Synthesis, vasorelaxant activity and 2D-QSAR study of some novel pyridazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.12.015

Novel 3,6-disubstituted pyridazines were synthesized by facile method and screened for their vasorelaxant properties utilizing isolated thoracic rat aortic rings. Compounds 8a and 11a exerted potent vasorelaxant activity (IC₅₀ = 198 and 177 μM, respectively) relative to doxazosin mesylate (used reference standard, IC₅₀ = 226 μM), that, they may represent promising hits for treatment of cardiovascular disorders. The observed activity was validated by a statistically significant QSAR model (N = 32, n = 6, R² = 0.811782, R²_cvOO = 0.7153, R²_cvMO = 0.7209, F = 17.9708, s² = 9.65226 × 10^-8) that was obtained employing CODESSA-Pro software.

Full text of DOI:10.1016/j.ejmech.2015.12.015

Accepted Manuscript
Synthesis, vasorelaxant activity and 2D-QSAR study of some novel pyridazine
derivatives
Riham F. George, Dalia O. Saleh
PII:
S0223-5234(15)30397-4
10.1016/j.ejmech.2015.12.015
EJMECH 8255
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2015
Revised Date: 8 December 2015
Accepted Date: 9 December 2015
Please cite this article as: R.F. George, D.O. Saleh, Synthesis, vasorelaxant activity and 2D-QSAR
study of some novel pyridazine derivatives, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2015.12.015.
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ACCEPTED MANUSCRIPT
Synthesis, vasorelaxant activity and 2D-QSAR study of some novel pyridazine
derivatives
Riham F. George a,*, Dalia O. Salehb

ACCEPTED MANUSCRIPT
Synthesis, vasorelaxant activity and 2D-QSAR study of some novel pyridazine
derivatives
Riham F. George a,*, Dalia O. Salehb
a Pharmaceutical Chemistry department, Faculty of Pharmacy, Cairo University, ElKasr El-Aini street,
11562 Cairo, Egypt
b Pharmacology Department, National Research centre, Dokki, Giza 12622, Egypt
Abstract
Novel 3,6-disubstituted pyridazines were synthesized by facile method and screened for
their vasorelaxant properties utilizing isolated thoracic rat aortic rings. Compounds 8a
and 11a exerted potent vasorelaxant activity (IC50 = 198 and 177 ꢀM, respectively)
relative to doxazosin mesylate (used reference standard, IC50 = 226 ꢀM), that, they may
represent promising hits for treatment of cardiovascular disorders. The observed activity
was validated by a statistically significant QSAR model (N=32, n=6, R2=0.811782,
R2
= 0.7153, R2
= 0.7209, F=17.9708, s2=9.65226x10-8) that was obtained
cvOO
cvMO
employing CODESSA-Pro software.
Keywords: pyridazines, vasodilator, QSAR
* Corresponding author. Tel.: +2 01222979898, E-mail address:
rihamfgeorge@yahoo.com
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1.
Introduction
Cardiovascular diseases (CVDs), in particular coronary heart disease and stroke,
are the leading cause of mortality in the developed countries. According to the World
Health Organization (WHO), 17 million people die every year by CVDs, accounting for
almost one third of deaths worldwide per year [1]. Among cardiovascular disorders,
hypertension is known as a silent killer as it is the most common risk factor that can
cause coronary disease, myocardial infarction, stroke and sudden death. In addition, it is
the major contributor to cardiac failure and renal insufficiency [2,3]. Moreover,
hypertension is not only responsible for high morbidity and mortality, but it impacts
negatively the quality of life of a huge number of people across the world. Therefore,
prevention and treatment of hypertension is an important public health challenge. Over
the past decades, great efforts have been made to discover many antihypertensive drugs
that act through different mechanisms such as: diuretics [4], angiotensin-converting
enzyme inhibitors [5,6], angiotensin II receptor blockers [6,7], calcium channel blockers
[8], centrally sympathetic α2-adrenoreceptors stimulants [9], and drugs that prevent the
action of peripheral sympathetic activity as β-adrenergic [10,11] and α-adrenergic
blocking agents [12]. It is noteworthy that the reduction in blood pressure achieved with
most of the aforementioned classes of drugs is directly or indirectly related to the
relaxation of vascular smooth muscles; which makes vasorelaxation one of important
strategies in controlling hypertension [13]. Several agents have been developed; however
they are all associated with side effects such as fatigue, mood change, sleep disturbances,
etc [14]. Therefore, there is a continuous need to explore and develop new vasorelaxant
agents with minimal side effects.
The pyridazine nucleus represents a versatile scaffold to develop new
pharmacologically active compounds. This heterocyclic system has a wide range of
biological activities and can also be used to link other pharmacophoric groups [15–18].
For instance, the 6-(aryl or heteroaryl)pyridazinone derivatives represent an important
core with a wide pharmacological profile that includes interesting activities on
cardiovascular system, such as cardiotonic effects [19,20], antihypertensive activity [21]
and platelet aggregation inhibition [22]. Zardaverine I, levosimendan II and motapizone
III are some characteristic drugs bearing this pharmacophoric moiety [23–25] (Fig. 1).
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Most of the described pyridazinone derivatives, showing activity on cardiovascular
system, possess aryl residues at position 6 of the pyridazine ring [23,26,27]. Interestingly,
6-hydrazino-3-phenylpyridazine IV which exhibited strong hypotensive properties, is the
structural analog of the well known vasodilator hydralazine V that possesses a phenyl
ring attached to the pyridazine nucleous instead of being fused to it as in hydralazine [28]
(Figure 1). Encouraged by these findings; this work focused on design and synthesis of
some phenylpyridazines substituted with a heterocyclic ring, namely, pyrrolidine,
imidazole or pyrazole, (general structure A, Figure 2). These ring systems can be
considered as pharmacophoric moieties in many vasorelaxant agents [9,19,21,25,29–31].
Moreover, 6-hydrazino-3-phenylpyridazine IV was used for further structural
modification through derivatization of the hydrazine moiety into a semicarbazide group
(general structure B), or an aryl hydrazone (general structure C, Figure 2) in order to
study their effect on the modulation of the activity. Additionally, quantitative structure-
activity relationship (QSAR) studies were considered in the present work to validate the
obtained vasorelaxant activity and detect the most important structural parameters
controlling it.
Figure 1
Figure 2
2- Results and discussion
2.1. Chemistry
The targeted pyridazines were synthesized according to the general procedures
outlined in Schemes 1 and 2. Thus, the 6-aryl-3-pyridazinones 3a-d were synthesized
from the reaction of the appropriate acetophenone 1a-d and glyoxilic acid 2 followed by
cyclisation with hydrazine hydrate [32]. Chlorination of 3a-d with phosphorous
oxychloride afforded 3-chloro-6-arylpyridazines 4a-d which were further treated with
hydrazine hydrate in absolute ethanol to obtain the 3-hydrazino derivatives 5a-d [33].
The target compounds 6a-d and 7a-c were obtained from the precursor chloropyridazines
4a-d through a nucleophilic substitution reaction with imidazole and pyrrolidine,
respectively (Scheme 1). The structures of the obtained compounds 6a-d were confirmed
by 1H NMR which revealed three broad singlet signals at 7.30-7.32, 7.81-7.83 and 8.49-
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8.53 ppm corresponding to the imidazole protons. Compound 6b revealed triplet
corresponding to the imidazole proton at C2 at 7.83 ppm due to long range coupling with
protons at C4, C5 of the imidazole ring. However, the pyridazine protons of 6a-d
appeared as two doublets at 7.59-7.68 and 7.97-8.24 ppm, while the phenyl ring protons
of 6a revealed two multiplets at 7.53-7.59 and 8.10-8.12 ppm. In case of the p-substituted
derivatives 6b, 6d, two doublets appeared at the range 7.07-7.56 and 8.07 ppm. On the
other hand, the o-substituted derivative 6c showed doublet at 7.08 ppm corresponding to
the proton at C3 that coupled with H of C4, two doublet triplets at 7.18 and 7.49 ppm
corresponding to protons at C4 and C5 which coupled with protons at C3, C5, C6 and C4,
C6, C3, respectively, and one doublet of doublet at 8.04 ppm due to the proton at C6 that
coupled with protons at C5 and C4. Moreover, the appearance of multiplet signals in the
ranges 2.06-2.12 and a broad singlet at 3.63 ppm corresponding to the pyrrolidine protons
attested the obtained compounds 7a,b. Regarding compound 7c, the pyrrolidine protons
appeared as a multiplet at 1.98-2.01 ppm and a triplet at 3.56 ppm. Meanwhile,
cyclocondensation of the hydrazine derivatives 5a-d with acetyl acetone in absolute
ethanol afforded 3-(3,5-dimethyl-1H-pyrazol-1-yl)-6-arylpyridazines 8a-d. 1H NMR
spectra of 8c,d confirmed the obtained structure through the presence of three singlet
signals at 2.34, 2.81-2.82 and 3.89-3.90 ppm corresponding to two methyl and one
methoxy groups, respectively, in addition to a singlet attributed to the pyrazole proton at
6.08-6.09 ppm. Similarly, the reaction of 5a-d with either ethoxymethylene malononitrile
or ethyl ethoxymethylene cyanoacetate in absolute ethanol gave the 5-aminopyrazole
derivatives 9a-d and 10a-d, respectively. The IR spectra of 9b-d and 10 b-d showed the
NH2 stretching vibration as two bands at 3391-3285 and 3453-3312 cm-1, in addition
1
to the CN band at 2230-2210 cm-1 in case of compounds 9b-d. Moreover, H NMR
spectra of 10b-d revealed the triplet-quartet pattern of the C2H5 protons at 1.37-1.39 and
4.31-4.34 ppm along with two singlet signals at 7.60-7.62 and 7.82-7.84 ppm
corresponding to NH2 and pyrazole proton, respectively. The former signal for NH2 was
disappeared upon deuteration with D2O. On the other hand, nucleophilic addition of
hydrazines 5a-d to the appropriate isocyanate in methylene chloride in the presence of
triethylamine afforded the semicarbazides 11a-c, whose structures were confirmed by the
appearance of three NH bands at 3329-3300, 3250-3223 and 3138-3134 cm-1 in the IR
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1
spectra. In addition, H NMR showed three singlet signals corresponding to three NH
groups that were exchanged with D2O. Additionally, the condensation of the hydrazine
derivatives 5a-d with different aromatic aldehydes in absolute ethanol in the presence of
catalytic amount of glacial acetic acid revealed the corresponding phenyl hydrazones
12a-h. These derivatives were characterized by their spectral and elemental data, where
1H NMR spectra revealed the presence of the characteristic singlet signal of methylidene
proton at 8.09-8.43 ppm in addition to a singlet signal of NH at 7.84-7.95 or 11.40-11.93
ppm that was disappeared upon treatment with D2O.
Scheme 1
Scheme 2
2.2. In-vitro vasorelaxant activity
Vasorelaxant properties of the synthesized pyridazines were investigated in vitro
using isolated thoracic aortic rings of rats precontracted with norepinephrine
hydrochloride standard according to the reported procedure [34-40] and compared with
doxazosin mesylate as a reference standard. The observed data (Table 1) revealed that all
pyridazine derivatives exhibited remarkable vasodilating activity with IC50 values
ranging from 177–323 ꢀM compared to the reference standard doxazosin mesylate (IC50
= 226 ꢀM). It is noteworthy that thirteen compounds (5a, 6c, 8a, 8b, 10c, 10d, 11a, 11c,
12a, 12c, 12d, 12e, 12h) showed higher activity than doxazosin mesylate (Table 1).
Structure activity relationship (SAR) based on the observed vasorelaxant activity
indicated that the impact of the electronic nature of the substituent X viz H, 4-Cl, 2-
OCH3, 4-OCH3 upon the activity is not absolute. However, substitution with different R
resulted in either positive or negative change in the observed activity. Thus, considering
the hydrazine derivatives 5a–d as the prototypes of the target compounds, it could be
concluded that best results were obtained when the hydrazine group was structurally
extended to the semicarbazides 11a–c and hydrazones 12a–h. Moreover, replacement of
the hydrazine moiety with rigid azacyclic groups was sometimes in favor of activity as in
some
imidazolyl
6b,c,
3,5-dimethylpyrazolyl
8a,b
and
5-amino-4-
ethoxycarbonylpyrazolyl derivatives 10b–d. However, with other ring system such as the
pyrrolidinyl 7a–c and 5-amino-4-cyanopyrazolyl 9a–d, the activity was diminished.
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2.3. 2D- QSAR modeling
2.3.1. Dataset
QSAR is capable of generating a relationship between the chemical structure of an
organic compound and its aximu-chemical properties. The vasorelaxant active
pyridazines 5a-d, 6a-d, 7a,c, 8a–d, 9a–d, 10a–c, 11a–c and 12a–h were used as a
training set in the present QSAR study (homogeneous dataset of common heterocyclic
scaffold). The QSAR study was undertaken using comprehensive descriptors for
structural and statistical analysis (CODESSA-Pro) software.
2.3.2. Methodology
Geometry of the training set compounds was optimized using the molecular mechanics
force field (MM+) followed by the semi-empirical AM1 method implemented in the
HyperChem 8.0 package. The structures were fully optimized without fixing any
parameters, thus bringing all geometric variables to their equilibrium values. The energy
minimization protocol employed the Polak–Ribiere conjugated gradient algorithm.
Convergence to a local minimum was achieved when the energy gradient was ≤0.01 kcal
mol-1. The RHF (Restricted Hartree-Fock) method was used in spin pairing for the two
semi-empirical tools [41–50]. The resulting output files were exported to CODESSA-Pro
that includes MOPAC capability for the final geometry optimization.
CODESSA-Pro calculated 725 molecular descriptors including constitutional,
topological, geometrical, charge-related, semi-empirical, thermodynamic, molecular-
type, atomic-type and bond-type descriptors for the exported 32 bio-active pyridazine-
based compounds which were used as training set in the present study. Different
mathematical transformations of the experimentally observed vasodilation
property/activity (IC50, ꢀM) of the training set compounds were utilized for the QSAR
modeling determination including property (IC50, ꢀM), 1/property, log(property) and
1/log(property) values in searching for the best QSAR model.
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2.3.3. QSAR modeling
The best multi-linear regression (BMLR) technique was utilized which is a stepwise
search for the best n-parameter regression equations (where n stands for the number of
descriptors used), based on the highest R2 (squared correlation coefficient), R2cvOO
(squared cross-validation “leave one-out, LOO” coefficient), R2cvMO (squared cross-
validation “leave many-out, LMO” coefficient), F (Fisher statistical significance criteria)
values, and s2 (standard deviation). The QSAR models up to 6 descriptor model
describing the bio-activity of the vasorelaxant pyridazine-based active agents were
generated (obeying the thumb rule of 5:1, which is the ratio between the data points and
the number of QSAR descriptor). The observed and predicted values of the training set
compounds 5a–d, 6a–d, 7a,c, 8a–d, 9a–d, 10a–c, 11a–c and 12a–h according to the
multilinear QSAR models are presented in Table 1.
The statistical characteristics of the BMLR-QSAR model attained are presented in
Table 2. The established QSAR model is statistically significant. The descriptors are
sorted in the descending order of the respective values of the Student’s t-criterion, which
is a widely accepted measure of statistical significance of individual parameters in
multiple linear regressions. Figure 3 shows the QSAR multi-linear model plot of
correlations representing the observed vs. predicted 1/IC50 (ꢀM) values for the
vasodilation pyridazine-based active agents. The scattered plots are uniformly distributed,
covering ranges, observed 0.0031-0.0056, predicted 0.0030-0.0056 log (IC50, ꢀM) units.
Figure 3
2.3.4. Molecular descriptors
Molecular descriptors are the aximu-chemical parameters used to correlate the chemical
structure and the bio-property value expressed as 1/IC50, ꢀM. The descriptors were
obtained based on the BMLR method. The first descriptor controlling the attained
BMLR-QSAR model based on its level of significance (t-criterion = 6.4616) is FHACA
fractional HACA (HACA/TMSA) (MOPAC PC) which is a charge-related descriptor..
Fractional hydrogen bonding acceptor ability of the molecule FHACA1 is determined by
equation (1) [51].
FHACA1 = ꢀꢁꢂꢁꢃ ……………….. (1)
ꢄꢅꢆꢁ
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Where, HASA1 is the hydrogen bonding acceptor ability, TMSA is the total molecular
surface area. The second most important descriptor controlling the BMLR-QSAR model
(t = 4.6771) is shadow plane YZ, which is a geometrical descriptor reflecting molecular
size. Most geometrical descriptors are calculated directly from the (x,y,z) coordinates and
other quantities derived from the coordinates, e.g. interatomic distances or distances from
a specified origin (e.g. the molecule bary-center) [52,53]. The third and fourth descriptors
of QSAR model based on their t-value (4.1118, -4.9294, respectively) are HA dependent
HDSA-1 (Zefirov PC) (all) and HA dependent HDSA-1/TMSA (MOPAC PC) (all),
which are a charge-related descriptors. The hydrogen bonding acceptor ability of the
molecule HASA1 is determined by equation (2) [51].
∑
HASA1 = ꢁ ꢇꢁ A ∈ XH-acceptor ……………….. (2)
Where, sA stands for solvent-accessible surface area of H-bonding acceptor atoms. The
fifth descriptor of the attained QSAR model (t = -5.7082) is structural information
content (order 2), which is a topological descriptor determined by equations (3, 4) [51].
kSIC = kIC / log2 n ……………………. (3)
ꢈ
ꢍ
k
ꢉ log2 ꢉ ……………………. (4)
ꢊ
∑
IC = ‒
ꢋꢌꢃ
ꢍ
ꢈ
Where ni stands for number of atoms in the ith class, n is the total number of atoms in the
molecule, k is the number of atomic layers in the coordination sphere around a given
atom that are accounted for. The last descriptor controlling the BMLR-QSAR is
8aximum nucleophilic reaction index for atom C, which is a semi-empirical descriptor.
Molecular descriptor values controlling the attained BMLR-QSAR model are presented
in Table 3.
2.3.5. Validation of the BMLR-QSAR model
2.3.5.1. Internal validation
Internal validation is applied by the CODESSA-Pro technique employing both leave one
out (LOO), which involves developing a number of models with one example omitted at
a time, and leave many out (LMO), which involves developing a number of models with
many data points omitted at a time (up to 20% of the total data points). The observed
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correlations by the internal validation technique are R2cvOO = 0.715, R2cvMO = 0.721,
respectively which are significantly correlated with the squared correlation coefficient of the
attained QSAR model (R2 = 0.812). Standard deviation of the regression (s2 = 9.652 x 10-
8) is also a measurable value for the attained model together with the Fisher test value (F
= 17.971) that reflects the ratio of the variance explained by the model and the variance
due to their errors. A high value of the F-test relative to the s2 value is also validation of
the model.
The predicted vasodilation properties due to the attained QSAR model of most of
the synthesized pyridazines are compatible with their experimentally observed values
preserving their relative potencies (Table 1). The predicted IC50 value of compound 11a,
which is considered the most potent analogue among all the synthesized pyridazines (IC50
= 179 ꢀM) is compatible to its experimentally observed value (IC50 = 177 ꢀM) with
minor error (error “difference between the observed and predicted values” = -2). The
same words for the second most potent pyridazine analogue 8a (IC50 = 198, 210 ꢀM for
the observed and predicted values, respectively, error = -12). Compounds 5a, 6c, 8b, 10c,
11c, 12a, 12c and 12e which exhibit experimental potency higher than that of doxazosin
mesylate (standard reference used), reveal predicted properties correlated with their bio-
data (IC50 = 206, 205, 213, 225, 207, 209, 217, 205; 214, 213, 216, 232, 198, 215, 219,
201 for the observed and predicted values of 5a, 6c, 8b, 10c, 11c, 12a, 12c and 12e ꢀM,
respectively). Although high error values due to the observed and predicted vasodilation
properties of compounds 12d and 12h (which are considered high potent analogue) are
slightly high, their bio-properties are still preserved (IC50 = 200, 221; 214, 244 ꢀM for the
observed and predicted values of 12d and 12h, respectively; error = -14, -23 for
compounds 12d and 12h, respectively). Additionally, correlated predicted bio-data were
also revealed due to the QSAR modeling by the promising vasodilation active
pyridazines (IC50 = 230-323, 226-331 ꢀM for the observed and predicted values) of
observed potencies lower than that of doxazosin mesylate.
2.3.5.2. External validation
Compounds 7b and 10d were used as an external test set not only for validating the
attained QSAR model but also for examining its predicative ability. The test set
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analogues experimentally exhibit higher and lower vasodilation potencies relative to the
used standard reference (doxazosin mesylate). The variation in potency can indicate the
predication capabilities of the attained BMLR-QSAR model. Table 4 reveals the
observed and predicted IC50 values of the test set compounds. From the observed data, it
has been noticed that compound 10d which is considered a high potent agent (IC50 = 216
ꢀM) reveals a predicted IC50 = 214 ꢀM with minimum error value = 2. Compounds 7b
that is considered a low potent vasorelaxant active analogue (IC50 = 300 ꢀM) afforded
predicted IC50 = 283 ꢀM (error value = 17).
All the above internal and external validation observations give good sign for the
predictive capability of the attained BMLR-QSAR model and support the previous
statement concerning its predictive power due to the statistical values.
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Table 1. Observed and predicted vasorelaxant activity (IC50, ꢀM) of the training set compounds 5a–d, 6a–d, 7a,c, 8a–d, 9a–d, 10a–c,
11a–c and 12a–h and doxazosin mesylate.
Entry Compd.
R
X
Observed IC50, ꢀM Predicted IC50, ꢀM Error
1
2
3
4
5
6
7
8
NHNH2
H
206
275
279
235
273
255
205
259
214
257
259
252
256
257
213
269
-8
5a
5b
5c
5d
6a
6b
6c
6d
NHNH2
4-Cl
18
20
-17
17
-2
NHNH2
2-OCH3
4-OCH3
H
NHNH2
1-imidazolyl
1-imidazolyl
1-imidazolyl
1-imidazolyl
4-Cl
2-OCH3
4-OCH3
-8
-10
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9
1-pyrrolidinyl
H
235
284
198
213
299
323
287
310
316
260
254
230
225
177
253
253
210
216
316
315
291
276
331
235
248
268
232
179
-18
31
-12
-3
7a
10
11
12
13
14
15
16
17
18
19
20
21
22
1-pyrrolidinyl
2-OCH3
H
7c
3,5-dimethyl-1H-Pyrazol-1-yl
3,5-dimethyl-1H-Pyrazol-1-yl
3,5-dimethyl-1H-Pyrazol-1-yl
3,5-dimethyl-1H-Pyrazol-1-yl
8a
4-Cl
8b
8c
2-OCH3
4-OCH3
-17
8
8d
9a
5-amino-4-cyano-1H-pyrazol-1-yl
5-amino-4-cyano-1H-pyrazol-1-yl
5-amino-4-cyano-1H-pyrazol-1-yl
5-amino-4-cyano-1H-pyrazol-1-yl
5-amino-4-ethoxycarbonyl-1H-pyrazol-1-yl
H
-4
4-Cl
2-OCH3
4-OCH3
H
34
-15
25
6
9b
9c
9d
10a
10b
10c
11a
5-amino-4-ethoxycarbonyl-1H-pyrazol-1-yl 4-Cl
5-amino-4-ethoxycarbonyl-1H-pyrazol-1-yl 2-OCH3
-38
-7
C6H11
4-Cl
-2
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23
24
25
26
27
28
29
30
31
32
33
C6H11
2-OCH3
4-Cl
266
207
209
248
217
200
205
259
255
221
226
244
198
215
226
219
214
201
230
262
244
----
22
9
11b
11c
12a
12b
12c
12d
12e
12f
C6H5
4-CF3
4-Cl
-6
4-CF3
2-OCH3
4-Cl
22
-2
4-OCH3
4-OCH3
2-OCH3
4-Cl
-14
4
2,4- (OCH3)2
2,4- (OCH3)2
3,4,5-(OCH3)3
3,4,5-(OCH3)3
------------
2-OCH3
4-Cl
29
-7
12g
12h
2-OCH3
-----
-23
-----
Doxazosin
mesylate
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Table 2. Descriptor of the BMLR-QSAR model for the vasodilatory active pyridazines.
N = 32, n = 6, R2 = 0.812, R2cvOO = 0.715, R2cvMO = 0.721, F = 17.971, s2 = 9.652 x 10-8
Entry ID
Coefficient
0.0096
s
t
Descriptor
1
2
3
4
5
6
7
0
0.0009
0.0065
10.1478
6.4616
Intercept
D1
D2
D3
D4
D5
D6
0.0417
FHACA Fractional HACA (HACA/TMSA) (MOPAC PC)
Shadow plane YZ
5.4842 x 10-5 1.1726 x 10-5 4.6771
3.3666 x 10-5 8.1877 x 10-6 4.1118
HA dependent HDSA-1 (Zefirov PC) (all)
HA dependent HDSA-1/TMSA (MOPAC PC) (all)
Structural information content (order 2)
Max. nucleoph. React. Index for atom C
-0.0245
-0.0002
-0.0662
0.0050
3.5491 x 10-5 -5.7082
0.0105 -6.2774
-4.9294
1/IC50 = 0.0096 + (0.0417 x D1) + [(5.4842 x 10-5) x D2] + [(3.3666 x 10-5) x D3] – (0.0245 x D4) – (0.0002 x D5) – (0.0662 x D6)
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Table 3. Molecular descriptor values of the BMLR-QSAR model for the vasodilatory
active pyridazines.
Descriptors*
Entry Compd.
D1
D2
D3
D4
D5
D6
1
0.01996 20.8
0.01899 20.42
101.1514
117.851
0.22673
0.27135
0.25586
0.25483
0.20305
0.22665
0.21346
0.22294
0.14203
0.16894
0.14995
0.18407
0.1944
19.1222
0.01396
5a
2
20.51033 0.01251
24.09871 0.01404
23.68268 0.0177
22.71714 0.01462
23.63504 0.01243
27.61804 0.01192
27.21434 0.0172
23.92709 0.01841
28.89063 0.01856
25.34215 0.01317
26.60386 0.01089
30.27769 0.01304
29.89377 0.01807
25.44222 0.03215
26.73928 0.02933
30.31351 0.02928
29.92039 0.01221
32.45125 0.02267
33.66402 0.02149
37.26924 0.01691
33.74725 0.01374
37.48443 0.01397
29.75213 0.0236
5b
3
0
32.48
134.0734
127.3935
92.5631
5c
4
0.02653 20.86
0.01231 21.58
0.01147 21.5
0.01273 33.52
0.01789 21.4
5d
5
6a
6
111.6483
127.8707
126.9164
62.98108
101.1514
83.97477
114.5111
97.33439
115.4653
109.7398
109.7398
164.6096
165.5639
132.1648
162.224
6b
7
6c
8
6d
9
0
0
0
0
0
0
31.84
38.8
7a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
7c
35.94
31.84
36.62
30.14
8a
8b
8c
0.19416
0.20324
0.19697
0.27947
0.27831
0.21973
0.25276
0.25226
0.16984
0.18985
0.14897
0.11377
0.22227
0.19712
8d
0.02014 27.86
0.02249 28.08
0.02006 35.28
0.02829 28.34
0.03798 33.08
0.03055 32.76
0.03443 39.2
0.03008 54.34
0.02384 38.48
0.02546 45.86
0.00534 33.04
9a
9b
9c
9d
10a
10b
10c
11a
11b
11c
12a
12b
12c
176.0607
107.3541
134.5505
91.60884
74.90931
153.6357
127.8707
29.853
0.01069
0.0079
42
33.79116 0.01112
30.91076 0.01098
0.01256 35.1
15

ACCEPTED MANUSCRIPT
28
29
30
31
32
0.01251 41.26
0.01348 43.2
129.3021
167.4724
151.25
0.19786
0.22933
0.22414
0.24847
0.25802
32.49193 0.01013
34.16959 0.01059
34.81357 0.01263
35.55921 0.01719
36.45079 0.01442
12d
12e
12f
0.018
40.9
0.01261 41.94
0.01479 46.84
171.7666
176.0607
12g
12h
* D1 = FHACA Fractional HACA (HACA/TMSA) (MOPAC PC), D2 = Shadow plane
YZ, D3 = HA dependent HDSA-1 (Zefirov PC) (all), D4 = HA dependent HDSA-
1/TMSA (MOPAC PC) (all), D5 = Structural information content (order 2), D6 = Max.
nucleoph. React. Index for atom C.
16

ACCEPTED MANUSCRIPT
Table 4. Observed, predicated and molecular descriptor values of external test set compounds 7b and 10d according to the BMLR-
QSAR model.
Descriptors*
Observed
IC50, ꢀM
300
Predicted
IC50, ꢀM
283
Entry Compd.
Error
D1
D2
D3
D4
D5
D6
1
2
17
2
0
25.32
32.34
72.52367
178.4464
0.15211
0.24968
25.2
0.01664
0.00912
7b
216
214
0.03495
36.89834
10d
* D1 = FHACA Fractional HACA (HACA/TMSA) (MOPAC PC), D2 = Shadow plane YZ, D3 = HA dependent HDSA-1 (Zefirov PC)
(all), D4 = HA dependent HDSA-1/TMSA (MOPAC PC) (all), D5 = Structural information content (order 2), D6 = Max. nucleoph.
React.
Index
for
atom
C.
17

ACCEPTED MANUSCRIPT
3- Conclusion
In summary, different pyridazines substituted with different aryl/heterocyclic
moieties at position 3 and 6 were synthesized by facile methods. All the targeted
compounds were screened for their in vitro vasorelaxant activity using isolated thoracic
aortic rings of rats precontracted with norepinephrine hydrochloride standard reported
procedure and compared with doxazosin mesylate, which was used as a reference
standard. All synthesized pyridazine derivatives exhibited remarkable vasodilating
activity with IC50 values ranges from 177-323
doxazosin mesylate (IC50 = 226 M). Compounds 8a and 11a exerted the highest activity
with IC50 values 198 and 177 M, respectively, that, they may represent promising leads
ꢀM compared to the reference standard
ꢀ
ꢀ
for future optimization. 2D-QSAR study was undertaken utilizing CODESSA-Pro
software in order to validate the vasorelaxant observed bio-data and determine the most
important parameters controlling bio-activity. Statistically significant robust QSAR
model describing the pyridazines bio-properties was obtained. External validation
technique utilizing high and promising potent synthesized agents, support the predictive
power of the attained QSAR model in addition to the internal validation parameters.
Homogeneity of the training set analogues (the same chemical scaffold) may be the main
factor for the success of the QSAR model.
4. Experimental
4.1. Chemistry
Melting points were recorded on Stuart SMP10 digital melting point apparatus.
IR spectra (KBr disc) were recorded on a Shimadzu FT-IR 8400S infrared
spectrophotometer. NMR spectra were recorded on a Bruker Ascend 400/ R (1H: 400,
13C: 100 MHz) spectrometer. Some 13C NMR spectra were recorded on a Varian Mercury
VX 300 spectrometer (75 MHz). Mass spectra were recorded on a Shimadzu GCMS-QP
1000 EX (EI, 70 eV) spectrometer. Elemental analyses were carried out at the Regional
center for Mycology and Biotechnology, Al-Azhar University, Egypt. Compounds 3a–d,
4a–d and 5a–d were prepared according to the reported procedures [32,33].
18

ACCEPTED MANUSCRIPT
4.1.1. General procedure for preparation of 6a–d and 7a–c
An equimolar amount of the appropriate secondary amine (2 mmol) and sodium
hydride (60% in mineral oil) in dry dimethylformamide (5 ml) was stirred at R.T. for 1 h.
The appropriate chloropyridazine derivative 4a–d was added and the mixture was heated
at 100˚C for 6 h. The reaction mixture was cooled and poured on ice water. The obtained
precipitate was filtered off and washed with water and crystallized from ethanol.
4.1.1.1. 3-(1H-Imidazol-1-yl)-6-phenylpyridazine 6a
Yield 78% (0.43 g), mp 155-157˚C. IR: νmax/ cm-1 3059, 1589, 1562, 1481. 1H NMR
(CDCl3, 400 MHz):
δ 7.31 (br s, 1H, imidazole proton), 7.53-7.59 (m, 3H, arom.
protons), 7.68 (d, 1H, J= 9.20 Hz, pyridazine proton), 7.83 (br s, 1H, imidazole proton),
8.04 (d, 1H, J= 9.20 Hz, pyridazine proton), 8.10-8.12 (m, 2H, arom. protons), 8.53 (br
13
s, 1H, imidazole proton). C NMR (CDCl3, 100 MHz):
δ 116.1, 117.4, 126.6, 126.9,
129.2, 130.5, 131.4, 134.9, 135.1, 150.8, 158.5 (arom. carbons). Anal.Calcd. for
C13H10N4 (222.25): C, 70.26; H, 4.54; N, 25.21. Found: C, 70.49; H, 4.61; N, 25.42.
4.1.1.2. 3-(4-Chlorophenyl)-6-(1H-imidazol-1-yl)pyridazine 6b
1
Yield 88% (0.50 g), mp 206-208˚ C. IR: νmax/ cm-1 3042, 1593, 1557, 1489, 1441. H
NMR (CDCl3, 400 MHz):
δ 7.32 (br s, 1H, imidazole proton), 7.56 (d, 2H, J= 8.64 Hz,
arom. protons), 7.67 (d, 1H, J= 9.20 Hz, pyridazine proton), 7.83 (t, 1H, J=1.24 Hz,
imidazole proton), 8.02 (d, 1H, J= 9.20 Hz, pyridazine proton), 8.07 (d, 2H, J= 8.64 Hz,
13
arom. protons), 8.51 (br s, 1H, imidazole proton). C NMR (CDCl3, 75 MHz):
δ 115.9,
117.3, 125.8, 126.2, 128.0, 129.4, 131.5, 134.7, 138.4, 158.9, 159.0 (arom. carbons). MS:
m/z (%) 256 (M+, 100), 258 (M++2, 34). Anal.Calcd. for C13H9ClN4 (256.69): C, 60.83;
H, 3.53; N, 21.83. Found: C, 60.98; H, 3.50; N, 21.98.
4.1.1.3. 3-(1H-Imidazol-1-yl)-6-(2-methoxyphenyl)pyridazine 6c
Yield 80% (0.46 g), mp 159-161˚C. IR: νmax/ cm-1 3050, 2955, 2924, 2853, 1601, 1560,
1
1491, 1481, 1443. H NMR (CDCl3, 400 MHz):
δ 3.92 (s, 3H, OCH3), 7.08 (d, 1H, J=
8.28 Hz, arom. proton), 7.18 (dt, 1H, J= 0.74, 7.52, 11.24 Hz, arom.proton), 7.32 (br s,
1H, imidazole proton), 7.49 (dt, 1H, J=1.36, 7.86, 10.69 Hz, arom. proton), 7.59 (d, 1H,
19

ACCEPTED MANUSCRIPT
J= 9.20 Hz, pyridazine proton), 7.83 (br s, 1H, imidazole proton), 8.04 (dd, 1H, J= 1.68,
7.68 Hz, arom. proton), 8.24 (d, 1H, J= 9.20 Hz, pyridazine proton), 8.49 (br s, 1H,
imidazole proton). MS: m/z (%) 252 (M+, 34), 253 (M++1, 7), 185 (100). Anal.Calcd. for
C14H12N4O (252.27): C, 66.65; H, 4.79; N, 22.21. Found: C, 66.98; H, 4.84; N, 22.37.
4.1.1.4. 3-(1H-Imidazol-1-yl)-6-(4-methoxyphenyl)pyridazine 6d
Yield 60% (0.27 g), mp 178-180˚C. IR: νmax/ cm-1 3049-3009, 2965-2837, 1607, 1582,
1508, 1487. 1H NMR (CDCl3, 400 MHz):
δ 3.91 (s, 3H, OCH3), 7.07 (d, 2H, J= 8.84 Hz,
arom. protons), 7.30 (br s, 1H, imidazole proton), 7.62 (d, 1H, J= 9.20 Hz, pyridazine
proton), 7.81 (br s, 1H, imidazole proton), 7.97 (d, 1H, J= 9.24 Hz, pyridazine proton),
13
8.07 (d, 2H, J= 8.84 Hz, arom. proton), 8.49 (br s, 1H, imidazole proton). C NMR
(CDCl3, 100 MHz):
δ 55.46 (OCH3), 114.6, 116.0, 117.4, 125.9, 127.6, 128.3, 131.4,
134.8, 150.4, 158.1, 161.65 (arom. carbons). Anal.Calcd. for C14H12N4O (252.27): C,
66.65; H, 4.79; N, 22.21. Found: C, 66.81; H, 4.85; N, 22.43.
4.1.1.5. 6-(Pyrrolidin-1-yl)phenylpyridazine 7a
Yield 65 % (0.39 g), mp 135-137˚C. IR: νmax/ cm-1 3044, 2924, 2851, 1605, 1543, 1462.
1H NMR (CDCl3, 400 MHz):
δ 2.07-2.11 (m, 4H, pyrrolidine protons), 3.65 (br s, 4H,
pyrrolidine protons), 6.80 (d, 1H, J= 9.44 Hz, pyridazine proton), 7.38-7.42 (m, 1H,
arom. proton), 7.45-7.49 (m, 2H, arom. protons), 7.69 (d, 1H, J= 9.48 Hz, pyridazine
13
proton), 8.00-8.06 (m, 2H, arom. protons). C NMR (CDCl3, 100 MHz):
δ 25.4, 46.9
(pyrrolidine carbons), 119.7, 125.7, 126.3, 127.4, 128.3, 128.7, 129.1, 131.6, 141.8, 149.5
(arom. carbons). MS: m/z (%) 259 (M+, 69), 261 (M++2, 23), 70 (100). Anal.Calcd. for
C14H15N3 (225.30): C, 74.64; H, 6.71; N, 18.65. Found: C, 74.81; H, 6.79; N, 18.84.
4.1.1.6. 3-(4-Chlorophenyl)-6-(pyrrolidin-1-yl)pyridazine 7b
Yield 66% (0.39 g), mp 213-215˚C. IR: νmax/ cm-1 3042, 2957, 2855, 1605, 1549, 1487,
1
1458. H NMR (CDCl3, 400 MHz):
δ 2.06-2.12 (m, 4H, pyrrolidine protons), 3.63 (br s,
4H, pyrrolidine protons), 6.70 (d, 1H, J= 9.48 Hz, pyridazine proton), 7.43 (d, 2H, J=
8.52 Hz, arom. protons), 7.58 (d, 1H, J= 9.44 Hz, pyridazine proton), 7.94 (d, 2H, J=
8.56 Hz, arom. protons). MS: m/z (%) 259 (M+, 69), 261 (M++2, 23), 70 (100).
20

ACCEPTED MANUSCRIPT
Anal.Calcd. for C14H14ClN3 (259.73): C, 64.74; H, 5.43; N, 16.18. Found: C, 64.85; H,
5.52; N, 16.26.
4.1.1.7. 3-(2-Methoxyphenyl)-6-(pyrrolidin-1-yl)pyridazine 7c
Yield 57% (0.33 g), mp 130-132˚C. IR: νmax/ cm-1 3055, 2972, 2849, 1603, 1541, 1493,
1470, 1456. 1H NMR (CDCl3, 400 MHz):
δ 1.98-2.01 (m, 4H, pyrrolidine protons), 3.56
(t, 4H, J= 6.70 Hz, pyrrolidine protons), 3.77 (s, 3H, OCH3), 6.64 (d, 1H, J= 9.44 Hz,
pyridazine proton), 6.91 (d, 1H, J= 8.08 Hz, arom. proton), 6.97-7.02 (m, 1H,
arom.proton), 7.29 (t, 1H, J= 7.20 Hz, arom. proton), 7.73 (d, 1H, J= 9.44 Hz, pyridazine
proton), 7.85 (dd, 1H, J= 1.24, 7.60 Hz, arom. proton). Anal.Calcd. for C15H17N3O
(255.31): C, 70.56; H, 6.71; N, 16.46. Found: C, 70.81; H, 6.78; N, 16.63.
4.1.2. General procedure for the preparation of 8a–d
A solution of the hydrazide derivative 5a–d (2.15 mmol) and acetylacetone (2.30 mmol)
in absolute ethanol (5 ml) was heated under reflux for 2 h. The crystals separated on
cooling were filtered off, washed and recrystallized from ethanol.
4.1.2.1. 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-phenylpyridazine 8a
Yield 41% (0.22 g), mp 109-111˚C (reported 106-107˚C) [54].
4.1.2.2. 3-(4-Chlorophenyl)6-(3,5-dimethyl-1H-pyrazol-1-yl)pyridazine 8b
Yield 60% (0.36 g), mp 177-179˚C (reported 174-175˚C) [55].
4.1.2.3. 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(2-methoxyphenyl)pyridazine 8c
Yield 64% (0.39 g), mp 114-116˚C. IR: νmax/ cm-1 3005, 2978, 2924, 2839, 1605, 1575,
1543, 1435. 1H NMR (CDCl3, 400 MHz): δ 2.34 (s, 3H, CH3), 2.81 (s, 3H, CH3), 3.89 (s,
3H, OCH3), 6.08 (s, 1H, pyrazole proton), 7.05 (d, 1H, J= 8.28 Hz, arom. proton), 7.15 (t,
1H, J= 7.20 Hz, arom. proton), 7.45-7.49 (m, 1H, arom. proton), 8.01 (dd, 1H, J= 1.68,
7.64 Hz, arom. proton), 8.13-8.18 (m, 2H, pyridazine protons. Anal.Calcd. for C16H16N4O
(280.33): C, 68.55; H, 5.75; N, 19.99. Found: C, 68.78; H, 5.83; N, 20.17.
21

ACCEPTED MANUSCRIPT
4.1.2.4. 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-methoxyphenyl)pyridazine 8d
Yield 65% (0.39 g), mp 148-150˚C. IR: νmax/ cm-1 3003, 2986, 2970, 2924, 2837, 1609,
1
1571, 1543, 1516, 1468. H NMR (CDCl3, 400 MHz): δ 2.34 (s, 3H, CH3), 2.82 (s, 3H,
CH3), 3.90 (s, 3H, OCH3), 6.09 (s, 1H, pyrazole proton), 7.06 (d, 2H, J= 8.84 Hz, arom.
proton), 7.92 (d, 1H, J= 9.28 Hz, pyridazine proton), 8.08 (d, 2H, J= 8.84 Hz, arom.
13
protons), 8.18 (d, 1H, J= 9.28 Hz, pyridazine proton). C NMR (CDCl3, 100 MHz): δ
13.7 (CH3), 15.0 (CH3), 55.4 (OCH3), 110.0, 114.4, 120.5, 125.5, 128.2, 128.3, 142.4,
151.2, 155.4, 156.6, 161.3 (arom. carbons). Anal.Calcd. for C16H16N4O (280.33): C,
68.55; H, 5.75; N, 19.99. Found: C, 68.82; H, 5.81; N, 20.23.
4.1.3. General procedure for the preparation of 9a–d and 10a–d
An equimolar mixture of the hydrazine derivative 5a-d and ethoxymethylene
malononitrile or ethyl ethoxymethylene cyanoacetate (2.30 mmol) in absolute ethanol
(5ml) was refluxed for 2 h. The formed precipitate was filtered off on hot and washed
with ethanol to give 9a-d or 10a-d, respectively in pure form.
4.1.3.1. 5-Amino-1-(6-phenylpyridazin-3-yl)-1H-pyrazole-4-carbonitrile 9a
Yield 84% (0.51 g), mp 254-255˚C (reported 251-252˚C) [56].
4.1.3.2. 5-Amino-1-[6-(4-chlorophenyl)pyridazin-3-yl]-1H-pyrazole-4-carbonitrile 9b
Yield 75% (0.51 g), mp 294-296˚C. IR: νmax/ cm-1 3397, 3294, 3078, 3063, 2230, 1622,
1
1595, 1560, 1551, 1524, 1493. H NMR (DMSO-d6, 400 MHz): δ 7.65 (d, 2H, J= 8.60
Hz, arom. protons), 8.04 (s, 1H, pyrazole proton), 8.19-8.23 (m, 3H, 2 arom. protons +
pyridazine proton), 8.26 (s, 2H, NH2 exchanged with D2O), 8.49 (d, 1H, J= 9.40 Hz,
13
pyridazine proton). C NMR (DMSO-d6, 100 MHz): δ 74.1 (C-CN), 114.6 (CN), 120.0,
128.4, 129.0, 130.0, 134.4, 135.7, 144.4, 153.6, 156.1, 156.8 (arom. carbons).
Anal.Calcd. for C14H9ClN6 (296.72): C, 56.67; H, 3.06; N, 28.32. Found: C, 56.84; H,
3.04; N, 28.49.
22

ACCEPTED MANUSCRIPT
4.1.3.3. 5-Amino-1-[6-(2-methoxyphenyl)pyridazin-3-yl]-1H-pyrazole-4-carbonitrile 9c
Yield 69% (0.47 g), mp 266-268˚C. IR: νmax/ cm-1 3387, 3285, 3096, 3082, 3069, 3007,
2980, 2947, 2918, 2839, 2218, 1636, 1603, 1584, 1549, 1524, 1495. 1H NMR (DMSO-d6,
400 MHz): δ 3.86 (s, 3H, OCH3), 7.14 (t, 1H, J= 7.46 Hz, arom. proton), 7.23 (d, 1H, J=
8.32 Hz, arom. proton), 7.53 (t, 1H, J= 7.44 Hz, arom. proton), 7.79 (d, 1H, J= 7.24 Hz,
arom. proton), 8.02 (s, 1H, pyrazole proton), 8.16 (d, 1H, J= 9.32 Hz, pyridazine proton),
8.25 (s, 2H, NH2 exchanged with D2O), 8.28 (d, 1H, J= 9.40 Hz, pyridazine proton).
Anal.Calcd. for C15H12N6O (292.30): C, 61.64; H, 4.14; N, 28.75. Found: C, 61.87; H,
4.19; N, 28.93.
4.1.3.4. 5-Amino-1-[6-(4-methoxyphenyl)pyridazin-3-yl]-1H-pyrazole-4-carbonitrile 9d
Yield 97% (0.65 g), mp 294-295˚C. IR: νmax/ cm-1 3397, 3296, 3078, 3038, 3005, 2970,
2918, 2843, 2210, 1622, 1605, 1580, 1551, 1512, 1449. 1H NMR (DMSO-d6, 400 MHz):
δ 3.85 (s, 3H, OCH3), 7.13 (d, 2H, J= 8.80 Hz, arom. protons), 8.02 (s, 1H, pyrazole
proton), 8.13-8.17 (m, 3H, 2 arom. protons + pyridazine proton), 8.23 (s, 2H, NH2
exchanged with D2O), 8.42 (d, 1H, J= 9.40 Hz, pyridazine proton). Anal.Calcd. for
C15H12N6O (292.30): C, 61.64; H, 4.14; N, 28.75. Found: C, 61.83; H, 4.21; N, 28.97.
4.1.3.5. Ethyl 5-amino-1-(6-phenylpyridazin-3-yl)-1H-pyrazole-4-carboxylate 10a
Yield 79% (0.56 g), mp 188-190˚C (reported 190-191˚C) [57].
4.1.3.6. Ethyl 5-amino-1-[6-(4-chlorophenyl)pyridazin-3-yl]-1H-pyrazole-4-carboxylate
10b
Yield 68% (0.54 g), mp 209-211˚C. IR: νmax/ cm-1 3443, 3333, 3084, 3073, 2986, 2941,
1
2916, 2903, 1674, 1607, 1593, 1541, 1493. H NMR (CDCl3, 400 MHz): δ 1.39 (t, 3H,
J= 7.12 Hz, CH3), 4.34 (q, 2H, J= 7.12 Hz, CH2), 7.53 (d, 2H, J= 8.48 Hz, arom.
protons), 7.61 (s, 2H, NH2 exchanged with D2O), 7.84 (s, 1H, pyrazole proton), 7.98-
8.02 (m, 3H, 2 arom. protons + pyridazine proton), 8.26 (d, 1H, J= 9.32 Hz, pyridazine
proton). 13C NMR (CDCl3, 75 MHz):
δ 14.4 (CH3), 59.6 (OCH2), 119.0, 125.8, 126.4,
127.9, 129.3, 133.8, 136.5, 138.4, 142.7, 152.0, 155.8 (arom. carbons), 164.0 (C=O). MS
23

ACCEPTED MANUSCRIPT
(m/z, %): 343 (M+, 100), 344 [M++ 1], 297 (50). Anal.Calcd. for C16H14ClN5O2 (343.77):
C, 55.90; H, 4.11; N, 20.37. Found: C, 56.07; H, 4.15; N, 20.49.
4.1.3.7.
Ethyl
5-amino-1-[6-(2-methoxyphenyl)pyridazin-3-yl]-1H-pyrazole-4-
carboxylate 10c
Yield 64% (0.50 g), mp 137-139˚C. IR: νmax/ cm-1 3424, 3312, 3080, 2990, 2959, 2918,
2839, 1688, 1618, 1601, 1545, 1493. 1H NMR (CDCl3, 400 MHz): δ 1.37 (t, 3H, J= 7.12
Hz, CH3), 3.87 (s, 3H, OCH3), 4.31 (q, 2H, J= 7.10 Hz, CH2), 7.03 (d, 1H, J= 8.32 Hz,
arom. proton), 7.12 (t, 1H, J= 7.48 Hz, arom. proton), 7.41 (t, 1H, J= 8.48 Hz, arom.
proton), 7.62 (s, 2H, NH2 exchanged with D2O), 7.82 (s, 1H, pyrazole proton), 7.90 (dd,
1H, J= 1.24, 7.60 Hz, arom. proton), 8.14 (s, 2H, pyridazine protons). 13C NMR (CDCl3,
100 MHz): δ 14.6 (CH3), 55.6 (OCH3), 59.7 (CH2), 95.5, 111.4, 117.5, 121.3, 125.0,
130.5, 130.8, 131.3, 142.5, 152.1, 156.2, 156.5, 157.1 (arom. carbons), 164.2 (C=O).
Anal.Calcd. for C17H17N5O3 (339.36): C, 60.17; H, 5.05; N, 20.64. Found: C, 60.34; H,
5.08; N, 20.87.
4.1.3.8.
Ethyl
5-amino-1-[6-(4-methoxyphenyl)pyridazin-3-yl]-1H-pyrazole-4-
carboxylate 10d
Yield 79% (0.62 g), mp 198-200˚C. IR: νmax/ cm-1 3453, 3331, 3082, 3046, 2988, 2965,
2922, 2901, 2833, 1676, 1612, 1580, 1545, 1516. 1H NMR (CDCl3, 400 MHz): δ 1.39 (t,
3H, J= 7.12 Hz, CH3), 3.90 (s, 3H, OCH3), 4.33 (q, 2H, J= 7.12 Hz, CH2), 7.06 (d, 2H,
J= 8.84 Hz, arom. protons), 7.60 (br s, 2H, NH2 exchanged with D2O), 7.84 (s, 1H,
pyrazole proton), 7.96 (d, 1H, J= 9.36 Hz, pyridazine proton), 8.02 (d, 2H, J= 8.84 Hz,
arom. protons), 8.20 (d, 1H, J= 9.36 Hz, pyridazine proton). 13C NMR (CDCl3, 75
MHz): δ 14.4 (CH3), 55.3 (OCH3), 59.6 (OCH2), 114.5, 118.9, 126.1, 127.8, 128.1, 142.4,
151.9, 156.2, 156.5, 161.4 (arom. carbons), 164.1 (C=O). Anal.Calcd. for C17H17N5O3
(339.36): C, 60.17; H, 5.05; N, 20.64. Found: C, 60.31; H, 5.12; N, 20.91.
4.1.4. General procedure for the preparation of 11a–c
To a mixture of the hydrazine derivative 5b,c (1.65 mmol) and triethylamine (0.25 ml) in
dry methylene chloride (5 ml), the appropriate isocyanate (1.65 mmol) was added
24

ACCEPTED MANUSCRIPT
dropwise. The reaction was stirred at room temperature for 6 h, the obtained precipitate
was filtered off, washed and crystallized from ethanol.
4.1.4.1.
2-[6-(4-Chlorophenyl)pyridazin-3-yl]-N-cyclohexylhydrazine-1-carboxamide
11a
Yield 43% (0.24 g), mp 211-213˚C. IR: νmax/ cm-1 3300, 3242, 3134, 3051, 3034, 3015,
2930, 2853, 1645, 1614, 1601, 1558, 1464. 1H NMR (CDCl3, 400 MHz):
1.01-1.09 (m,
δ
2H, cyclohexyl protons), 1.23-1.33 (m, 2H, cyclohexyl protons), 1.51-1.64 (m, 4H,
cyclohexyl protons), 1.85 (br d, 2H, J= 9.4 Hz, cyclohexyl protons), 3.56-3.66 (m, 1H,
cyclohexyl proton), 5.59 (s, 1H, NH exchanged with D2O), 5.61 (s, 1H, NH exchanged
with D2O), 6.77 (s, 1H, NH exchanged with D2O), 7.13 (d, 1H, J= 9.24 Hz, pyridazine
proton), 7.42 (d, 2H, J= 8.68 Hz, arom. protons), 7.66 (d, 1H, J= 9.24 Hz, pyridazine
proton), 7.81 (d, 2H, J= 8.28 Hz, arom. protons). Anal.Calcd. for C17H20ClN5O (345.83):
C, 59.04; H, 5.83; N, 20.25. Found: C, 59.25; H, 5.91; N, 20.41.
4.1.4.2. N-Cyclohexyl-2-[6-(2-methoxyphenyl)pyridazin-3-yl]hydrazine-1-carboxamide
11b
Yield 74% (0.42 g), mp 197-198˚C. IR: νmax/ cm-1 3306, 3223, 3073, 2999, 2928, 2853,
1
1659, 1599, 1580, 1541, 1493, 1460. H NMR (CDCl3, 400 MHz):
δ 1.08 (td, 2H, J=
2.87, 11.89, 14.80 Hz, cyclohexyl protons), 1.21-1.31 (m, 2H, cyclohexyl protons), 1.50-
1.56 (m, 2H, cyclohexyl protons), 1.61-1.65 (m, 2H, cyclohexyl protons), 1.85 (dd, 2H,
J= 2.92, 12.16 Hz, cyclohexyl protons), 3.53-3.61 (m, 1H, cyclohexyl proton), 3.78 (s,
3H, OCH3), 3.81 (s, 1H, NH exchanged with D2O), 3.82 (s, 1H, NH exchanged with
D2O), 5.95 (s, 1H, NH exchanged with D2O), 6.94 (d, 1H, J= 8.32 Hz, arom. proton),
7.02 (td, 1H, J= 0.83, 7.53, 8.33 Hz), 7.20 (d, 1H, J= 9.24 Hz, pyridazine proton), 7.36
(td, 1H, J= 1.74, 7.86, 9.12), 7.70 (dd, 1H, J= 1.64, 7.60 Hz, arom.proton), 7.88 (d, 1H,
J= 9.32 Hz, pyridazine proton. 13C NMR (CDCl3, 75 MHz):
δ 24.8, 25.4, 33.5, 48.7
(cyclohexyl carbons), 55.4 (OCH3), 111.2, 112.0, 121.1, 125.7, 130.5, 130.7, 138.5,
156.8, 160.0 (arom. carbons), 158.1 (C=O). Anal.Calcd. for C18H23N5O2 (341.42): C,
63.32; H, 6.79; N, 20.51. Found: C, 63.48; H, 6.85; N, 20.67.
25

ACCEPTED MANUSCRIPT
4.1.4.3. 2-[6-(4-Chlorophenyl)pyridazin-3-yl]-N-phenylhydrazine-1-carboxamide 11c
Yield 64% (0.35 g), mp 237-239˚C. IR: νmax/ cm-1 3329, 3250, 3138, 3061, 3007, 2984,
1659, 1601, 1597, 1557, 1448. 1H NMR (DMSO-d6, 400 MHz): δ 6.95 (t, 1H, J=7.34Hz,
arom. proton), 7.08 (d, 1H, J= 9.36 Hz, pyridazine proton), 7.25 (t, 2H, J= 7.94 Hz, arom.
protons), 7.51 (d, 2H, J= 7.92 Hz, arom. protons), 7.56 (d, 2H, J= 8.64 Hz, arom.
protons), 8.02 (d, 1H, J= 9.36 Hz, pyridazine proton), 8.05 (d, 2H, J= 8.68 Hz, arom.
protons), 8.34 (s, 1H, NH exchanged with D2O), 8.91 (s, 1H, NH exchanged with D2O),
13
8.96 (s, 1H, NH exchanged with D2O). C NMR (DMSO-d6, 75 MHz):
δ 113.2, 118.6,
121.8, 125.6, 127.4, 128.5, 128.8, 135.5, 139.6, 151.1, 160.5 (arom. carbons), 156.1
(C=O). Anal.Calcd. for C17H14ClN5O (339.78): C, 60.09; H, 4.15; N, 20.61. Found: C,
60.18; H, 4.19; N, 20.89.
4.1.5. General procedure for the preparation of 12a–h
To an equimolar mixture of the hydrazine derivative 5b,c and the appropriate aldehyde in
absolute ethanol (25ml) was added glacial acetic acid (0.50 ml). The reaction was heated
under reflux for 3 h. The obtained precipitate was filtered off and washed with ethanol to
give 12a–h in a pure form.
4.1.5.1. 3-(4-Chlorophenyl)-6-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}pyridazine
12a
1
Yield 87% (0.59 g), mp > 300˚C. IR: νmax/ cm-1 3120, 3050, 1611, 1593, 1549, 1489. H
NMR (DMSO-d6, 400 MHz):
δ 7.59 (d, 2H, J= 7.04 Hz, arom. protons), 7.72-7.79 (m,
3H, 2 arom. protons + pyridazine proton), 7.95 (d, 2H, J= 7.24 Hz, arom. protons), 8.11-
8.16 (m, 3H, 2 arom. protons + pyridazine proton), 8.23 (s, 1H, N=CH), 11.93 (s, 1H, NH
exchanged with D2O). Anal.Calcd. for C18H12ClF3N4O (376.76): C, 57.38; H, 3.21; N,
14.87. Found: C, 57.49; H, 3.23; N, 15.01.
4.1.5.2.
3-(4-Methoxyphenyl)-6-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}
pyridazine 12b
Yield 77% (0.53 g), mp 262-264˚C. IR: νmax/ cm-1 3125, 3060, 2940, 2839, 1611, 1589,
1
1545, 1493. H NMR (CDCl3, 400 MHz):
δ 3.83 (s, 3H, OCH3), 6.98 (d, 1H, arom.
26

ACCEPTED MANUSCRIPT
proton), 7.05 (t, 1H, J= 7.50 Hz, arom. protons), 7.42 (t, 1H, J= 7.74 Hz, arom. protons),
7.60 (d, 3H, J= 7.84 Hz, arom. protons+ pyridazine proton), 7.70 (d, 1H, J= 6.64 Hz,
arom. proton), 7.77 (d, 2H, J= 7.92 Hz, arom. protons), 7.95 (s, 1H, NH, exchanged
with D2O), 8.09 (d, 1H, J= 9.12 Hz, pyridazine proton), 8.39 (s, 1H, N=CH). Anal.Calcd.
for C19H15F3N4O (372.34): C, 61.29; H, 4.06; N, 15.05. Found: C, 61.43; H, 4.12; N,
15.23.
4.1.5.3. 3-(4-Chlorophenyl)-6-[2-(4-methoxybenzylidene)hydrazinyl]pyridazine 12c
Yield 82% (0.63 g), mp 272-273˚C. IR: νmax/ cm-1 3125, 3060, 2951, 2916, 2835, 1618,
1603, 1551, 1508, 1489. 1H NMR (DMSO-d6, 400 MHz):
δ 3.81 (s, 3H, OCH3), 7.00 (d,
2H, J= 7.36 Hz, arom. proton), 7.56-7.68 (m, 5H, arom. protons), 8.09 (d, 4H, J= 8.28
Hz, arom. protons + N=CH), 11.53 (s, 1H, NH, exchanged with D2O). Anal.Calcd. for
C18H15ClN4O (338.79): C, 63.81; H, 4.46; N, 16.54. Found: C, 63.96; H, 4.53; N, 16.80.
4.1.5.4. 3-[2-(4-Methoxybenzylidene)hydrazinyl] 6-(4-methoxyphenyl)pyridazine 12d
Yield 80% (0.62 g), mp 239-241˚C. IR: νmax/ cm-1 3120, 3005, 2930, 2906, 2831, 1611,
1593, 1541, 1508, 1493. 1H NMR (DMSO-d6, 400 MHz):
δ 3.81 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 7.00 (d, 2H, J= 8.64 Hz, arom. protons), 7.09 (t, 1H, J= 7.38 Hz, arom.
proton), 7.17 (d, 1H, J= 8.24 Hz, arom. proton), 7.43 (t, 1H, J= 7.16 Hz, arom. proton),
7.57 (d, 1H, J= 9.32 Hz, pyridazine proton), 7.65 (d, 2H, J= 8.64 Hz, arom. protons),
7.71 (d, 1H, J= 6.44 Hz, arom. proton), 7.86 (d, 1H, J= 9.36 Hz, pyridazine proton), 8.11
(s, 1H, N=CH), 11.40 (s, 1H, NH, exchanged with D2O). 13C NMR (DMSO-d6, 75 MHz):
δ
55.2 (OCH3), 55.6 (OCH3), 111.0, 111.8, 114.2, 120.5, 126.2, 128.2, 129.7, 141.0,
151.5, 156.5, 158.0, 160.0 (arom. carbons). Anal.Calcd. for C19H18N4O2 (334.38): C,
68.25; H, 5.43; N, 16.76. Found: C, 68.47; H, 5.49; N, 16.94.
4.1.5.5. 3-(4-Chlorophenyl)-6-[2-(2,4-dimethoxybenzylidene)hydrazinyl]pyridazine 12e
Yield 79% (0.53 g), mp 225-226˚C. IR: νmax/ cm-1 3150, 3003, 2968, 2940, 2928, 2835,
1
1609, 1568, 1539, 1504, 1489. H NMR (CDCl3, 400 MHz):
δ 3.77 (s, 3H, OCH3), 3.79
(s, 3H, OCH3), 6.39 (d, 1H, J= 1.96 Hz, arom. proton), 6.48 (dd, 1H, J= 1.86, 8.66 Hz,
arom. proton), 7.39 (d, 2H, J= 8.48 Hz, arom. protons), 7.70-7.87 (m, 6H, 2 pyridazine
27

ACCEPTED MANUSCRIPT
13
protons + 3 arom. protons + NH exchanged with D2O), 8.43 (s, 1H, N=CH). C NMR
(CDCl3, 100 MHz): 55.5 (OCH3), 55.6 (OCH3), 98.1, 105.8, 116.1, 126.5, 127.1, 127.4,
δ
129.1, 135.2, 140.3, 145.0, 153.5, 156.5, 157.0 (arom. carbons). Anal.Calcd. for
C19H17ClN4O2 (368.82): C, 61.87; H, 4.65; N, 15.19. Found: C, 62.13; H, 4.69; N, 15.35.
4.1.5.6.
3-[2-(2,4-Dimethoxybenzylidene)hydrazinyl]-6-(2-methoxyphenyl)pyridazine
12f
Yield 72% (0.48 g), mp 214-216˚C. IR: νmax/ cm-1 3200, 3013, 3003, 2955, 2918, 2897,
2833, 1607, 1566, 1539, 1503, 1491. 1H NMR (CDCl3, 400 MHz):
3.73 (s, 3H, OCH3),
δ
3.78 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.38 (d, 1H, J= 2.20 Hz, arom. proton), 6.47
(dd, 1H, J= 2.16, 8.64 Hz, arom. proton), 6.94 (d, 1H, J= 8.28 Hz, arom. proton), 7.02 (t,
1H, J= 7.46 Hz, arom. proton), 7.31-7.35 (m, 1H, arom. proton), 7.59 (d, 1H, J= 9.40 Hz,
pyridazine proton), 7.81-7.84 (m, 4H, pyridazine proton + 2 arom. protons + NH
exchanged with D2O), 8.36 (s, 1H, N=CH). 13C NMR (CDCl3, 75 MHz):
δ 55.3 (OCH3),
55.4 (OCH3), 55.5 (OCH3), 98.1, 105.4, 111.2, 112.1, 117.0, 121.0, 125.8, 126.8, 129.7,
130.5, 130.6, 138.2, 151.8, 156.9, 158.4, 158.7, 161.6 (arom. carbons). Anal.Calcd. for
C20H20N4O3 (364.41): C, 65.92; H, 5.53; N, 15.38. Found: C, 66.11; H, 5.64; N, 15.52.
4.1.5.7.
3-(4-Chlorophenyl)-6-[2-(3,4,5-trimethoxybenzylidene)hydrazinyl]pyridazine
12g
Yield 83% (0.60 g), mp 294-296˚C. IR: νmax/ cm-1 3219, 3086, 2997, 2924, 2851, 2833,
1
1614, 1599, 1539, 1506, 1491. H NMR (DMSO-d6, 400 MHz):
δ 3.70 (s, 3H, OCH3),
3.86 (s, 6H, 2OCH3), 7.03 (s, 2H, arom. protons), 7.57 (d, 2H, J=8.16 Hz, arom. protons),
7.70 (d, 1H, J= 9.36 Hz, pyridazine proton), 8.08-8.10 (m, 4H, pyridazine proton + 2
arom. protons + N=CH), 11.70 (s, 1H, NH exchanged with D2O). Anal.Calcd. for
C20H19ClN4O3 (398.85): C, 60.23; H, 4.80; N, 14.05. Found: C, 60.47; H, 4.87; N, 14.13.
4.1.5.8. 3-(2-Methoxyphenyl)-6-[2-(3,4,5-trimethoxybenzylidene)hydrazinyl]pyridazine
12h
Yield 75% (0.55 g), mp 252-254˚C. IR: νmax/ cm-1 3200, 3086, 2997, 2955, 2926, 2837,
1601, 1589, 1576, 1506, 1491. 1H NMR (CDCl3, 400 MHz):
δ 3.81 (s, 6H, 2OCH3), 3.82
28