Design and synthesis of novel PARP-1 inhibitors based on pyridopyridazinone scaffold

Article abstract of DOI:10.1016/j.bioorg.2019.03.068

Various pyridopyridazinone derivatives were designed as Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. The pyridopyridazinone scaffold was used as an isostere of the phthalazine nucleus of the lead compound Olaparib in addition to some modifications in the tail part of the molecule. Preliminary biological evaluation indicated that most compounds possessed inhibitory potencies comparable to Olaparib in nanomolar level. The best PARP-1 inhibitory activity was observed for compound 8a with (IC₅₀ = 36 nM) compared to Olaparib as a reference drug (IC₅₀ = 34 nM). Molecular modeling simulation revealed that, the designed compounds docked well into PARP-1 active site and their complexes are stabilized by three key hydrogen bond interactions with both Gly863 and Ser904 as well as other favorable π-π and hydrogen-π stacking interactions with Tyr907 and Tyr896, respectively. Computational ADME study predicted that the target compounds 8a and 8e have proper pharmacokinetic and drug-likeness properties. These outcomes afford a new structural framework for the design of novel inhibitors for PARP-1.

Full text of DOI:10.1016/j.bioorg.2019.03.068

Accepted Manuscript
Design and synthesis of novel PARP-1 inhibitors based on pyridopyridazinone
scaffold
Ghada F. Elmasry, Enayat E. Aly, Fadi M. Awadallah, Samir M. El-Moghazy
PII:
S0045-2068(19)30308-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.068
YBIOO 2894
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
25 February 2019
24 March 2019
25 March 2019
Please cite this article as: G.F. Elmasry, E.E. Aly, F.M. Awadallah, S.M. El-Moghazy, Design and synthesis of
novel PARP-1 inhibitors based on pyridopyridazinone scaffold, Bioorganic Chemistry (2019), doi: https://doi.org/
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0.1016/j.bioorg.2019.03.068
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Design and synthesis of novel PARP-1 inhibitors based on
pyridopyridazinone scaffold
Ghada F. Elmasry*, Enayat E. Aly, Fadi M. Awadallah and Samir M. El-Moghazy
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, El-Kasr
El-Eini Street, P.O. Box 11562 Cairo, Egypt
Abstract
Various pyridopyridazinone derivatives were designed as Poly(ADP-ribose)
polymerase-1 (PARP-1) inhibitors. The pyridopyridazinone scaffold was used as
an isostere of the phthalazine nucleus of the lead compound Olaparib in addition
to some modifications in the tail part of the molecule. Preliminary biological
evaluation indicated that most compounds possessed inhibitory potencies
comparable to Olaparib in nanomolar level. The best PARP-1 inhibitory activity
was observed for compound 8a with (IC = 36 nM) compared to Olaparib as a
50
reference drug (IC = 34 nM). Molecular modeling simulation revealed that, the
50
designed compounds docked well into PARP-1 active site and their complexes
are stabilized by three key hydrogen bond interactions with both Gly863 and
Ser904 as well as other favorable - and hydrogen- stacking interactions with
Tyr907 and Tyr896, respectively. Computational ADME study predicted that the
target compounds 8a and 8e have proper pharmacokinetic and drug-likeness
properties. These outcomes afford a new structural framework for the design of
novel inhibitors for PARP-1.
_____
Keywords: Pyridopyridazinones; PARP-1 inhibitors; Olaparib; Molecular
docking.
*
Corresponding
author
Tel.:
+201061761234;
e-mail:
ghada.elmasry@pharma.cu.edu.eg
1

1. Introduction
Cancer is a disease characterized by uncontrolled growth of abnormal cells
disregarding the normal cell division rules. Normal cells are continually subject
to signals that regulate the cell division, differentiation or death. Cancer cells
deregulate these signals, leading to uncontrolled division and proliferation [1].
Poly(ADP-ribose) polymerase (PARP) inhibitors represent an interesting
novel class of targeted cancer therapies [2,3]. Poly(ADP-ribose)polymerase-1
(PARP-1) is a chromatin-bound nuclear enzyme capable of DNA repair as it
recognizes and binds rapidly to DNA single or double strand breaks [4,5].
Activated PARP-1 utilizes NADP to synthesize poly (ADP)-ribose upon
association with DNA, either on itself or on a variety of nuclear target proteins
such as histones, topoisomerases, DNA ligases and DNA polymerases [6,7]. This
leads to the formation of highly negatively charged nuclear proteins that
facilitate the recruitment of the damaged DNA through the base excision repair
(BER) machinery. Thus, inhibition of PARP enhances the effects of cytotoxic
drugs such as topoisomerase I inhibitors and DNA alkylating agents as well as
ionizing radiation [8,9]. Furthermore, some PARP inhibitors have been shown to
display single agent activity against tumors exhibiting BRCA1- or BRCA2-
mutations [10-12]. Accordingly, development of PARP inhibitors is an attractive
research area providing advanced cancer treatment possibilities.
Few PARP-1 inhibitors have been discovered and FDA approved e.g.,
Olaparib II [13], Niraparib III [14], Rucaparib IV [15] and Talazoparib V [16].
Moreover, several phthalazinones have been considered as effective orally
bioavailable PARP inhibitors [17,18] such as KU58948 VI and AZD 2461 VII
(
Figure 1).
Nevertheless, effort of pursuing new inhibitors with high potency, significant
aqueous solubility and good oral bioavailability against PARP-1 is still needed.
2

Figure 1. Nicotinamide-based PARP-1 inhibitors
Surveying the literature revealed that the design of PARP-1 inhibitors
generally depends on the nicotinamide moiety of NAD+ to mimic the ligand-
protein interaction of NAD+ with PARP-1 [19] and thus the common
3

pharmacophoric features are aromatic ring and carboxamide moiety, which can
form hydrogen bonds and -stacking interactions in PARP-1 catalytic domain
[20-22].
X-Ray crystal structures of PARP-1 with bound inhibitors [23] and
molecular modeling studies [21,24] indicate that there are three crucial
hydrogen bond interactions between the carboxamide moiety and two critical
amino acid residues in the PARP active site, Gly863 (NH to Gly C=O and C=O
to Gly NH) and Ser904 (C=O to Ser OH). In addition, the phenyl moiety of
Tyr907 makes - stacking interaction with the nicotinamide of NAD+.
Attempts to improve PARP inhibitors’ binding affinity have been made by
restriction of the carboxamide group which is usually free to rotate. Locking of
the carboxamide group into the required conformation can be made by either
inserting on the aromatic ring heteroatoms or groups able to give an
intramolecular hydrogen bond with the amide NH, or enclosing the amide group
into a bicyclic system [20].
Based on these findings, the design of the new compounds relied on the
use of the phthalazinone derivatives (II, VI and VII) as lead compounds where
two main modifications were conducted. The first structural modification
involved an isosteric replacement of the phthalazinone nucleus with the
pyridopyridazinone scaffold. Literature review revealed that the majority of
PARP-1 inhibitors are phthalazinones, quinazolinones or isoquinolinones
[25,26], and to our knowledge none of them featured the nicotinamide moiety
i.e. the pyridine ring and the carboxamide group (in the form of lactam) at its 3-
position. This urged us to mimic the nicotinamide moiety and incorporate it as a
bicyclic system namely, pyridopyridazine. This also aimed to maintain low lipid
solubility and blood brain barrier permeability similar to Olaparib to avoid
possible penetration and CNS side effects [27,28]. In silico pharmacokinetic
study proved that if our compounds possessed a phthalazinone instead of the
pyridopyridazinone nucleus they would have high blood brain barrier
4

penetration. Moreover, it was reported that the presence of nitrogen in the
tetrahydro-pyridopyridazinone scaffold of compound VIII improved the
pharmacokinetic properties over similar carbon based analogues [21]. The
second structural modification pertained to attaching a variety of functional
pharmacophoric fragments to the pendant benzyl group at position 4 of the
pyridopyridazinone scaffold. These fragments were attached through an amide
linkage at position 4 of the benzyl group to block possible metabolism at this
position and extend half-life.
In summary, the current design featured the following pharmacophoric
elements: 1) a hetero aromatic ring (pyridyl ring was taken instead of phenyl of
the phthalazinone to mimic the nicotinamide moiety, seek new possibilities and
explore alternative chemical templates), 2) a lactam moiety with NH group
locked into the required conformation, this was achieved with ring closure of the
carboxamide into bicyclic system, 3) an auxiliary appendage with a shorter or
longer chain is generally attached to the polycyclic core as a solvent accessory
region (the substitutions at the para position of benzyl contribute to block
possible metabolism and extend half-life) [18] as shown in Figure 2.
5

Figure 2. Design approach of the targeted pyridopyridazinone derivatives 8a-g,
a,b, 10a-d and 11a-g as PARP-1 inhibitors
9
To verify the rationale of the design and to approach the development of
potent inhibitors, the molecular docking study of all of the compounds to the
catalytic domain of PARP-1 was first conducted. Affinity of the compounds to
the target enzyme was judged by comparing the binding free energy (docking
score) and the binding mode of the target compounds with that of the co-
crystallized ligand Olaparib. Analysis of the docking results showed that all the
designed compounds fit into the active site of PARP-1, most of them displayed
6

comparable docking scores and binding modes similar to that of Olaparib and
were able to reproduce all the main interactions accomplished by the co-
crystallized ligand with the key amino acids in the active site. Docking of
Olaparib, docking of compound 8a, and alignment of the co-crystallized ligand
(Olaparib) and compound 8a in the catalytic domain of human PARP-1 are
shown in Figures 3-5.
Figure 3. 2D interaction diagram showing Olaparib docking pose interactions
with the key amino acids in the PARP-1 active site (distances in Å).
7

Figure 4. 2D diagram of compound 8a showing its interactions with the
PARP-1 active site (distances in Å).
Figure 5. X-Ray Co-crystal structure of Olaparib (green) overlaid with the
proposed binding mode of compound 8a (magenta) in the catalytic domain of
8

human PARP-1 showing the main interactions with the key amino acids in the
active site.
2. Results and discussion
2.1. Chemistry
The new pyridopyridazinone derivatives were synthesized, as depicted in
schemes 1 and 2. At first, the quinolinic anhydride 2 was prepared as reported by
the reaction of acetic anhydride with quinolinic acid 1 [29,30]. Next, reduction
of quinolinic anhydride with NaBH in dry tetrahydrofuran in the presence of
4
acetic acid was done to obtain the intermediate 2-hydroxymethyl-3-
pyridinecarboxylic acid 3, which spontaneously lactonized upon addition of
acetic anhydride to yield the furo[3,4-b]pyridin-5(7H)-one 4 [33,34]. Reaction of
compound 4 with 4-carboxy benzaldehyde using sodium methoxide in methanol
and ethyl acetate gave the corresponding 4-(5-hydroxy-7-oxo-7H-
cyclopenta[b]pyridin-6-yl)benzoic acid 5, which was then reacted with excess
hydrazine hydrate at 100 °C to obtain the key compound 4-((5-oxo-5,6-
dihydropyrido[2,3-d]pyridazin-8-yl)methyl)benzoic acid 6.
The synthesis of the target compounds is illustrated in scheme 2 where
one pot amide synthesis was adopted in which the acid chloride 7, obtained from
the reaction of the precursor acid derivative 6 with thionyl chloride, was reacted
with the appropriate amino compounds viz, secondary amines, 2-
aminobenzothiazoles, 2-aminophenylthiazoles, anilines and sulfanilamide in dry
DMF in the presence of triethylamine to give 8a-g, 9a,b, 10a-d and 11a-g,
respectively. The postulated structures of the products were proved by the
disappearance of carboxylic OH band in the IR spectra and its exchangeable
1
1
signals in the H-NMR spectra. Furthermore, the H-NMR showed characteristic
signals at δ 1.58-3.41 ppm region attributed to the morpholino, piperidino,
9

pyrrolidino or piperazino protons in compounds 8a-g, in addition to signals
1
attributed to the additional aromatic protons in compounds 8d-g. H-NMR
spectra of compounds 9a,b, 10a-d and 11a-g showed additional deuterium
exchangeable singlet signals around δ 10.06-10.47 ppm corresponding to the
new NH proton, in addition to the aromatic protons signals of the attached
13
moieties. C NMR supported the carbon skeleton of the target compounds.
Scheme 1. Preparation of the key starting compound 6.
1
0

Scheme 2. Synthesis of the designed compounds 8a-e, 9a,b, 10a-d and 11a-g.
1
1

2.2. In vitro PARP-1 inhibitory activity
In order to gain a more clear insight to the structure–activity relationship,
all final compounds were evaluated for their inhibitory activity on PARP-1. The
IC values of all the tested compounds against PARP-1 expressed in (nM
50
concentration) and pIC values (-log IC in molar) compared to the reference
50
50
drug Olaparib are summarized in Table 1.
Table 1. In vitro inhibitory activity of the synthesized compounds against
PARP-1.
Compound
IC (nM)
pIC ±S.D
5
0
50
8
a
36.74
133.03
165.08
71.58
7.43±0.02
6.87±0.015
6.78±0.015
7.15±0.015
7.31±0.015
6.74±0.015
6.85±0.02
7.20±0.015
7.16±0.015
7.34±0.02
6.66±0.02
7.27±0.02
7.22±0.015
6.20±0.015
7.19±0.02
6.55±0.015
8
b
8
c
8
d
8
e
48.90
8
f
180.92
142.001
63.81
8
9
g
a
9
b
69.79
1
0a
0b
0c
0d
1a
1b
1c
45.52
1
218.78
53.83
1
1
60.87
1
634.88
64.33
1
1
283.85
1
2

1
1d
1e
1f
1g
Olaparib
54.18
269.19
82.94
56.46
34.15
7.27±0.015
6.57±0.02
7.08±0.02
7.25±0.02
7.47±0.015
1
1
1
Interestingly, the target pyridopyridazinones 8a-g, 9a,b, 10a-d and 11a-g
exhibited a wide range of PARP-1 inhibitory activity but all in the nanomolar
range with IC in the range of 36-634 nM. It can therefore be claimed that a
50
wide variety of substituents can be tolerated at the terminal position of the
molecule. This observation can be rationalized if these side chains are
approaching the solvent surface and do not significantly interact with the
enzyme. However, it was noted that hydrophobic residues are preferred at this
position particularly. Concerning the shorter derivatives 8a-c of the substituted
carbonyl series 8a-g, the pyrrrolo derivative 8a was found to be the most active
and the order of activity was pyrrolo > piperidino > morpholino. It is worth to
mention that 8a displayed the highest activity among the all the target
compounds (IC = 36 nM) approaching the activity of the control Olaparib (IC
50
50
=
34 nM). For the compounds bearing phenyl piperazine moities 8d-g,
introduction of an electron-donating group on the benzene ring increased the
inhibitory activity as depicted by the methoxy derivative 8e, while the electron-
withdrawing substituted derivatives (Cl and CF ) (8f and 8g, IC = 180 and 142
3
50
nM, respectively) displayed much lower potency compared to the unsubstituted
analogue (8d, IC = 48 nM). Comparing the benzothiazole derivatives 9a,b and
50
their phenylthiazole counterparts 10a-d revealed that the fusion of the thiazole
moiety with the phenyl ring did not elicit significant changes in the inhibitory
activity. Moreover, substituted and unsubstituted benzothiazole congeners were
nearly equipotent. Regarding the phenylthiazole series, the unsubstituted
1
3

phenylthiazole 10a derivative showed high potency (IC = 48 nM). On the
50
contrary, the methyl congener 10b displayed much lower activity (IC = 218
50
nM). Replacement of the methyl group with methoxy or bromo substituents
enhanced the activity where compounds 10c and 10d showed IC = 53 and 60
50
nM, respectively. With respect to the anilino derivatives 11a-f, the unsubstituted
compound 11a exhibited the lowest activity among all the synthesized
compounds. Exploring the effect of different substituents at position 4 on the
benzamide moiety, revealed that the introduction of electron donating groups
enhanced the inhibitory potency viz the hydroxy 11b and methoxy 11d
derivatives rather than electron withdrawing groups. Finally, the sulfonamide
derivative 11g showed good potency.
3. In silico studies
3.1. Molecular docking
The molecular docking approach for all of the compounds was used to
support the design hypothesis prior synthesis as mentioned before.
Crystallographic structure of the human PARP-1 catalytic domain (PDB code:
5DS3) was obtained from the Protein Data Bank (PDB) database. The key
interactions in the PARP-1 active site, consistent with previous literature reports,
were highlighted. The lactam amide group of almost all the designed molecules
displayed three key hydrogen-bond interactions with both Ser904 and Gly863;
which involved the interaction of the pyridazinone carbonyl group as H-bond
acceptor with the backbone carbonyl of Gly863 -NH group and Ser904 -OH
group, besides the interaction of the pyridazinone ring NH as H-bond donor with
Gly863. Moreover, a specific -stacking interaction for the designed PARP
inhibitors was observed between the pyridazinone ring and the phenyl moiety of
Tyr907. These interactions are also displayed in the binding of Olaparib to the
PARP-1 active site which suggested the potential activity of the novel
1
4

compounds. The docking results of the most active compounds and Olaparib
with amino acids of the active site are summarized in Table 2. The highest
inhibition of PARP-1 detected for compounds 8a, 10a, 8e, 10c, 11d and 11g
suggested a detailed analysis of their possible binding modes in the catalytic
domain of PARP-1. The pyridopyridazinones 8a and 8e showed slightly lower
binding energy score compared to Olaparib while compounds 10a (Figure 6),
10c, 11d and 11g displayed comparable or even higher binding to PARP-1. Both
Ser904 and Gly863 were engaged in main hydrogen bond interactions with the
carboxamide group of the target derivative 8a. The pyridazinone ring of 8a
showed clear -stack interaction with Tyr-907. Additionally, 8a was involved in
hydogen- interactions with Tyr896 and Lys903 in a similar manner to Olaparib.
It is worth to mention that the key hydrogen bonds displayed by 8a characteristic
of this class of inhibitors were nearly of the same length of that of Olaparib or
even shorter and this may in part account for its high biological activity despite
the lower binding energy. The methoxy group attached to the piperazine moiety
of 8e showed additional hydrogen- interaction with Arg878. Compounds 8e
and 10a were stabilized through three hydrogen– interactions with Tyr896 and
Arg878. Compound 10c was involved in a new hydrogen- interaction with
Pro881. Moreover, compound 11d formed an additional hydrogen bond to
Arg878, similar to that of Olaparib via the oxygen of methoxy group, in addition
to a characteristic hydrogen- interaction with the same amino acid. Finally, the
pyridopyridazinone 11g achieved two hydrogen bonds with Arg878 via its
sulphone moiety. These findings revealed a good correlation between the
molecular modeling studies and the biological results.
-1
Table 2. Docking scores as “S” (Kcal mol ) and bond interactions of the most
active derivatives with key amino acids of PARP-1.
S
No. of
Distance Amino acids
(Aº) involved
Cpd. No.
-1
Interacting groups
(
Kcal mol ) bonds
1
5

8
a
-29.08
-29.32
6
7
2.87
Gly863
Gly863
Ser904
Tyr907
Tyr896
Lys903
H-bond with NH of pyridazinone
H-bond with C=O of pyridazinone
H-bond with C=O of pyridazinone
- stack with pyridazinone ring
H- interaction with pyridyl ring
H- interaction with pyridyl ring
H-bond with NH of pyridazinone
H-bond with C=O of pyridazinone
H-bond with C=O of pyridazinone
- stack with pyridazinone ring
H- interaction with pyridyl ring
H- interaction with phenyl ring
H- interaction with methoxyphenyl
H-bond with NH of pyridazinone
H-bond with C=O of pyridazinone
H-bond with C=O of pyridazinone
- stack with pyridazinone ring
H- interaction with pyridyl ring
H-interaction with phenyl ring
H- interaction with thiazole ring
H-bond with NH of pyridazinone
H-bond with C=O of pyridazinone
H-bond with C=O of pyridazinone
- stack with pyridazinone ring
H- interaction with pyridyl ring
H- interaction with methoxy phenyl ring
H-bond with NH of pyridazinone
H-bond with C=O of pyridazinone
H-bond with C=O of pyridazinone
- stack with pyridazinone ring
H- interaction with pyridyl ring
H-bond with O of methoxy
2
2
.99
.77
8
e
3.14
Gly863
Gly863
Ser904
Tyr907
Tyr896
Tyr896
Arg878
2
3
.92
.25
1
0a
0c
-35.05
7
2.85
Gly863
Gly863
Ser904
Tyr907
Tyr896
Tyr896
Arg878
3
2
.17
.95
1
-32.39
-32.35
6
7
2.91
Gly863
Gly863
Ser904
Tyr907
Tyr896
Pro881
2
2
.95
.99
1
1d
2.90
Gly863
Gly863
Ser904
Tyr907
Tyr896
Arg878
Arg878
2
3
.97
.17
3
.00
H- interaction with methoxy phenyl
H-bond with NH of pyridazinone
H-bond with C=O of pyridazinone
H-bond with C=O of pyridazinone
- interaction with pyridazinone ring
H- interaction with pyridyl ring
H-bond with O of sulfone
1
1g
-31.19
-31.95
7
8
2.98
Gly863
Gly863
Ser904
Tyr907
Tyr896
Arg878
Arg878
Gly863
Gly863
Ser904
Tyr907
Tyr896
Tyr896
Lys903
Arg878
2
3
.93
.04
2
3
.93
.19
H-bond with amino of sulfone
Olaparib
2.81
H-bond with NH of pyridazinone
H-bond with C=O of pyridazinone
H-bond with C=O of pyridazinone
- stack with pyridazinone ring
H-bond with C=O linker
2
2
.92
.77
2
2
.83
.89
H- interaction with phenyl of phthalazinone
H- interaction with phenyl of phthalazinone
H-bond with C=O linker
1
6

Figure 6. 3D diagram of compound 10a showing its interaction with the PARP-
active site
1
3
.2. Drug-likeness properties
The drug-likeness profiles for Olaparib II and the most active compounds
a and 8e were predicted using SwissADME server [31]. The results of the
8
Swiss ADME prediction of drug likeness of these compounds are shown in
Supplemantary Table 1. Compounds 8a and 8e showed no violation to Lipinski
and other pharmacokinetics filters, and contained no alerts for Pan Assay
Interfering Substances (PAINS). Furthermore, the pyridopyridazinones 8a and
8
e exhibited a predicted consensus log Po/w value of 2.41 and 2.92, respectively
with good to moderate water solubility, high GIT absorption and no BBB
permeability which correlated with minimal predicted CNS adverse effects.
Moreover, the oral bioavailability radar chart of compound 8a and 8e indicated
1
7

their good predicted oral bioavailability and promising pharmacokinetic
properties (For details see Supplementary Figure 1 and Figure 2).
4. Conclusion:
This research work aimed at discovering novel PARP-1 inhibitors with a
significant advantage of prospective improved physicochemical properties and
where a wide variety of substituents can be tolerated. The design of the target
compounds was based on the Olaparib template model which was modified
through replacement of its phthalazine ring with an isosteric pyridopyridazine
nucleus and through appending of various chemical fragments to its benzyl side
chain. Most compounds possessed inhibitory potencies comparable to, or slightly
lower than Olaparib. Molecular docking study was performed prior to synthesis
to assess the design of the new compounds and after biological testing to have a
deep insight of the SAR of the compounds. Drug likeness of the compounds was
also predicted in silico and revealed promising pharmacokinetic properties of the
newly synthesized compounds. Therefore, this study represents an introduction
of a new ring system (pyridopyridazinone) in the field of NAD-like PARP-1
inhibitors. As a preliminary study, a success was achieved in presenting
compounds with activity in the nanomolar level. We thus believe that these
compounds represent good hits for further study of structure–activity
relationship and optimization.
5
5
5
. Experimental
.1. Chemistry
.1.1 General
Starting materials and solvents were purchased from commercial suppliers
and were used without further purification. NMR and Infrared spectra were
performed at the Microanalytical Unit-Faculty of Pharmacy, Cairo University.
1
8

1
1
HNMR spectra were recorded on a Bruker Ascend 400 MHz spectrometer and
3
C spectra were run at 100 MHz in deuterated dimethylsulfoxide (DMSO‑ d6).
Chemical shift values (δ) are given in parts per million (ppm). Coupling
constants are given in hertz (Hz) and spin multiplicities are given as s (singlet), d
(
doublet), dd (doublet of doublet), t (triplet), q (quartet) or m (multiplet). Infrared
spectra (IR) were recorded as potassium bromide discs on Schimadzu FT-IR
400S spectrophotometer (Kyoto, Japan) and expressed in wave number (υmax,
8
-1
cm ). TLC was performed using silica gel/TLC cards DC-Alufolien-Kiesel gel
Vilber GmbH, Germany) with fluorescent indicator UV254 using chloroform :
(
methanol 8.5 : 1.5 as the eluting system and the spots were visualized using
Vilber Lourmet ultraviolet lamp (Vilber GmbH, Germany) at = 254 nm. Melting
points were determined by open capillary tube method using Electrothermal
9100 melting point apparatus (Staffordshire, UK) and are uncorrected. Mass
spectral data are given as m/z (relative abundance %). Elemental analyses were
carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar
University, Egypt. Compounds 2, 3 [29,30] and 4 [32,33] were synthesized as
reported in the literature.
5.1.2. 4-(5-Hydroxy-7-oxo-7H-cyclopenta[b]pyridin-6-yl)benzoic acid 5
A cold solution of CH ONa (0.8 g, 35 mmol) in MeOH (24 mL) was added
3
dropwise to a mixture of compound 4 (1 g, 7.4 mmol) and 4-
carboxybenzaldehyde (1.12 g, 8 mmol) in ethyl acetate (8 mL). The reaction
mixture was stirred at room temperature for 1 h, heated to reflux for 1 h, then the
solvent was distilled off under reduced pressure. The residue was poured into ice
water, acidified with glacial acetic acid to pH 2-3 and separated by filtration to
-
1
give compound 5. Yield: 65%, m.p.: >300 °C, I.R. (KBr, cm ): υ
3537 (OH),
max
3
1
097 (CH aromatic), 2638-2515 (carboxylic OH), 1678 (C=O), 1600 (C=N),
1
535 (C=C), H-NMR: δ 7.19-7.22 (m, 1H, pyridyl-H), 7.48 (dd, 1H, J = 1.4, 7.1
Hz, pyridyl-H), 7.75 (d, 2H, J = 8.7 Hz, phenyl-H), 8.37 (dd, 1H, J = 1.4, 5.1 Hz,
1
9

pyridyl-H), 8.62 (d, 2H, J = 8.7 Hz, phenyl-H), 12.26 (s, 2H, 2 OH exchanged by
D O). Anal. Calcd. for C H NO (267.24): C, 67.42; H, 3.39; N, 5.24. Found: C,
2
15
9
4
67.14; H, 3.62; N, 5.49.
5.1.3. 4-((5-Oxo-5,6-dihydropyrido[2,3-d]pyridazin-8-yl)methyl)benzoic acid 6
A solution of 99% hydrazine hydrate (3 mL, 94 mmol) and compound 5 (1 g,
.75 mmol) was stirred at 100 °C for 5 h. The reaction mixture was allowed to
3
cool and the separated product was filtered off, washed with ethanol and left to
dry. The obtained solid was dissolved in distilled water and the solution was
acidified with acetic acid. The resulting precipitate was filtered off, washed with
water and dried to give compound 6 which was recrystallized from DMF. Yield:
-
1
4
2
1
7
0%, m.p.: 230-231 °C, I.R (KBr, cm ): υ
3244 (NH), 3086 (CH aromatic),
max
931 (CH aliphatic), 2931-2854 (carboxylic OH), 1701 (2C=O), 1593 (C=N),
1
550 (C=C), H-NMR: δ 4.40 (s, 2H, CH ), 7.43 (d, 2H, J = 8.1 Hz, phenyl-H),
2
.83-7.87 (m, 3H, pyridyl-H and phenyl-H), 8.35 (d, 1H, J = 8.24 Hz, pyridyl-
H), 9.04 (d, 1H, J = 4.3 Hz, pyridyl-H), 12.50 (s, 1H, carboxylic OH exchanged
13
by D O), 12.84 (s, 1H, NH exchanged by D O). C-NMR: δ 37.5 (CH carbon),
2
2
2
126.9, 128.4, 129.4, 129.6, 130.1, 134.6, 143.5, 144.2, 144.9 (Ar-Cs), 154.1
(
C=N), 159.1 (C=O), 167.6 (C=O). Anal. Calcd. for C H N O (281.27): C,
15
11
3
3
64.05; H, 3.94; N, 14.94. Found: C, 63.89; H, 4.09; N, 15.21.
5.1.4. 4-((5-Oxo-5,6-dihydropyrido[2,3-d]pyridazin-8-yl)methyl)benzoyl
chloride 7
Compound 6 (0.5 g, 1.8 mmol) was heated under reflux with thionyl chloride (5
mL) and DMF (3 drop) for 2 h., excess thionyl chloride was distilled off under
reduced pressure and residual thionyl chloride was removed azeotropically with
dry benzene. The acid chloride thus obtained was used for further reactions
without purification.
2
0

5.1.5. General procedure for the synthesis of compounds 8a-g, 9a,b, 10a-d, 11a-f
and 12
The crude acid chloride 7 obtained in the previous step was cooled to 0 °C and
dissolved in dry DMF (2 mL). To this solution, the appropriate amine (1.65
mmol) and triethylamine (1 mL, 10 mmol) were added. The reaction mixture
was stirred at 100 °C for 6 hours. The solution was poured over crushed ice and
the precipitated solid was filtered off, washed with water, dried and
recrystallized from the suitable solvent.
5.1.5.1. 8-(4-(Pyrrolidine-1-carbonyl)benzyl)pyrido[2,3-d]pyridazin-5(6H)-one
8
a
-1
Yield: 60%, m.p.: 185-187 °C, I.R (KBr, cm ): υ
3425 (NH), 3159 (CH
max
aromatic), 2970 (CH aliphatic), 1690, 1654 (2C=O), 1612 (C=N), 1562 (C=C),
1
H-NMR: δ 1.75-1.85 (m, 4H, pyrrolidinyl-H), 3.33 (t, 4H, pyrrolidinyl-H, J =
6
8
.28 Hz), 4.36 (s, 2H, CH ), 7.34 (d, 2H, J = 8.0 Hz, phenyl-H), 7.44 (d, 2H, J =
2
.2 Hz, phenyl-H), 7.82-7.88 (m, 1H, pyridyl-H), 8.37 (dd, 1H, J = 1.2, 8.2 Hz,
pyridyl-H), 9.04 (dd, 1H, J = 1.2, 4.4 Hz, pyridyl-H), 12.86 (s, 1H, NH
13
exchanged by D O). C-NMR: δ 24.3, (pyrrolidine-Cs), 37.4 (CH carbon), 45.8
2
2
(
(
(
pyrrolidine-Cs), 127.7, 128.3, 128.9, 134.6, 135.9, 139.9, 144.3, 145.1, 154.1
Ar-Cs), 159.0 (C=N), 161.1 (C=O), 168.4 (C=O). Anal. Calcd. for C H N O
19
18
4
2
334.38): C, 68.25; H, 5.43; N, 16.76. Found: C, 68.47; H, 5.66; N, 16.91.
5.1.5.2. 8-(4-(Piperidine-1-carbonyl)benzyl)pyrido[2,3-d]pyridazin-5(6H)-one
8
b
-1
Yield: 71%, m.p.: 168-170 °C, I.R (KBr, cm ): υ
3444 (NH), 3124 (CH
max
aromatic), 2931(CH aliphatic), 1680, 1670 (2C=O), 1620 (C=N), 1504 (C=C),
1
H-NMR: δ 1.48-1.74 (m, 10H, piperidinyl-H), 4.36 (s, 2H, CH ), 7.28 (d, 2H, J
2
=
8.04 Hz, phenyl-H), 7.37 (d, 2H, J = 8.2 Hz, phenyl-H), 7.85-7.88 (m, 1H,
pyridyl-H), 8.4 (d, 1H, J = 1.2, 7.92 Hz, pyridyl-H), 9.05 (d, 1H, J = 3.2 Hz,
2
1

13
pyridyl-H), 12.79 (s, 1H, NH exchanged by D O). C-NMR: δ 24.5, 26.4
2
(
piperidine-Cs), 37.4 (CH carbon), 50.1 (piperidine-Cs), 126.3, 127.4, 128.2,
2
1
1
29.1, 131.3, 134.6, 135.1, 139.7, 145.1 (Ar-Cs), 154.0 (C=N), 159.0 (C=O),
+
69.2 (C=O). MS m/z (%): 348 (M , 21.36%), 84 (100%). Anal. Calcd. for
C H N O (348.41): C, 68.95; H, 5.79; N, 16.08. Found: C, 69.23; H, 5.87; N,
20
20
4
2
16.24.
5.1.5.3. 8-(4-(Morpholine-4-carbonyl)benzyl)pyrido[2,3-d]pyridazin-5(6H)-one
8
c
-1
Yield: 45%, m.p.: 180-182 °C, I.R (KBr, cm ): υ
3464 (NH), 3100(CH
max
1
aromatic), 2920 (CH aliphatic), 1666 (2C=O), 1620 (C=N), 1543 (C=C), H-
NMR: δ 3.41 (t, 2H, J = 5.00 Hz, morpholinyl-H), 3.59 (t, 2H, J = 5.12 Hz,
morpholinyl -H), 3.67 (t, 2H, J = 4.56 Hz, morpholinyl -H), 3.72 (t, 2H, J =
3
.88 Hz, morpholinyl -H), 4.33 (s, 2H, CH ), 7.32 (d, 2H, J = 8.0 Hz, phenyl-
2
H), 7.37 (d, 2H, J = 8.2 Hz, phenyl-H), 7.66-7.69 (m, 1H, J = 3.8, 7.4 Hz,
pyridyl-H), 8.03 (d, 1H, J = 1.2, 6.2 Hz, pyridyl-H), 9.09 (d, 1H, J = 3.3 Hz,
pyridyl-H), 11.41 (s, 1H, NH exchanged by D O). Anal. Calcd. for C H N O
2
19 18
4
3
(350.38): C, 65.13; H, 5.18; N, 15.99. Found: C, 65.28; H, 5.34; N, 16.21.
5
.1.5.4.
(6H)-one 8d
Yield: 70%, m.p.: 136-138 °C, I.R (KBr, cm ): υ
8-(4-(4-Phenylpiperazine-1-carbonyl)benzyl)pyrido[2,3-d]pyridazin-
5
-1
3553 (NH), 3089 (CH
max
aromatic), 2920 (CH aliphatic), 1680, 1666 (2C=O), 1620 (C=N), 1496 (C=C),
1
H-NMR: δ 3.35 (s, 8H, piperazinyl-H), 4.37 (s, 2H, CH ), 6.81 (d, 2H, J = 6.7
2
Hz, phenyl piperazine-H), 6.94 (d, 2H, J = 8.2 Hz, phenyl piperazine-H), 7.23 (t,
3
H, J = 7.6 Hz, 2 phenyl-H and 1 phenyl piperazine-H), 7.39 (d, 2H, J = 5.2,
phenyl -H), 7.87-7.90 (m, 1H, pyridyl-H), 8.40 (d, 1H, J = 8.1 Hz, pyridyl-H),
.05 (d, 1H, J = 4.1 Hz, pyridyl-H), 12.86 (s, 1H, NH exchanged by D O). Anal.
9
2
2
2

Calcd. for C H N O (425.49): C, 70.57; H, 5.45; N, 16.46. Found: C, 70.39; H,
25
23
5
2
5.63; N, 16.80.
5.1.5.5.
8-(4-(4-(2-Methoxyphenyl)piperazine-1-
carbonyl)benzyl)pyrido[2,3-d]pyridazin-5(6H)-one 8e
-1
Yield: 65%, m.p.: 150-152 °C, I.R (KBr, cm ): υ
3444 (NH), 3062 (CH
max
1
aromatic), 2924 (CH aliphatic), 1666 (2C=O), 1624 (C=N), 1500 (C=C), H-
NMR: δ 2.95 (s, 8H, piperazinyl-H), 3.77 (s, 3H, methoxy-H), 4.37 (s, 2H, CH2),
6
7
=
.88-6.95 (m, 4H, methoxy phenyl-H), 7.38 (d, 4H, J = 8.84 Hz, phenyl-H),
.87-7.90 (m, 1H, pyridyl-H), 8.40 (d, 1H, J = 8.1 Hz, pyridyl-H), 9.05 (d, 1H, J
13
3.4 Hz, pyridyl-H), 12.86 (s, 1H, NH exchanged by D O). C-NMR: δ 37.4
2
(
CH carbon), 46.2, 50.7 (piperazine-Cs), 55.8 (OCH carbon) 112.3, 118.8,
2
3
1
1
21.2, 123.4, 126.5, 127.9, 128.4, 128.8, 129.1, 134.6, 139.9, 141.1, 144.3,
45.1, 152.5 (Ar-Cs), 154.1 (C=N), 159.1 (C=O), 169.2 (C=O). Anal. Calcd. for
C H N O (455.52): C, 68.56; H, 5.53; N, 15.37. Found: C, 68.79; H, 5.80; N,
26
25
5
3
15.61.
5.1.5.6.
8-(4-(4-(4-Chlorophenyl)piperazine-1-carbonyl)benzyl)pyrido[2,3-
d]pyridazin-5(6H)-one 8f
Yield: 77%, m.p.: 188-190 °C, I.R (KBr, cm ): υ
-1
3421 (NH), 3066 (CH
max
1
aromatic), 2924 (CH aliphatic), 1670 (2C=O), 1624 (C=N), 1496 (C=C), H-
NMR: δ 3.14 (s, 8H, piperazinyl-H), 4.37 (s, 2H, CH ), 6.94-6.97 (m, 2H, chloro
2
phenyl-H), 7.23-7.27 (m, 4H, chloro phenyl and phenyl-H), 7.36-7.41 (m, 2H,
phenyl-H), 7.87-7.90 (m, 1H, pyridyl-H), 8.40 (dd, 1H, J = 1.3, 8.3 Hz, pyridyl-
H), 9.05 (dd, 1H, J = 1.3, 4.7 Hz, pyridyl-H), 12.85 (s, 1H, NH exchanged by
13
D O). C-NMR: δ 37.3 (CH carbon), 48.5, 48.7 (piperazine-Cs), 117.7, 123.3,
2
2
126.5, 127.8, 128.3, 129.1, 129.2, 134.3, 134.6, 139.9, 144.3, 145.11, 149.99
+
(
Ar-Cs), 154.1 (C=N), 159.1 (C=O), 169.3 (C=O). MS m/z (%): 459 (M ,
1
0.16%), 461 (M+2, 4.14%), 67 (100%). Anal. Calcd. for C H ClN O
25
22
5
2
(459.93): C, 65.29; H, 4.82; N, 15.23. Found: C, 65.17; H, 4.98; N, 15.41.
2
3

5.1.5.7.
8-(4-(4-(4-(Trifluoromethyl)phenyl)piperazine-1-
carbonyl)benzyl)pyrido[2,3-d]pyridazin-5(6H)-one 8g
-1
Yield: 65%, m.p.: 120-122 °C, I.R (KBr, cm ): υ
3464 (NH), 3116 (CH
max
1
aromatic), 2924 (CH aliphatic), 1670 (2C=O), 1635 (C=N), 1550 (C=C), H-
NMR: δ 3.35 (s, 8H, piperazinyl-H), 4.37 (s, 2H, CH ), 7.18-7.24 (m, 2H,
2
trifluorophenyl-H), 7.37-7.45 (m, 6H, trifluorophenyl-H and phenyl-H), 7.87-
7.90 (m, 1H, pyridyl-H), 8.40 (d, 1H, J = 8.2 Hz, pyridyl-H), 9.05 (d, 1H, J = 3.5
Hz, pyridyl-H), 12.86 (s, 1H, NH exchanged by D O). Anal. Calcd. for
2
C H F N O (493.49): C, 63.28; H, 4.49; N, 14.19. Found: C, 63.56; H, 4.65;
26
22
3
5
2
N, 14.53.
5
.1.5.8.
d]pyridazin-8-yl)methyl)benzamide 9a
Yield: 70%, m.p.: 200-202 °C, I.R (KBr, cm ): υ
N-(Benzo[d]thiazol-2-yl)-4-((5-oxo-5,6-dihydropyrido[2,3-
-1
3421 (2NH), 3066 (CH
max
1
aromatic), 2978 (CH aliphatic), 1666 (2C=O), 1593 (C=N), 1531(C=C), H-
NMR: δ 4.42 (s, 2H, CH ), 7.49 (d, 2H, J = 8.0 Hz, phenyl-H), 7.80 (d, 2H, J =
2
8
.6 Hz, phenyl-H), 7.87-7.92 (m, 2H, 1 benzothiazolyl-H and 1 pyridyl-H), 8.04
t, 2H, J = 7.0 Hz, benzothiazolyl -H), 8.38 (t, 1H, J = 8.4 Hz, benzothiazolyl -
H), 8.66 (s, 1H, pyridyl-H), 9.05 (d, 1H, J = 4.0 Hz, pyridyl-H), 10.45 (s, 1H,
(
13
NH exchanged by D O), 12.86 (s, 1H, NH exchanged by D O). C-NMR: δ 37.3
2
2
(
CH carbon), 112.3, 113.2, 120.3, 123.2, 126.4, 128.3, 128.5, 129.2, 133.6,
2
1
1
1
34.5, 134.6, 144.3, 145.1, 149.8, 154.1, 155.4, 159.1 (Ar-Cs), 160.3 (C=O),
66.0 (C=O). Anal. Calcd. for C H N O S (413.45): C, 63.91; H, 3.66; N,
22
15
5
2
6.94. Found: C, 63.75; H, 3.89; N, 17.21.
.1.5.9. N-(6-Ethoxybenzo[d]thiazol-2-yl)-4-((5-oxo-5,6-
5
dihydropyrido[2,3-d]pyridazin-8-yl)methyl)benzamide 9b
-1
Yield: 65%, m.p.: 185-187 °C, I.R (KBr, cm ): υ
3421 (2NH), 3066 (CH
max
1
aromatic), 2924 (CH aliphatic), 1670 (2C=O), 1604 (C=N), 1550 (C=C), H-
NMR: δ 1.34 (t, 3H, J = 8.0 Hz, CH CH ), 4.06 (q, 2H, J = 6.9 Hz, CH CH ),
3
2
3
2
2
4

4
.42 (s, 2H, CH ), 6.90 (dd, 1H, J = 2.4, 8.8 Hz, benzothiazolyl -H), 7.27-7.56
2
(
m, 3H, 2 benzothiazolyl-H and 1 pyridyl-H), 7.64 (d, 2H, J = 8.8 Hz, phenyl -
H), 7.86 (d, 2H, J = 7.8 Hz, phenyl -H), 8.54 (s, 1H, pyridyl-H), 9.04 (d, 1H, J =
+
4
6
.8 Hz, pyridyl-H), 12.86 (s, 2H, NH exchanged by D O). MS m/z (%): 457 (M ,
2
4.98%), 64 (100%). Anal. Calcd. for C H N O S (457.51): C, 63.01; H, 4.19;
24
19
5
3
N, 15.31. Found: C, 63.29; H, 4.37; N, 15.48.
5
.1.5.10. 4-((5-Oxo-5,6-dihydropyrido[2,3-d]pyridazin-8-yl)methyl)-
N-(4-phenylthiazol-2-yl)benzamide 10a
Yield: 66%, m.p.: 199-201 °C, I.R (KBr, cm ): υ
-1
3421 (2NH), 3124 (CH
max
aromatic), 2924 (CH aliphatic), 1732, 1681 (2C=O), 1620 (C=N), 1543 (C=C),
1
H-NMR: δ 4.40 (s, 2H, CH ), 7.34-7.42 (m, 5H, phenyl-H), 7.44 (s, 1H,
2
thiazolyl -H), 7.78-7.88 (m, 5H, 4 phenyl-H and 1 pyridyl-H), 8.32 (s, 1H, J =
7.96 Hz, pyridyl-H), 9.04 (d, 1H, J = 4.3 Hz, pyridyl-H), 12.85 (s, 2H, NH
+
exchanged by D O). MS m/z (%): 439 (M , 22.44%), 43 (100%). Anal. Calcd.
2
for C H N O S (439.49): C, 65.59; H, 3.90; N, 15.94. Found: C, 65.83; H, 4.12;
24
17
5
2
N, 15.78.
5
.1.5.11. 4-((5-Oxo-5,6-dihydropyrido[2,3-d]pyridazin-8-yl)methyl)-N-
4-(p-tolyl)thiazol-2-yl)benzamide 10b
Yield: 50%, m.p.: 160-162 °C, I.R (KBr, cm ): υ
(
-1
3421 (2NH), 3024 (CH
max
aromatic), 2924 (CH aliphatic), 1732, 1666 (2C=O), 1612 (C=N), 1465 (C=C),
1
H-NMR: δ 2.50 (s, 3H, CH ), 4.39 (s, 2H, CH ), 7.16 (d, 4H, J = 8.12 Hz,
3
2
methylphenyl-H), 7.34 (s, 1H, thiazolyl -H), 7.66 (d, 1H, J = 7.8 Hz, pyridyl-H),
.77 (d, 4H, J = 7.9 Hz, phenyl-H), 8.36 (s, 1H, pyridyl-H), 9.04 (s, 1H, pyridyl-
H), 12.86 (s, 2H, NH exchanged by D O). Anal. Calcd. for C H N O S
7
2
25 19
5
2
(453.13): C, 66.21; H, 4.22; N, 15.44. Found: C, 66.48; H, 4.39; N, 15.61.
5.1.5.12.
N-(4-(4-Methoxyphenyl)thiazol-2-yl)-4-((5-oxo-5,6-
dihydropyrido[2,3-d]pyridazin-8-yl)methyl)benzamide 10c
2
5

-1
Yield: 52%, m.p.: 145-147 °C, I.R (KBr, cm ): υ
3444 (2NH), 3116 (CH
max
1
aromatic), 2999 (CH aliphatic), 1670 (2C=O), 1620 (C=N), 1485 (C=C), H-
NMR: δ 3.78 (s, 3H, OCH ), 4.43 (s, 2H, CH ), 6.90 (d, 2H, J = 8.8 Hz,
3
2
methoxyphenyl-H), 7.00 (d, 2H, J = 8.4 Hz, methoxyphenyl-H), 7.25 (s, 1H,
thiazolyl-H), 7.81 (d, 2H, J = 8.4 Hz, phenyl-H), 7.88 (d, 2H, J = 8.3 Hz, phenyl-
H), 8.07 (d, 1H, J = 8.0 Hz, pyridyl-H), 8.39 (d, 1H, J = 4.0 Hz, pyridyl-H), 9.05
(
d, 1H, J = 4.1 Hz, pyridyl-H), 12.86 (s, 2H, NH exchanged by D O). Anal.
2
Calcd. for C H N O S (469.52): C, 63.95; H, 4.08; N, 14.92. Found: C, 64.23;
25
19
5
3
H, 4.21; N, 15.13.
5.1.5.13..
N-(4-(4-Bromophenyl)thiazol-2-yl)-4-((5-oxo-5,6-
dihydropyrido[2,3-d]pyridazin-8-yl)methyl)benzamide 10d
-1
Yield: 55%, m.p.: 142-144 °C, I.R (KBr, cm ): υ
3417, 3425 (2NH), 3116
max
(CH aromatic), 2920 (CH aliphatic), 1666 (2C=O), 1620 (C=N), 1469 (C=C),
1
H-NMR: δ 4.42 (s, 2H, CH ), 7.08 (s, 1H, thiazolyl-H), 7.50-7.59 (m, 5H, 4
2
bromophenyl-H and 1 pyridyl-H), 7.74 (d, 2H, J = 8.3 Hz, phenyl-H), 7.84 (d,
2
1
1
1
H, J = 8.2 Hz, phenyl-H), 8.37 (s, 1H, pyridyl-H), 9.04 (s, 1H, pyridyl-H),
13
2.88 (s, 2H, NH exchanged by D O). C-NMR: δ 37.5 (CH carbon), 127.9,
2
2
28.2, 128.9, 129.4, 129.6, 131.4, 131.8, 132.1, 134.5, 137.9, 144.0, 146.9,
50.1, 151.2, 153.9, 157.5, 159.3, (Ar-Cs), 162.4 (C=O), 163.3 (C=O). Anal.
Calcd. for C H BrN O S (518.39): C, 55.61; H, 3.11; N, 13.51. Found: C,
24
16
5
2
55.89; H, 3.27; N, 13.74.
5
.1.5.14.
yl)methyl)-N-phenylbenzamide 11a
Yield: 50%, m.p.: 205-207 °C, I.R (KBr, cm ): υ
4-((5-Oxo-5,6-dihydropyrido[2,3-d]pyridazin-8-
-1
3417 (2NH), 3055 (CH
max
1
aromatic), 2924 (CH aliphatic), 1666 (2C=O), 1597 (C=N), 1539 (C=C), H-
NMR: δ 4.40 (s, 2H, CH ), 7.07-7.09 (m, 3H, aniline-H), 7.32 (d, 2H, J = 7.1 Hz,
2
aniline-H), 7.47 (d, 2H, J = 7.4 Hz, phenyl-H), 7.75 (d, 2H, J = 8.0 Hz, phenyl-
H), 7.88 (d, 2H, J = 7.5 Hz, pyridyl-H), 9.02 (d, 1H, J = 9.0 Hz, pyridyl-H),
2
6

1
0.19 (s, 1H, NH exchanged by D O), 12.88 (s, 1H, NH exchanged by D O).
2
2
Anal. Calcd. for C H N O (356.38): C, 70.77; H, 4.53; N, 15.72. Found: C,
21
16
4
2
70.89; H, 4.67; N, 15.89.
5
.1.5.15.
d]pyridazin-8-yl)methyl)benzamide 11b
Yield: 77%, m.p.: 239-241 °C, I.R (KBr, cm ): υ
N-(4-Hydroxyphenyl)-4-((5-oxo-5,6-dihydropyrido[2,3-
-1
3433 (2NH), 3267 (OH),
max
3070 (CH aromatic), 2935 (CH aliphatic), 1666 (2C=O), 1608 (C=N), 1512
1
(
C=C), H-NMR: δ 4.42 (s, 2H, CH ), 7.23 (d, 1H, J = 8.6 Hz, hydroxy phenyl-
2
H), 7.43-7.55 (m, 3H, hydroxy phenyl-H), 7.81 (d, 2H, J = 8.7 Hz, phenyl-H),
.83-7.89 (m, 2H, phenyl-H), 8.06 (d, 1H, J = 7.8 Hz, pyridyl-H), 8.37 (d, 1H, J
7.9 Hz, pyridyl-H), 9.05 (s, 1H, pyridyl-H), 9.24 (s, 1H, OH exchanged by
D O), 10.29 (s, 1H, NH exchanged by D O), 12.86 (s, 1H, NH exchanged by
7
=
2
2
13
D O). C-NMR: δ 37.4 (CH carbon), 115.4, 122.3, 122.4, 126.4, 128.3, 128.7,
2
2
129.3, 129.8, 130.6, 134.8, 141.3, 143.6, 145.8 (Ar-Cs), 154.3 (C=N), 159.5
(
C=O), 166.4 (C=O). Anal. Calcd. for C H N O (372.38): C, 67.73; H, 4.33;
21
16
4
3
N, 15.05. Found: C, 68.01; H, 4.50; N, 15.32.
5
.1.5.16.
d]pyridazin-8-yl)methyl)benzamide 11c
Yield: 70%, m.p.: 263-265 °C, I.R (KBr, cm ): υ
N-(4-Fluorophenyl)-4-((5-oxo-5,6-dihydropyrido[2,3-
-1
3425, 3313 (2NH), 3070
max
(CH aromatic), 2927 (CH aliphatic), 1701 (2C=O), 1612 (C=N), 1527 (C=C),
1
H-NMR: δ 4.42 (s, 2H, CH ), 7.18 (t, 2H, J = 8.7 Hz, fluoro phenyl-H), 7.47 (d,
2
2H, J = 7.9 Hz, fluoro phenyl-H), 7.75-7.78 (m, 3H, 1 pyridyl-H and 2 phenyl-
H), 7.87 (d, 2H, J = 8.9 Hz, phenyl-H), 8.37 (d, 1H, J = 8.0 Hz, pyridyl-H), 9.04
(
d, 1H, J = 3.7 Hz, pyridyl-H), 10.24 (s, 1H, NH exchanged by D O), 12.87 (s,
2
1
3
1
1
H, NH exchanged by D O). C-NMR: δ 37.5 (CH carbon), 115.5, 115.7,
2
2
22.5, 122.6, 126.4, 128.3, 128.4, 129.2, 133.6, 134.6, 142.1, 144.3, 145.0 (Ar-
Cs), 154.1 (C=N), 159.1 (C=O), 165.7 (C=O). Anal. Calcd. for C H FN O
21
15
4
2
(374.38): C, 67.37; H, 4.04; N, 14.97. Found: C, 67.55; H, 4.21; N, 15.13.
2
7

5
.1.5.17.
d]pyridazin-8-yl)methyl)benzamide 11d
Yield: 72%, m.p.: 248-250 °C, I.R (KBr, cm ): υ
N-(4-Methoxyphenyl)-4-((5-oxo-5,6-dihydropyrido[2,3-
-1
3464, 3047 (2NH), 3055
max
(CH aromatic), 2931 (CH aliphatic), 1700 (2C=O), 1600 (C=N), 1512 (C=C),
1
H-NMR: δ 3.74 (s, 3H, OCH ), 4.41 (s, 2H, CH ), 6.91 (d, 2H, J = 8.8 Hz,
3
2
methoxy phenyl-H), 7.45 (d, 2H, J = 8.0 Hz, methoxy phenyl-H), 7.64 (d, 2H, J
8.8 Hz, phenyl-H), 7.87 (d, 3H, J = 8.0 Hz, phenyl-H and pyridyl-H), 8.37 (d,
H, J = 8.1 Hz, pyridyl-H), 9.04 (d, 1H, J = 3.9 Hz, pyridyl-H), 10.06 (s, 1H,
NH exchanged by D O), 12.86 (s, 1H, NH exchanged by D O). Anal. Calcd. for
=
1
2
2
C H N O (386.41): C, 68.38; H, 4.70; N, 14.50. Found: C, 68.54; H, 4.59; N,
22
18
4
3
14.67.
5.1.5.18.
N-(4-Chlorophenyl)-4-((5-oxo-5,6-dihydropyrido[2,3-
d]pyridazin-8-yl)methyl)benzamide 11e
Yield: 73%, m.p.: 253-255 °C, I.R (KBr, cm ): υ
-1
3475, 3417 (2NH), 3055
max
(CH aromatic), 2927 (CH aliphatic), 1671 (2C=O), 1593 (C=N), 1527 (C=C),
1
H-NMR: δ 4.42 (s, 2H, CH ), 7.40 (d, 2H, J = 8.8 Hz, chloro phenyl-H), 7.47
2
(
d, 2H, J = 8.0 Hz, chloro phenyl-H), 7.79 (d, 2H, J = 8.8 Hz, phenyl-H), 7.87
d, 3H, J = 8.0 Hz, phenyl-H and pyridyl-H), 8.37 (d, 1H, J = 7.9 Hz, pyridyl-H),
(
9
.04 (d, 1H, J = 3.7 Hz, pyridyl-H), 10.30 (s, 1H, NH exchanged by D O), 12.86
2
13
(
s, 1H, NH exchanged by D O). C-NMR: δ 37.5 (CH carbon), 122.2, 126.4,
2
2
127.6, 128.2, 128.3, 128.5, 128.9, 129.2, 133.5, 134.6, 138.6, 142.3, 145.0 (Ar-
Cs), 154.1 (C=N), 159.1 (C=O), 165.9 (C=O). Anal. Calcd. for C H ClN O
21
15
4
2
(390.83): C, 64.54; H, 3.87; N, 14.34. Found: C, 64.77; H, 4.08; N, 14.18.
5.1.5.19.
N-(3-Chloro-4-fluorophenyl)-4-((5-oxo-5,6-dihydropyrido[2,3-
d]pyridazin-8-yl)methyl)benzamide 11f
Yield: 79%, m.p.: 210-212 °C, I.R (KBr, cm ): υ
-1
3421 (2NH), 3066 (CH
max
1
aromatic), 2927 (CH aliphatic), 1670 (2C=O), 1600 (C=N), 1500 (C=C), H-
NMR: δ 4.42 (s, 2H, CH ), 7.41 (t, 1H, J = 9.1 Hz, chloro-fluoro phenyl-H), 7.48
2
2
8

(
d, 2H, J = 8.2 Hz, chloro-fluoro phenyl-H), 7.69-7.71 (m, 2H, phenyl-H), 7.87
d, 3H, J = 8.3 Hz, phenyl-H and pyridyl-H), 7.97 (d, 1H, J = 5.5 Hz, pyridyl-H),
(
9
.05 (s, 1H, pyridyl-H), 10.31 (s, 1H, NH exchanged by D O), 12.86 (s, 1H, NH
2
exchanged by D O). Anal. Calcd. for C H ClFN O (408.82): C, 61.70; H,
2
21 14
4
2
3.45; N, 13.70. Found: C, 61.59; H, 3.67; N, 14.02.
5.1.5.20. 4-((5-Oxo-5,6-dihydropyrido[2,3-d]pyridazin-8-yl)methyl)-
N-(4-sulfamoylphenyl)benzamide 11g
Yield: 71%, m.p.: 151-153 °C, I.R (KBr, cm ): υ
-1
3448, 3425 (NH , 2NH),
max
2
3070 (CH aromatic), 2924 (CH aliphatic), 1662 (2C=O), 1593 (C=N), 1531
1
(
C=C), 1319, 1145 (SO ), H-NMR: δ 4.42 (s, 2H, CH ), 7.27 (s, 2H, NH
2
2
2
exchanged by D O), 7.48 (d, 2H, J = 8.1 Hz, phenyl-H), 7.74 (d, 2H, J = 8.7 Hz,
2
phenyl-H), 7.88-7.91 (m, 5H, 4 sulfamoyl phenyl-H and 1 pyridyl-H), 8.37 (d,
1H, J = 8.2 Hz, pyridyl-H), 9.04 (d, 1H, J = 3.60 Hz, pyridyl-H), 10.47 (s, 1H,
13
NH exchanged by D O), 12.87 (s, 1H, NH exchanged by D O). C-NMR 100
2
2
MHz: δ 35.5 (CH carbon), 120.2, 127.2, 128.6, 129.2, 132.7, 134.5, 136.9,
2
1
1
1
34.6, 139.3, 141.3, 146.7, 149.8, 151.4 (Ar-Cs), 154.1 (C=N), 158.6 (C=O),
60.1 (C=O). Anal. Calcd. for C H N O S (435.46): C, 57.92; H, 3.94; N,
21
17
5
4
6.08. Found: C, 58.14; H, 4.07; N, 16.32.
5.2. In vitro PARP-1 inhibitory assay
PARP-1 enzyme inhibition activity was measured for pyridopyridazinones
(8a-g, 9a,b, 10a-d, 11a-f and 12) using a colorimetric 96-well PARP-1 assay kit
(catalog no. 80580) (BPS Bioscience), according to the manufacturer's protocol.
Briefly, the histone mixture was diluted 1:5 with 1x PBS, 50 µl of histone
solution was added to each well and incubated at 4 ºC overnight. The plate was
washed three times using 200 µl PBST buffer (1x PBS containing 0.05% Tween-
20) per well. Liquid was removed from the wells by tapping the strip wells on
clean paper towels. To each well, 200 µl of Blocking buffer was added, followed
2
9