%. Yellow crystals. Mp
203–205.5 °C (acetone).
TLC (m. ph. I): Rf = 0.42. IR (KBr, cmꢀ1):
= 1186 (C@S); 1475
~
m
(S@C–N–); 1354, 1528 (-NO2); 2923, 2951 (–CH–); 3145 (–NH–).
1H NMR (CDCl3, ppm): d = 1.58–1.86 (m, 4H, C(6)H2/C(7)H2);
2.73–2.83 (m, 1H, C(5)H); 2.96 (m, 1H, C(5)H); 3.18 (br t, 1H,
3J = 10 Hz, C(8)H); 4.41 (d, 1H, 3J = 14 Hz, C(8)H); 5.29 (s, 1H,
C(3)H); 7.33 (s, 1H, –NH–); 7.58 (t, 1H, 3J = 8 Hz, arom. C(4)H);
7.82 (d, 1H, 3J = 8 Hz, arom. C(6)H); 8.24 (m, 2H, arom.
C(5)H + C(3)H). 13C NMR (CDCl3, ppm): d = 23.16 and 23.31 (2 C,
C6 + C7); 45.42 (C8); 53.45 (C5); 79.32 (C3); 122.58 (arom. C4);
124.49 (arom. C2); 129.95 (arom. C5); 133.35 (arom. C6); 139.32
(arom. C1); 148.52 (arom. C2); 176.11 (C@S). MS (70 eV, 210 °C):
m/z (%): 277.9 (100) [M+]; 217.8 (27) [M+–CH2NS]; 156.0 (53)
[M+–C6H4-NO2]; 88.9 (12); 85.0 (100) [C4H9N2+]; 56.3 (25); 55.3
(14); 41.0 (19); 30.0 (34); 28.0 (17). Elemental analysis
[C12H14N4O2S (278.3), %]: Calcd C, 51.78; H, 5.07; N, 20.13. Found:
C, 51.52; H, 4.85; N, 19.62.
4.2.1.2.12.
3-(3-Chlorophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2k. With m-chlorobenzalde-
hyde (0.211 g). Reaction time 15 min. Yield 70%. Yellowish crystals.
Mp 190–199 °C (acetone).
TLC (m. ph. I): Rf = 0.63. IR (KBr, cmꢀ1):
¼ 1076 (–Cl); 1183
~
m
(C@S); 1472 (S@C–N–); 2929, 2950 (–CH–); 3169 (–NH–). 1H
NMR (CDCl3, ppm): d = 1.60–1.82 (m, 4H, C(6)H2/C(7)H2); 2.63–
2.75 (m, 1H, C(5)H); 2.95 (m, 1H, C(8)H); 3.18 (br t, 1H, C(5)H);
4.38 (d, 1H, 3J = 6 Hz, C(8)H); 5.13 (s, 1H, C(3)H); 6.85 (s, 1H, –
NH–); 7.31–7.37 and 7.47 (m + s, 4H, arom.). 13C NMR (CDCl3,
ppm): d = 23.17 and 23.25 (2C, C6 + C7); 45.47 (C8); 53.16 (C5);
79.98 (C3); 125.68 (arom. C4); 127.70 (arom. C2); 129.87 (arom.
C5); 130.12 (arom. C6); 134.85 (arom. C1); 138.67 (arom. C3);
176.30 (C@S). MS (70 eV, 205 °C): m/z (%): 268.9 (37) [M+]; 206.9
(24) [M+–CH2NS]; 156.0 (22) [M+–C6H4-Cl]; 88.9 (10); 85.0 (57)
[C4H9N2+]; 56.3 (13); 41.0 (13); 30.1 (17); 28.0 (29). Elemental
analysis [C12H14ClN3S (267.8), %]: Calcd C, 53.82; H, 5.27; N,
15.76. Found: C, 53.82; H, 5.35; N, 15.69.
4.2.1.2.16. 3-(4-Nitrophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]-[1,2-a]
pyridazine-1-thione 2o. With p-nitrobenzaldehyde (0.166 g).
Reaction time 20 min. Yield 84%. Red-brown crystals. Mp 205–
210 °C (acetone).
TLC (m. ph. I): Rf = 0.40. HPꢀL1C (m. ph. IV): RP-Select B, t0 = 1.97;
~
m = 1188 (C@S); 1475 (S@C–N–);
tR = 4.90; k0 = 1.49. IR (KBr, cm ):
1349, 1526 (–NO2); 2927, 2957 (–CH–); 3156 (–NH–). 1H NMR
(CDCl3, ppm): d = 1.66–1.88 (m, 4H, C(6)H2/C(7)H2); 2.73–2.83 (m,
1H, C(5)H); 2.95 (m, 1H, C(5)H); 3.17 (br t, 1H, 3J = 10 Hz, C(8)H);
4.43 (d, 1H, 3J = 12 Hz, C(8)H); 5.28 (s, 1H, C(3)H); 6.98 (s, 1H,
–NH–); 7.61–7.68 (m, 2H, arom. C(2)H + C(6)H); 8.22–8.28 (m, 2H,
arom C(3)H + C(5)H). 13C NMR (CDCl3, ppm): d = 23.15 and 23.34
(2 C, C6 + C7); 45.46 (C8); 53.53 (C5); 79.29 (C3); 124.05 (2C, arom.
C3 + C5); 128.38 (2 C, arom. C2 + C6); 143.71 (arom. C1); 148.79
(arom. C4); 176.25 (C@S). MS (70 eV, 205 °C): m/z (%): 277.9 (30)
[M+]; 155.9 (13) [M+–C6H4-NO2]; 85.0 (29) [C4H9N2+]; 32.0 (22);
30.0 (14); 28.0 (100). Elemental analysis [C12H14N4O2S (278.3), %]:
Calcd C, 51.78; H, 5.07; N, 20.13. Found: C, 51.77; H, 4.69; N, 19.06.
4.2.1.2.17. 3-(2-Hydroxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2p. With o-hydroxybenzalde-
hyde (0.134 g). Reaction time 15 min. Yield 57%. Yellowish crystals.
Mp. 193–195.5 °C (acetone).
4.2.1.2.13.
3-(4-Chlorophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2l. With p-chlorobenzalde-
hyde (0.155 g). Differing from the given prescription it was heated
between 125 and 130 °C. Reaction time 110 min. Yield 53%. Light
beige crystals. Mp 178–179 °C (acetone).
TLC (m. ph. I): Rf = 0.89. HPLC (m. ph. IV): RP-Select B, t0 = 1.99;
tR = 6.19; k0 = 2.11. IR (KBr, cmꢀ1):
m = 1088 (–Cl); 1184 (C@S);
~
1479 (S@C–N–); 2930, 2955 (–CH–); 3186 (–NH–). 1H NMR (CDCl3,
ppm): d = 1.58–1.80 (m, 4H, C(6)H2/C(7)H2); 2.62–2.72 (m, 1H,
C(5)H); 2.93 (m, 1H, C(5)H); 3.18 (br t, 1H, C(8)H); 4.39 (br d,
1H, 3J = 6 Hz, C(8)H); 5.12 (s, 1H, C(3)H); 6.49 (s, 1H, –NH–);
7.34–7.43 (m, 4H, arom.). 13C NMR (CDCl3, ppm): d = 23.16 and
23.23 (2C, C6 + C7); 45.56 (C8); 52.99 (C5); 80.14 (C3); 128.99 (2
C, arom. C3 + C5); 129.11 (2 C, arom. C2 + C6); 134.92 (arom. C4);
135.80 (arom. C1); 176.52 (C@S). GC/MS (220 °C): m/z (%): 156.3
(15) [M+–C6H4-Cl]; 125.3 (20); 111.2 (10) [ꢃC6H10N3S]; 85.4 (100)
[C4H9N2+]; 70.4 (20); 56.4 (81). Elemental analysis [C12H14ClN3S
(267.8), %]: Calcd C, 53.82; H, 5.27; N, 15.76. Found: C, 52.33; H,
4.35; N, 15.12.
TLC (m. ph. I): Rf = 0.44. IR (KBr, cmꢀ1):
= 1186 (C@S); 1428,
~
m
1460 (–OH); 1475 (S@C–N–); 2945, 2958 (–CH–); 3040 (–NH–);
3409 (–OH). 1H NMR (CDCl3, ppm): d = 1.60–1.87 (m, 4H, C(6)H2/
C(7)H2); 2.72 (dt, 1H, 3J = 4 Hz, 3J = 12 Hz, C(5)H); 3.13 (m, 2H,
C(8)H); 4.47 (d, 1H, 3J = 12 Hz, C(5)H); 5.26 (s, 1H, C(3)H); 6.58
(s, 1H, –NH–); 6.88 (?, 2H, arom. C(3)H + C(5)H); 7.14 (dd, 1H,
3J = 2 Hz, 3J = 8 Hz, arom. C(6)H); 7.27 (dt, 1H, 3J = 2 Hz, 3J = 8 Hz,
arom. C(4)H); 8.9 (br s, 1H, –OH). 13C NMR (CDCl3, ppm):
d = 22.91 and 23.02 (2 C, C6 + C7); 46.18 (C8); 52.59 (C5); 80.17
(C3); 117.50 (arom. C3); 118.05 (arom. C5); 120.07 (arom. C4);
129.22 (arom. C6); 131.33 (arom. C1); 156.42 (arom. C2); 178.54
(C@S). MS (70 eV, 190 °C): m/z (%): 249.0 (100) [M+]; 247.0 (20);
156.0 (12) [M+–C6H4-OH]; 120.0 (12); 85.0 (77) [C4H9N2+]; 77.3
(19); 70.9 (17); 56.3 (14); 41.0 (16); 30.1 (21); 28.1 (43). Elemental
analysis [C12H15N3OS (249.3), %]: Calcd C, 57.80; H, 6.06; N, 16.93.
Found: C, 57.08; H, 6.03; N, 16.41.
4.2.1.2.14.
3-(2-Nitrophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2m. With o-nitrobenzalde-
hyde (0.166 g). Reaction time 20 min. Yield 69%. Yellow-beige crys-
tals. Mp 171–172 °C (acetone).
TLC (m. ph. I): Rf = 0.62. IR (KBr, cmꢀ1):
= 1186 (C@S); 1477
~
m
(S@C–N–); 1356, 1529 (–NO2); 2924, 2952 (–CH–); 3144 (–NH–
).1H NMR (CDCl3, ppm): d = 1.63–1.88 (m, 4H, C(6)H2/C(7)H2);
2.79 (m, 1H, C(5)H); 2.98 (m, 1H, C(5)H); 3.17 (bt, 1H, 3J = 12 Hz,
C(8)H); 4.44 (d, 1H, 3J = 14 Hz, C(8)H); 5.28 (s, 1H, C(3)H); 6.84
(s, 1H, –NH–); 7.60 (t, 1H, 3J = 12 Hz, arom. C(4)H); 7.83 (m, 1H,
arom. C(6)H); 8.23–8.28 (m, 2H, arom. C(3)H + C(5)H). 13C NMR
(CDCl3, ppm): d = 23.13 and 23.29 (2 C, C6 + C7); 45.49 (C8);
53.44 (C5); 79.40 (C3); 122.62 (arom. C3); 124.56 (arom. C4);
129.99 (arom. C6); 133.32 (arom. C5); 139.23 (arom. C1); 148.54
(arom. C2); 176.26 (C(1)@S). MS (70 eV, 300 °C): m/z (%): 277.8
4.2.1.2.18. 3-(3-Hydroxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2q. With m-hydroxybenzalde-
hyde (0.134 g). Reaction time 15 min. Yield 64%. Pale gray crystals.
Mp 198–205 °C (acetone).

5528
U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
TLC (m. ph. I): Rf = 0.36. IR (KBr, cmꢀ1):
m
= 1182 (C@S) 1416,
4.38 (br d, 1H,3J = 12 Hz, C(8)); 5.12 (s, 1H, C(3)H); 6.39 (s, 1H,
~
1445 (–OH); 1475 (S@C–N–); 2933, 2954 (–CH–); 3172 (–OH/–
NH–). 1H NMR (DMSO-d6, ppm): d = 1.37–1.68 (m, 4H, C(6)H2/
C(7)H2); 2.49–2.63 (m, 1H, C(5)H ? beneath solvent signal); 2.83–
2.97 (m, 2H, C(5)H/C(8)H); 4.18 (d, 1H, 3J = 12 Hz, C(8)H); 5.14 (s,
1H, C(3)H); 6.72–6.83 (m, 3H, arom. C(2)H + C(3)H + C(4)H); 7.17
(t, 1H, 3J = 8 Hz, arom. C(6)H); 9.26 (s, 1H, –NH–); 9.42 (s, 1H, –
OH). 13C NMR (DMSO-d6, ppm): d = 23.50 and 23.53 (2 C, C6 + C7);
45.06 (C8); 52.93 (C5); 79.39 (C3); 114.35 (arom. C2); 116.29 (arom.
C4); 118.36 (arom. C6); 129.83 (arom. C5); 139.78 (arom. C1);
157.87 (arom. C3); 176.807 (C@S). MS (70 eV, 205 °C): m/z (%):
249.0 (8) [M+]; 206.9 (20); 156.0 (62) [M+–C6H4-OH]; 88.9 (16);
85.0 (100) [C4H9N2+]; (23); 55.4 (12); 41.0 (18); 30.1 (33); 28.0
(21). Elemental analysis [C12H15N3OS (249.3),%]: Calcd C, 57.80; H,
6.06; N, 16.93. Found: C, 57.42; H, 5.56; N, 16.85.
NH); 6.98–7.39 (m, 9H, arom.). 13C NMR (CDCl3, ppm): d = 23.16
and 23.21 (C6 + C7); 45.49 (C5 + C8); 80.38 (C3); 117.82; 119.67;
122.04; 123.73; 130.24 (C20, C40, C50, C60, C400); 119.14
(C200 + C600); 129.90 (C300 + C500); 138.33 (C10); 156.66; 157.84
(C30 + C100); 176.44 (C@S). MS [(ESI) m/z]: Calcd for
[C18H19N3OS+H]+: 326.1293. Found [M+H+]: 326.129. Elemental
analysis [C18H19N3OS (325.4), %]: Calcd C, 66.43; H, 5.88; N,
12.91. Found: C, 65.96; H, 5.86; N, 12.45.
4.2.1.2.22. 3-(4-Dimethylaminophenyl)-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]tri-azolo[1,2-a]pyridazine-1-thione 2u. With p-dimethyl-
aminobenzaldehyde (0.164 g). Differing from the given prescrip-
tion it was heated between 100 °C and 105 °C. Reaction time
20 min. Yield 70%. Colorless crystals. Mp 196–202 °C (acetone).
~
TLC (m. ph. I): Rf = 0.53. IR (KBr, cmꢀ1):
m = 813 (p-subst. aro-
4.2.1.2.19. 3-(4-Hydroxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2r. With p-hydroxybenzalde-
hyde (0.134 g). Reaction time 15 min. Yield 53%. Yellowish crystals.
Mp 210–214 °C (acetone).
matic ring); 1182 (C@S); 1436 (–CH–); 1469 (S@C–N–); 1528;
1568; 1614 (-CN-); 2803, 2842, 2910, 2948 (–CH–); 3177
(–NH–). 1H NMR (DMSO-d6, ppm): d = 1.37–1.73 (m, 4H, C(6)H2/
C(7)H2); 2.43–2.55 (m, 1H, C(5)H ? beneath solvent signal);
2.76–3.04 (m, 8H, C(5)H/C(8)H/–N(CH3)2); 4.11 (br d, 1H,
3J = 12 Hz, C(8)H); 5.03 (s, 1H, C(3)H); 6.70 (d, 2H, 3J = 8 Hz, arom.
C(3)H + C(5)H); 7.20 (d, 2H, 3J = 8 Hz, arom. C(2)H + C(6)H); 9.17(s,
1H, –NH–). 13C NMR (DMSO-d6, ppm): d = 23.43 and 23.49 (2 C,
C6 + C7); 40.14 and 40.57 (2 C, arom. N-CH3); 45.28 (C8); 52.30
(C5); 80.04 (C3); 112.38 (2C, arom. C3 + C5); 128.78 (2C, arom.
C2 + C6); 130.49 (arom. C1); 151.48 (arom. C4); 177.23 (C@S).
MS (70 eV, 205 °C): m/z (%): 276.0 (39) [M+]; 191.9 (13); 190.9
(100). Elemental analysis [C14H20N4S (276.4), %]: Calcd C, 60.83;
H, 7.29; N, 20.27. Found: C, 60.32; H, 7.14; N, 20.60.
Preparation by heating of 1a with benzaldehydes in a microwave
reactor - general procedure: Hexahydropyridazine-1-carbothioa-
mide 1a (1 mmol, 0.145 g)) and the appropriate aldehyde
(1.1 mmol) were reacted in a microwave reactor under the de-
scribed conditions (power, temperature, pressure) without or with
a solvent and for the time, respectively, which are given below.
Subsequently, the mixture was cooled for 24 h at 4 °C. The solvent
was evaporated and the residue was suspended in a small amount
of acetone. The formed crystalline product was collected, washed
careful with acetone and dried.
TLC (m. ph. I): Rf = 0.35. HPLC (m. ph. IV): RP-Select B, t0 = 1.97;
tR = 3.16; k0 = 0.60. IR (KBr, cmꢀ1):
m = 1184 (C@S); 1436, 1456 (–
~
OH); 1494 (S@C–N–); 2855, 2940 (–CH–); 3135 (–OH). 1H NMR
(DMSO-d6, ppm): d = 1.37–1.67 (m, 4H, C(6)H2/C(7)H2); 2.50 (m,
1H, C(5)H ? beneath solvent signal); 2.78–2.99 (m, 2H, C(5)H/
C(8)H); 4.14 (d, 1H, 3J = 12 Hz, C(8)H); 5.07 (s, 1H, C(3)H); 6.76
(d, 2H, 3J = 8 Hz, arom. C(3)H + C(5)H); 7.20 (d, 2H, 3J = 8 Hz, arom.
C(2)H + C(6)H); 9.20 (s, 1H, –NH–); 9.60 (s, 1H, –OH). 13C NMR
(DMSO-d6, ppm): d = 23.44 and 23.48 (2 C, C6 + C7); 45.19 (C8);
52.48 (C5); 79.68 (C3); 115.54 (2 C, arom. C3 + C5); 129.18 (2 C,
arom. C2 + C6); 131.22 (arom. C1); 160.78 (arom. C4); 177.02
(C@S). MS (70 eV, 25 °C): m/z (%): 249.0 (75) [M+]; 189.0 (18)
[M+–CH2NS]; 164.0 (23); 120.0 (11); 106.5 (10); 85.0 (100)
[C4H9N2+]; 77.3 (11); 56.4 (21); 41.0 (13); 39.1 (10); 30.0 (44);
28.0 (13). Elemental analysis [C12H15N3OS (249.3), %]: Calcd C,
57.80; H, 6.06; N, 16.93. Found: C, 57.24; H, 5.64; N, 16.73.
4.2.1.2.20. 3-(4-Methoxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2s. With p-methoxybenzalde-
hyde (0.150 g). Reaction time 75 min. Yield 35%. Pale beige crystals.
Mp 136–138 °C (acetone).
TLC (m. ph. I): Rf = 0.59. HPLC (m. ph. IV): RP-Select B, t0 = 1.97;
In this manner the following compounds were obtained:
tR = 4.71; k0 = 1.39. IR (KBr, cmꢀ1):
= 1184 (C@S); 1587; 1611
4.2.1.2.23. 3-Phenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-
a] pyridazine-1-thione 2f. With benzaldehyde (0.117 g). Without
solvent. 300 W, 130 °C, 15 bar, Reaction time 3 min. Yield 63%.
~
m
(arom. ring); 2834, 2927, 2948 (–CH2–); 3164 (–NH–). 1H NMR
(CDCl3, ppm): d = 1.63-1.75 (m, 4H, C(6)H2, + C(7)H2); 2.60 (br t;
1H, C(5)H); 2.94 (br d, 1H, 3J = 10 Hz, C(5)H); 3.16 (br s, 1H,
C(8)H); 3.82 (s, 3H, OCH3); 4.37 (bs, 1H, C(8)H); 5.10 (s, 1H;
(C3)H); 6.36 (s, 1H, NH); 6.93 (d, 2H, 3J = 10 Hz, arom. C(2)H + arom.
C(6)H); 7.39 (d, 2H, 3J = 8 Hz, arom. C(3)H + arom. C(5)H). 13C NMR
(CDCl3, ppm): d = 18.51 (C6); 18.75 (C7); 41.03 (C8); 48.07 (C5)
50.85 (-OCH3); 76.17 (C3); 109.57 and 109.79 (arom. C2 + arom.
C6); 123.49 (arom C1); 124,45 and 124,67 (arom. C3 + arom. C5);
156.29 (arom. C4); 172.08 (C@S). MS (70 eV, 160 °C) m/z (%): 263.5
(71) [M+ꢂ]; 203.5 (13) [M+–NH-C@S]; 178.4 (33) [M+–C4H8N2];
156.2 (4) [M+–C6H4-OCH3]; 134.3 (12); 85.2 (100) [CH-N2-C@S+];
56.1 (20) [C4H8+]; 30.0 (33) [–N-CH2+]. HRMS [(ESI) m/z (Intens.
0.35 ꢁ 104)]: 264.114713 [M+H]+. Elemental analysis [C13H17N3OS
(263.4), %]: Calcd C, 59.28; H, 6.51; N, 15.95; S, 12.17; O, 6.07. Found:
C, 59.35; H, 6.51; N, 15.97; S, 12.19; O, 6.08.
4.2.1.2.24.
3-(4-Chlorophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2l. With p-chlorobenzalde-
hyde (0.155 g). Solvent ethanol (1 mL). Double-stage, stage 1:
200 W, 15 bar, 180 °C, Reaction time 1 min; stage 2: 300 W,
15 bar, 180 °C, Reaction time: 5 min. Yield 50%.
4.2.1.2.25.
3-(4-Nitrophenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2o. With p-nitrobenzaldehyde
(0.166 g). Solvent ethanol (1 mL). Double-stage, stage 1: 200 W,
15 bar, 180 °C, Reaction time 1 min; stage 2: 300 W, 15 bar,
180 °C, Reaction time 5 min. Yield 64%.
4.2.1.2.26. 3-(4-Hydroxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2r. With p-hydroxybenzalde-
hyde (0.134 g). Solvent ethanol (1 mL). 300 W, 130 °C, 15 bar,
Reaction time 3 min. Yield 77%.
4.2.1.2.21. 3-(3-Phenoxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2t. With m-phenoxybenzalde-
hyde (0.218 g). Reaction time 10 min. Yield 74%. Pale beige crystals.
Mp 146–148 °C (acetone).
4.2.1.2.27. 3-(4-Methoxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2s. With p-methoxybenzalde-
hyde (0.150 g). Without solvent. 300 W, 15 bar, 130 °C. Reaction
time 3 min. Differing from the given prescription ethyl acetate
was used for working up. Yield 69%.
4.2.1.2.28. 3-(3-Phenoxyphenyl)-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo-[1,2-a]pyridazine-1-thione 2t. With m-phenoxybenzalde-
hyde (0.218 g). Solvent acetone (1 mL). 300 W, 130 °C, 15 bar.
Reaction time 3 min. Yield 84%.
TLC (m. ph. I): Rf = 0.63. HPLC (m. ph. IV): RP-Select B, t0 = 1.97;
tR = 9.49; k0 = 3.82. IR (cmꢀ1):
m
= 1183 (C@S); 1478 (S@C–N–);
~
1583 (C@N) 2841, 2959 (–CH–); 3176 (–NH). 1H NMR (CDCl3,
ppm): d = 1.55–1.78 (m, 4H, C(6)H2 + C(7)H2); 2.60–2.69 (m, 1H,
C(5)H); 2.96 (d, 1H, 3J = 10 Hz, C(5)H); 3.18 (br s, 1H, C(8)H);

U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
5529
4.2.1.3.
2,3-Diphenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazol-
A mixture of N-phenylhexa-
134.57 (arom. C10); 143.24 (olefin. CH@CH); 155.60 (C@N);
176.82 (C@S). Elemental analysis [C14H15N3S (257.4) ꢁ H2O
(275.4),%]: Calcd C, 61.06; H, 6.22; N, 15.26. Found: C, 59.82; H,
5.55; N, 15.00.
Method 2: A mixture of hexahydropyridazine-1-carbothioamide
1a (1 mmol, 0.145 g), cinnamaldehyde (1 mmol, 0.132 g), and p-tol-
uenesulfonic acid monohydrate (0.03 mmol, 0.006 g) was heated to
between 125 °C and 130 °C for 4 h. Subsequently, the cooled mass
was taken up in acetone and solvent was removed. The formed crys-
tals were collected, washed with a little methanol, and dried in va-
cuo. Yield 7%. Pale yellow crystals. Mp 246–250 °C (acetone).
o[1,2-a]-pyridazine-1-thione 6.
hydropyridazine-1-carbothioamide 1d (2 mmol, 0.443 g) and
benzaldehyde (20 mL) was allowed to stand at room temperature
for 2 weeks. Then ethanol (15 mL) was added, the precipitated
product was isolated, washed with ethanol, and dried in vacuo.
Yield 90%. Colorless crystals. Mp 145–148 °C (ethanol).
~
TLC (m. ph. IX): Rf = 0.23. IR (KBr, cmꢀ1):
m = 3034 (arom. H);
2948, 2932, 2854, 2830 (–CH2–); 1684; 1596; 1497; 1395; 1260
(C@S). 1H NMR (CDCl3, ppm) d = 1.80 (br s, 4H, C(6)H2 + C(7)H2);
2.73 (br m, 1H, C(5)H); 3.00 (br d, 1H, 3J = 10 Hz, C(5)H); 3.26
(br t, 1H, C(8)H); 4.61 (br d, 1H, C(8)H); 5.35 (s, 1H, C(3)H);
7.24–7.32 (m, 10H, arom.). 13C NMR (CDCl3, ppm): d = 23.23,
23.63 (C6 + C7); 46.48 (C8); 53.30 (C5); 87.01 (C3); 127.03 (2 arom.
C); 127.30 (1 arom. C); 128.36 (2 arom. C); 128.64 (2 arom. C);
128.86 (2 arom. C); 129.70 (1 arom. C); 135.47 (1 arom. C);
138.29 (1 arom. C); 176.73 (C@S). MS (70 eV, 180 °C):
m/z (%): 309.4 (37) [M+]; 308.5 (93) [M–H]+; 261.2 (52); 246.3
(98); 231.7 (50); 192.2 (16); 175.4 (79); 174.3 (100); 134.7 (16);
130.6 (17); 125.8 (21); 124.8 (11); 117.7 (32); 103.9 (22); 90.8
(41); 76.8 (65); 69.9 (46); 68.9 (27); 57.0 (18); 55.0 (25); 51.0
(22); 43.0 (17); 42.0 (17); 41.0 (27). Elemental analysis
[C18H19N3S (309.4), %]: Calcd C, 69.87; H, 6.20; N, 13.58. Found:
C, 69.32; H, 6.35; N, 13.35
4.2.1.4.3.
3-[2-(2-Nitrophenyl)ethenyl)]-5,6,7,8-tetrahydro-1H-
[1,2,4]tri-azolo[1,2-a]pyridazine-1-thione 3c. A well prepared mix-
ture of hexahydropyridazine-1-carbothioamide 1a (0.5 mmol,
0.073 g), 20-nitrocinnamaldehyde (0.55 mmol, 0.097 g), and p-tolu-
enesulfonic acid monohydrate (0.03 mmol, 0.006 g) was heated to
between 125 °C and 130 °C for 20 min. After cooling the mass was
treated with hot acetone, the crystals were collected by suction fil-
tration, washed with methanol and dried in vacuo. Yield: 20%. Yel-
low-orange crystals. Mp 253–256.5 °C (acetone).
TLC (m. ph. I): Rf = 0.83. HPLC (m. ph. X): RP-18, t0 = 1.90;
tR = 4.29; k0 = 1.26. IR (KBr, cmꢀ1):
m = 1256 (C@S); 1441; 1501,
~
1523 (NO2); 1571 (C@N); 1604 (CH@CH); 2868, 2957 (aliph. H);
3011 (olefin. H); 3047 (arom. H). 1H NMR (DMSO-d6, ppm):
d = 1.98 (m, 4H, C(6)H2/C(7)H2); 4.00 (m, 2H, C(8)H2); 4.31 (m,
2H, C(5)H2); 7.30 (d, 1H, 3J = 16 Hz, olefin. CH@CH); 7.70 (t, 1H,
4.2.1.4. 5,6,7,8-Tetrahydro-1H-[1,2,4]triazolo-[1,2-a]pyridazine-
1-thiones 3. 4.2.1.4.1. 3-Methyl-5,6,7,8-tetrahydro-1H-[1,2,4]
triazolo[1,2-a]pyridazine-1-thione 3a. A mixture of N-[(2-acetyl-
1,2,3,4,5,6-hexahydropyridazine-1-yl)thiocarbonyl]ethan-amide
5b (1 mmol, 0.229 g), methanol (5 mL), and p-toluenesulfonic
acid monohydrate (0.1 mmol, 0.019 g) was refluxed for 24 h.
Subsequently, the solvent was slowly evaporated in vacuo, the
resulting crystals were washed with a little ethanol, collected,
and dried in vacuo. Yield: 60%. Colorless crystals. Mp 210–
213 °C (methanol).
3
3J = 8 Hz, arom. C(40)H); 7.83 (t, 1H, J = 8 Hz, arom. C(50)H); 8.02
3
(d, 1H, = 16 Hz, olefin. CH@CH); 8.04–8.10 (m, 2H, arom. C(30)H,
C(60)H). 13C NMR (DMSO-d6, ppm): d = 19.35 (C6); 19.79 (C7);
45.43 (C8); 45.66 (C5); 115.09 (olefin. C); 124.60 (arom. C30);
128.85 (arom. C6); 129.53 (arom. C40), 130.55 (arom. C10); 133.56
(arom. C50); 133.99 (olefin. C); 148.27 (arom. C20); 153.19 (C@N);
174.70 (C@S). Elemental analysis [C14H14N4O2S, 302.4),%]: Calcd
C, 55.61; H, 4.67; N, 18.53. Found: C, 53.79; H, 3.96; N, 18.00.
4.2.1.4.4. 3-Phenyl-5,6,7,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]
pyridazine-1-thione 3d. Substance was obtained as filter cake
from a recrystallization of larger amounts of 2f from acetone.
Yield: 25 %. Colorless crystals. Mp 262–268 °C.
TLC (m. ph. III): Rf = 0.18. HPLC (m. ph. V): RP-select B, t0 = 1.87;
tR = 2.41; k0 = 0.29. IR (KBr, cmꢀ1):
m = 1177 (C@S); 1520; 1632
~
(C@N); 2876 (CH3); 2944, 2960 (CH2). 1H NMR (DMSO-d6, ppm):
d = 1.91 (br s, 4H, C(6)H2, C(7)H2); 2.28 (s, 3H; (C3)CH3); 3.91 (br
s, 2H, C(8)H2); 4.03 (br s, 2H, C(5)H2). 13C NMR (DMSO-d6, ppm):
d = 11.45 (CH3); 19.60 (C6); 19.79 (C7); 45.21 (C8); 45.34 (C5);
155.48 (C@N); 174.62 (C@S). Elemental analysis [C7H11N3S
(169.2), %]: Calcd C, 49.68; H, 6.55; N, 24.83; S, 18.94. Found: C,
49.73; H, 6.56; N, 24.85; S, 18.96.
TLC (m. ph. XIII): Rf = 0.71. IR (cmꢀ1):
= 1189 (C@S); 1421 (CH);
~
m
1459 (C@N); 1533, 1604 (C@C, arom.). 1H NMR (CDCl3, ppm):
d = 2.03–2.17 (m, 4H, C(6)H2 u. C(7)H2); 4.18 (t, 2H, 3 = 11.6 Hz,
C(5)H2); 4,28 (t, 2H, 3 = 12.4 Hz, C(8)H2); 7,49–7,58 (m, 3H, arom.
C(3)H, C(4)H u. C(5)H); 7,73–7,75 (m, 2H, arom. C(2)H u. C(6)H).
13C NMR (CDCl3, ppm): d = 20.82 u. 21.15 (C6 u. C7); 46.43 (C8);
48.70 (C5); 125.52 (arom. C1); 129.30 u. 129.38 (arom. C2, C3, C5
u. C6); 132.11 (arom. C4); 158.55 (C3); 176.24 (C(1)@S; very weak
signal). HRMS [(ESI) m/z]: Calcd for [C12H15N3S+H]+: 234.1059.
Found for [M+H+]: 234.1066.
4.2.1.4.2. 3-(2-Phenylethenyl)-5,6,7,8-tetrahydro-1H-[1,2,4]triaz-
olo[1,2-a]-pyridazine-1-thione 3b. Method 1: A mixture of hexa-
hydropyridazine-1-carbothioamide 1a (0.1 mmol, 0.145 g),
cinnamaldehyde (1 mmol, 0.132 g), and p-toluenesulfonic acid
monohydrate (0.03 mmol, 0.006 g) was allowed to stand at room
temperature for 2 h. Subsequently, a small amount of acetone
was added and the crystallization of the product was initiated by
rubbing with a glass stick. The crystals were collected by suction
filtration, washed with a little acetone, and dried in vacuo. Yield:
35%. Pale yellow crystals. Mp 255–259 °C (acetone, mp. of the
monohydrate).
4.2.1.4.5. 3-(4-Chlorophenyl)-5,6,7,8-tetrahydro-1H-[1,2,4]triazol-
o[1,2-a]-pyridazine-1-thione 3e. The substance precipitated after
several days from a solution of 2l in DMSO. To obtain the solid
the suspension was centrifuged and the supernatant removed.
The remaining DMSO was gently gathered in filter paper (as far
as possible). Yield: 30 %. Colorless crystals. Mp 205–215 °C
(DMSO).
TLC (m. Ph. XIII): Rf = 0.76. IR (cmꢀ1):
= 1171 (C@S); 1468
~
m
TLC (m. ph. I): Rf = 0.68. HPLC (m. ph. X): RP-18, t0 = 1.90;
~
tR = 4.47; k0 = 1.35. IR (KBr, cmꢀ1):
m
= 1251 (C@S); 1458; 1504;
(C@N); 1522, 1575, 1595 u. 1656 (C@C, arom.); 2874, 2916, 2964
u. 3060 (CH). 1H NMR (CDCl3, ppm): d = 2.06–2.17 (m, 4H,
C(6)H2) u. C(7)H2); 4.18–4.23 (m, 4H, C(5(H2) u. C(8)H2); 7.45–
7.49 (m,2H, arom. C(3)H u. C(5)H); 7.58–7.71 (m, 2H, arom.
C(2)H u. C(6)H). 13C NMR (CDCl3, ppm): d = 20.62 u. 21.01 (C6 u.
C7); 46.39 (C8); 48.64 (C5); 123,95 (arom. C1); 129.58 (arom. C3
u. C5); 130.68 (arom. C2 u. C6); 138.50 (arom. C4); 157.19 (C3);
176.26 (C(1)@S). HRMS [(ESI) m/z]. Calcd for [C12H11N3SCl+H+]:
266.0513. Found for [M+H+]: 266.0524.
1578; 1634 (C@N, CH@CH); 2973 (aliph. H) 3023 (olefin. H);
3054 (arom. H); 3443 (H2O). 1H NMR (CDCl3, ppm): d = 1.61 (s,
2H, H2O); 2.09 (m, 4H, C(6)H2/C(7)H2); 4.15 (bs, 4H, C(5)H2/
C(8)H2); 6.68 (d, 1H, 3J = 16 Hz, olefin. CH@CH); 7.38–7.41 (m,
3H, arom. C(30)H, C(40)H, C(50)H); 7.52–7.55 (m, 2H, arom. C(20)H,
C(60)H); 8.06 (d, 1H, 3J = 14 Hz, CH@CH). 13C NMR (CDCl3, ppm):
d = 20.68 (C6); 20.72 (C7); 45.85 (C5, C8); 108.03 (olefin. CH@CH);
128.02 (arom. C30, C50); 129.07 (arom. C20, C60); 130.51 (C40);

5530
U. Schulz et al. / Bioorg. Med. Chem. 21 (2013) 5518–5531
4.3. Biology
4.3.6. Statistics
Normality was tested and if positive F- and t-test were per-
4.3.1. Cell culture materials
formed. Otherwise Mann–Whitney-test was executed. Statistical
All chemicals (sulfanilamide, N-(1-naphthyl)ethylenediamine
dihydrochloride, MTT, dimethyl sulfoxide, aminoguanidine hydro-
chloride) as well as interleukin-1b, fetal calf serum and trypane
blue were from Sigma Aldrich. Cell culture plastics, PBS, trypsin/
significance was assumed for p <0.05 or lower.
4.4. 3D structures
EDTA and interferon-
RPMI1640 and penicillin/streptomycin from Lonza.
c
were obtained from Biochrom AG,
3D structure models of compounds 2c, 2d and 2f were obtained
with the program MOE 2011.1076 by using MMFF94x force field.
4.3.2. Cell line
Acknowledgements
The insulin-producing rat insulinoma cell line RIN5F
(ECACC catalogue No. 95090402) was used. The adherent cells
were grown under standard conditions (95% humidity, 37 °C, 5%
The authors gratefully thank Mr. Franz studener, pharmacist
and head of the pharmacy, ’Mohren-Apotheke’, Neumarkt 8,
99947 Bad Langensalza, Germany, for the financial basis of this
work. They also wish to sincerely thank Prof. Dr. Thomas von woe-
dtke (Campus PlasmaMed) and Dr. Kai masur (head of the work-
group ‘Cellular effects’, Centre of Innovation Competence
plasmatis), both of them INP Greifswald e.V., for the generous tech-
nical support of the biological works of this project. Finally, the
authors are grateful for the scholarship of the ‘Studienstiftung
des deutschen Volkes’, that made the continuative working on this
project possible.
CO2) in RPMI1640 supplemented with L-glutamine (2 mmol/L),
10% heat-inactivated FCS, penicillin (100 I.E.) and streptomycin
(100 I.E.). Cells were trypsinized once in a week, washed with
FCS containing media and then counted. Vitality was determined
with trypane blue staining.
4.3.3. iNOS expression and inhibition
Tests were performed with 1 ꢁ 105 cells per well in 200
lL cul-
ture media (as defined above) in 96-well plates at standard culture
conditions. Twenty-four hours after cells had been seeded out,
iNOS was induced by adding 1 ng (500 I.E.)/mL IL-1b (human, re-
Supplementary data
combinant) and 10 ng (30 I.E.)/mL IFN-
the primary screening, test compounds were prepared as
62.5 mM stock solution in DMSO and added to the cells at concen-
trations of 78 and 39 M with a resulting final concentration of
c (rat, recombinant). For
a
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.064.
l
0.5% DMSO in the well. The DMSO concentration did not affect
the NO production and lowered the cell viability only marginally
(about 10%) and was for that reason acceptable for the cells. The
reference inhibitor aminoguanidine was tested at the same con-
centrations (added in DMSO) on every plate as well. DMSO was
added to the controls at the same concentration. Selected sub-
stances with significantly higher inhibition of iNOS than amino-
guanidine were chosen for IC50 determination. Therefore,
concentrations that result in 10 to 90% inhibition of iNOS (plus
one to two values under or above) were selected for each individ-
ual compound. IC50 was calculated from linear regression of loga-
rithmized concentrations versus percentage of inhibition.
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