Synthesis and structure elucidation of 2,3,5,6,7,8-hexahydro-1h-[1,2,4]triazolo[1,2-a]pyridazine-1-thione, 3,3-disubstituted and 2-substituted derivatives and evaluation of their inhibitory activity against inducible nitric oxide synthase

Article abstract of DOI:10.1691/ph.2014.4589

The 3-monosubstituted 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thiones 3 (R¹, R³ =H) were recently reported to possess inhibitory activity against inducible nitric oxide synthase in a cell based assay (Schulz et al. 2013). The 3,3-disubstituted 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thiones 3 and 4 (R² ,R³ ≠ H) were synthesized by cyclocondensation of the hexahydropyridazine-1-carbothioamides 1 with ketones. In order to access the 3,3-unsubstituted 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thiones, the unsubstituted parent system of these compounds, several synthetic routes were studied. By these methods the desired heterocyclic system 2a as well as new a-anellated and N-substutited hexahydropyridazines were obtained. The biological evaluation of the title compounds confirmed the previously made finding that an aromatic moiety in position 3 of the substance is important for an inducible nitric oxide synthase (iNOS) inhibitory activity.

Full text of DOI:10.1691/ph.2014.4589

ORIGINAL ARTICLES
Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry, Ernst Moritz Arndt University, Greifswald, Germany
Synthesis and structure elucidation of 2,3,5,6,7,8-hexahydro-
1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thione, 3,3-disubstituted and
2-substituted derivatives and evaluation of their inhibitory activity against
inducible nitric oxide synthase*
U. Schulz, M. Freitag, K. Schmidt, M. Witetschek, M. Polzin, O. Morgenstern
Received March 14, 2014, accepted May 30, 2014
Privat-Dozent Dr. Olaf Morgenstern, Ernst Moritz Arndt University Greifswald, Institute of Pharmacy –
Pharmaceutical/Medicinal Chemistry, Friedrich Ludwig Jahn-Straße 17, D-17487 Greifswald, Germany
omorgens@uni-greifswald.de
Dedicated to Prof. Dr. Peter H. Richter, Bad Saarow-Pieskow, Germany, on the occasion of his 75th birthday.
∗N,N-Coupled heterobicycles from cyclic hydrazine derivatives. Part 15. Part 14: Schulz et al. (2013)
Pharmazie 69: 731–744 (2014)
doi: 10.1691/ph.2014.4589
The 3-monosubstituted 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thiones 3 (R1, R3 = H)
were recently reported to possess inhibitory activity against inducible nitric oxide synthase in a cell
based assay (Schulz et al. 2013). The 3,3-disubstituted 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-
a]pyridazine-1-thiones
3
and
4
(R2,R3 =/ H) were synthesized by cyclocondensation of the
hexahydropyridazine-1-carbothioamides 1 with ketones. In order to access the 3,3-unsubstituted
2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thiones, the unsubstituted parent system of
these compounds, several synthetic routes were studied. By these methods the desired heterocyclic sys-
tem 2a as well as new a-anellated and N-substutited hexahydropyridazines were obtained. The biological
evaluation of the title compounds confirmed the previously made finding that an aromatic moiety in position
3 of the substance is important for an inducible nitric oxide synthase (iNOS) inhibitory activity.
1. Introduction
Ever since the discovery of one of the smallest molecules in
the human body, nitric oxide and its role to various diseases
have been the focus of intense medicinal and pharmaceutical
research. The inducible nitric oxide synthase (iNOS) is a
mediator for many inflammatory processes, e.g. COPD or
asthma. Although enzyme inhibition would appear to be
an attractive approach to manage such diseases, a drug for
this target is not yet available. Recently, we reported the
synthesis of 3-monosubstituted 2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thiones (compounds 3, R1,
R3 = H, Scheme 1) and their biological evaluation as inhibitors
of inducible nitric oxide synthase in a cell based assay. In that
work an overview of the current state of the relevant research
is given (Schulz et al. 2013). A result of these investigations
was that compounds with aryl substituents in position 3, and
particularly those with cinnamyl radicals, were the most active.
In continuation of these studies, further changes in the sub-
stitution pattern of the heterocyclic parent system (Scheme 1)
on the inhibitory activity were investigated in an estab-
lished biological test model. In this context the unsubstituted
2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2–a]pyridazine-1-
thione 2a (R1 = H, Scheme 1), representing the parent structure
of compounds 3 and 4, was of particular interest.
Scheme 1: General synthesis route for the 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazo-
lo[1,2-a]pyridazine-1-thiones 2 (R1 = H, CH3, C6H5, CO-C6H4-4-NO2),
3 and 4 (for both of them R2 = alkyl, aryl; R3 = alkyl, aryl) starting from
hexahydropyridazine-1-carbothioamides 1.
tigated. Additionally, for comparative chemical studies the
syntheses of the 2-substituted [1,2,4]triazolo[1,2-a]pyridazine-
1-thiones 2 (R1 =/ H) and the 4, starting from the N-substituted
hexahydropyridazine-1-carbothioamides 1b and 1c, were inves-
tigated.
Moreover, in the present work the effects of the introduc-
tion of a second substituent in position 3, formally leading to
the 3,3-disubstituted [1,2,4]triazolo[1,2-a]pyridazine-1-thiones
3 (R2, R3 = alkyl, aryl), on the inhibitory activity was inves-
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Scheme 2: Results of cyclocondensation reactions of hexahydropyridazine-1-carbothioamides 1 with formaldehyde under different conditions.
(a) 5a from 1a, 2b from 1b, and 2c from 1c: CH3OH, 30% aqueous HCHO solution, refluxing, 3 h
(b) 5b from 1b: 30% aqueous HCHO solution, room temperature, 2 days
(c) 6 from 1c: method 1 - paraformaldehyde, molecular sieve, 140–150 ◦C, 15 min; method 2 - CH3OH, 30% aqueous HCHO solution, refluxing, 6 xh, (d) 7 from 1a:
paraformaldehyde, molecular sieve, 140–150 ◦C, 25 min
2. Investigations, results and discussion
are linked by a methylene group (Scheme 2). The reaction of
paraformaldehyde with 1c also resulted in the formation of 6
in a slightly better yield as already observed utilizing aque-
ous formaldehyde. For 6 a methylene brigded structure was
assigned.
2.1. Synthesis
Analogously to the synthesis of the 3-monosubstituted
2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2–a]pyridazine-
1-thiones (Schulz et al. 2013), the title compounds 2, 3 and
4 were obtained by reacting the hexahydropyridazine-1-
carbothioamides 1 (Morgenstern et al. 2004) with carbonyl
compounds (Scheme 1). In this context the results of the
investigations to obtain compound 2a (R1 = H) are reported
here as well.
An attempt to generate 2a via cyclization of the piperidazin-1-
ylmethane amine derivative 9a (derived from 9b by hydrolysis)
or by elimination of the nitrobenzoyl residue from compound
2d (Scheme 3) also failed. Although the piperidin-1-ylmethane
amine 10a has already been reported (Takeuchi et al. 1986,
Takeuchi et al. 1988), the aza analogue 9a is not yet known.
The same applies for 10b (Foldeak et al. 1962) and 9b. In con-
trast to 9a, compound 9b could be directly synthesized from 8
hydrochloride. The hydrochloride of 9b was obtained in anal-
ogy to 10b by Mannich reaction utilizing 4-nitrobenzamide as
acidic component. In this context, 10b was prepared for compar-
ison purposes. The 1H and 13C NMR data, until now unknown
in literature, is reported here for the first time. However, all
attempts to obtain 10a and 9a failed by targeted hydrolysis of
both 10b and 9b. Instead, 4-nitrobenzamide was isolated in any
case.
2.1.1. 3-Unsubstituted compounds 2
To synthesize 2a, the hexahydropyridazine-1-carbothioamide
1a (R1 = H) (Morgenstern et al. 2004) was reacted with
formaldehyde under different conditions. When heat-
ing 1a in aqueous formaldehyde solution the reaction
occurred rapidly and uniformly. However, the isomeric 1-
imino[1,3,4]thiadiazolo[1,2–c]pyridazine 5a (Scheme 2) was
isolated exclusively.
The substituted carbothioamides 1b,c (Morgenstern et al. 2004)
actually yielded the desired [1,2,4]-triazolo[1,2–a]pyridazines
2b and 2c (Scheme 2) under the same conditions.
However, the latter was obtained in considerably better
yields. Applying milder conditions, 1b reacted to the 1-
phenylimino[1,3,4]thiadiazolo[1,2–c]pyridazine 5b (isomer of
2b). Heating the N-methylcarbothioamide 1c in aqueous
formaldehyde solution in the presence of methanol, com-
pound 6 was formed from two molecules of the starting
material. The suitability of paraformaldehyde as C1 reactant
was alternatively tested. Unfortunately, the reaction of 1a and
paraformaldehyde in a melt did also not lead to 2a. Although
the ring closure gave the desired 2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2–a]pyridazine system, the reaction proceeded
most likely in a second step to 7, where two molecules 2a
The attempt to cyclize 9b with carbon disulphide by
heating did not lead to 2d but to [1,3,4]thiadiazolo
[1,2-c]pyridazine-1,3-dithione 11, the structure of which was
confirmed also by an alternative synthesis starting from 8.
Under mild conditions, however, 9b was transformed into the 2-
(4-nitrobenzoyl)-[1,2,4]triazolo[1,2-a]pyridazine-1-thione 2d.
Although 2d was somewhat stable, its conversion into the
desired 2a could not be realized.
Alternatively, it was examined whether 9c can be synthesized
and converted into 9a. While 10c, which is easily acces-
sible by Mannich reaction, has long been known (Feldman
and Wagner 1942), compound 9c has not yet been reported.
For comparative purposes, 10c was synthesized and the 1H
and 13C NMR spectra were recorded. However, all attempts
to synthesize 9c in the same manner failed, but gave instead
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ORIGINAL ARTICLES
Scheme 3: Investigated synthetic routes to obtain suitable precursors for 2a, the parent system of 2,3,5,6,7,8-hexahydro-1H [1,2,4]triazolo[1,2-a]pyridazine-1-thiones 2.
(a) 8 hydrochloride, potassium phthalimide, 30% aqueous HCHO, microwave irradiation (90 W, 130 ◦C, 3.3 bar), 5 min
(b) 8 hydrochloride, phthalimide, C2H5OH, 30% aqueous HCHO, (C2H5)3N, refluxing, 5 h
(c) 8 hydrochloride 4-nitrobenzamide, paraformaldehyde, C2H5OH, refluxing, 5 h.
(d) 8 hydrochloride, potassium phthalimide, H2O, 30% aqueous HCHO, refluxing, 6 h
(e) 8 hydrochloride, CH3OH, (C2H5)3N, CS2, 80–90 ◦C, 30 min
(f) 9b hydrochloride, C2H5OH, (C2H5)3N, CS2, 80–90 ◦C, 6 h
(g) 9b hydrochloride, CH3OH, (C2H5)3N, CS2, room temperature, 5 d
(h) 10a-c are accessible starting from piperidine instead of 8 hydrochloride - 10a (Takeuchi et al. 1986, Takeuchi et al. 1988) 10b (Foeldea´k et al. 1962), and 10c
(Feldman, Wagner 1942)
1,2-bis(phthalimidomethyl)piperidazine 12. When using potas-
sium phthalimide instead of phthalimide the tetrazine 13 was
obtained. The microwave-assisted reaction to obtain 9c was also
not successful; instead, the 2,3-tetramethylene phthalazine-1,4-
dione 14 (Csampai et al. 1991; Rink and Grabowski 1956) was
isolated.
The unsubstituted parent compound 2a was finally obtained
by the synthesis route presented in Scheme 4. Compound
16, which was easily isolated from the reaction mixture, is
rapidly formed by the heating of 1a in formic acid. The
formal didehydro analog is formed via the intermediate 15,
which was isolated and characterized. The formation of this
intermediate is a suitable marker for optimizing the yield of
compound 16 (Scheme 4). The hydrogenation of the 2,3-double
bond of 16 went smoothly by modification of a procedure
utilizing sodium borohydride under mild conditions (Irwin
1972). In this way the 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]-pyridazine-1-thione 2a was obtained in good yield.
even when using a large excess of the carbonyl compound [ace-
tone (3a), ethyl methyl ketone (3b), and diethyl ketone (3 g)
(Table)]. In contrast, with only a small excess of cyclohexanone
the 3,3-pentamethylene derivative 3f was obtained within a short
time and in good yield.
Another exception is the 3-cyclopropyl-3-methyl derivative 3c,
formation of which takes place only under special conditions.
In this case, the use of aluminum chloride as a catalyst and the
constant removal of the water that formed (through addition of
molecular sieves) at room temperature led to an almost complete
conversion of 1a. However, the yield of 3c was only sparingly
because of its chemolability.
As already observed with the reaction of benzaldehyde with
the N-phenyl derivative 1b (Schulz et al. 2013), this substituted
semicyclic compound reacts as easily as 1a with acetone to the
analogous 3,3-dimethyl derivative 4a. Standing a solution of 1b
in acetone for several weeks proceeded to 4a in a good yield.
2.1.2. 3,3-Disubstituted compounds 3 and 4
The reaction of hexahydropyridazine-1-carbothioamide 1a with
different ketones provided the 3,3-disubstituted title com-
pounds 3a-k (Scheme 1, Table) mostly in good yields. The
reactants were heated without any solvent and under acid
catalysis analogously as described for the 3-monosubstituted
[1,2,4]triazolo[1,2-a]pyridazine-1-thiones (Schulz et al. 2013).
Heating 1b in acetone in the presence of aluminium chloride
lead to a completion of the desired synthesis of 4a within two
hours.
2.1.3. 3,3-Dialkyl derivatives 3a-c,g, 3,3-pentamethylene
derivative 3f, and 4a
2.1.4. 3-Aryl and 3,3-diaryl derivatives 3d,e,h-k
The related alkylaryl or diaryl ketones were reacted with
hexahydropyridazine-1-carbothioamide 1a between 150 and
155 ◦C, as described for the aromatic aldehydes (Schulz et al.
2013). Depending on the reactivity of the ketones, the time until
full conversion differs in comparison with the aldehydes [e.g.
for benzaldehyde 75 min (Schulz et al. 2013)] in the expected
order (Table).
The mentioned compounds 3 were usually synthesized by
refluxing the carbothioamide 1a (with the exception of 3c, see
below) with an excess of the respective ketone in the presence of
small amounts of p-toluenesulfonic acid monohydrate. The time
required for full conversion of 1a was usually much longer (>
20 h) compared to various benzaldehydes and aromatic ketones,
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ORIGINAL ARTICLES
Table: 3, 3-Disubstituted [1, 2, 4]triazolo[1,2-a]pyridazine-1-thiones 3a-k
1
2
a,b
c
3
R
R
Reaction time (Yield [%]) conventional
Reaction time (Yield [%]) microwave-assisted
a
b
c
d
e
f
g
h
i
CH3
CH3
CH3
CH3
CH3
33 h (81)
44 h (66)
6 month (10)
4 h (75)
40 min (66)
CH2CH3
cyclopropyl
C6H5
30 min (49)
CH3
4-CH3O-C6H4
6 h (50)
-(CH2)5-
C2H5
C6H5
C6H5
C6H5
C6H5
45 min (60)
20 h (76)
13 h (75)
15 h (38)
21 h (52)
14 h (40)
C2H5
C6H5
2-Cl-C6H4
4-Cl-C6H4
4-CH3O-C6H4
30 min (61)
10 min (8)
j
k
a
b
c
◦
C
3a,b,g: refluxing 1a in the appropriate ketone; 3d,e,h-k: heating the educts at 150–155
3c: allowing to stand the educts with AlCl /molecular sieve at room temperature
3
additional data are given in the experimental section
Although the yields after work-up are clearly lower (for 3i: sig-
nificantly), the microwave-assisted synthesis has the advantage
that the reaction time are shortened in some cases, especially
in the synthesis of 3a (see Table for the preparation of the
3,3-disubstituted thiones 3).
Because of the poor solubility of compound 5a, the hydrochlo-
ride had to be prepared for recording the 1H and 13C NMR
spectra. A singlet at 5 ppm in the 1H NMR spectrum of 5a
hydrochloride was an indication of the successful cyclization, as
it can only be assigned to the protons at the C3 atom. In contrast
to 2a, no signal appears that would indicate a thioamide proton.
In the 13C NMR spectrum of 2a and 5a the expected six sig-
nals are found. But as for 2a, comparable to the [1,2,4]triazolo
[1,2-a]pyridazine-1-thiones (Schulz et al. 2013), the signal for
C1 is found at about 178 ppm indicating the C = S carbon atom,
the spectrum of 5a showed a signal for C1 (C = N) at 169 ppm
giving evidence for the existence of the thiadiazolo isomer.
2.2. Structural investigations
The main focus of the structural studies was to verify the struc-
tures assigned to compounds 2, 3, and 4. In this context, the
exclusion of possible alternative monocyclic or bicyclic struc-
tures was of particular interest.
2.2.1. Compounds 2a and 5a
2.2.2. Compounds 2b and 5b, 2c
The isomeric compounds 2a (Scheme 4) and 5a (Scheme 2)
were obtained by different synthesis routes. In the IR spectrum
of 2a, the appearance of only one NH band at 3100 cm-1 is
already a clear indication for the preservation of the bicyclic
system in the process of the hydrogenation of 16. The IR spec-
trum of 5a compared to that of 2a showed additional bands
between 1500 cm−1 and 1600 cm−1, indicating the simultaneous
presence of a C = N- and NH-structure. The 1H NMR spec-
trum of 2a showed all characteristic signals as reported for the
[1,2,4]triazolo[1,2-a]pyridazine-1-thiones (Schulz et al. 2013).
Especially mentioned should be the signal splitting for the pro-
tons on C3, C5 and C8 indicating a magnetic nonequivalence
due to different spatial positions (Fig. 1) and the singlet for NH
in the hitherto observed normal range for this class.
In the IR spectra of the compounds 2b, 5b and 2c (Scheme 2)
no NH vibrational bands are present, indicating a success-
ful cyclization reaction. Analogously to 2a, in the 1H NMR
spectrum of 2b the characteristics of the class (magnetic
nonequivalence, see before) are found. Compared to the struc-
turally isomeric 5b, this signal for the C3 methylene group
of 2b appears at higher field at 4 ppm, caused by a lack of
influence of the phenyl ring. In the 13C NMR spectrum for
both 2b and 5b, the expected signals for 10 carbon atoms
are present. The signals for the methylene carbon atom C3
clearly differ for the compounds 2b and 5b (77.23 ppm vs.
58.11 ppm) due to the differences in electron attraction of the
adjacent structures phenyl and sulfur, respectively. Addition-
ally, the positions of the signal for the C1 atom in 2b and
Scheme 4: Synthesis of 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thione 2a via the two isolable intermediates 15 and 16.
(a) 15: HCOOH, refluxing, 45 min
(b) 16 (directly from 1a): HCOOH, refluxing, 1 h
(c) NaBH4, propan-2-ol, room temperature,18 h
734
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ORIGINAL ARTICLES
Fig. 1: Spatial representation of compound 2a for visualization of the arrangement of the protons at the C3 atom.
5b differ as observed before (171.35 ppm vs. 160.73 ppm) for
C = S and C = N, respectively. For the 2-methyl derivative 2c
(C1 at 178.87 ppm indicating C = S; Scheme 2), both alterna-
tive ring open and 1-methylimino-thiadiazolo[1,2-a]pyridazine
structures can be excluded. Comparable to 2b, the signal for
the C3 atom is found at 72.68 ppm. In an alternative thiadiazolo
[1,2-a]pyridazine structure it would be expected to appear at
higher field (see above, 5b).
NMR, IR, and MS gave evidences for these or other alternative
structures. The same applies for 4a.
Consistent with these findings, signals for the acidic proton
1
at the N2 appear between 6 and 8 ppm in the H NMR spec-
tra and at about 176 ppm for the C = S carbon atom in the
13C NMR spectra, which speaks for the formation of the 3.
The values are in accordance with those of 2a, which was
obtained in an alternative synthesis route (see above), and the
related 3-monosubstituted [1,2,4]triazolo[1,2-a]pyridazine-1-
thiones (Schulz et al. 2013), respectively. It is worth mentioning
that the methyl substituents in position 3 of the derivatives 3a,c
appear in the spectra as very flat, broad peaks (barely visible)
even after an exceptionally high rate of spectra accumulations,
even though the corresponding methyl proton signals in the
2.2.3. Compounds 2d and 11
The successful formation of the [1,2,4]thiadiazolo
[1,2-a]pyridazine-1,3-dithione 11 from 9b and carbon disul-
phide at higher temperature was already indicated by the IR and
NMR spectra (lack of complete 4-nitrobenzoylaminomethyl
partial structure, molecular symmetry) and could be verified by
targeted synthesis using 8-hydrochloride and carbon disulfide.
In contrast, under mild conditions 2d was formed instead.
Thus, an additional signal at 198.03 ppm in comparison to
compound 9b appears in the 13C NMR spectrum, supporting
evidence for the insertion of the C = S fragment. Again, the
signal for the protons at C3 appears split in the 1H NMR
spectrum, but compared to the previous 2, the position of this
comparable signal for 2d at 5 ppm is shifted a little to lower
field by the anisotropic effect of the adjacent carbonyl group
at the C2 atom. The IR spectrum shows all the characteristic
bands that are expected for 2d. These findings finally confirmed
the formation of compound 2d. Unfortunately, in all spectra
appear additional signals caused by an impurity formed from
the starting compound 9b, which is generated from 2b due to
its relatively low stability. Nevertheless, the HRMS confirmed
the elemental composition calculated for 2d.
1
related H NMR spectra are visible clearly and sharply. Inter-
estingly, in the 1H NMR of the 3-(2-chlorophenyl)-3-phenyl
derivative 3i occurred two broad singulets for the N(2)H. This
finding could be explained with a different spatial alignment
of the substituent in position 3. In particular different approx-
imations of the 2-chlorophenyl moiety to the N(2)H should be
possible. The behavior of the 3,3-disubstituted thiones 3a-k in
the EI mass spectrometry is comparable to those observed for
the 3-monosubstituted compounds (Schulz et al. 2013). Unlike
the other compounds with general structure 3, the molecular
ion peak for the 3-(2-chlorophenyl)-3-phenyl derivative 3i is
very weak, indicating its low stability. Ultimately, the chemical
behavior of compounds 3 is also consistent with the assigned
structure.
All recorded analytical data of 4a including the absence of a
NH signal (phenyl in position 2) in the 1H NMR spectrum and
NH bands in the IR spectrum, respectively, verify the expected
ring closure. Interestingly, the carbon atoms of the two methyl
groups in position 3, as observed with 3a,c (see above), are also
extremely difficult to detect. The same applies to for the carbon
atom of the thioxo group.
2.2.4. Compounds 3 and 4a
The IR and the 13C NMR spectra of all compounds 3 did not give
anyindicationsfortheexistenceofaC = Npartialstructureofthe
conceivable alternatives [1,3,4]thiadiazolo[3,4-a]-pyridazin-1-
imines of type A and a ring-open structures B, respectively
(Fig. 2). In addition, none of the other analytical data yielded by
2.3. Biological results
The biological tests were performed as described before (Schulz
et al. 2013) using the rat insulinoma cell line RIN5F stimulated
Fig. 2: Title compounds 3 and their possible alternative isomeric [1,3,4]thiadiazolo[3,4-a]pyridazin-1-imine (A) and open-ring substituted
hexahydropyridazine-1-carbothioamide (B) structure.
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ORIGINAL ARTICLES
with IL-1␤ and IFN-␥ to induce inducible nitric oxide synthase
unsubstituted parent structure and a number of 3,3-disubstituted
derivatives. While investigating different synthesis routes for
compound 2a, several other new substances possessing a
hexahydropyridazine ring were obtained. The biological inves-
tigations strengthen the previously proposed hypothesis that an
aromatic substitution at position 3 and its orientation are essen-
tial for iNOS inhibitory activity. The results also revealed that
the insertion of a second substituent in position 3 is disadvan-
tageous. For the further development of this substance class
computational chemistry methods may be useful.
(iNOS). The amount of produced NO was determined by mea-
suring the oxidation product nitrite based on the Griess reaction
(Griess 1879). Cytotoxicity was investigated by using a MTT
based assay (Mosmann 1983). Due to the poor solubility of most
of the substances only relatively low test concentrations (78 and
39 ␮M giving about 20 % resp. 10 % inhibition for the reference
inhibitor AG) could be used.
To determine whether a change in the substitution pattern influ-
ences the potency as an iNOS inhibitor, several synthesized
bicyclic thiones were exemplary evaluated. Thus, only sub-
stances with a modification of the substitution pattern in position
3 adjacent to an unsubstituted N2 atom were selected to ensure
the comparability of the results with those of our previous inves-
tigations (Schulz et al. 2013).
3. Experimental
3.1. General
As can be seen in Fig. 3A, the results show no activity
for compound 2a, the parent structure of the 3-substituted
[1,2,4]triazolo[1,2-a]pyridazine-1-thiones 3, and the tested
thiones with one or two aliphatic substituents, respectively.
As the results of our previous investigations on monosubsti-
tuted thiones clearly indicated that a phenyl moiety is needed
for iNOS inhibitory activity, it was assumed that this applies
for disubstituted derivatives as well. The data obtained sup-
port this thesis. The inactivity of the 3,3-disubstituted methyl
phenyl derivative 3d might be explained by the fact that the aro-
matic structure is located in a very opposite direction to one
of our hitherto best hits from our previous investigations. This
was a 3-cinnamyl derivative (IC50 = 9.81 2.21 ␮M) that had an
ethenylene group acting as spacer between the position 3 and the
phenyl ring and determining the spatial orientation of the aro-
matic moiety (Fig. 4 B, structures aligned). This implies that for
compounds3theoptimalorientationofthearomaticringisinflu-
enced by an adjacent aliphatic substituent at the same atom (C3).
Considering the iNOS inhibitory potential only the 3,3-diaryl
substituted thiones 3 h (R2, R3 = phenyl) and 3k (R2 = phenyl,
R3 = 4-methoxyphenyl) were significantly more active than AG.
Interestingly the 3-(2-chlorphenyl), 3-phenyl derivate 3i is not
active. It is possible that the chlorine atom sterically blocks
interactions with the N(2)H the binding site or prevents inter-
actions with the proton by forming intramolecular hydrogen
bonds. The low-energy conformations of the three diphenyl sub-
stituted thiones 3 h,i,k are quite similar (see Fig. 4 A, structures
aligned). For the two candidates that were identified in the pri-
mary screening, an IC50 value had been determined which was
for 3 h at 65.88 19.84 ␮M and for 3k at 68.04 38.23 ␮M
(mean SD; n = 17). Compared to aminoguanidine with an IC50
value of 132.01 14.96 ␮M (mean SD; n = 7) (Schulz et al.
2013) the tested substances are only slightly more active, espe-
cially in comparison to the activity of the best hit from previous
investigations (IC50 = 9.81 2.21 ␮M). In addition, the deter-
mination of the IC50 gave relatively large fluxuations, as noted
by the high SD, for the inhibitory concentrations were at the
borderline solubility of the compounds.
The reported melting temperature ranges were determined on a
Kofler-BoëtiusapparatustypePHMK81/3035(VEBWägetech-
nik 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
IntelliVentTM 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-d, 5a, 6, 10c,
12-16 only IR 200 FT-IR (Thermo Electron Corporation Nico-
let), ATR technique (diamond); H, H,1H-COSY, DEPT-135,
13C NMR spectra: AVANCE DPX 200 and for compounds 15
and 16 only the above named and additionally HSQC- and
HMBC spectra: FT-NMR-Spektrometer Avance IIITM 400 (both
spectrometers from Bruker Analytische Messtechnik GmbH);
temperature 25◦C, solvents used are specified in the data
of the related compound; internal standard tetramethylsilane;
chemical shift ␦ in ppm. To verify the signal assignments 2D
NMR techniques as HSQC, HMBC and 1H,1H-COSY were
used. Mass spectra: EI, M 40 AMD (Intectra GmbH), elec-
tron impact, energy 70 eV, (with the exception of the molecular
ion peaks normally only peaks > 10% are listed). High Res-
olution Mass Spectra: for the compounds 2a, 2c, 2b, 2d, 5a
hydrochloride, 5b, 6, 9b hydrochloride 10b, 10c, 12, 13, 14, 15:
ESI, Shimadzu High Performance Liquid Chromatograph/Mass
Spectrometer LCMS-IT-TOF with the system characterized
as following: solvent delivery module 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 Prominence,
evaporative light scattering detector ESD-LT II, spectromet-
ric detector RF-10A XL, LCMS-IT-TOF workstation, software
LCMS solution Version 3.41; column Chromolith® SpeedRod
RP-18 endcapped, 50 mm; mobile phases (m. ph.) used for the
compounds:
1
1
The results obtained strengthen the hypothesis that an aromatic
substituent in position 3 of the 2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thiones, is essential for an
iNOS inhibitory activity. It was repeatedly demonstrated that
the orientation of the phenyl moiety plays an important role.
However, a second substituent at this position is not advanta-
geous. On the contrary, it is clearly detrimental to inhibitory
potential.
Finally, like thereferencesubstance aminoguanidine(AG) itself,
also none of the tested compounds showed a cytotoxic effect
(Fig. 3B).
In summary, it could be shown that the new class
of 2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-
1-thiones can be successfully extended to include the
m. ph. IX: acetonitrile/water 1:1 (0.1% HCOOH in H2O)
(v/v), flow rate 0.2 mL·min−1 (compounds 2a, 2c, 2b, 2d,
5a hydrochloride, 10c, 13, 14)
m. ph. X: acetonitrile/water from 2:8 to 9:1 within 15 min
(0.1% HCOOH in H2O) (v/v), flow rate 0.2 mL·min−1 (com-
pound 12)
m. ph. XI: methanol/water 1:1 (0.1% HCOOH in H2O) (v/v),
flow rate: 0.4 mL·min−1 (compound 15)
m. ph. XII: methanol/water 6:4 (0.1% HCOOH in H2O)
(v/v), flowrate:0.4 mL·min−1 (compound9bhydrochloride,
10b); flow rate: 0.2 mL·min−1 (compound 6)
m. ph. XIII: methanol/water 8:2 (0.1% HCOOH in H2O)
(v/v), flow rate: 0.2 mL·min−1 (compound 5b)
736
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ORIGINAL ARTICLES
Fig. 3: Concentration-dependent inhibition of iNOS by aminoguanidine (AG), 3-unsubstituted thione 2a, and 3,3-disubstituted thiones 3 (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 as the reference inhibitor was tested in every concentration as well. Mean SD; n = 6 (> 30 for AG,
as control on every plate).
Fig. 4: (A) compound 3 h, 3i and 3k superposed; (B) compound 3d superposed with one of the best hits from previous investigations, the 3-phenylethenyl derivative of the parent
structure 2a.
High Resolution Mass Spectra: for the compounds 3d, 11, 16:
ESI, BrukerDaltonicsMicroOTOF-LC(ESI-TOF);externalcal-
ibration (Agilent ESI TuneMix); column Zorbax RP-C18; 2.1 x
30 mm, 3.5 ␮m; mobile phase (m. ph.): m. ph. XVII: gradient
propan-2-ol/water 20% Ba’ 50% B.; flow rate: 0.3 mL/min.
For silica gel aluminum 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:
m. ph. VII: acetonitrile/water 1:1 (v/v)
m. ph. VIII: 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.
3.2. Chemistry
3.2.1. Hexahydropyridazine-1-carbothioamides 1, 6 and 15
Hexahydropyridazine-1-carbothioamides 1a-c
Synthetic procedures and experimental data are already reported
(Schulz et al. 2013).
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. IV: dichloromethane/acetone/triethylamine
10:10:1 (v/v/v)
N,N’-Dimethyl-2,2’-methylenedi(hexahydropyridazine-1-car-
bothioamide) 6
Method 1: - from 1c and paraformaldehyde: A well prepared
mixture of 0.7 mmol (0.112 g) N-methylhexahydropyridazine-
1-carbothioamide (1c) and 0.8 mmol (0.024 g) paraformalde-
hyde was heated together with 0.2 g of dried molecular sieve in a
metal bath at 140-150 ◦C until the reaction was terminated (TLC
control, about 15 min). The cooled solid melt was dissolved in a
little acetone and the solution was slowly concentrated at room
temperature. The formed crystals were collected, washed twice
with a little methanol and dried in vacuo. Yield 10%. Colorless
crystals. M.r. 244-246 ◦C (acetone).
Each mobile phase (m. ph.) used is specified in the data of
the related compound. The substances were detected with
UV radiation (␭ = 254 nm) or Munier spray reagent (Munier
1953) ENREF 1 and iodine azide reagent by awe (Awe 1948).
The HPLC investigations were done under the following condi-
tions:
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 (␭ = 220 nm 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
TLC (m. P. II): Rf = 0.42. HPLC (m. P. VI): RP-18, t0 = 1.77;
tR = 4.98; k’ = 1.81. IR (cm−1): ν˜ = 1185 (C = S); 1522 (NH);
1
2854 (CH3); 2941 (aliph. H); 3325 (NH). H NMR (DMSO-
d6, ppm): ␦ = 1.30-1.84 (bm, 10H, heterocycl. CH2); 2.73 (bt,
2H, heterocycl. CH2); 2.93 (d, J = 4.6 Hz, 6H, 2 x -CH3); 3.01
(bt, 1H, heterocycl. CH2); 3.19 (bd, 1H, heterocycl. CH2); 3.85
m. ph. V: acetonitrile/0.02 M KH2PO4 (pH 2.85) 1:1 (v/v)
m. ph. VI: acetonitrile/0.02 M KH2PO4 (pH 2.85) 3:7 (v/v)
Pharmazie 69 (2014)
737

ORIGINAL ARTICLES
(d, J = 11.0 Hz, 1H, heterocycl. CH2); 4.08 (d, J = 11.6 Hz, 1H,
Elemental analysis [C6H9N3S (155.2), %]: calcd. C, 46.43; H,
5.84; N, 27.07; S 20.65; found C, 47.25; H, 5.95; N, 27.55; S,
21.02.
heterocycl. CH2); 5.05 and 5.11 (bs, 2 x 1H, CH2); 7.97 (q,
J = 4.6 Hz, 2H, 2 x NH). 13C NMR (DMSO-d6, ppm): ␦ = 17.70
(2 x C5); 23.38 (2 x C4); 31.79 (2 x CH3); 40.76 (2 x C3);
48.16 (2 x C6); 69.41 (CH2); 181.11 (2 x C = S). MS (70 eV,
180 ◦C) m/z (%): 171.7 (54) [M+· - .CH2-(NC4H8N)-CS-NH-
CH3]; 97.9 (100) [C4H8N2-CH2+]; 84.9 (23) [C4H8N2+]; 70.8
(12); 57.3 (13); 42.0 (14); 28.0 (50). HRMS [(ESI) m/z (%)]:
Compound 6: calcd. [C13H26N6S2 + Na]+: 353.1553; found:
353.1555 (35) (calcd. for [C13H26N6S2]+: 330.1660; found:
330.1663) and compound 2c as a splitting product, calcd.
[C7H13N3S + H]+: 172.0903; found 172.0899 (100). Elemental
analysis [C13H26N6S2 (330.5), %]: calcd. C, 47.24; H, 7.93; N,
25.43; S, 19.04; found C, 46.57; H, 8.00; N, 24.62; S, 18.64.
3.2.3. 2,3,5,6,7,8-Hexahydro-1H-[1,2,4]triazolo[1,2-a]py-
ridazine-1-thiones 2a—d, 7
2,3,5,6,7,8-Hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-
thione (2a)
A
mixture of 1 mmol (0.155 g) tetrahydro-1H-[1,2,4]
triazolo[1,2-a]pyridazine-1-thione (16), 3.8 mmol (0.144 g)
NaBH4, and 4.5 mL propan-2-ol was stirred at room temper-
ature for 18 h. Subsequently, 7 mL water were added and the
resulting solution was three times extracted each with 8 mL
dichloromethane and the combined organic phases were dried
over sodium sulphate. After filtration the organic phase was
carefully concentrated, whereby crystals formed. These were
sucked off and dried at room temperature in vacuo. Yield 91%.
Colorless crystals. M.r. 85-93 ◦C (dichloromethane).
TLC (m. P. III): Rf = 0.76. HPLC (m. P. VII): RP-18, t0 = 1.75;
tR = 2.37; k’ = 0.35. IR (cm−1): ν˜ = 1174 (C = S); 1486 (NH);
2834, 2912, 2959 (–CH2–); 3173 (NH). 1H NMR (CDCl3, ppm):
␦ = 1.64 (bs, 2H, C(7)H2); 1.80–1.84 (m, 2H, C(6)H2); 2.82
(bs, C(5)H2); 3.19 (bs, 1H, C(8)H); 4.20 (bs, 1H, C(3)H);
4.54 (bs, 2H, C(8)H u. C(3)H); 6.69 (bs, 1H, NH).13C NMR
(CDCl3, ppm): ␦ = 23.18 (C7); 23.69 (C6); 45.21 (C8); 53.40
(C5); 67.03 (C3); 178.49 (C(1) = S). HRMS [(ESI) m/z]: calcd.
for C6H11N3S + H+: 158.0746; found [M + H+]: 158.0707.
Method 2: - from 1c and aqueous formaldehyde solution:
To a suspension of 1 mmol N-methylhexahydropyridazine-1-
carbothioamide (1c)(0.159 g) in 1 mL methanol was added 0.1 g
30% aqueous formaldehyde solution (approximately 1 mmol
formaldehyde) and the reaction mixture was refluxed for 7 h
(bath temperature 120 ◦C). After cooling overnight, the formed
crystals were separated, washed with a little methanol and dried
on a clay plate. Yield 3%. Colorless crystals. M.r. 226-229 ◦C
(water).
2-Formyl-hexahydropyridazine-1-carbothioamide (15)
This compound is an intermediate in the synthesis of compound
16 (synthetic procedure see below).
A
mixture of 5 mmol (0.726 g) hexahydropyridazine-1-
carbothioamide (1a) and 2 mL 99% formic acid was refluxed for
45 min. Subsequently, the formed solution was concentrated in
a warm air flow. The resulting crystals were recrystallized from
ethanol whereby compound 16 was included in the residue. The
mother liquor was concentrated again in a warm air flow and the
formed solid was recrystallized from water. The yielded crystals
were collected and washed with a little acetone. Yield 5%. Light
beige crystals. M.r. 166-168 ◦C (water).
2-Phenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione (2b)
A mixture of 1 mmol (0.221 g) N-phenylhexahydropyridazine-
1-carbothioamide (1b), 1 mL methanol and 0.1 g 30% aqueous
formaldehyde solution (approximately 1 mmol formaldehyde)
was refluxed for 6 h (bath temperature 120 ◦C). After cooling
overnight, the formed crystals were separated and washed with
a little methanol and dried on a clay plate. Yield 3%. Colorless
crystals. M.r. 218-221 ◦C (methanol).
TLC (m. P. II): Rf = 0.86. HPLC (m. P. V): RP-18, t0 = 1.75;
tR = 12.64; k’ = 6.22. IR (cm−1): ν˜ = 1172 (C = S); 1588, 1686
(arom. ring). 1H NMR (CDCl3, ppm): ␦ = 1.80 (bs, 4H, C(6)H2
and C(7)H2); 3.23 (t, J = 10.4, 2H, C(5)H2); 4.06 (t, J = 10.6, 2H;
C(8)H2); 6.81-6.92 (t, J = 15.4 Hz, 3H, arom. H from C10, C12
and C14); 7.14 (t, J = 15.4 Hz, 2H, arom. H an C11and C13);
7.32-7.34 (d, 2H, C(3)H2). 13C NMR (CDCl3, ppm): ␦ = 22.52,
22.57 (C6 and C7); 45.41 (C8); 48.17 (C5); 77.23 (C3); 121.55
(2 arom. C); 122.44 (1 arom. C); 128.89 (2 arom. C); 134.03
(1 arom. C); 146.62 (C3); 171.35 (C(1) = S). HRMS [(ESI)
m/z]: calcd. for C12H15N3S + H+: 234.1059; found [M + H+]:
234.1052.
TLC (m. P. III): Rf = 0.70. IR (cm−1): ν˜ = 1184 (C = S); 1607
(NH2); 1670 (C = O); 2852, 2930, 2958 (aliph. and formyl H);
1
3170; 3255, 3347 (NH2). H NMR (CDCl3, ppm): ␦ = 1.25 (s,
3H, CH3); 1.76 (m, 3H; C(4)H2 and C(5)H); 1.89 (m, 1H,
C(5)H); 2.89 (dt, 1H, C(3)H); 3.10 (dt, 1H, C(6)H); 4.39 (dd, 1H,
C(3)H); 5.48 (dd, 1H, C(6)H); 6.32 (bs, 2H, NH2); 8.30 (s, 1H,
COH). 13C NMR (CDCl3, ppm): ␦ = 22.7 (C4); 23.0 (C5); 42.0
(C3); 51.6 (C6); 164.9 (C = O); 184.2 (C = S). HRMS [(ESI)
m/z]: calcd. [C6H11N3OS + H]+: 174.0696; found [M + H]+:
174.0733.
3.2.2. 5,6,7,8-Tetrahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione 16
A
mixture of 5 mmol (0.726 g) hexahydropyridazine-1-
2-Methyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
carbothioamide (1a) and 2 mL 99% formic acid (approximately
52.5 mmol) was refluxed for 1 h. After the excess of formic
acid was removed the formed crystals were recrystallized from
ethanol, filtered off, washed with ethanol and dried in vacuo.
Yield 80%. Colorless crystals. M.r. 276–279 ◦C (ethanol).
TLC (m. P. III): Rf = 0.14. HPLC (m. P. VI): RP-select B,
t0 = 2.10; tR = 2.37; k’ = 0.13. IR (KBr, cm−1): ν˜ = 1178 (C = S);
1505 (C = N); 1849 (S = C-N = C); 2865, 2925, 2959 (CH2);
[1,2-a]pyridazine-1-thione (2c)
A mixture of 1 mmol (0.159 g) N-methylhexahydropyridazine-
1-carbothioamide (1c), 1 mL water, and 0.1 g 30% aqueous
formaldehyde solution (approximately 1 mmol formaldehyde)
was refluxed for 4 h (bath temperature 120 ◦C). After cooling
overnight, the formed crystals were separated, washed with a
little water and dried at room temperature in vacuo. Yield 71%.
Colorless crystals. M.r. 32-36 ◦C (water).
TLC (m. P. II): Rf = 0.31. HPLC (m. P. VII): RP-18, t0 = 1.75;
tR = 3.17; k’ = 0.81. IR (cm−1): ν˜ = 1186 (C = S); 2853, 2924,
2950(–CH3, –CH2–). 1HNMR(CDCl3, ppm):␦ = 1.62-1.67(m,
2H, C(6)H2); 1.77-1.84 (m, 2H; C(7)H2); 2.76 (t, J = 10.2 Hz,
4H, C(5)H2 and C(8)H2); 3.11 (s, 3H, CH3); 4.20-4.34 (bd, 2H,
C(3)H2). 13C NMR (CDCl3, ppm): ␦ = 22.91 (C6); 23.78 (C7);
32.76(CH3);45.98(C8);53.30(C5);72.68(C3);178.87(C = S).
1
3072 (=C-H). H NMR (DMSO-d6, ppm): ␦ = 1.95 (bm, 4H,
C(6)H2 and C(7)H2); 3.93 (t, J = 6.0 Hz, 2H, C(5)H2); 4.14 (t,
J = 5.2 Hz, 2H, C(8)H2); 8.48 (s, 1H, C(3)H). 13C NMR (DMSO-
d6, ppm): ␦ = 19.42 (C6); 20.03 (C7); 45.18 (C5); 46.65 (C8);
146.60 (C = N); 175.20 (C = S). MS (70 eV, 350 ◦C) m/z (%):
154.8 (100) [M+·]; 126.9 (35) [M+ – CH2 = CH2]; 99.9 (33); 72.7
(15); 68.9 (17); 55.3 (14); 42.1 (16); 41.0 (41); 28.0 (48); 27 (10).
738
Pharmazie 69 (2014)

ORIGINAL ARTICLES
HRMS [(ESI) m/z]: calcd. for C7H13N3S + H+: 172.0903; found
In this manner the following compounds were obtained:
[M + H+]: 172.0903.
3,3-Dimethyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
2-(4-Nitrobenzoyl)-2,3,5,6,7,8-hexahydro-1H-
triazolo[1,2-a]pyridazine-1-thione (3a)
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (2d)
With 25 mL (approximately 340 mmol) acetone. Reaction time
33 h. Yield 81%. Light beige crystals. M.r. 156–157 ◦C (propan-
2-ol).
To a solution of 1 mmol (0.300 g) 4-nitro-N-(hexahydro-
pyridazin-1-ylmethyl)benzamide hydrochloride (9b) HCl in
10 mL methanol were added 1 mmol (0.101 g) triethylamine and
45 mmol (3.426 g) carbon disulphide. The reaction mixture was
allowed to stand for 5 d at room temperature. The formed crys-
tals were separated, washed with methanol and dried on a clay
plate. Yield 19%. Yellow crystals. M.r. 208-215 ◦C (methanol).
TLC (m. P. IV): Rf = 0.93. HPLC (m. P. VII): RP-18, t0 = 1.75;
tR = 14.75; k’ = 7.43. IR (cm−1): ν˜ = 1190 (C = S); 1544 (-NO2);
1515, 1598 (arom. –C = C–); 1652 (C = O). 1H NMR (DMSO-
d6, ppm): ␦ = 1.43-1.98 (m, 4H, C(6)H2 and C(7)H2); 3.13 (bt,
4H, C(5)H2 and C(8)H2); 4.76-4.96 (m, 2H, C(3)H2); 8.05 (d,
J = 8.8 Hz, 2H, arom. H); 8.30 (d, J = 8.8 Hz, 2H, arom. H). 13C
NMR (DMSO-d6, ppm): ␦ = 18.30 (C6); 23.44 (C7); 44.56 (C8);
50.45 (C5); 55.68 (C3); 123.37 (2 arom. C); 128.94 (2 arom. C);
139.62 (1 arom. C); 149.08 (1 arom. C); 165.33 (C = O); 198.03
(C = S). HRMS [(ESI) m/z]: calcd. for C13H14N4O3S + H+:
307.0859; found [M + H+]: 307.0807.
TLC (m. P. I) Rf = 0.32. IR (KBr, cm−1): ν˜ = 1172 (C = S);
1460;1485; 1626 (NH); 2850, 2923, 2942, 2962, 2975 (aliph.
C-H); 3158 (NH).1H NMR (CDCl3, ppm): ␦ = 1.40 (s, 6H,
C(3)(CH3)2); 1.64 (m, 2H, C(6)H2); 1.82 (m, 2H, C(7)H2);
2.76 (t, 2H, C(8)H2); 3.78 (s, 2H, C(5)H2); 6.1 (s, 1H, (N(2)H).
13C NMR (CDCl3, ppm): ␦ = 23.15; 23.71 (C6 and C7); 23.94
(broad and flat signal, 2 x CH3); 45.3 (C8); 47.5 (C5); 77.3
(C3); 176.4 (C = S). MS (70 eV, 115 ◦C) m/z (%): 185.1 (80)
[M+·]; 170.0 (100) [M+·–·CH3]; 115.5 (17); 100.0 (17); 85.0
(65) [C4H9N2+]; 70.9 (35); 56.3 (37); 55.3 (32); 41.1 (58);
30.0 (56); 28 (67). Elemental analysis [C8H15N3S (185.3), %]:
calcd. C, 51.86; H, 8.16; N, 22.68; found C, 51.77; H, 8.44;
N, 22.39.
3-Ethyl-3-methyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione (3b)
With 10 mL (approximately 112 mmol) ethyl methyl ketone.
Reaction time 44 h. Yield 66%. Colorless crystals. M.r. 103-
104 ◦C (propan-2-ol).
2,2’-Methylene di(2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione) (7)
TLC (m. P. I) Rf = 0.53. IR (KBr, cm−1): ν˜ = 1180 (C = S); 1419;
1449; 1482; 1635 (NH), 2846 (CH3); 2914, 2956 (aliph. C-H);
3167 (NH).1H NMR (CDCl3, ppm): ␦ = 0.95 (t, 3H, -CH2CH3);
1.35 (s, 3H, CH3); 1.65 (m, 4H, C(7)H2 and -CH2CH3); 1.80 (m,
2H, C(6)H2) 2.73 (t, 2H, C(5)H2); 3.45 (bs, 1H, C(8)H2); 4.19
(bd, 1H, C(8)H2); 6.5 (s, 1H, NH). 13C NMR (CDCl3, ppm):
␦ = 8.26 (-CH2CH3); 19.56 (CH3); 23.24, 23.78 (C6 and C7);
31.06 (-CH2CH3); 45.25 (C8); 47.52 (C5); 80.01 (C3); 175.88
(C = S). MS ·(70 eV, 180 ◦C) m/z (%):· 199.1 (40) [M+·]; 184.0
(15) [M+· – CH3]; 170 (100) [M+· – C2H5]; 115.5 (11); 85.0
(26) [C4H9N2+]; 70.9 (12); 69.9 (11); 56.3 (22); 55.3 (38); 42.1
(27); 41.1 (24); 32.0 (11); 30.0 (21); 29 (14); 28 (62); 27 (12).
Elemental analysis [C9H17N3S (199.3), %]: calcd. C, 54.24; H,
8.60; N, 21.08; found C, 53.29; H, 8.71; N, 20.21.
A mixture of a well prepared trituration of 0.7 mmol (0.102 g)
hexahydropyridazine-1-carbothioamide (1a) with 0.8 mmol
(0.024 g) paraformaldehyde and 0.2 g molecular sieve was
heated at 140–150 ◦C until the reaction was completed (TLC
monitoring, about 25 min). After cooling, the formed solid was
dissolved in a little acetone and the solution was slowly con-
centrated. The formed crystals were sucked off, washed twice
with a little methanol and dried in vacuo. Yield 15%. Colorless
crystals. M.r. 154–157 ◦C (acetone).
TLC (m. P. II): Rf = 0.74. HPLC (m. P. VI): RP-select B,
t0 = 2.10; tR = 3,86; k’ = 0.84. IR (KBr, cm−1): ν˜ = 1188
(C = S); 1480; 2842, 2851, 2941 (aliph. H); 3260 (ass. OH). 1H
NMR (CDCl3, ppm): ␦ = 1.63 (bs, 4H, C(6)H2 and C(6’)H2);
1.82 (m; 4H, C(7)H2 and C(7’)H2); 2.74 (bs, 4H, C(5)H2
and C(5’)H2); 3.19 (bs, 2H, je 1H of C(8)H2 and C(8’)H2);
4.51 (bs, 6H, je 1H von C(8)H2 and C(8’)H2 and je 2H of
3,3-Diethyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo[1,2-
a]pyridazine-1-thione (3 g)
C(3)H2 and C(3’)H2); 5.4 (s, 2H, ring-linking CH2-group). 13
C
With 10 mL (94 mmol) diethyl ketone. Reaction time 20 h. Yield
76%. Colorless crystals. M.r. 127-129 ◦C (propan-2-ol).
TLC (m. P. I) Rf = 0.73. IR (KBr, cm−1): ν˜ = 1184 (C = S); 1421;
1441; 1473; 1634 (NH); 2849, 2880, 2929, 2954, 2972 (aliph.
C-H); 3175 (NH).1H NMR (CDCl3, ppm): ␦ = 0.92 (t, 6H, (2 x
-CH2CH3); 1.69 (m, 8H C(6)H2, C(7)H2 and 2 x -CH2CH3);
2.75 (t, 2H, C(5)H2); 3.80 (bt, 2H, C(8)H2); 6.47 (s, 1H, NH).
13C NMR (CDCl3, ppm): ␦ = 8.11 (2 x CH2CH3); 23.15, 23.71
(C6 and C7); 25.95 (2 x -CH2CH3); 45.25 (C8); 47.24 (C5);
82.39 (C3); 175.67 (C = S). MS (70 eV, 150 ◦C) m/z (%): 213.0
NMR (CDCl3, ppm): ␦ = 23.01 (C7 and C7‘); 23.85 (C6 and
C6‘); 45.88 (C5 and C5‘); 53.70 (C8 and C8‘); 55.58 (-CH2-);
71.09 (C3 and C3‘); 178.64 (2 x C = S). MS (70 eV, 295 ◦C)
m/z (%): 325.7 (35) [M+·]; 169.8 (41) [2,3,5,6,7,8-hexahydro-
1H-[1,2,4]triazolo[1,2-a]pyridazine-1-thion-3-ylmethyl+];
155.8
(100)
[2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thionyl+]; 96.9 (46); 72.6 (15); 55.2
(17); 42.1 (28); 32.0 (11), 30.0 (12); 28.0 (59). Elemental
.
analysis [C13H22N6S2 (326.5) H2O [344.5], %]: calcd. C,
45.33; H, 7.02; N, 24.40; S, 18.61; found C, 44.62; H, 6.70; N,
24.12; S, 19.22.
(27) [M+·]; 185.1 (10); 184.0 (100) [M+· – C2H5]; 85.0 (15)
·
[C4H9N2+]; 83 (11); 56.3 (20); 55.3 (19); 41.1 (21); 30.0 (12);
29 (12); 28 (32). Elemental analysis [C10H19N3S (213.3), %]:
calcd. C, 56.30; H, 8.98; N, 19.70; found C, 56.66; H, 8.69; N,
19.43.
3.2.4. 3,3-Disubstituted 2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo[1,2–a]pyridazine-1–thiones 3 and 4a
3-Cyclopropyl-3-methyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (3c)
2,3,5,6,7,8-Hexahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-1-
thiones 3a,b,g
A mixture of 0.5 mmol (0.073 g) hexahydropyridazine-1-
carbothioamide (1a), 1 mL (approximately 11 mmol) cyclo-
propyl methyl ketone, 0.05 mmol (0.007 g) anhydrous alu-
minium chloride, and 0.1 g fresh dehydrated molecular sieve
was allowed to stand at room temperature. After the starting
compound 1a was completely reacted (about 6 month), the sus-
pension was passed through a glass sinter frit and the resulting
A
mixture of 5 mmol (0.726 g) hexahydropyridazine-1-
carbothioamide (1a), the given amount of the appropriate
ketone, and 0.25 mmol (0.48 g) p-toluenesulfonic acid monohy-
drate was refluxed until the starting compound was completely
reacted (TLC monitoring). Then the excess of the ketone was
removedatroomtemperature. Theformedcrystals, werewashed
with a little ice-cold propan-2-ol, separated and dried in vacuo.
Pharmazie 69 (2014)
739

ORIGINAL ARTICLES
2H, arom. H). 13C NMR (CDCl3, ppm): ␦ = 23.14 (C6); 23.81
(C7); 24.35 (-CH3) 45.49 (C8); 48.71 (C5); 55.30 (-OCH3);
80.99 (C3); 113.87 127.55, 159.84 (arom. C); 176.62 (C = S).
MS (70 eV, 270 ◦C) m/z (%): 277.0 (41) [M+·]; 262.1 (18); 191.9
clear filtrate was concentrated at room temperature. The remain-
ing dark and viscous residue was digested several times with
little boiling hexane. During cooling of the hexane phase crystals
were formed which were separated, washed with hexane, and
dried in vacuo. Yield 10%. Colorless crystals. M.r. 112-114 ◦C
(hexane).
.
(77) [M+. - C4H9N2]; 161.9 (11); 134.0 (13); 91.0 (17); 85.0
(100) [C4H9N2+]; 56.3 (20); 41.0 (13); 32.0 (13); 30.0 (32);
28.0 (66). Elemental analysis [C14H19N3OS (277.4), %]: calcd.
C, 60.62; H, 6.90; N, 15.15; found C, 60.56; H, 6.85; N, 14.62.
TLC (m. P. I) Rf = 0.57. IR (KBr, cm−1): ν˜ = 1202 (C = S);
1434; 1476; 1583; 1634 (NH); 2839, 2924, 2947, 2981; 3016
(aliph. H); 3177 (NH).1H NMR (CDCl3, ppm): ␦ = 0.29–0.33
(m, 1H, cyclopropyl); 0.52–0.55 (m, 3H cyclopropyl); 1.31
(s, 3H, CH3); 1.60 -1.64 (m, 3H, 2H of C(6)H2 and 1H of
cyclopropyl);1.79–1.83 (m, 2H, C(7)H2; 2.72–2.88 (m, 2H,
C(5)H2); 3.81 (bs, 2H, C(8)H2); 5.98 (s, 1H, NH). 13C NMR
(CDCl3, ppm): ␦ = 1.51 (C2 and C3 cyclopropyl), 17.15 (very
broad, less intensively, C1 cyclopropyl), 21.58 (very broad, less
intensively, CH3), 23.51 (C6), 24.02 (C7), 45.42 (C5), 48.45
(C8), 79.94 (C3), 176.06 (C = S). MS·(70 eV, 115 ◦C) m/z (%):
3,3-Pentamethylen-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo[1,2-a]pyridazine-1-thione (3f)
With cyclohexanone (0.540 g). Reaction time 45 min. Yield
60%. Colorless crystals. M.r. 184–185 ◦C (propan-2-ol).
TLC (m.P. I): Rf = 0.62. IR (KBr, cm−1): ν˜ = 1186 (C = S);
1485; 1633 (NH); 2852, 2921 (aliph. C-H); 3193 (NH). 1H NMR
(CDCl3, ppm): ␦ = 1.30–1.84 (bm, 14H); 2.72 (t, 2H, CH2 of the
cyclohexane ring); 3.94 (bs, 2H, C(8)H2), 7.26 (s, 1H, NH). 13
C
211.0 (52) [M+·]; 196.0 (30) [M+· – CH3]; 170.0 (39) [M+·
–
NMR (CDCl3, ppm): ␦ = 22.80, 23.39, 23.84, 25.05, 32.73 (5
pentamethylene C); 23.38 (C7); 23.85 (C6); 45.07 (C8); 46.89
(C5); 79.21 (C3); 175.90 (C = S). MS (70 eV, 180 ◦C) m/z (%):
225.1 (48) [M+·]; 182.0 (86); 169.0 (18); 85.0 (44) [C4H9N2+];
81.0 (18); 70.9 (16); 56.3 (15); 55.3 (22); 42.1 (13); 41.0 (35);
39.1 (10); 32.0 (20); 30.0 (23); 28.0 (100). Elemental analysis
[C11H19N3S (225.4), %]: calcd. C, 58.63; H, 8.50; N, 18.65;
found C, 57.95; H, 8.26; N, 18.38.
·C3H5]; 85.0 (100) [C4H9N2+]; 70.9 (11); 68.9 (12); 67.9 (13);
56.3 (30); 55.3 (27); 42.1 (31); 41.1 (48); 39.1 (22); 42.1 (31);
32.0 (15); 30.1 (43); 29 (11); 28 (86); 27 (14). Elemental analy-
sis [C10H17N3S (211.3), %]: calcd. C, 56.84; H, 8.11; N, 19.88;
found C, 55.79; H, 8.91; N, 19.89.
3,3-Disubstituted
2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1, 2-a]pyridazine-1-thiones 3d-f,h-k
3,3-Diphenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione (3h)
Method 1: Preparation by conventional heating of 1a with
ketones - general procedure: A well prepared mixture of
5 mmol (0.726 g) hexahydropyridazine-1-carbothioamide 1a,
5.5 mmol of the appropriate ketone, and 0.25 mmol (0.048 g)
p-toluenesulfonic acid monohydrate was heated at 150–155 ◦C
until the starting compound 1a was completely reacted (TLC
monitoring). Subsequently, the cooled mass was dissolved in a
little methanol, and the solution was concentrated at room tem-
perature whereby crystals formed. The formed crystals were
washed with little cold propan-2-ol, separated, and dried in
vacuo.
With benzophenone (1.002 g). Reaction time 13 h. The synthe-
sis differed from the general method by washing the isolated
crystals with a little methanol. Yield 75%. Colorless crystals.
M.r. 252–255 ◦C (methanol).
TLC (m. P. I): Rf = 0.84. IR (KBr, cm−1): ν˜ = 1166 (C = S);
1452; 1472; 1634 (NH); 2853; 2922; 2944; 2960 (aliph. C-
1
H); 3060 (arom. C-H); 3152 (NH). H NMR (CDCl3, ppm):
␦ = 1.58–1.86(m, 4H, C(6)H2 andC(7)H2);2.25(t, 2H, C(8)H2);
3.98 (s, 2H, C(5)H2); 7.28–7.41 (m, 10H, arom. H); 7.94 (s, 1H,
NH). 13C NMR (CDCl3, ppm): ␦ = 23.22; 24.08 (C6, C7); 45.12
(C8); 49.90 (C5); 85.78 (C3); 127.27, 128.33, 128.41, 139.81
(all arom. C); 175.96 (C = S). MS (70 eV, 250 ◦C) m/z (%):
309.5 (65) [M+·]; 232.4 (16) [M+· – ·C6H5]; 224.4 (28); 165.3
(37); 121.3 (12); 103.9 (15); 85.2 (100) [C4H9N2+]; 77.5 (16);
56.6 (14); 41.2 (12) 30.1 (27). Elemental analysis [C18H19N3S
(309.4), %]: calcd. C, 69.87; H, 6.19; N, 13.58; found C, 69.56;
H, 5.81; N, 13.53.
In this manner the following compounds were obtained:
3-Methyl-3-phenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]
triazolo[1,2-a]pyridazine-1-thione (3d)
With acetophenone (0.661 g). Reaction time 4 h. Yield 75%.
Colorless crystals. M.r. 144–146 ◦C (propan-2-ol).
TLC (m. P. I): Rf = 0.64. IR (KBr, cm−1): ν˜ = 1204 (C = S);
1477; 1493; 1582, 1627 (NH); 2952; 2970; 2987 (aliph. C-H);
3057 (ar-H); 3154 (NH). 1H NMR (CDCl3, ppm): ␦ = 1.60–1.76
(m, 10H, C(6)H2 and C(7)H2 and C(3)–CH3); 2.49 (s, C(8)H2);
3-(2-Chlorophenyl)-3-phenyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (3i)
3.83(s, C(5)H2);7.14(s, 1H, NH), 7.32–7.54(m, 5arom. H). 13
C
NMR (CDCl3, ppm); ␦ = 23.17 (C7); 23.84 (C6); 24.21 (CH3);
45.39 (C8); 48.68 (C5); 81.32 (C3); 126.07, 128.57, 128.54,
139.97 (all arom. C); 176.63 (C = S). MS (70 eV, 180 ◦C) m/z
(%):247.2(39)[M+·];232.1(21)[M+· –·CH3];161.9(11);102.8
(17); 91.0 (11); 85.1 (100) [C4H9N2+]; 77.4 (22); 56.4 (25);
41.1 (15); 30.0 (40); 28.0 (49). Elemental analysis [C13H17N3S
(247.4), %]: calcd. C, 63.12; H, 6.93; N, 16.99; found C, 62.35;
H, 6.38; N, 16.62.
With o-chlorobenzophenone (1.192 g). Reaction time 15 h. The
synthesis differed from the general method by treating the melt
with acetone and washing the formed crystals with a little
acetone. Yield 38%. Colorless crystals. M.r. 244–246 ◦C (ace-
tone).
TLC (m. P. I): Rf = 0.89. IR (KBr, cm−1): ν˜ = 753 (ar–Cl);
1166 (C = S); 1443; 1460; 1632 (NH); 2840; 2926; 2937;
1
2956 (aliph. C-H); 3066 (arom. C-H); 3222 (NH). H NMR
(CDCl3, ppm): ␦ = 1.63–1.78 (m, 4H, C(6)H2 and C(7)H2);
1.94–2.01; 2.38–2.48; 3.33; 4.59 (m and m and t and d, 8H;
C(8)H2; C(5)H2); 7.27–7.38 (m, 9H, arom. H); 8.13 and 8.17
(each bs, 0.5 H, NH). 13C NMR (CDCl3, ppm): ␦ = 23.06 (C7);
24.14 (C6); 45.17 (C8); 49.83 (C5); 85.12 (C3); 126.55, 127.83,
128.39, 128.67, 128.97, 129.94, 131.45, 131.95 (all arom. C);
176.58 (C = S). MS (70 eV, 300 ◦C) m/z (%): 343.0 (3) [M+·];
3-(4-Methoxyphenyl)-3-methyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (3e)
With p-methoxyacetophenone (0.826 g). Reaction time 6 h.
Yield 50%. Colorless crystals. M.r. 137–138 ◦C (propan-2-ol).
TLC (m. P. I): Rf = 0.70. IR (KBr, cm−1): ν˜ = 1178 (C = S);
1513; 1584; 1610 (broad peak basis, arom. ring and NH); 2850,
1
2930, 2953 (aliph. C-H); 3000 (arom. C-H); 3176 (NH). H
308.0 (21) [M+· – Cl]; 165.0 (11); 85.0 (45) [C4H9N2+]; 32.0
·
NMR (CDCl3, ppm): ␦ = 1.61 (q, 3H, C(6)H2 and C(7)H); 1.64
(s, 3H, -CH3); 1.71 (s, 1H, C(7)H); 2.40 (q, 2H, C(5)H2); 3.72
(bs, 1H, C(8)H); 3.81 (s, 3H, -OCH3); 3.99 (bs, 1H, C(8)H); 6.56
(s, 1H, NH); 6.90 (d, J = 12 Hz, 2H, arom. H); 7.41 (d, J = 12 Hz,
(23); 30.0 (12); 28.0 (100). Elemental analysis [C18H18ClN3S
(343.9), %]: calcd. C, 62.87; H, 5.28; N, 12.22; found C, 60.54;
H, 5.76; N, 12.43.
740
Pharmazie 69 (2014)

ORIGINAL ARTICLES
3-(4-Chlorophenyl)-3-phenyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (3j)
With 2-chlorobenzophenone (0.238 g). 300 W, 130 ◦C, 15 bar.
Reaction time 10 min. Ethyl acetate as solvent for working up.
Yield 8%.
With p-chlorobenzophenone (1.192 g). Reaction time 21 h. The
synthesis differed from the general method by dissolving the
melt in propan-2-ol and subsequent concentrating the solu-
tion. Thereby formed crystals, which were washed with a
little acetone. Yield 52%. Colorless crystals. M.r. 252–254 ◦C
(propan-2-ol/acetone).
3,3-Dimethyl-2-phenyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (4a)
A mixture of 0.5 mmol (0.073 g) hexahydropyridazine-1-
carbothioamide 1a, 5 mL acetone, and 0.5 mmol (0.067 g)
anhydrous aluminium chloride was refluxed for 2 h. Subse-
quently, the batch was carefully concentrated, and the residue
was treated with 1 mL water, whereby crystals formed. The
crystals were sucked off, washed twice with a little water and
then once with a little cold propan-2-ol, and dried. Yield 64%.
Colorless crystals. M.r. 138–140 ◦C (acetone).
TLC (m. P. I) Rf = 0.82. IR (KBr, cm−1): ν˜ = 1167 (C = S);
1450; 1472; 1593; 1632 (NH); 2853, 2944, 2962, (aliph. C-H);
3158 (broad band, arom. C-H and NH).1H NMR (CDCl3, ppm):
␦ = 1.64 (m, 2H, C(6)H2) and 1.82 (m, 2H, C(7)H2); 2.21 (m,
2H, C(5)H2); 3.83 (bs, 1H, C(8)H2); 4.07 (bs, 1H, C(8)H2); 7.32
(m, 9H, arom. H); 8.17 (s, 1H, (NH). 13C NMR (CDCl3, ppm):
␦ = 23.18 (C7); 24.03 (C6); 45.17 (C7); 49.90 (C8); 85.42
(C3); 127.34, 128.49, 128.55, 128.58, 128.75, 134.34, 138.84
(all arom. C); 175.93 (C = S). MS (70 eV, 250 ◦C) m/z (%):
342.8 (14) [M+·]; 258.0 (8); 164.9 (16); 85.0 (100) [C4H9N2+];
28 (50). Elemental analysis [C18H18ClN3S (343.9), %]: calcd.
C, 62.87; H, 5.28; N, 12.22; found C, 62.24; H, 5.05; N,
12.04.
TLC (m. P. I): Rf = 0.70. IR (KBr, cm−1): ν˜ = 1179 (C = S);
1497, 1597 (arom. ring); 2846, 2933, 2948, 2965, 2980 (aliph.
H); 3032, 3063 (arom. H). 1H NMR (CDCl3, ppm): ␦ = 1.35 (s,
6H, 2 x CH3); 1.68 (m, 2H, C(6)H2); 1.88 (m, 2H, C(7)H2);
2.82 (t, 2H, C(5)H2); 4.03 (bs, 2H, C(8)H2); 7.23–7.28 (m, 2H,
arom. H); 7.38–7.50 (m, 3H, arom. H). 13C NMR (CDCl3, ppm):
␦ = 22.13 (broad and flat signal, 2 x CH3); 22.97 (C7); 23.94
(C6); 45.89 (C8); 47.59 (C5); 82.66 (C3); 128.37, 129.11,
130.31, 137.12 (all arom. C); 177.50 (C = S). MS (70 eV, 320 ◦C)
m/z (%): 260.9 (56) [M+·]; 245.8 (100) [M+· - .CH3]; 126.0 (18);
117.7 (10); 102.7 (15), 77.3 (19); 70.8 (21); 41.9 (11); 41.0 (12);
27.9 (38). Elemental analysis [C14H19N3S (261.4), %]: calcd. C,
64.33; H, 7.33; N, 16.08; found C, 63.58; H, 7.52; N, 15.88.
3-(4-Methoxyphenyl)-3-phenyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (3k)
With p-methoxybenzophenone (1.167 g). Reaction time 14 h.
Yield 40%. Colorless crystals. M.r. 218–220 ◦C (propan-2-ol).
TLC (m. P. I): Rf = 0.78. IR (KBr, cm−1): ν˜ = 1176 (C = S);
1509; 1583; 1609 (boad basis; arom. ring and NH); 2842,
1
2958 (aliph. C-H); 3007 (arom. C-H); 3165 (NH). H NMR
(CDCl3, ppm): ␦ = 1.63 (q, 2H, C(6)H2); 1.77 (q, 2H, C(7)H2);
2.22 (q, 2H, C(5)H2); 3.78 (s, 3H, -OCH3); 4.04 (bs, 2H,
C(8)H2); 6.82 (d, 2H, arom. H); 7.28 (d, 7H, arom. H); 7.76
(s, 1H, NH). 13C NMR (CDCl3, ppm): ␦ = 23.24 (C6); 24.09
(C7); 45.15 (C8); 49.86 (C5); 55.27 (-OCH3); 85.52 (C3);
113.66, 127.10, 128.28, 128.78, 131.28, 140.48, 159.64 (all
arom. C); 175.81 (C = S). MS (70 eV, 225 ◦C) m/z (%): 339.0
(27) [M+·]; 254.0 (100); 85.0 (51) [C4H9N2+]; 56.3 (12); 41.0
(13); 32.0 (12); 30.1 (67) 28 (11); 26 (15). Elemental analysis
[C19H21N3OS (339.5), %]: calcd. C, 67.23; H, 6.24; N, 12.38;
found C, 66.81; H, 5.96; N, 12.13.
3.2.5. 5,6,7,8-Tetrahydro-[1,3,4]thiadiazolo[3,4-a]
pyridazin-1-imines 5
5,6,7,8-Tetrahydro-[1,3,4]thiadiazolo[3,4-a]pyridazin-1-imine
(5a)
A
mixture of 1 mmol (0.145 g) hexahydropyridazine-1-
carbothioamide (1a), 1 mL methanol, and 0.1 g 30% aqueous
formaldehyde solution (approximately 1 mmol formaldehyde)
was refluxed (bath temperature 120 ◦C) for 3 h. After cooling,
overnight the resulting viscous mass was triturated with some
methanol and the formed crystals were separated. The crystals
were washed with a little methanol and dried on a clay plate.
Yield 23%. Colorless crystals. M.r. 209–213 ◦C (methanol).
TLC (m. P. II): Rf = 0.73. IR (cm−1): ν˜ = 1526 (NH); 1563
(C = N); 2854, 2940 (CH2); 3183 (NH). MS (70 eV, 255 ◦C):
m/z (%): 157.1 [M+.] (24); 105.0 (14); 98 (19); 97.0 (30); 87.0
(21); 77.1 (13); 75.0 (12); 74.1 (28); 70.0 (16); 57.1 (21); 56.1
(12); 55.0 (25); 43.0 (34); 42.0 (30); 41.0 (30); 31.9 (25); 28.9
(19); 28.9 (15); 27.8 (100); 26.9 (14).
Method 2: Preparation by heating of 1a with ketones in
a microwave reactor - general procedure: 1 mmol (0.145 g)
hexahydropyridazine-1-carbothioamide 1a and 1.1 mmol of the
appropriate ketone were reacted in a microwave reactor under
the described conditions (power, temperature, pressure). Subse-
quently, the mixture was cooled at 4 ◦C for 24 h. The product
was suspended in a small amount of the solvent given below.
The formed crystalline product was collected, washed carefully
with the mentioned solvent and dried.
Hydrochloride of 5a:
In this manner the following compounds were obtained:
0.1 mmol (0.016 g) 5a was dissolved in concentrated hydrochlo-
ric acid (some drops) and after dilution with methanol (about
2 mL) the solution was slowly concentrated at room tempera-
ture. The formed crystalline 5a hydrochloride was filtered off,
washed with a little methanol and dried at room temperature in
vacuo. It was utilized for the following analytical investigations.
Yield 85%. Yellow crystals. M.r. 205–230 ◦C (methanol).
HPLC (m. P. V): RP-Select B, t0 = 1.98; tR = 2.55; k’ = 0.29.
1H NMR (DMSO-d6), ppm): ␦ = 1.57 (bs, 2H, C(7)H2); 1.79 (t,
J = 10.4 Hz, 2H, C(6)H2); 2.95 (t, J = 10.0 Hz, 4H, C(5(H2 and
C(8)H2); 4.89 (s, 2H, C(3)H2); 10.02 (bs, 1H, –NH2+); 10.47
(bs, 1H, –NH2+). 13C NMR (DMSO-d6), ppm): ␦ = 22.33(C6);
22.66 (C7); 46.19 (C8); 51.45 (C5); 58.49 (C3); 169.20 (C = N).
HRMS [(ESI) m/z]: calcd. for C6H11N3S (base) + H+: 158.0746;
found [M (base) + H+]: 158.0783
3,3-Dimethyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione (3a)
With acetone (0.063 g). 150 W, 125 ◦C, 10 bar. Reaction time
40 min. Acetone as solvent for working up. Yield 66%.
3-Methyl-3-phenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione (3d)
With acetophenone (0.132 g). 300 W, 130 ◦C, 15 bar. Reaction
time 30 min. Methanol as solvent for working up. Yield 49%.
3,3-Diphenyl-2,3,5,6,7,8-hexahydro-1H-[1,2,4]triazolo
[1,2-a]pyridazine-1-thione (3h)
With benzophenone (0.200 g). 300 W, 130 ◦C, 15 bar. Reaction
time 30 min. Methanol as solvent for working up. Yield 61%.
3-(2-Chlorophenyl)-3-phenyl-2,3,5,6,7,8-hexahydro-1H-
[1,2,4]triazolo[1,2-a]pyridazine-1-thione (3i)
N-Phenyl-5,6,7,8-tetrahydro-[1,3,4]thiadiazolo
[3,4-a]pyridazin-1-imine (5b)
Pharmazie 69 (2014)
741

ORIGINAL ARTICLES
A
mixture of 0.5 mmol (0.111 g) N-phenyl-hexahydro-
NMR (DMSO-d6, ppm): ␦ = 1.06 (bs, 2H, heterocycl. C(4)H2);
1.79 (m, 4H, heterocycl. C(3)H2 and C(5)H2); 2.96 (bs, 2H,
heterocycl. C(2)H2 and C(6)H2) and 3.34 (bs, 2H, hetero-
pyridazine-1-carbothioamide 1b and 0.070 g 30% aqueous
formaldehyde solution (approximately 0.7 mmol formaldehyde)
was allowed to stand at room temperature for 2 days in a sealed
reaction vessel. Subsequently, 5 mL water were added and the
formed crystals were separated, washed with some water, and
dried in vacuo. Yield 75%. Colorless crystals. M.r. 59-62 ◦C
(water).
TLC (m. P. I): Rf = 0.70. HPLC (m. P. VIII): RP-select B,
t0 = 1.95; tR = 7.61; k’ = 2.90. IR (KBr, cm−1): ν˜ = 1487, 1586
(arom. ring); 1612 (C = N); 2824, 2851, 2938, 2993 (aliph. H);
3028, 3053, 3076 (arom. H). 1H NMR (CDCl3, ppm): ␦ = 1.61
(m) and 1.87 (m, 4H, C(6)H2 and C(7)H2); 2.72 (bs, 1H, C(5)H);
3.03 (bs, 1H, C(5)H); 3.34 (bd, 1H, C(8)H); 4.23 (bs, 1H,
C(3)H2) and 4.35 (bs, 1H, C(3)H2); 4.73 (bs, 1H, C(8)H);
6.93–7.10 (m, 3H, arom. H); 7.26–7.39 (m, 2H, arom. H). 13C
NMR (CDCl3, ppm): ␦ = 20.60 (C6); 24.02 (C7); 45.66 (C8);
50.35 (C5); 58.11 (C3); 121.52, 123.39, 128.93, 152.91 (6
arom. C) 160.73 (C = N). MS (70 eV, RT) m/z (%): 232.9 (23)
[M+·]; 134.9 (3) [C6H5-N = C = S+]; 98.0 (100) [M+· – ·C6H5-
N = C = S]; 96.9 (35); 77.3 (10) [C6H5+]; 42.1 (21); 28 (13).
HRMS [(ESI) m/z): calcd. for [C12H15N3S + H]+: 234.1059;
found [M + H]+: 234.1060
3
cycl. C(2)H2 and C(6)H2); 4.61 (bdd, JH(N)H = 6.4 Hz, 2H,
3
-N-CH2-NH-); 8.22 (d, JHH = 9.0 Hz, 2H, arom. C(2’)H and
C(6’)H); 8.38 (d, 3JHH = 9.0 Hz, 2H, arom. C(3’) and C(5’)H).
3
10.03 (t, JH(N)H = 6.4 Hz, 1H, amide NH); 10.23 (bs, 1H, het-
erocycl. NH+). 13C NMR (DMSO-d6, ppm); ␦ = 21.08 (C4),
22.11 (C3 and C5), 49.98 (C2 and C6) (all heterocycl. C);
59.27 (N–CH2–NH-); 123.57, 129.34, 138.22, 149.60 (all arom.
C); 166.24 (C = O). HRMS [(ESI) m/z]: calcd. for [M for
C12H16N4O3 (base) + H+]: 264.1336; found [M (base) + H+]:
264.1343. Elemental analysis [C13H17N3O3 (263.3), %]: calcd.
C, 59.30; H, 6.51; N, 15.96; found C, 58.07; H, 6.08; N, 16.16.
Hydrochloride of 10b:
Colorless crystals. M.r. 191–201 ◦C (ethanol) ((Foldeak et al.
1962): 215 ◦C).
3.2.7. N-Substituted phthalimide derivatives 10c and 12
2-(Piperidin-1-ylmethyl)-1,3-dihydroisoindoline-1,3-dione
(10c)
Prepared by the described procedure (Feldman, Wagner
1942) however, in a small batch containing 1 mmol piperi-
dine (0.085 g), 0.1 g 30% aqueous formaldehyde solution
(approximately 1 mmol formaldehyde) and 1 mmol (0.147 g)
phthalimide and utilizing ethanol as a solvent. Yield 86%. Col-
orless crystals. M.r. 121–123 ◦C (ethanol) (Feldman, Wagner
1942): 119–119.5 ◦C (ethanol)).
TLC (m. P. IV): Rf = 0.88. HPLC (m. P. VII): RP-18, t0 = 1.75;
tR = 2.78; k’ = 0.59. IR (cm−1): ν˜ = 1463; 1611; 1702, 1769
(C = O); 2811, 2852 (aliph. H); 2928 (peak of an absorp-
tion band, arom. H). 1H NMR (CDCl3, ppm): ␦ = 1.37 (m,
2H, piperidino C(4)H2); 1.57 (m, 4H, piperidino C(3)H2 and
C(5)H2); 2.61 (t, 3JH(C3/5)H = 10.4 Hz, 4H, piperidino C(2)H2 and
C(6)H2); 4.63 (s, 2H, N-CH2-N); 7.74 (m, 2H, arom. H); 7.88
(m, 2H, arom. H). 13C NMR (CDCl3, ppm): ␦ = 23.79 (C4),
25.99 (C3 and C5), 51.95 (C2 and C6) (all piperidino C); 60.35
(N-CH2-N); 123.40 (arom. C4’ and C7’); 132.05 (arom. C3a’,
C7a’); 134.06 (arom. C5’ and C6’); 169.42 (2 x C = O). HRMS
[(ESI) m/z]: calcd. for [C14H16N2O2 + H]+: 245.1285; found:
245.1248 [M + H]+.
3.2.6. 4-Nitrobenzamides 9b und 10b
4-Nitro-N-(hexahydropyridazin-1-ylmethyl)benzamide
hydrochloride (9b HCl)
A mixture of 5 mmol (0.981 g) 8 hydrochloride, 5 mmol
(0.831 g) 4-nitrobenzamide, 5 mmol (0.150 g) paraformalde-
hyde and 10 mL ethanol was refluxed for 5 h. Subsequently, the
reaction mixture was allowed to stand for 12 h at room tempera-
ture. The formed crystals were filtered off, washed with acetone,
and recrystallized from water. The formed crystals were sepa-
rated and washed once again with acetone. Yield 62%. Colorless
crystals. M.r. 189–192 ◦C (water/acetone).
TLC (m. P. IV): Rf = 0.50. HPLC (m. P. V): RP-select B,
t0 = 1.75; tR = 2.82; k’ = 0.61.
IR (KBr, cm−1): ν˜ = 1344, 1518 (NO2); 1530 (NH); 1602;
1669 (C = O); 2448, 2533, 2558, 2715, 2795, 2854, 2933,
2953 (peaks of a broad absorption band, aliph. H); 3038,
3069 (arom. H); 3105, 3246 (NH). 1H NMR (DMSO-d6, ppm):
␦ = 1.70 (bs, 4H, heterocycl. C(4)H2 and C(5)H2); 2.95 (bs, 2H;
heterocycl. C(6)H2); 3.17 (bs, 2H, heterocycl. C(3)H2); 4.56
2,2’-(Hexahydropyridazin-1,2-ylendimethylene)-di(1,3-
dihydroisoindoline-1,3-dione) (12)
3
3
(d, JHH = 6.2 Hz, 2H, -N-CH2-NH-); 8.23 (d, JHH = 8.8 Hz,
3
2H, arom. C(2’)H and C(6’)H); 8.36 (d, JHH = 8.8 Hz, 2H,
A mixture of 1 mmol (0.123 g) 8 hydrochloride, 1 mmol
(0.147 g) phthalimide, 5 mL ethanol, 0.1 g 30% aqueous
formaldehyde solution (approximately 1 mmol formaldehyde)
and 1 mmol (0.101 g) triethyl amine was refluxed (bath tem-
perature 120 ◦C) for 5 h. After cooling and during standing
overnight, crystals were formed, which were separated, washed
with ethanol, and dried on a clay plate. Yield 5%. Pale yellow
crystals. M.r. 194–196 ◦C (ethanol).
TLC (m. P. IV): Rf = 0.93. HPLC (m. P. VII): RP-18, t0 = 1.75;
tR = 7.19; k’ = 3.11. IR (cm−1): ν˜ = 1706, 1772 (C = O);
2858 (aliph. H)., 2940 (arom. H). 1H NMR (CDCl3, ppm):
␦ = 1.65 (t, J = 7.0 Hz, 4H, hexahydropyridazine C(4)H2 and
C(5)H2); 3.16 (bs, 4H, hexahydropyridazine C(3)H2 and
C(6)H2); 4.56 (s, 4H, 2 x N-CH2-N); 7.47–7.60 (m, 8H,
3
arom. C(3’) and C(5’)H); 9.65 (t, JHH = 6.2 Hz, 1H, amide
NH); 10.28 (bs, 2H, heterocycl. NH2+). 13C NMR (DMSO-
d6, ppm); ␦ = 19.49 (C4), 21.96 (C5), 48.69 (C6), 48.72 (C3)
(all heterocycl. C); 59.17 (-N-CH2-NH-); 123.38 (C3’, C5’),
129.29 (C2’, C6’), 139.18 (C1’), 149.28 (C4’); 166.28 (C = O).
MS (70 eV, 345 ◦C) m/z (%): 263.9 (5) [M+·Base]; 165.9 (15)
[M+– CH2C4H8N2]; 149.0 (28); 103.7 (11); 98.0 (21); 85.0
(22) [C4H9N2+]; 36.0 (15); 32.0 (22); 30.0 (13); 28 (100).
HRMS [(ESI) m/z]: calcd. for [M for C12H16N4O3 (base) + H+]:
265.1292; found [M (base) + H+]: 265.1295. Elemental anal-
ysis [C12H17ClN4O3 (300.74), %]: calcd. C, 47.92; H, 5.70;
N, 18.63; found C, 47.69; H, 5.74; N, 18.90.
4-Nitro-N-(piperidin-1yl-methyl)benzamide (10b)
Prepared by the procedure as described before (Foldeak et al.
1962). Yield 78%. Yellowish crystals. M.r. 122-128 ◦C (ethanol)
((Foldeak et al. 1962): 205 ◦C).
all arom. H). 13C NMR (CDCl3, ppm): ␦ = 19.97 (hexahy-
dropyridazine C4 and C5); 43.37 (hexahydropyridazine C3
and C6); 54.95 (2 x N-CH2-N); 123.00 (arom. C4’, C7’,
C4”, C7”); 132.04 (arom. C3a’, C7a’ C3a”, C7a”); 133.48
(arom. C4’, C5’, C4”, C5”); 167.55 (C = O, C1’, C3’, C1”,
C3”). MS (70 eV, 180 ◦C) m/z (%): 405.1 (8) [M + H+]; 244.7
(51) [C13H14N3O2+]; 160.4 (100) [C9H6NO2+]; 133.3 (13);
TLC (m. P. IV): Rf = 0.12. IR (KBr, cm−1): ν˜ = 1523 (NO2);
1534 (NH); 1599; 1660 (C = O); 2703, 2796, 2850, 2914,
2930, 2969 (peaks of a broad absorption band, aliph. and
arom. H,); 3168 (amide NH); 3286 (heterocycl. NH+). 1H
742
Pharmazie 69 (2014)

ORIGINAL ARTICLES
105.2 (10); 104.2 (17); 97.2 (69); 77.2 (24); 76.2 (17). HRMS
there are transformed into ␦ values here: 1.65 (m, 8H); 2.39
(m, 4H, –N–CH2–C); 2.78 (d, J = 11 Hz, 4H, –N–CH2–CH2–);
3.05 (d, J = 9 Hz, 2H, –N–CH2–N–); 3.55 (d, J = 9 Hz, 2H,
–N–CH2–N–]. 13C NMR (CDCl3, ppm): ␦ = 23.84 (C2, C3,
C9, C10); 52.60 (C1, C4, C8, C11); 79.77 (C6 and C13).
HRMS [(ESI) m/z): calcd. for [C10H20N4 + H]+: 197.1761;
found [M + H]+: 197.1698
[(ESI) m/z]: calcd. for [C22H20N4O4 + H]+: 405.1557; found:
405.1549 [M + H]+.
3.2.8. Anellated hexahydropyridazine derivatives 11,13,
and 14
1,2,3,4,6,11-Hexahydropyridazino[1,2-b]phthalazine-6,11-
dione (14)
5,6,7,8-Tetrahydro[1,3,4]thiadiazolo[3,4-a]pyridazine-1,3-
dithione (11)
To a solution of 1 mmol (0.123 g) 8 hydrochloride and 1 mmol
(0.185 g) potassium salt of phthalimide in 2 mL water 0.1 g
30% aqueous formaldehyde solution (approximately 1 mmol
formaldehyde) was added and the mixture was irradiated for
5 min in a microwave reactor (90 W, 130 ◦C, 3.3 bar). After cool-
ing, the resulting crystals were collected, washed with water, and
dried in vacuo at room temperature.
Method 1 - from 8 hydrochloride and carbon disulphide: A
mixture of 1 mmol (0.123 g) 8 hydrochloride, 5 mL methanol,
1 mmol (0.101 g) triethyl amine, and 15 mmol (1.142 g) car-
bon disulphide was heated (bath temperature 80–90 ◦C). Within
10 min fine needles crystallized. After the reaction was com-
pleted (TLC monitoring, about 30 min), the crystals were filtered
off and washed with some ethanol. Yield 82%. Colorless nee-
dles. M.r. 188–190 ◦C (ethanol).
Yield 9%. Yellowish crystals. M.r. 135-141 ◦C (water) [(Rink
and Grabowski 1956):158-160 ◦C (water); (Clement 1960): two
forms of crystals were isolated - swords 145.5-146.5 ◦C (water)
and fine needles 156-156.5 ◦C (water)].
TLC(m. P. I):Rf = 0.65. HPLC(m. P. VII):RP-selectB, t0 = 1.88;
tR = 6.94; k’ = 2.69. IR (KBr, cm-1): ν˜ = 1191 (C = S, extraor-
1
dinary intensively); 1403; 2858, 2929, 2952 (CH2). H NMR
TLC (m. P. IV): Rf = 0.86. HPLC (m. P. VII): RP-18,
t0 = 1.75; tR = 3.01; k’ = 0.53. IR (cm-1): ν˜ = 1602; 1633;
1664; 1710 (C = O); 2850 (aliph. H); 2949 (arom. H). 1H
NMR (CDCl3, ppm): ␦ = 1.97 (m, 4H, C(2)H2, C(3)H2); 4.19
(t, 3J = 1.4 Hz, 4H, C(1)H2, C(4)H2); 7.28–7.85 (m, 2H,
C(7)H, C(10)H); 8.26–8.35 (m, 2H, C(8)H, C(9)H). 13C NMR
(CDCl3, ppm): ␦ = 22.14 (C2, C3); 44.32 (C1, C4); 127.70 (C7,
C10), 128.85 (C6a, C10a); 133.39 (C8, C9); 158.51 (C = O,
C6, C11). HRMS [(ESI) m/z]: calcd. for [C12H12N2O2 + H]+:
217.0972; found [M + H]+: 217.0964.
(CDCl3, ppm): ␦ = 2.05 (m, 4H, C(6)H2, C(7)H2); 4.20 (m, 4H;
C(5)H2, C(8)H2). 13C NMR (CDCl3, ppm): ␦ = 20.73 (C6, C7);
49.76 (C5, C8); 181.97 (2 x C = S). MS (70 eV, 60 ◦C) m/z (%):
204.1 (100) [M+·]; 84.1 (6) [C4H8N2+]; 72.0 (27); 41.1 (29); 32
(14), 29 (12); 27.9 (65). Elemental analysis [C6H8N2S3 (204.3),
%]: calcd. C, 35.27; H, 3.95; N, 13.71; S, 47.07; found C, 35.96;
H, 3.91; N, 13.59; S, 46.66.
Method 2 - as a product of the attempt of the cyclization of
9b hydrochloride with carbon disulphide: To a suspension of
0.33 mmol (0.099 g) 9b hydrochloride in 5 mL ethanol 1 mmol
(0.101 g) triethyl amine and 15 mmol (1.142 g) carbon disul-
phide were added. The mixture was heated (bath temperature
80–90 ◦C) for 6 h. Subsequently, the solution was concen-
trated and with the resulting product (about 50 mg) was done
a column chromatographic separation (solid phase silica gel H
Merck; mobile phase petroleum ether/ethyl acetate 5:1 (v/v),
column: length 10 cm, diameter 1 cm). The first fraction (TLC
monitoring) was evaporated to dryness, the resulting crystals
were collected, washed with some acetone and dried in vacou.
Yield 31%. Pale yellow crystals. M.r. 180–184 ◦C [petroleum
ether/ethyl acetate 5:1 (v/v)].
3.3. Biology
Material and methods are described in detail in a previous pub-
lication (Schulz et al. 2013). Here are the key parameters given.
3.3.1. Cell culture materials
All chemicals as well as interleukin-1␤, fetal calf serum and try-
pane blue were from Sigma Aldrich. Cell culture plastics, PBS,
trypsin/EDTA and interferon-␥ were obtained from Biochrom
AG, RPMI1640 and penicillin/streptomycin from Lonza.
TLC (m. P. I): Rf = 0.65. Mixed melting temperature with the
substance yielded by method 1: 184–189 ◦C.
1,2,3,4,8,9,10,11-Octahydrodipyridazino
[1,2-a:1’,2’-d][1,2,4,5]tetrazine (13)
3.3.2. Cell line
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% CO2) in RPMI1640 supplemented with L-glutamine
(2 mmol/L), 10% heat-inactivated FCS, penicillin (100 I.E.) and
streptomycin (100 I.E.).
A mixture of 1 mmol (0.123 g) 8 hydrochloride, 1 mmol
(0.185 g) potassium salt of phthalimide, 2 mL water, and 0.1 g
30% aqueous formaldehyde solution (approximately 1 mmol
formaldehyde) was refluxed for 6 h (bath temperature 120 ◦C).
After cooling overnight, the mixture was allowed to concentrate
at room temperature. The resulting solid was digested several
times with anhydrous diethyl ether. The combined diethyl ether
fractions were evaporated to dryness, and the resulting product
was dried at room temperature. Yield 13% Yellowish crystals.
M.r. 150–160 ◦C (diethyl ether, impurities by phthalimide and
8 hydrochloride). ((Schulz et al. 2013): 168 ◦C (ethanol/diethyl
ether)).
TLC (m. P. IV): Rf = 0.80. HPLC (m. P. VII): RP-18, t0 = 1.75;
tR = 3.02; k’ = 0.53. IR (cm-1): ν˜ = 2696, 2735, 2776, 2848,
2936 (aliph. H). 1H NMR (CDCl3, ppm): ␦ = 1.64 (bs, 8H,
C(2)H2, C(3)H2, C(9)H2 and C(10)H2); 2.37 (bs, 4H, je 1H
of C(1)H2, C(4)H2, C(8)H2, C(11)H2); 2.78 (d, 4H, J = 10.2 Hz,
je 1H of C(1)H2, C(4)H2, C(8)H2, C(11)H2); 3.06 (d, J = 8.4 Hz,
2H, je 1H von C(6)H2, C(13)H2); 3.53 (d, J = 8.4 Hz, 2H, je 1H
of C(6)H2, C(13)H2) [(Nelsen, Hintz 1972), the ␶ values given
3.3.3. iNOS expression and inhibition
Tests were performed with 1 × 105 cells per well in 200 ␮L
culture media in 96-well plates at standard culture conditions.
Twenty-four hours after cells had been seeded out, iNOS was
induced by adding 1 ng/mL IL-1␤ (human, recombinant) and
10 ng/mL IFN-␥ (rat, recombinant). For the primary screening,
test compounds were added to the cells at concentrations of
78 ␮M and 39 ␮M with 0.5% DMSO as solutizer. The reference
inhibitor aminoguanidine was tested at the same concentrations.
DMSO was added to the controls as well. Selected substances
with considerably higher inhibition of iNOS than aminoguani-
dine were chosen for determination of IC50 that was calculated
Pharmazie 69 (2014)
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ORIGINAL ARTICLES
from linear regression of logarithmized concentrations versus
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3.3.4. NO assay
Nitrite concentration, an indication for the degree of NO pro-
duction, was determined from 50 ␮L cell-free supernatant (two
samples from each cavity) using the Griess reaction (Green et al.
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amide solution in 5% phosphoric acid and an aqueous 0.01%
N-(1-naphthyl)ethylenediamine dihydrochloride solution were
separately pipetted to the supernatant and incubated for 5 min
in the dark. Afterwards, the absorption was measured in a plate
reader at a wavelength of 550 nm. For calibration, a dilution
series of nitrite (0 to 100 ␮M) was included on every plate.
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After 4 h incubation at 37 ◦C medium was carefully removed
and 100 ␮L DMSO were added. After dissolution of the for-
mazan crystals the absorption was immediately measured at a
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Acknowledgements: The authors gratefully thank Mr. Franz Studener, head
pharmacist at “Mohren-Apotheke”, Neumarkt 8, 99947 Bad Langensalza,
Germany, for generous financial support. The authors also wish to sincerely
thank Prof. Dr. Thomas von Woedtke (Campus PlasmaMed) and Dr. Kai
Masur (Centre of Innovation Competence plasmatis), of the INP Greifswald
e.V., for the generous technical support in the biological part of this project.
Finally, U.S. is grateful for the scholarship from the “Studienstiftung des
deutschen Volkes“, that helped make the project possible.
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