New pyridazine-fluorine derivatives: Synthesis, chemistry and biological activity. Part II

Article abstract of DOI:10.1016/j.ejmech.2013.04.069

A comprehensive study concerning synthesis, structure and biological activity of new pyridazine-fluorine (PYF) derivatives is presented. The first synthesis of PYF derivatives in phase-transfer catalysis (PTC) under microwave (MW) and conventional thermal

Full text of DOI:10.1016/j.ejmech.2013.04.069

Accepted Manuscript
New Pyridazine–Fluorine Derivatives: Synthesis, Chemistry and Biological Activity.
Part II
Roxana-Angela Tucaliuc, Valeriu V. Cotea, Marius Niculaua, Cristina Tuchilus, Dorina
Mantu, Ionel I. Mangalagiu
PII:
S0223-5234(13)00422-4
10.1016/j.ejmech.2013.04.069
EJMECH 6282
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 January 2013
Revised Date: 12 March 2013
Accepted Date: 24 April 2013
Please cite this article as: R.-A. Tucaliuc, V.V. Cotea, M. Niculaua, C. Tuchilus, D. Mantu, I.I.
Mangalagiu, New Pyridazine–Fluorine Derivatives: Synthesis, Chemistry and Biological Activity. Part II,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.069.
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ACCEPTED MANUSCRIPT
•
•
•
•
synthesis and antimicrobial activity of new pyridazine–fluorine (PYF)
the first synthesis of PYF in phase-transfer catalysis under microwave
regiochemistry and chorochemistry involved in these reactions are discussed
trifluoromethyl group on pyridazine skeleton increase the antimicrobial activity

ACCEPTED MANUSCRIPT
New Pyridazine–Fluorine Derivatives: Synthesis, Chemistry and
Biological Activity. Part II
Roxana-Angela Tucaliuc,1 Valeriu V. Cotea,1 Marius Niculaua,1 Cristina
Tuchilus,2 Dorina Mantu,3 Ionel I. Mangalagiu3*
1. University of Agriculture and Veterinary Medicine "Ion Ionescu de la Brad" Iasi, Aleea Mihail
Sadoveanu, Iasi, Romania,
2. “Gr.T.Popa” University of Medicine and Pharmacy, Iasi, School of Pharmacy, Str. Universitatii
16, 700115 Iasi, Romania.
3. "Al. I. Cuza" University of Iasi, Organic Chemistry Department, Bd. Carol 11, 700506 Iasi,
Romania; e-mail: ionelm@uaic.ro
Abstract
A comprehensive study concerning synthesis, structure and biological activity of new pyridazine–fluorine
(PYF) derivatives is presented. The first synthesis of PYF derivatives in phase-transfer catalysis (PTC)
under microwave (MW) and conventional thermal heating (TH) is reported. Under MW irradiation the
consumed energy decreases considerably, the amount of used solvent in liquid phase is at least five-fold
less comparative with conventional TH, while PTC did not use solvents. Consequently these reactions
could be considered environmentally friendly. Also, the reaction time decrease substantially and, in some
cases, the yields are higher. A feasible explanation for MW efficiency is presented. Regiochemistry and
chorochemistry involved in these reactions are also discussed; the reactions are regioselective and
chorospecific or choroselective, respectively. Ten new pyridazine–fluorine cycloadducts are obtained.
The in vitro antibacterial and antifungal activities of the PYF compounds were tested. Introduction of a
trifluoromethyl moiety on the pyridazine skeleton is leading to an increasing of the antimicrobial activity.
Structure - activity correlationships have been done.
Graphical abstract
The design, synthesis and in vitro antimicrobial activity of new pyridazine–fluorine (PYF) derivatives are
presented. The first synthesis of PYF derivatives in phase-transfer catalysis (PTC) under microwave
(MW) and conventional thermal heating (TH) is also reported. Introduction of a trifluoromethyl moiety
on the pyridazine skeleton is leading to an increasing of the antimicrobial activity.
Keywords: Pyridazine; Fluorine; Chorochemistry; Microwaves; Biological activities.
Article Outline
1. Introduction
2. Results and discussion
2.1. Chemistry
2.2. Design and biological activity
3. Conclusion
4. Experimental protocols
4.1. Chemistry
4.1.1. General procedure for [3+2] dipolar cycloaddition in the liquid phase
1

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4.1.2. General procedure for [3+2] dipolar cycloaddition in phase-transfer
catalysis
2,2,2-Trifluoroethyl
b]pyridazine-5-carboxylate (3a)
2,2,2-Trifluoroethyl
b]pyridazine-5-carboxylate (3b)
2,2,2-Trifluoroethyl
7-(4-fluorobenzoyl)-4a,5,6,7-tetrahydropyrrolo[1,2-
7-(4-chlorobenzoyl)-4a,5,6,7-tetrahydropyrrolo[1,2-
7-(4-methylbenzoyl)-4a,5,6,7-tetrahydropyrrolo[1,2-
b]pyridazine-5-carboxylate (3c)
Ethyl
7-(4-fluorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo
[1,2-b] pyridazine -6-carboxylate (4a)
Ethyl
7-(4-chlorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo
[1,2-b] pyridazine-6-carboxylate (4b’)
Ethyl
7-(4-chlorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo
[1,2-b] pyridazine-6-carboxylate (4b’’)
Ethyl 7-(4-methylbenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo
[1,2-b] pyridazine-6-carboxylate (4c)
Ethyl
carboxylate (5a)
Ethyl
7-(4-fluorobenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-b]pyridazine-6-
7-(4-chlorobenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-b]pyridazine-6-
carboxylate (5b)
Ethyl 7-(4-methylbenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-b]pyridazine-6-
carboxylate (5c)
4.2. Microbiology
Acknowledgements
References
1. Introduction
Over the last decades, pyridazine and its derivatives have been extensively
investigated because of their important role especially in medicinal chemistry, a large
variety of biological activities being described: antibacterial, antifungus,
antituberculosis, antiviral, anti-inflammatory, anticancer, cardiovascular disorders,
etc.[1,2]. On the other hand, the importance of fluorine in medicinal chemistry is well
recognized, more than 150 drugs from the pharmaceuticals market being fluorinated
compounds [3-7].
A simple and flexible method to generate fused pyridazine derivatives, involves 1,3-
dipolar cycloaddition of cycloimmonium ylides to various symmetric and non-
symmetric dipolarophiles [8-16]. The reaction of ylides with non-symmetrical
dipolarophiles raises supplementary issues regarding regiochemistry and
chorochemistry, being a problem widely discussed and far away from being solved
[8,9].
Within the past decade, microwave (MW) assisted reactions have became a widely
accepted method in organic synthesis [11,14-19]. Furthermore, phase-transfer catalytic
(PTC) reactions under MW conditions have the great advantage of using small amounts
of solvents, or even no organic solvents (‘solvent free’), thus being environmentally
friendly [17,18].
In continuation of our work in the field of biologically active pyridazine
[11,13,20,21], we decided to synthesize new pyridazine-fluorine (PYF) derivatives in
2

ACCEPTED MANUSCRIPT
order to test their respective biological potentials. In equal measure we were interested
in developing a new environmentally friendly method for preparation of these
derivatives by using MW technologies in PTC, and in study in the regiochemistry and
chorochemistry involved in the cycloaddition reactions.
2. Results and Discussion
2.1. Chemistry
In a previous paper [11], we designed a general and straightforward synthetic route
for obtaining fused pyrrolopyridazine using MW irradiation. In order to reach our goal
we decided to adapt this strategy, thus pyridazinium ylides 2 (generated ‘in situ’ from
the corresponding cycloimmonium salts, 1) were treated with various dipolarophiles
containing fluorine moiety (non-symmetrically substituted Z-alkenes and Z-alkynes) via
a [3+2] dipolar cycloaddition leading to fused PYF derivatives (Scheme 1).
11
11
10
11
5'
10
R
10
Cl
11
Cl
O
O
10
H
O
7
1
R
C
6
1
C
6
O
H 7
1
H
6' 6'
C
H 7
6'
6'
N
N
N
COOCH2CH3
CF3
COOCH2CH3
C
7
H
N
N
N
1
H
H
+
H
H
H
H
N
Hb
Ha
H
Hb
6
4a
N
4a
5'
4a
5
5
H
5
4
4
4
Hb
CF3
COOCH2CH3
6
CF3
4a
5
5'
4
4a. R = F
4c. R = CH3
4b'. R = Cl; 50%
4b''. R = Cl; 50%
COOCH2CF3
COOC2H5
H
H
3a-c. R= F, Cl, CH3
F3C
CH
H2C
COOCH2CF3
N
N
Br
1
N
N
6
CH2
TEA
F3C
COOC2H5
O
9
7
C
CH
COC6H4Rꢀ4
11
12
R
10
O
C
R
COC6H4Rꢀ4
2a-c. R= F, Cl, CH3
N
1
N
N
COOC2H5
1a-c. R = F, Cl, CH3
6' 6'
N
6
COOCH2CH3
5
CF3
4
CF3
5a-c. R=F, Cl, CH3
i
Scheme 1. Reaction pathway for preparation of pyridazine-fluorine derivatives
The cycloaddition reaction of the ylides 2 with 2,2,2- trifluoroethyl acrylate took
place in accordance with our expectation; we obtained a single isomer, the
tetrahydropyrrolopyridazine PYF 3. Because a single regioisomer is obtained, these
types of reactions are considered highly regioselective under charge control in
accordance with the usual electronic effects in dipolarophiles: one bond is formed
between the ylide carbanion and the non-substituted carbon atom of the dipolarophile
and the second bond between the positively charged carbon of the ylide and the
substituted carbon of the dipolarophile). For the saturated structure proposed for
tetrahydropyrrolopyridazine PYF 3 the main spectral data confirm the proposed
structure.
3

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The structures of the compounds were proved by elemental and spectral analysis
1
[IR, H-NMR, 13C NMR, two-dimensional experiments 2D-COSY, 2D-HETCOR
(HMQC), long range 2D-HETCOR (HMBC) and MS (ES) - see Supporting
1
Information]. In the H NMR spectrum of the PYF derivatives 3, the most informative
signals are those of the H5, H6a, H6b, H7 and H4a atoms. H7 appears as a doublet of
doublet at 5.60 ppm (3a), 5.57 ppm (3b) and 5.64 ppm (3c), having two different
coupling constants (J7,6a = 8.4 Hz, J7,6b = 3.6 Hz), which proves the E-configuration to
H6a proton and Z- configuration to H6b proton. The H6a (dtd) proton which appears at
2.33 ppm (3a), 2.32 ppm (3b) and 2.32 (3c), has three different coupling constants
(J6a,6b =13.2 Hz, J6a,7 = 8.8 Hz, J6a,5 = 4.8 Hz) which proves the Z-configuration with
respect to H5. An additional evidence for the assigned structures is the great chemical
shifts difference between the protons: H7 gives the signal around 5.60 ppm, H4a around
4.30 ppm, H5 around 3.30 ppm, H6b around 2.40 ppm and H6a around 2.30 ppm. In the
13C NMR spectra the carbons C4a, C5, C6 and C7 appear at chemical shifts in accordance
with the saturated structures proposed for the adducts 3 (e.g. (3a): C4a at 57.18 ppm, C5
at 50.12 ppm, C6 at 25.47 ppm and C7 at 71.19 ppm).
When the dipolarophile was ethyl 4,4,4-trifluorocrotonate (E-isomer, non-
symmetrically olefine) the reactions involved additional stereo- and regiochemical
problems, that is chorochemistry.7 While for ylides 2a and 2c the reaction occur
chorospecifically, for ylide 2b (R = Cl) it occur choroselectively, after flash
chromatography and crystallization from an appropriate solvent, we recovered an
inseparable mixture of two regisomers (4b’and 4b’’, ratio 1:1).
In the 1H NMR spectrum of the tetrahydropyrrolopyridazine PYF derivatives 4a and 4c,
the position of the signals for H7 protons (d, 5.76 ppm and 5.77 ppm) and the coupling
constants (J7,6b = 7.2 Hz) prove the E-configuration with H6b. Also, the different
coupling constants for the H4a (dd) proton (J4a,5 = 8.8 Hz, J4a,4 = 5.2 Hz) proves the Z-
configuration with H5. In the 13C NMR spectra carbons C4a, C5, C6 and C7 appear at
chemical shifts in accordance with the saturated structures proposed for the adducts 4a
and 4c (4a: C4a at 56.40 ppm, C5 at 48.47 ppm, C6 at 51.10 ppm and C7 at 69.54 ppm).
1
For the PYF regisomers 4b’ and 4b’’ the structure was elucidated by H-NMR, 13C
NMR, and two-dimensional experiments 2D-COSY, 2D-HETCOR (HMQC), long
range 2D-HETCOR (HMBC) - all signals are recurrent and in accordance with the
proposed structures.
The reaction with ethyl 4,4,4-trifluorobutinoate leads to the aromatized
pyrrolopyridazine PYF derivatives 5. Aromatisation of the initially hydrogenated
diazine i occurs spontaneously and could be explained by oxidative dehydrogenation.
For the pyrrolopyridazines PYF 5, the main spectral data confirms the proposed
structures. The lack of signals until 7.04 ppm and the position of H4 proton which
appears at 8.23-8.21 ppm as a doublet in the proton 1H NMR spectra, combined with the
fact that in the 13C NMR spectra the carbons C4a, C5, C6 and C7 appear at high chemical
shifts are a good evidence of the proposed structures for the aromatized
pyrrolopyridazines PYF 5.
The major disadvantages of the synthesis carried out under conventional thermal
heating (TH) include long reaction time (180 min), high energy consumption, low to
moderate yields, etc. As an alternative route we have performed the synthesis under
MW irradiation (it was used a monomode reactor STAR-2, CEM corporation, USA).
Table 1 lists the optimized reaction conditions, under TH and MW irradiation.
4

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Using MW irradiation, in liquid phase, the best results were obtained by applying a
constant irradiation power (25% of the full power of the magnetron, 800 W) while for
phase-transfer catalysis (PTC) the best results were obtained using a constant
o
temperature (50 C) and varying the irradiation power. As far as the PTC reactions, a
mix of potassium fluoride and N-(p-R-phenacyl)-pyridazinium bromides was used as
solid phase, while the liquid phase consisted of dipolarophiles dissolved in trioctyl-
methyl-ammonium chloride–Aliquat 336.
Table 1: The synthesis of PYF derivatives under conventional TH and MW irradiation
Conventional TH
Microwaves
Liquid phase PTC (KF-Aliquat)
Compd.
Reaction
Yield %
time/min
Reaction
Reaction
Yield %
Yield %
time/min
time/min
180
180
180
180
180
180
180
180
180
22
30
23
14
16
9
38
41
46
5
5
5
5
5
5
5
5
5
16
24
19
10
11
7
59
59
68
15
15
15
-
-
-
15
15
15
14
21
17
-
-
-
56
52
58
3a
3b
3c
4a
4b' + 4b’’
4c
5a
5b
5c
As indicated in Table 1, under MW irradiation these reactions could be considered
environmentally friendly because the consumed energy decreased considerably, the
amount of used solvent in liquid phase is at least five-fold less comparative with
conventional TH, while PTC does not use solvents. The reaction time decreases
substantially and, in some cases, the yields are higher. We presume that the MW heating
approach is more effective in [3+2] dipolar cycloaddition reactions due to two factors: i)
the mode of action under MW irradiation, and ii) the structure of the ylide intermediate.
It is well known that the magnetic field component of MW radiation is responsible for
the dielectric heating effect [19]. The greater the dipole moment of the molecule, the
larger the effect of the MW energy will be. The ylides having a 1, 2- dipolar structure
are excellent dipoles and, therefore, the efficiency of MW heating increases
considerably when compared with TH.
2.2. Design and biological activity
In a previously published paper [11] we proved the antibacterial and antifungal activity
of pyridazine derivatives which contain a pyridazine–acetophenone skeleton (1’) as
pharmacophoric moiety.
N
N
CF3
N
N
O
O
Considering
the
antimicrobial potential of
fluorine moiety [3-5], we
decided to introduce a
R
R
F3C
1'
1"
trifluoromethyl group, in order to combine and increase the respective biological
potentials. The synthesized compounds were tested, at a 4 mg/disk concentration, for
their in vitro antimicrobial activity against different strains of Gram-positive, Gram-
5

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negative bacteria and fungus. The primary screen was carried out by the disc diffusion
method [22, 23] using a nutrient agar medium (Mueller Hinton agar for antibacterial
tests and Sabouraud agar for antifungal tests). Chloramphenicol (antibiotic) and Nysatin
(antimycotic) were used as control drugs. Table 2 summarizes the antimicrobial activity
of the compounds and control drugs. The results are expressed as zone diameter (mm).
Table 2. The inhibition zone (diameter mean, in mm) as a criteria of antibacterial and
antifungal activity for some diazine derivatives described in the text.
S.
S.
B.
P.
E. coli
ATCC
25922
Candida
albicans
ATCC
Strain→
Product and
reference drug
↓
aureus luteea subtillis
ATCC ATCC
25923 9341
Aeruginosa
ATCC
27853
10231
Chloramphenicol
30 µg/disc
Nysatin,
25
30
25
-
19
29
-
-
-
-
-
29
100 µg/disc
28
30
30
28
35
29
30
29
49
31
33
39
38
42
38
31
38
36
19
20
20
21
19
21
16
18
19
26
35
28
31
38
27
34
42
40
27
37
30
29
39
31
29
26
32
55
58
59
58
61
61
41
44
60
3a (R=F)
3b (R=Cl)
3c (R=CH3)
4a (R=F)
4(b’+b”) (R=Cl)
4c (R=CH3)
5a (R=F)
5b (R=Cl)
5c (R=CH3)
The comparative analysis of the data from Table 2, leads to the following main
conclusions:
- the PYF derivatives have a spectacular nonselective antimicrobial activity against
Gram positive germs Sarciria Luteea and B. subtillis;
- against the remaining Gram-positive bacteria, compounds bearing a methyl group in
para position of acetophenone moiety (3c, 4c and 5c), show a higher activity tendency;
- the PYF derivatives 3 and 4 have a better antibacterial activity than 5;
- compounds bearing a methyl group in para position of acetophenone moiety (3c, 4c
and 5c), seem to be more active;
- only the PYF derivatives (3b) and (4b’+b’’) have a significant activity against fungus
Candida albicans.
Taking into consideration the above results, we decided to determine the MICs for
compounds bearing a methyl group in para position of the acetophenone moiety. The
compounds MICs against the sensitive microbial strains were determined using
Mueller–Hinton Broth (MHB) culture media in comparison to chloramphenicol [24],
Table 3.
6

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Table 3. The antibacterial activities measured by MICs of some diazine derivatives
described in the text
S. luteea
ATCC
9341
B. subtillis
S. aureus
ATCC
25923
Strain→
Product and
reference drug
↓
0.25
0.11
0.09
0.10
0.25
0.18
0.20
0.22
0.25
3.80
4.20
0.16
Chloramphenicol, µg/mL
3c (R=CH3) , mg/mL
4c (R=CH3) , mg/mL
5c (R=CH3) , mg/mL
These results show again that our pyridazine–fluorine derivatives have a good
antibacterial activity, especially against Gram positive germs Sarciria Luteea and B.
subtillis.
Having in view the above results, the following structure-activity relationships
correlations could be done:
- if we compare the antimicrobial activity of PYF derivatives, with our previously
reported results [11], we can see an increasing effect of the trifluoromethyl group
concerning antibacterial activity;
- the PYF derivatives with the hydrogenated pyrrolo ring (3 and 4) have a better
antibacterial activity that their aromatized analogues (5);
- there is a certain influence of the isoster R substituent from the para- position of the
benzoyle ring, the antibacterial activity decreasing in the order Me > Cl > F.
3. Conclusion
Here we report a straightforward method for preparation of new pyridazine–fluorine
(PYF) derivatives, by using PTC synthesis under MW and TH. Under MW irradiation
the reactions could be considered as environmentally friendly, because the consumed
energy decreased considerably and the amount of used solvent in liquid phase is at least
five-fold less comparative with conventional TH, while PTC does not use solvents.
Also, the reaction time decreased substantially under MW and, in some cases, the yields
are higher. We presume that the MW efficiency in [3+2] dipolar cycloaddition reactions
can be explained by two factors: the mode of action under MW irradiation and the
structure of the ylide intermediate. The regiochemistry studies indicate that the reactions
occur highly regioselective (a single regioisomer being formed), under charge control.
The chorochemistry studies show that the reactions occurs either chorospecifically,
either choroselectively, according with the influence of the para-R-phenyl-substituent in
the 7- position of the PYF moiety. The in vitro antimicrobial activity of the PYF
compounds indicated that the introduction of a trifluoromethyl moiety on the pyridazine
skeleton is beneficial for antimicrobial activity. Practically, all the PYF derivatives have
7

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a spectacular antimicrobial activity against Gram positive germs, Sarciria Luteea and B.
subtillis, and very good activity against Gram negative germ S. aureus. Regarding
fungus Candida albicans no significant activity of PYF derivatives was observed.
4. Experimental protocols
4.1. Chemistry
1H and 13C NMR spectra were recorded on a Bruker Avance 400 DRX spectrometer
operating at 400/100 MHz, downfield from an internal standard, SiMe4 in CDCl3.
Chemical shifts are given in ppm (δ-scale), coupling constants (J) in Hz. IR spectra
were recorded with a Shimadzu Prestige 8400s FTIR spectrophotometer in KBr. Mass
spectral (MS) data were obtained using a Brucker APEX-Q IV 7T system. A monomode
reactor (STAR-2, CEM corporation, USA, (800 W) was used for carrying out the
reaction under MW. Melting points were determined using a MELTEMP II apparatus
and are uncorrected.
4.1.1. General procedure for [3+2] dipolar cycloaddition in the liquid phase
A mixture of cycloimmonium salt 2 (2 mmol) and dipolarophile (2.2 mmol) was
suspended in chloroform (50 mL under conventional TH and 10 mL under MW
heating). Then, triethylamine (2.2 mmol) was added.
Under conventional TH, the solution was refluxed for 3 h on an oil bath. Using MW
irradiation, the best results were obtained using a constant irradiation power (25% from
the full power of the magnetron, 800 W) and varying the temperature (‘power control’).
Under MW heating, the solution mixture was placed in the reaction vessel (Pyrex glass)
and exposed to microwaves for 5 min. Once the heating cycle was completed, the tube
was cooled to ambient temperature and the resulting mixture was washed thoroughly
three times with water, dried with sodium sulfate, filtered and the solvent evaporated.
The crude product was purified by chromatography on silica gel with dichloromethane
/methanol (99/1) as eluent.
4.1.2. General procedure for [3+2] dipolar cycloaddition in phase-transfer catalysis
The corresponding cycloimmonium salt 2 (2 mmol), dipolarophile (2.2 mmol), KF (2
mmol) and 0.1 ml trioctyl-methyl-ammonium chloride – Aliquat 336, were grounded in
an agate mortar until a fine homogeneous mixture was obtained. The mixture was
exposed to MW for 15 min; the best results were obtained by applying a constant
o
temperature (50 C) and varying the irradiation power (‘temperature control’). The
resulting mixture was washed thoroughly three times with chloroform. The crude
product was purified by chromatography on silica gel with dichloromethane/methanol
(99/1) as eluent.
2,2,2-Trifluoroethyl
7-(4-fluorobenzoyl)-4a,5,6,7-tetrahydropyrrolo[1,2-
o
b]pyridazine-5-carboxylate (3a): White crystals, mp: 94-95 C. IR (KBr): ν/cm-1:
1
3092, 2903, 1740, 1686, 1596, 1504, 1481, 1266. H NMR (400 MHz, CDCl3): δppm
:
2.35-2.31 (1H, dd, JHH = 4.4 Hz, 8.4 Hz, 13.2 Hz, H6a), 2.45-2.42 (1H, dd, JHH = 3.6 Hz,
8.8 Hz, 13.2 Hz, H6b), 3.32-3.26 (1H, ddd, JHH = 3.6 Hz, 4.4 Hz, 8.4 Hz, H5), 4.26-4.23
8

ACCEPTED MANUSCRIPT
(1H, dd, JHH = 4.8 Hz, 7.6 Hz, H4a), 4.39-4.34 (1H, dd, JHH = 8.4 Hz, 12.8 Hz, H5’),
4.56-4.51 (1H, dd, JHH = 8.4 Hz, 12.8 Hz, H5’), 5.61-5.58 (1H, dd, JHH = 3.2 Hz, 8.4 Hz,
H7), 5.87-5.84 (1H, dd, JHH = 3.6 Hz, 10.0 Hz, H3), 6.00-5.96 (1H, dd, JHH = 4.8 Hz,
10.0 Hz, H4), 6.69-6.68 (1H, dd, JHH = 3.2 Hz, H2), 7.17-7.13 (2H, dd, JHH = 8.8 Hz,
H11), 8.21-8.18 (2H, dd, JHH = 8.8 Hz, H10). 13C NMR (100 MHz, CDCl3): δppm: 25.47
(C6), 50.12 (C5), 57.18 (C4a), 61 (CH2-5’), 71.19 (C7), 115.91-115.69 (C11), 118.67 (C3),
121.52 (CF3), 124.27 (C4), 131.56 (C12), 132.17 (C10), 134.07 (C2), 164.76 (C9), 172.13
(C5’, keto ester), 195.01 (C8, keto). Anal. Calcd. C17H14F4N2O3: C, 55.14; H, 3.81; N,
7.57; Found: C, 55.19; H, 3.77; N, 7.41. MS (ESI) m/z: 371 (M+1), 372 (M+2), 393
(M+Na+), 394 [(M+1)+Na+], 763 (2M+Na+).
2,2,2-Trifluoroethyl
7-(4-chlorobenzoyl)-4a,5,6,7-tetrahydropyrrolo[1,2-
o
b]pyridazine-5-carboxylate (3b): Yellow crystals, mp: 118-120 C. IR (KBr): ν/cm-1:
1
3094, 2914, 1746, 1687, 1592, 1498, 1266. H NMR (400 MHz, CDCl3): δppm: 2.34-
2.31 (1H, dd, JHH = 4.8 Hz, 8.8 Hz, 13.2 Hz, H6a), 2.44-2.41 (1H, dd, JHH = 3.6 Hz, 8.8
Hz, 13.2 Hz, H6b), 3.31-3.26 (1H, ddd, JHH = 3.6 Hz, 4.4 Hz, 8.4 Hz, H5), 4.24-4.21 (1H,
dd, JHH = 4.8 Hz, 7.2 Hz, H4a), 4.39-4.34 (1H, dd, JHH = 8.4 Hz, 12.8 Hz, H5’), 4.56-4.50
(1H, dd, JHH = 8.4 Hz, 12.8 Hz, H5’), 5.58-5.55 (1H, dd, JHH = 3.2 Hz, 8.4 Hz, H7), 5.86-
5.83 (1H, dd, JHH = 2.4 Hz, 10.0 Hz, H3), 5.99-5.95 (1H, dd, JHH = 6.4 Hz, 10.0 Hz, H4),
6.68-6.67 (1H, d, JHH = 3.2 Hz, H2), 7.47-7.44 (2H, d, JHH = 8.8 Hz, H11), 8.11-8.09
(2H, dd, JHH = 8.4 Hz, H10). 13C NMR (100 MHz, CDCl3): δppm: 25.31 (C6), 49.84 (C5),
57.19 (C4a), 61 (CH2-5’), 71.24 (C-7), 118.68 (C3), 121.72 (CF3), 125.13 (C4), 128.39
(C11), 134.29 (C10), 131.42 (C12), 134.29 (C2), 146.07 (C9), 172.45 (C5’, keto ester),
195.12 (C8, keto). Anal. Calcd. C17H14ClF3N2O3: C, 52.79; H, 3.65; N, 7.24; Found: C,
52.77; H, 3.67; N, 7.18.
2,2,2-Trifluoroethyl
7-(4-methylbenzoyl)-4a,5,6,7-tetrahydropyrrolo[1,2-
o
b]pyridazine-5-carboxylate (3c): Yellow crystals, mp: 147-149 C. IR (KBr): ν/cm-1:
1
3092, 2914, 1741, 1679, 1607, 1503, 1436, 1271. H NMR (400 MHz, CDCl3): δppm
:
2.41 (3H, s, CH3-12), 2.36-2.34 (1H, dd, JHH = 4.8 Hz, 8.0 Hz, 13.2 Hz, H6a), 2.39-2.37
(1H, dd, JHH = 4.0 Hz, 8.8 Hz, 13.2 Hz, H6b), 3.31-3.26 (1H, ddd, JHH = 3.2 Hz, 4.8 Hz,
8.4 Hz, H5), 4.33-4.30 (1H, dd, JHH = 4.8 Hz, 8.0 Hz, H4a), 4.42-4.34 (1H, dd, JHH = 8.4
Hz, 12.8 Hz, H5’), 4.58-4.48 (1H, dd, JHH = 8.4 Hz, 12.8 Hz, H5’), 5.65-5.62 (1H, dd,
JHH = 4.0 Hz, 8.4 Hz, H7), 5.87-5.83 (1H, dd, JHH = 3.2 Hz, 10.0 Hz, H3), 5.99-5.95 (1H,
dd, JHH = 5.2 Hz, 10.0 Hz, H4), 6.92-6.68 (1H, dd, JHH = 3.2 Hz, H2), 7.29-7.26 (2H, d,
JHH = 8.0 Hz, H11), 8.05-8.03 (2H, dd, JHH = 8.0 Hz, H10). 13C NMR (100 MHz, CDCl3):
δppm: 21.66 (CH3-12), 27.56 (C6), 43.71 (C5), 57.31 (C4a), 61 (CH2-5’), 70.92 (C7),
117.08 (C3), 121.65 (CF3), 124.84 (C4), 129.47 (C11), 134.95 (C10), 133.52 (C12), 134.70
(C2), 145.38 (C9), 172.03 (C5’, keto ester), 195.26 (C8, keto). Anal. Calcd.
C18H17F3N2O3: C, 59.02; H, 4.68; N, 7.65; Found: C, 59.11; H, 4.61; N, 7.55. MS (ESI)
m/z: 389 (M+Na+), 390 [(M+1)+Na+], 755 (2M+Na+), 756 [(2M+1)+Na+].
Ethyl
7-(4-fluorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo[1,2-b]
o
pyridazine -6-carboxylate (4a): White crystals, mp: 141-142 C. IR (KBr): ν/cm-1:
1
3090, 2981, 1730, 1684, 1590, 1502, 1470, 1262. H NMR (400 MHz, CDCl3): δppm
:
1.29-1.26 (3H, t, JHH = 7.2 Hz, CH3-6’), 3.63-3.57 (1H, dd, JHH = 8.8 Hz, H6), 3.67-3.63
(1H, dd, JHH = 8.8 Hz, H5), 4.22-4.17 (2H, q, JHH = 7.2 Hz, CH26’), 5.02-4.99 (1H, dd,
JHH = 5.2 Hz, 8.4 Hz, H4a), 5.77-5.75 (1H, d, JHH = 7.2 Hz, H7), 5.88-5.85 (1H, dd, JHH
9

ACCEPTED MANUSCRIPT
= 3.2 Hz, 9.6 Hz, H3), 5.96-5.92 (1H, dd, JHH = 5.2 Hz, 10.0 Hz, H4), 6.78-6.77 (1H, d,
JHH = 3.2 Hz, H2), 7.29-7.27 (2H, dd, JHH = 8.0 Hz, H11), 7.95-7.92 (2H, dd, JHH = 8.0
Hz, H10). 13C NMR (100 MHz, CDCl3): δppm: 14.30 (CH3-5’), 48 (C5), 51.10 (C6), 56.40
(C4a), 61.49 (CH2-6’), 69.54 (C7), 118.67 (C3), 124.38 (C4), 127.58 (CF3), 128.11 (C11),
128.99 (C10), 136.61 (C2), 144.95 (C12), 158.27 (C9), 171.39 (C5’, keto ester), 194.93
(C9, keto). Anal. Calcd. C18H16F4N2O3: C, 56.25; H, 4.20; N, 7.29; Found: C, 56.31; H,
4.13; N, 7.21. MS (ESI) m/z: 407 (M+Na+), 791 (2M+Na+).
Ethyl
7-(4-chlorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo[1,2-b]
o
pyridazine-6-carboxylate (4b’): White crystals, mp: 117-118 C. IR (KBr): ν/cm-1:
1725, 1696, 1306, 1273, 1249, 1216, 1207, 1159 cm-1. H NMR (400 MHz, CDCl3):
1
δppm: 1.27-1.23 (3H, t, JHH = 6.4 Hz, CH3-6’), 3.63-3.56 (1H, dd, JHH = 9.2 Hz, 8.8 Hz,
H6), 3.69-3.63 (1H, dd, JHH = 9.2 Hz, 8.8 Hz, H5), 4.22-4.19 (2H, q, JHH = 7.2 Hz, CH2-
6’), 4.38-4.34 (1H, dd, JHH = 8.4 Hz, 5.6 Hz, H4a), 4.75-4.72 (1H, dd, JHH = 9.2 Hz, H3),
5.89-5.86 (1H, dd, JHH = 8.0 Hz, 4.0 Hz, H7), 5.97-5.94 (1H, dd, JHH = 10.0 Hz, 4.8
Hz, H4), 6.79-6.67 (1H, d, JHH = 7.2 Hz, H2), 7.45-7.43 (2H, d, JHH = 8.0 Hz, H11), 7.99-
7.97 (2H, d, JHH = 8.4 Hz, H10). 13C NMR (100 MHz, CDCl3): δppm: 14.18 (CH3-6’),
44.34 (CH2-6’), 50.18 (C6), 56.36 (C5), 61.60 (C4a), 69.65 (C7), 118.69 (C3), 124.30
(CF3), 124.89 (C4), 128.94 (C11), 134.24 (C10), 134.26 (C12), 136.85 (C2), 139.90 (C9),
170.44 (C6’, keto ester), 190.33 (C8, keto). MS (ESI) m/z: 423 (M+Na+), 425
[(M+2)+Na+], 425 [(M+3)+Na+], 823 (2M+Na+), 824 [(2M+1)+Na+].
Ethyl
7-(4-chlorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo[1,2-b]
o
pyridazine-6-carboxylate (4b’’): White crystals, mp: 117-118 C. IR (KBr): ν/cm-1:
1725, 1696, 1306, 1273, 1249, 1216, 1207, 1159 cm-1. H NMR (400 MHz, CDCl3):
1
δppm: 1.30 -1.27 (3H, t, JHH = 6.4 Hz, CH3-5’), 3.25-3.21 (1H, dd, JHH = 8.4 Hz, 5.2 Hz,
H6), 4.17-4.13 (1H, dd, JHH = 7.6 Hz, 3.6 Hz, H5), 4.26-4.22 (2H, q, JHH = 7.2 Hz, CH2-
5’), 4.96-4.93 (1H, dd, JHH = 8.8 Hz, 4.8 Hz, H4a), 5.72-5.71 (1H, dd, JHH = 7.2 Hz, H3),
5.91-5.89 (1H, dd, JHH = 8.0 Hz, 4.0 Hz, H7), 6.21-6.18 (1H, dd, JHH = 9.6 Hz, 5.6 Hz,
H4), 6.79-6.67 (1H, d, JHH = 7.2 Hz, H2), 7.47-7.45 (2H, d, JHH = 8.0 Hz, H11), 8.16-8.14
(2H, d, JHH = 7.6 Hz, H10). 13C NMR (100 MHz, CDCl3): δppm: 14.29 (CH3-5’), 48.38
(CH2-5’), 50.97 (C6), 57.76 (C5), 61.60 (C4a), 70.59 (C7), 118.78 (C3), 128.15 (CF3),
126.46 (C4), 129.15 (C11), 130.66 (C10), 134.38 (C12), 137.21 (C2), 140.52 (C9), 171.22
(C5, keto ester), 193.87 (C8, keto). MS (ESI) m/z: 423 (M+Na+), 425 [(M+2)+Na+], 425
[(M+3)+Na+], 823 (2M+Na+), 824 [(2M+1)+Na+].
Ethyl
7-(4-methylbenzoyl)-5-(trifluoromethyl)-4a,5,6,7-tetrahydropyrrolo[1,2-b]
o
pyridazine-6-carboxylate (4c): Beige crystals, mp: 140-142 C. IR (KBr): ν/cm-1:
1
3094, 2989, 1725, 1696, 1598, 1480, 1257. H NMR (400 MHz, CDCl3): δppm: 1.29-
1.22 (3H, t, JHH = 7.2 Hz, CH3-6’), 2,41 (3H, s, CH3-12), 3.61-3.57 (1H, dd, JHH = 8.8
Hz, H6), 3.68-3.63 (1H, dd, JHH = 8.4 Hz, H5), 4.22-4.17 (2H, q, JHH = 7.2 Hz, CH2-6’),
5.03-4.99 (1H, dd, JHH = 5.2 Hz, 8.8 Hz, H4a), 5.78-5.76 (1H, d, JHH = 7.2 Hz, H7), 5.88-
5.85 (1H, dd, JHH = 3.2 Hz, 10.0 Hz, H3), 5.96-5.92 (1H, dd, JHH = 4.8 Hz, 9.6 Hz, H4),
6.78-6.77 (1H, d, JHH = 3.2 Hz, H2), 7.29-7.27 (2H, d, JHH = 8.4 Hz, H11), 7.95-7.93
(2H, d, JHH = 8.4 Hz, H10). 13C NMR (100 MHz, CDCl3): δppm: 14.29 (CH3-5’), 21.73
(CH3-12), 48.46-47.59 (C5), 51.08 (C6), 56.40 (C4a), 61.49 (CH2-6’), 69.53 (C7), 118.66
(C3), 124.86 (C4), 128.10 (CF3), 128.98 (C11), 129.30 (C10), 133.50 (C12), 136.62 (C2),
150.75 (C9), 171.39 (C5’, keto ester), 194.68 (C9, keto). Anal. Calcd. C19H19F3N2O3: C,
10

ACCEPTED MANUSCRIPT
60.00; H, 5.03; N, 7.36; Found: C, 60.08; H, 5.00; N, 7.31. MS (ESI) m/z: 403
(M+Na+), 404 [(M+2)+Na+], 783 (2M+Na+), 784 [(2M+1)+Na+].
Ethyl
7-(4-fluorobenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-b]pyridazine-6-
o
carboxylate (5a): Yellow crystals, mp: 104-106 C. IR (KBr): ν/cm-1: 3106, 2958,
1
1690, 1669, 1599, 1506, 1457, 1457, 1217. H NMR (400 MHz, CDCl3): δppm: 1.44-
1.41 (3H, t, JHH = 7.2 Hz, CH3-6’), 4.46-4.41 (2H, q, JHH = 7.2 Hz, CH2-6’), 7.04-7.00
(1H, dd, JHH = 4.4 Hz, 9.2 Hz, H3), 7.16-7.12 (2H, d, JHH = 8.8 Hz, H11), 7.86-7.82 (2H,
d, JHH = 8.8 Hz, H10), 8.23-8.21 (1H, dd, JHH = 4.4 Hz, H4), 8.67-8.64 (1H, dd, JHH = 9.2
Hz, H2). 13C NMR (100 MHz, CDCl3): δppm: 14.13 (CH3-6’), 60.95 (CH2-6’), 102.45
(C6), 116.36 (C11), 116.81 (C3), 117.18 (C5), 123 (CF3), 126.35 (C7), 129.28 (C-4),
130.48 (C4a), 132 (C10), 133.22 (C12), 145.12 (C2), 162,20 (C9), 167.90 (C6’, keto ester),
185.80 (C8, keto). Anal. Calcd. C18H12F4N2O3: C, 56.85; H, 3.18; N, 7.37; Found: C,
56.96; H, 3.11; N, 7.24. MS (ESI) m/z: 381 (M+1), 423 (M+Na+), 783 (2M+Na+).
Ethyl
7-(4-chlorobenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-b]pyridazine-6-
carboxylate (5b): White crystals, mp: 124-126 oC. IR (KBr): ν/cm-1: 3100, 2960, 1705,
1676, 1585, 1502, 1467, 1218. 1H NMR (400 MHz, CDCl3): δppm: 1.45-1.41 (3H, t, JHH
= 8.0 Hz, CH3-6’), 4.47-4.41 (2H, q, JHH = 8.0 Hz, CH2-6’), 7.05-7.01 (1H, dd, JHH
=
4.0 Hz, 8.0 Hz, H3), 7.46-7.44 (2H, d, JHH = 8.0 Hz, H11), 7.77-7.75 (2H, d, JHH = 8.0
Hz, H10), 8.23-8.22 (1H, dd, JHH = 4.0 Hz, H4), 8.69-8.66 (1H, dd, JHH = 8.0 Hz, H2).
13C NMR (100 MHz, CDCl3): δppm: 14.12 (CH3-6’), 60.97 (CH2-6’), 102.47 (C6),
116.66 (C3), 117.18 (C5), 126.09 (CF3), 126.09 (C7), 129.30 (C4), 129.36 (C11), 130.52
(C4a), 130.91 (C10), 135.08 (C12), 145.14 (C9), 145.10 (C2), 162.20 (C6’, keto ester),
186.16 (C8, keto). Anal. Calcd. C18H12ClF3N2O3: C, 54.49; H, 3.05; N, 7.06; Found: C,
54.96; H, 3.00; N, 6.92. MS (ESI) m/z: 419 (M+Na+), 815 (2M+Na+).
Ethyl
7-(4-methylbenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-b]pyridazine-6-
carboxylate (5c): White crystals, mp: 138-139 oC. IR (KBr): ν/cm-1: 3194, 2958, 1706,
1674, 1600, 1502, 1444, 1220. 1H NMR (400 MHz, CDCl3): δppm: 1.26 -1.21 (3H, t, JHH
= 7.2 Hz, CH3-6’), 2.41 (3H, s, CH3-12), 4.46-4.41 (2H, q, JHH = 7.2 Hz, CH2-6’), 7.01-
6.98 (1H, dd, JHH = 4.4 Hz, 9.2 Hz, H3), 7.27-7.25 (2H, d, JHH = 8.4 Hz, H11), 7.72-7.70
(2H, d, JHH = 8.4 Hz, H10), 8.21-8.19 (1H, dd, JHH = 4.0 Hz, H4), 8.66-8.63 (1H, dd, JHH
= 9.2 Hz, H2). 13C NMR (100 MHz, CDCl3): δppm: 14.13 (CH3-6’), 21.86 (CH3-12),
60.87 (CH2-6’), 102.20 (C6), 116.50 (C3), 117.18 (C5), 126.95 (CF3), 126.93 (C7),
129.18 (C4), 129.70 (C11), 129.76 (C10), 130.35 (C4a), 134.26 (C12), 145.01 (C2), 145.81
(C9), 162.31 (C6’, ‘keto ester), 187.02 (C8, keto). Anal. Calcd. C19H15F3N2O3: C, 60.64;
H, 4.02; N, 7.44; Found: C, 60.66; H, 3.98; N, 7.39. MS (ESI) m/z: 399 (M+Na+), 401
[(M+2)+Na+], 775 (2M+Na+), 776 [(2M+1)+Na+].
4.2. Microbiology
Agar diffusion assay
The antibacterial and antifungal activity of the compounds were determined by the
disc diffusion method [22, 23]. The microbial suspension (0.5 McFarland turbidity
standard) was mixed (1/10 ratio) with agar nutrient (Mueller Hinton agar for
antibacterial tests, respectively Sabouraud agar for antifungal tests). Sterile filter paper
discs (5 mm diameter), moistened with the test compound solution in chloroform of
11

ACCEPTED MANUSCRIPT
specific concentration (4 mg/disc), were carefully placed on the agar culture plates
[filter paper discs were previously introduced in sterile stainless steel cylinders (5 mm
internal diameter, 10 mm height)]. After 24 h of incubation at 30 °C for bacteria and 48
h at 28 °C for fungi, the diameter of the inhibition zone (mm) was measured (Table 2).
Chloramphenicol and nysatin were purchased from the market (Himedia, Mumbai,
India) and used in a concentration of 30 µg/disc, respectively 100 µg/disc as control
drugs for antibacterial and antifungal activities.
Broth microdilution assay
The minimum inhibitory concentrations (MICs) were determined using a broth
microdilution assay [24]. The microbial suspensions were adjusted to the turbidity of a
0.5 McFarland standard. The test compound were dissolved in 50% dimethylsulfoxide
at a concentration of 250 mg/mL and then subjected (in the well plates) to a serial of
two-fold dilution in Mueller Hinton broth (antibacterial tests, concentration range from
250 to 0.12 mg/mL; final volume in each well 100 µL). Other 95 µL of broth and 5 µL
of microbial inoculums were further dispensed into each well. The plates were
incubated at 37 °C for 24 h. The test compounds were screened twice against each
microorganism. The lowest concentration of the tested compound that visibly inhibited
the growth of the microorganisms was recorded as the MIC. The MIC values of
chloramphenicol (µg/mL) towards bacteria strains were also evaluated.
Acknowledgements
This work was co-financed from the European Social Fund Sectoral Operational Human
Resources Development 2007-2013, project number POSDRU/I.89/1.5/S62371
„Postdoctoral School in Agriculture and Veterinary Medicine area”.
Supporting information
Supporting information associated with this article could be found, in the online version,
at……………..
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New Pyridazine–Fluorine Derivatives: Synthesis, Chemistry and Biological Activity. Part II
Roxana-Angela Tucaliuc,1 Valeriu V. Cotea,1 Marius Niculaua,1 Cristina Tuchilus,2 Dorina Mantu,3
Ionel I. Mangalagiu4*
1. University of Agriculture and Veterinary Medicine "Ion Ionescu de la Brad" Iasi, Aleea Mihail Sadoveanu, Iasi, Romania,
2. “Gr.T.Popa” University of Medicine and Pharmacy, Iasi, School of Pharmacy, Str. Universitatii 16, 700115 Iasi, Romania.
3. "Al. I. Cuza" University of Iasi, Organic Chemistry Department, Bd. Carol 11, 700506 Iasi, Romania; e-mail: ionelm@uaic.ro
1. 1H
NMR
spectrum
of
compound
2,2,2-Trifluoroethyl
7-(4-fluorobenzoyl)-4a,5,6,7-
tetrahydropyrrolo[1,2-b]pyridazine-5-carboxylate (3a).
1

2. 13C
tetrahydropyrrolo[1,2-b]pyridazine-5-carboxylate (3a).
NMR
spectrum
of
compound
2,2,2-Trifluoroethyl
7-(4-fluorobenzoyl)-4a,5,6,7-
ACCEPTED MANUSCRIPT
3. ES-MS spectrum of compound 2,2,2-Trifluoroethyl 7-(4-fluorobenzoyl)-4a,5,6,7-
tetrahydropyrrolo[1,2-b]pyridazine-5-carboxylate (3a).
2

1
ACCEPTED MANUSCRIPT
4. H NMR spectrum of compounds Ethyl 7-(4-chlorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-
tetrahydropyrrolo[1,2-b] pyridazine-6-carboxylate (4b’+4b”).
5. 13C NMR spectrum of compounds Ethyl 7-(4-chlorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-
tetrahydropyrrolo[1,2-b] pyridazine-6-carboxylate (4b’+4b”).
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ACCEPTED MANUSCRIPT
6. ES/MS- spectrum of compounds Ethyl 7-(4-chlorobenzoyl)-5-(trifluoromethyl)-4a,5,6,7-
tetrahydropyrrolo[1,2-b] pyridazine-6-carboxylate (4b’+4b”).
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ACCEPTED MANUSCRIPT
7. 1H NMR spectrum of compounds Ethyl 7-(4-chlorobenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-
b]pyridazine-6-carboxylate (5b)
8. 13C NMR spectrum of compounds Ethyl 7-(4-chlorobenzoyl)-5-(trifluoromethyl)pyrrolo[1,2-
b]pyridazine-6-carboxylate (5b)
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