Synthesis, Characterization, and Docking Evaluations of New Derivatives of Pyrimido[4,5-c]pyridazine as Potential Human AKT1 Inhibitors

Article abstract of DOI:10.1002/jhet.2296

(Chemical Equation Presented) A number of new derivatives of pyrimido[4,5-c]pyridazine have been synthesized from the treatment of 6-acetyl-3-amino-2,5-diphenyl-2,5-dihydropyridazine-4-carbonitrile (1) as precursor with various reactants obtained quantita

Full text of DOI:10.1002/jhet.2296

Month 2015 Synthesis, Characterization, and Docking Evaluations of New Derivatives
of Pyrimido[4,5-c]pyridazine as Potential Human AKT1 Inhibitors
Ayla Hazrathoseyni, Seyed Mohammad Seyedi,* Hossein Eshghi, Ali Shiri,* Mohammad Saadatmandzadeh,
and Ali Reza Berenji
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, 91775-1436 Mashhad, Iran
*
E-mail: smseyedi@um.ac.ir; alishiri@um.ac.ir
Received May 12, 2014
DOI 10.1002/jhet.2296
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
This article is dedicated to Prof. Mohammad Rahimizadeh.
A number of new derivatives of pyrimido[4,5-c]pyridazine have been synthesized from the treatment
of 6-acetyl-3-amino-2,5-diphenyl-2,5-dihydropyridazine-4-carbonitrile (1) as precursor with various reac-
tants obtained quantitatively the desired products (2), (5), (7), and (9a–e). The structures of all the syn-
thesized products have been elucidated thoroughly. The potential AKT1 inhibitory activities of these new
synthesized compounds have also been studied by docking calculations, which have been performed in
Gold 5.2 software using Genetic algorithm.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
On the other hand, AKT1 is a member of the serine/
threonine AGC protein kinase family. It is frequently
overexpressed and active in many types of human cancers
including cancers of colon, breast, brain, pancreas and
prostate as well as lymphomas and leukemias. Some
clinical trials are in progress to test the efficacy of AKT path-
way inhibitors in treating cancer. Inhibitor VIII as the most
reported effective compound for the inhibitory of AKT1
and the crystal structure of AKT1 complexed to inhibitor
VIII (PDB:3O96) have been reported in the literature [15].
Hence, these observations prompted us to synthesize
new derivatives of pyrimido[4,5-c]pyridazine and eval-
uating of them as potential AKT1 inhibitors.
A large number of biologically active compounds con-
taining pyrimido[4,5-c]pyridazine structure have been de-
scribed. These compounds are useful in human therapy
because of their inhibitory activities of AKT [1], monoamine
oxidase [2], phosphodiesterase 5 (PDE5) [3,4], selective
human A1 adenosine receptor ligands [5], and p38R [6].
Several synthetic routes for the construction of
pyrimidopyridazines have been found in the literature.
3
-Aminopyridazine was condensed with l,3-dicarbonyl
compounds in polyphosphoric acid to give the related
pyrimidopyridazine [7]. The reaction between bromo-
acetone and 6-hydrazinoisocytosine were afforded pyrimido-
pyridazine in low yield [8]. 6-(l-Alkylhydrazino)isocytosines
have been also reported to cyclize with 1,3-diketo esters or
with simple α-keto esters to give the corresponding
pyrimido[4,5-c]pyridazines in good yields [9,10]. Morrison
and co-workers were described the cyclizations of glyoxals
with 6-hydrazinoisocytosines [11] while the synthesis of
-(substituted)pyrimido[4,5-c]pyridazine-5,7(1H,6H)-diones
were performed by Turbiak through the condensation of
-methyl-6-(1-methylhydrazinyl)uracil with phenyl and
alkyl glyoxal monohydrates [12]. A series of new 4-aryl-6,
-dimethylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-dione de-
rivatives were synthesized via three component reactions of
,3-dimethylbarbituric acid with arylglyoxals in the presence
of hydrazinium dihydrochloride [13,14].
RESULTS AND DISCUSSION
Chemistry. According to the reported procedure [16–18],
the preparation of precursor (1) is started from the reaction of
benzenediazonium salt with ethyl 3-oxobutanoate to give
1-(2-phenylhydrazono)propan-2-one. On the other hand,
2-benzylidenemalonitrile is obtained from the treatment of
benzaldehyde and malononitrile. Consequently, the reaction
of the latter compound with 1-(2-phenylhydrazono)propan-
2-one in EtOH gives the precursor (1) quantitatively.
3
3
8
The capability of the precursor (1) in the synthesis of
new derivatives of pyrimido[4,5-c]pyridazines heterocyclic
ring system was initially carried out by treatment of
1
©
2015 HeteroCorporation

A. Hazrathoseyni, S. M. Seyedi, H. Eshghi, A. Shiri, M. Saadatmandzadeh, and A. R. Berenji
Vol 000
Table 1
which was removed on deuteration, a singlet signal at δ
5.25 ppm due to CH group in the pyridazine ring, a broad
singlet signal at δ 7.10 ppm due to C¼NH group, a multi-
plet signal at δ 7.20–7.70, which are corresponded to the
hydrogens of phenyl moieties. In IR spectrum, the
disappearing of stretching vibration band of CN group at
Experimental and theoretical chemical shift values of the compounds
shown in Figure 2, calculated at mPW1PW91/6-31G(d)-PCM.
a
b
c
Entry
C5
|Δδ|
C6
|Δδ|
8
8
179.35
152.49
179.68
168.31
142.74
169.32
168.35
142.75
169.23
168.38
142.62
169.26
168.42
142.6
0.33
27.19
—
1.01
26.58
—
0.88
26.48
—
0.88
26.64
—
0.87
26.69
—
0.66
22.88
—
165.47
168.24
176.61
167.92
172.01
169.25
167.61
172.01
168.83
167.59
172.02
168.87
167.58
172.03
168.92
167.34
172.04
168.29
11.14
8.37
—
1.33
2.76
—
1.22
3.18
—
1.28
3.15
—
1.34
3.11
—
0.95
3.75
—
*
ꢀ1
ꢀ1
2
194 cm and NH at 3412 and 3309 cm and appearing
2
Exp.
ꢀ
1
of a sharp peak due to NH group at 3125 cm also confirm
the new ring closure.
9
9
a
a
*
Exp.
Moreover, precursor (1) is refluxed in a mixture of
triethylorthoformate and anhydride acetic acid to give
compound (3). Then, the stirring of a mixture of (3) and
25% NH3(aq) in ethanol at room temperature affords (4).
The treatment of (4) with NaOEt in EtOH obtains quantita-
tively product (5) as another new synthesized heterocyclic
compound of pyrimido[4,5-c]pyridazine (Scheme 1).
9
9
b
b
*
Exp.
9
9
c
c
*
Exp.
9
9
d
d
*
1
Exp.
169.29
168.07
145.85
168.73
The H-NMR spectrum of compound (5) shows a singlet
9
9
e
e
signal at δ 2.33 ppm indicating the presence of CH group,
*
3
a singlet signal at δ 5.55 ppm due to CH group in the
pyridazine ring, a broad signal at δ 7.05 ppm due to NH2
group, a multiplet signal at δ 7.21–7.70, which are corre-
sponding to hydrogens of aromatic rings, and a singlet
signal at δ 8.05 ppm due to the hydrogen of the new fused
pyrimidine ring.
Exp.
a
*
Acetone-d
6
for compounds 8 and 8 and CDCl
3
for other compounds.
b
c
|
Δδ| = absolute deviation chemical shift for C5 from experimental data.
Δδ| = absolute deviation chemical shift for C6 from experimental data.
|
compound (1) with phenyl isocyanate in the mixture of
KOH in dry dimethylformamide at room temperature to
afford compound (2) (Scheme 1).
On the other hand, a mixture of compound (3) and
hydrazine hydrate is refluxed in dry ethanol to give first
the intermediate (6), which is isomerized to thermodynam-
ically stable product (7) through Dimroth rearrangement
(Scheme 1). In the protocol led to the formation of
compound (7), a nucleophilic condensation reaction on
1
The H-NMR spectrum of compound (2) shows a singlet
signal at δ 2.40 ppm indicating the presence of CH group,
3
a broad singlet signal at δ 5.05 ppm due to NH group,
Scheme 1
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Synthesis, Characterization, and Docking Evaluations of New Derivatives of
Pyrimido[4,5-c]pyridazine as Potential Human AKT1 Inhibitors
Figure 1. The chemical mechanism of the preparation of compound (7) via Dimroth rearrangement.
Scheme 2
the carbonyl group of compound (6) is taken place by
hydrazine. The spectral and microanalytical data of the
synthesized compound (7), summarized in the experimen-
tal section, confirm the desired structure.
The plausible mechanism of this rearrangement could be
explained as depicted in Figure 1. The similar results about
this rearrangement are in agreement with the reported
results for analogous compounds [19].
On the other hand, the reaction of precursor (1) with CS2
was carried out to obtain the related cyclized product,
which is consequently further alkylated with various
alkylhalides (Scheme 2).
According to the literature review, there are at least two
different published methods about the treatment with CS2,
which give the corresponding heterocyclized product
Furthermore, the chemical shifts of C5 and C6 identified
in Figure 2 are crucial in deducing of the real structures;
13
therefore, the C-NMR chemical shifts were also com-
puted and applied as another criterion. The calculated
13
C-NMR chemical shifts of (8) and (9a–e) compared with
the corresponding ones of (8*) and (9a–e)* are qualita-
tively in close agreement with the experimental data
(Table 1). Eventually, the depicted structures of com-
pounds (8) and (9a–e) in Scheme 2 are the true structures,
which are obtained through the reaction conditions.
The spectral and microanalytical data of all the newly
synthesized compounds (8) and (9a–e) are fully charact-
1
erized. The H-NMR spectrum of compound (8) shows a
[20,21]. As a good criterion, the density functional theory
calculations can be applied to evaluate the structures of
the products (8) and (9a–e) [22]. Hence, the mPW1PW91
method and 6-31G(d) basis set were utilized to calculate
the optimized structures (Fig. 2). The energy difference
between the proposed regioisomers (8) and (9a–e) and
the corresponding ones, (8)* and (9a–e)*, are listed in
Table 1. The calculated results show that compounds (8)
and (9a–e) are more stable than the other regioisomers.
Figure 2. The proposed structures for the computational calculations.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Hazrathoseyni, S. M. Seyedi, H. Eshghi, A. Shiri, M. Saadatmandzadeh, and A. R. Berenji
Vol 000
singlet signal at δ 2.38 ppm indicating the presence of CH3
group, a singlet signal at δ 5.89 ppm due to CH signal of
pyridazine ring, a multiplet peak at δ 7.21–7.70 ppm be-
longing to hydrogen signals of phenyl groups, two broad
singlet signals at δ 7.14 and 10.24 ppm corresponding to
two protons indicating the presence of two NH groups, which
The plausible mechanism for compound (8) can be
explained by Dimroth rearrangement, which is an isomeri-
zation that consists in a translocation of sulfur and nitrogen
through a ring-opening-ring-closure sequence as depicted
in Figure 3 [23].
Molecular docking.
Docking is carried out using
are disappeared by adding D O. In the IR spectrum, the dis-
Genetic Optimization for Ligand Docking (Gold)
software based on Goldscore fitness function that uses the
Genetic algorithm (GA). All water molecules and hetero
atoms are removed from the protein to evaluate the
scoring function in Goldsoftware. For each of the 50
independent GA runs, a maximum number of 100 000
GA operations are performed on a set with a population
size of 100 individuals. Operator weights are set to 95, 95,
and 10 for crossover, mutation, and migration, respectively.
Default cutoff values of 2.5 Å (dH-X) for hydrogen bonds
and 4.0 Å for Van der Waals distance are employed. The
root mean square deviation values for the docking
calculations are based on the root mean square deviation
matrix of the ranked solutions. We observed that the best
ranked solutions are always among the first 50 GA runs,
and the conformation of the molecules based on the best
fitness score is further analyzed.
2
appearance of stretching vibration band of CN group of the
ꢀ
1
ꢀ1
precursor (1) at 2194 cm and NH at 3412 and 3309 cm
2
ꢀ
1
and disclosing of a peak at 3125 cm for NH group also
confirm the occurrence of the desired heterocyclization.
1
On the other hand, the H-NMR spectrum of compound
(
2
9a), as an example, shows three singlet signals at δ 2.36,
.44, and 2.52 ppm corresponding to three CH groups, a
3
singlet signal at δ 5.47 ppm due to CH group in the
pyridazine ring, a multiplet signal at δ 7.22–7.67, which
is corresponded to ten hydrogens of aromatic rings. The
disappearance of two broad singlet signals of NH groups
of compound (8) is another reason to confirm the alkyl-
ation of compound (8). The disappearance of vibration
band of NH group in the IR spectrum of compound (9a)
as well as the occurrence of molecular ion peak at m/z
4
20 clearly confirms the synthesis of compound (9a).
Figure 3. The chemical mechanism of the preparation of compound (8) via Dimroth rearrangement.
Table 2
The data obtained from docking studies of compounds (2), (5), (7), (8), and 9 (a–e), on inhibitory activities of AKT1.
Entry
Goldscore
ΔG
d
(kJ/mol)
K
i
H-B
Pi-Pi
Pi-σ
VdW-B
2
5
7
8
68.37
67.96
65.98
81.19
69.81
75.32
85.75
76.51
99.32
87.89
ꢀ36.55
ꢀ23.76
ꢀ28.88
ꢀ24.66
ꢀ34.61
ꢀ31.61
ꢀ41
3.94e-07
6.86e-05
8.71e-06
4.77e-05
8.62e-07
2.90e-06
6.56e-08
3.60e-07
2.12e-09
5.02e-07
2
2
5
3
1
3
2
1
2
2
3
2
1
2
—
3
—
1
3
—
—
—
—
—
—
—
—
1
—
1
1
2
—
1
—
1
—
1
9
9
9
9
9
a
b
c
d
e
ꢀ36.78
ꢀ49.5
Co-Crystal
ꢀ35.96
5
—
ΔG
d
, final docked energy; K
i
, estimated inhibitory constant; H-B, the number of hydrogen bond; Pi-Pi, the number of Pi-Pi interactions, Pi-σ: the number
of Pi-σ interactions.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Synthesis, Characterization, and Docking Evaluations of New Derivatives of
Pyrimido[4,5-c]pyridazine as Potential Human AKT1 Inhibitors
Because of the lots of reported articles on the importance
of anticancer activities of pyridazine derived heterocyclic
compounds and our interests on the assessment of inhibi-
tory activities of AKT1, the docking of the newly synthe-
sized compounds (2), (5), (7), (8), and (9a–e) with AKT1
fitness function. The algorithm exhaustively searches the
entire rotational and translational space of the ligand with
respect to the receptors. The various solutions evaluate by
a score which is equivalent to the absolute value of the total
energy of the ligand in the protein environment. The best
docking solutions of Goldscore for each compound is
(PDB ID: 3O96) are performed by using of Goldscore
Figure 4. The best docked structure of (9e) in the active site pocket of AKT1 (PDB Code: 3O96, flexible residue: TRP80- ILE84- SER205- LYS268-
TYR272- ILE290- ASP292- CYS296 in stick (left) and solvent surface (right) views. [Color figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
Figure 5. The 2D representation of the interaction between compound (9e) in the crystal structure of AKT1 (PDB Code: 3O96) using of Goldscore fitness
function. Blue dashed line: hydrogen bond (THR A:78, THR A:77), brown line: P
i i
-P interaction (TRP A:76, LYS A:141, ARG A225), Pi-σ interaction
(THR A:78). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Hazrathoseyni, S. M. Seyedi, H. Eshghi, A. Shiri, M. Saadatmandzadeh, and A. R. Berenji
Vol 000
Figure 6. The 2D representation of the interaction between Co-Crystal in the crystal structure of AKT1 (PDB Code: 3O96) using of Goldscore fitness
function. Blue dashed line: hydrogen bond (THR A: 243, LYS A: 249), green line: Van der Waals bond (PHE A: 245), brown line: P -P interaction
i
i
(LYS A: 19, LYS A: 228, ARG A: 225). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
considered. Goldscore performs a force field based on
scoring function and is made up of four components:
with Co-crystal. The authors think the Pi-Sigma interaction
in compound (9e), which shows the best results in Table 2,
can be important on the potent inhibitory effect of AKT1 in
comparison with the other synthesized compounds and
even co-crystal (Figs. 4–6).
1
2
3
) Protein-ligand hydrogen bond energy (external H-bond).
) Protein-ligand Van der Waals energy (external vdw).
) Ligand internal Van der Waals energy (internal vdw).
4) Ligand interamolecular hydrogen bond energy (internal-H-
bond).
CONCLUSION
The external vdw score is multiplied by a factor of 1.375
The chemical procedures outlined provided very simple
and straightforward routes to obtain various derivatives of
pyrimido[4,5-c]pyridazine. The designed and synthesized
compounds were studied as potential AKT1 inhibitory
activity, and the results showed that compound (9e) had
better inhibitory effect than the other compounds and even
co-crystal. Further studies are in progress to improve our
knowledge of the SAR of this inhibitory activity.
when the total fitness score is computed. This is an empir-
ical correction to encourage protein-ligand hydrophobic
contact. The fitness function has been optimized for the
prediction of ligand binding positions. Goldscore = S
+
hb_ext)
S (vdw_ext) + S (hb_int) + S (vdw_int), where S (hb_ext)
(
is the protein-ligand hydrogen bond score, S(vdw_ext) is
the protein-ligand Van der Waals score, S(hb_int) is the
score from interamolecular hydrogen bond in the ligand,
and S(vdw_int) is the score from intramolecular strain in
the ligand. The possible active site is identified using
Accelrys DS Visualizer. Eight active site residues as
TRP80- ILE84- SER205- LYS268- TYR272- ILE290-
ASP292- CYS296 are found by the removal of inhibitor
VIII. Therefore, it is chosen as the most biologically favor-
able site for docking.
EXPERIMENTAL
Melting points were recorded on an Electrothermal type 9100
melting point apparatus. The IR spectra were obtained on Avatar
370 FT-IR Thermo Nicolet and only noteworthy absorptions are
1
13
listed. The H-NMR (400 MHz) and the C-NMR (100 MHz)
spectra were recorded on a Bruker Avance DRX-400 Fourier
transformer spectrometer. Chemical shifts are reported in ppm
downfield from TMS as internal standard. The mass spectra were
scanned on a Varian Mat CH-7 at 70 eV. Elemental analyses were
performed on a Thermo Finnigan Flash EA microanalyzer.
The estimated inhibitory constants of the mentioned
docked compounds as well as Goldscore, free energy of
binding, and the whole interactions among the moieties
of the products are shown in Table 2.
3-Acetyl-5-imino-1,4,6-triphenyl-4,5,6,8-tetrahydropyrimido
The evaluation of the results in Table 2 and the compar-
[
4,5-c]pyridazin-7(1H)-one (2). To a solution of (1) (10 mmol,
ison of Goldscores, ΔG , and K of the newly synthesized
d
i
3.16g) and phenyl isocyanate (10 mmol, 1.19 g) in dry
dimethylformamide (5 mL), KOH (12 mmol 0.67 g) were added,
and the mixture was stirred at room temperature for 5 h [monitored
compounds with Co-Crystal demonstrate that compounds
2) and (9c–e) have the considerable results in comparison
(
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Synthesis, Characterization, and Docking Evaluations of New Derivatives of
Pyrimido[4,5-c]pyridazine as Potential Human AKT1 Inhibitors
by thin-layer chromatography (TLC) using chloroform:methanol
:1]. Then, the reaction mixture was poured into water and
Anal. Calcd for C H N O: C, 69.96; H, 4.99; N, 20.40. Found:
20 17 5
C, 69.92; H, 4.95; N, 20.40.
9
neutralized with diluted HCl solution. The resulting solid was
filtered, washed with water (2 × 20 mL), dried, and recrystallized
5-Hydrazinyl-3-(1-hydrazonoethyl)-1,4-diphenyl-1,4-
dihydropyrimido[4,5-c]pyridazine (7). A mixture of compound
(3) (10 mmol, 3.72 g) and excess amount of hydrazine hydrate
(60 mmol, 1.50 g) was refluxed in absolute ethanol (20 mL) for 5 h.
After the completion of the reaction, which was monitored by TLC
using chloroform:methanol (9:1), the reaction mixture was poured
into an ice-water bath, and the resulting solid was filtered off, dried,
and recrystallized from ethanol. Yellow powder, yield = 85%; mp
from ethanol, respectively. Yellow powder, yield = 70%; mp
1
2
86–288°C; H-NMR (DMSO-d ): δ 2.40 (s, 3H, CH –CO), 5.05
6
3
(
br, 1H, NH), 5.25 (s, 1H, CH pyridazine), 7.10 (br s, 1H, C¼NH),
13
7
8
1
6
.20–7.7 (m, 15H, phenyl); C-NMR (DMSO-d ): δ 25.8, 36.3,
6.1, 120.7, 124.6, 124.8, 125.1, 126.6, 128.8, 129.4, 131.4, 135.4,
37.3, 141.6, 146.2, 151.6, 154.0, 166.0, 192.1; MS (EI): m/z
1
(
1
%) = 435 (M+), 391, 347; IR (KBr disk): ν 3383, 3183, 1679,
621, 1599, 1521, 1379, 1188, 694 cm ; Anal. Calcd for
266–269°C; H-NMR (DMSO-d ): δ 1.95 (s, 3H, CH ), 5.65 (s,
6
3
ꢀ
1
2
1H, CH pyridazine), 5.75 ( br s, 2H, NH ), 6.60 (br s, 1H, NH),
C
26
H
21
N
5
O
2
: C, 71.71; H, 4.86; N, 16.08. Found: C, 71.69; H,
6.90 (s, 2H, NH
2
), 7.11–7.60 (m, 10H, phenyl), 7.85 (s, 1H,
): δ, 10.3, 36.2, 97.5, 114.7, 114.8,
13
4.76; N, 16.06.
CH¼N); C-NMR (DMSO-d
6
Ethyl N-(6-acetyl-4-cyano-2,5-diphenyl-2,5-dihydropyridazin-
-yl)formimidate (3). The solution of compound (1) (10 mmol,
.16 g) and excess amount of triethylorthoformate (20 mL) in
125.9, 128.6, 128.8, 128.9, 129.1, 129.2, 129.3, 129.4, 136.3,
139.9, 141.9, 146.2, 153.9, 159.4, 166.1; MS (EI): m/z (%) = 372
(M+), 356, 341, 281, 77, 57; IR (KBr): v 3403, 3321, 3272, 1633,
3
3
ꢀ
1
anhydride acetic acid (5 mL) were refluxed for about 6 h. After the
completion of the reaction, which was monitored by TLC using
chloroform:methanol (9:1), the solvent was evaporated under
reduced pressure. The resulting precipitant was washed with water
1594, 745, 695 cm ; Anal. Calcd for C20
N, 30.09. Found: C, 64.49; H, 5.38; N, 30.05.
Synthesis of 1-(1,4-diphenyl-5,7-dithioxo-1,4,5,6,7,8-
hexahydropyrimido[4,5-c]pyridazin-3-yl)ethanone (8).
mixture of 6-acetyl-3-amino-2,5-diphenyl-2,5-dihydropyridazine-
4-carbonitrile (1) (10 mmol, 3.16 g) and CS (100mmol, 7.6 g) in
10% ethanolic sodium hydroxide solution (20mL) was heated
under reflux for 7 h. After the completion of the reaction
(monitored by TLC), the reaction mixture was cooled and added
on to crushed ice. The obtained precipitant was filtered, dried, and
20 8
H N : C, 64.50; H, 5.41;
A
(2 × 20 mL), dried, and recrystallized from ethanol, respectively.
1
Yellow powder, yield = 90%; mp 137–135°C; H-NMR (CDCl
.15 (t, 3H, CH CH ), 2.45 (s, 3H, CH –CO), 4.15 (q, 2H,
O–CH –), 5.05 (s, 1H, CH pyridazine), 7.20–7.60 (m, 10H,
3
): δ
2
1
2
3
3
2
13
phenyl), 7.91 (s, 1H, N¼CH–O); C-NMR (CDCl ): 14.2, 25.8,
3
3
1
1
2
8.8, 61.7, 105.8, 116.3, 122.4, 122.5, 125.2, 126.9, 127.0, 127.6,
27.7, 130.5, 132.8, 132,9, 141.3, 142.2, 149.3, 152.0, 166.9,
91.6; MS (EI): m/z (%) = 372 (M+); IR (KBr): ν 3068, 2982,
recrystallized from ethanol, respectively. Orange powder,
yield = 72%; mp >300°C; H-NMR (acetone-d ): δ 2.38 (s, 3H,
1
6
ꢀ1
202, 1686, 1637, 1198, 759, 702 cm ; Anal. Calcd for
CH –CO), 5.89 (s, 1H, CH pyridazine), 7.14 (br, 1H, NH),
3
13
C
22
H
20
N
4
O
2
: C, 70.95; H, 5.41; N, 15.04. Found: C, 70.90; H,
7.21–7.70 (m, 10H, phenyl), 10.24 (br s, 1H, NH); C-NMR
(acetone-d ): δ 24.1, 36.5, 104.5, 125.8, 125.9, 126.7, 126.8,
5.39; N, 15.02.
6
N′-(6-Acetyl-4-cyano-2,5-diphenyl-2,5-dihydropyridazin-3-
128.0. 128.1, 128.2, 128.3, 128.5, 128.6, 141.9, 142.3, 145.6,
150.5, 176.6, 179.7, 196.2; MS (EI): m/z (%) = 392 (M+), 318,
244, 135, 94, 76, 59, 43, 28; IR (KBr): ν 3125, 1683, 1533, 1478,
yl)formimidamide (4).
To a solution of compound (3)
(
10 mmol, 3.72 g) in absolute ethanol (20 mL), 25% ammonium
ꢀ
1
hydroxide solution (5 mL) was added. The solution was stirred
at room temperature until the precipitate was formed. The
resulting solid was washed with water (2 × 20 mL), dried, and
1375, 1269, 1141, 1122, 696 cm
;
Anal. Calcd for
20 16 4 2
C H N S O: C, 61.22; H, 4.11; N, 14.27; S, 16.34. Found: C,
61.20; H, 4.08; N, 14.24; S, 16.21.
recrystallized from ethanol, respectively. Yellow powder,
General procedure for the synthesis of 1-(5,7-bis(alkylthio)-
1,4-diphenyl-1,4-dihydropyrimido[4,5-c]pyridazin-3-yl)ethanone
1
yield = 93%; mp 240–242°C (decomp.); H-NMR (CDCl ): δ
3
2
.39 (s, 3H, CH
3
–CO), 4.32 (br s, 2H, NH
2
), 5.00 (s, 1H, CH
(9a–e).
A mixture of 1-(1,4-diphenyl-5,7-dithioxo-1,4,5,6,7,8-
pyridazine), 7.20–7.60 (m, 10H, phenyl and 1H, N¼CH–
hexahydropyrimido[4,5-c]pyridazin-3-yl)ethanone (8) (10 mmol,
3.92 g) and the appropriate alkyl halide ( 20 mmol) in 10%
ethanolic sodium hydroxide solution (40 mL) was heated under
reflux for 8 h. The reaction progress was monitored by TLC using
chloroform:methanol (9:1). After the completion of the reaction, the
mixture was cooled and poured into cold water. The obtained crude
solid was filtered, dried, and recrystallized from ethanol, respectively.
1-(5,7-Bis(methylthio)-1,4-diphenyl-1,4-dihydropyrimido
13
N); C-NMR (CDCl
3
): 25.8, 38.8, 106.4, 116.3, 122.4, 122.5,
25.2, 126.7, 126.8, 127.5, 127.6, 130.5, 132.7, 132.8, 141.3,
43.1, 146.4, 149.3, 152.5, 191.6; MS (EI): m/z (%) = 343 (M
); IR (KBr): ν 3412, 3330, 3191, 2198, 1671, 1651, 1595,
1
1
+
ꢀ
1
1
563, 1423, 1221, 1140, 697 cm
;
Anal. Calcd for
C H N O: C, 69.96; H, 4.99; N, 20.40. Found: C, 69.94; H,
20 17 5
4
.95; N, 20.39.
-(5-Amino-1,4-diphenyl-1,4-dihydropyrimido[4,5-c]
pyridazin-3-yl)ethanone (5). The mixture of compound (4)
10 mmol, 3.43 g) and sodium ethoxide (0.23 g sodium metal in
1
[4,5-c]pyridazin-3-yl)ethanone (9a).
Yellow powder, yield =
): δ 2.36 (s, 3H, CH -CO),
), 5.47 (s, 1H, CH
1
55%; mp 72–74°C; H-NMR (CDCl
2.44 (s, 3H, –SCH
3
3
(
3
), 2.52 (s, 3H, –SCH1
3
3
5
mL absolute ethanol) was refluxed for 5 h. The resulting
pyridazine), 7.22–7.67 (m, 10H, phenyl); C-NMR (CDCl ): δ
3
precipitate was filtered, washed with ethanol (2 × 20 mL), and
dried. Yellow powder, yield = 35%; mp 295–297°C; H-NMR
12.8, 14.1, 25.0, 35.9, 105. 8, 125.1, 125.2, 126.8, 127.6, 128.1,
128.2, 128.3, 128.4, 128.7, 128.8, 140.1, 141.3, 144.9, 151.4,
169.2, 169.3, 196.0; MS (EI): m/z (%)= 420 (M+), 405, 375, 341,
200; IR (KBr): v 2921, 1686, 1514, 1494, 1354, 1137, 1291,
1
(
6 3
DMSO-d ): δ 2.33 (s, 3H, CH –CO), 5.55 (s, 1H, CH
pyridazine), 7.05 (br s, 2H, NH
2
), 7.21–7.70 (m, 10H, phenyl),
1
3
ꢀ1
8
1
1
.05 (s, 1H, CH¼N); C-NMR (CDCl ): 24.5, 35.2, 97.2, 113.9,
694 cm ; Anal. Calcd for C H N S O: C, 62.83; H, 4.79; N,
3
22 20 4 2
14.0, 125.9, 127.9, 128.3, 128.4, 128.5, 128.6, 130.2, 130.3,
13.32; S, 15.25. Found: C, 62.80; H, 4.77; N, 13.28; S, 15.21.
35.8, 145.4, 148.7, 152.7, 164.3, 166.8, 193.4; MS (EI): m/z
1-(5,7-Bis(ethylthio)-1,4-diphenyl-1,4-dihydropyrimido[4,5-c]
(
3
%) = 343 (M+), 301, 266, 97, 77, 57; IR (KBr): ν 3402, 3329,
pyridazin-3-yl)ethanone (9b).
mp 55–58°C; H-NMR (CDCl
Yellow powder, yield = 53%;
): δ 1.24 (t, 3H, –SCH CH ),
ꢀ
1
1
178, 1671, 1655, 1564, 1487, 1423, 1222, 1141, 698 cm
;
3
2
3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Hazrathoseyni, S. M. Seyedi, H. Eshghi, A. Shiri, M. Saadatmandzadeh, and A. R. Berenji
Vol 000
interface for Gaussian programs, which gives
a visual
1
–
7
2
1
1
2
.34 (t, 3H, –SCH CH ), 2.44 (s, 3H, CH CO), 2.91 (q, 2H,
2
3
3
representation of the calculated data. The polarizable continuum
model [33] was chosen for the self-consistent reaction-field
calculations for different solutions. The polarizable continuum
model was applied within the mPW1PW91/6-31G(d) level to
predict the solvent effects on the structure.
SCH –), 3.20 (d of q, 2H, –SCH –), 5.48 (s, 1H, CH pyridazine),
2
2
13
3
.24–7.65 (m, 10H, phenyl); C-NMR (CDCl ): δ 14.4, 14.7, 24.4,
4.9, 25.3, 36.0, 105.9, 125.2, 125.3, 126.9, 127.6, 128.4, 128.5,
28.6, 128.7, 128.8, 128.9, 140.1, 141.3, 145.0, 151.8, 168.8,
69.2, 196.0; MS (EI): m/z (%) = 448 (M+), 419, 368; IR (KBr): v
ꢀ
1
Structure optimization.
Three dimensional structures of
921, 1690, 1514, 1493, 1355, 1133, 694 cm ; Anal. Calcd for
O: C, 64.26; H, 5.39; N, 12.49; S, 14.30. Found: C,
4.20; H, 5.31; N, 12.42; S, 14.20.
-(5,7-Bis(propylthio)-1,4-diphenyl-1,4-dihydropyrimido[4,5-c]
pyridazin-3-yl)ethanone (9c). Yellow powder, yield=48%; mp
compounds (2), (5), (7), and (9a–e) were simulated in Hyper
24 24 4 2
C H N S
6
+
ꢀ1
Chem 7.5 using MM method (RMS gradient= 0.1 kcal mol
)
(
HyperChem® Release 7, Hypercube Inc., http://www.hyper.
1
com). In the second optimization, output files were minimized
1
under Semi empirical AM1 methods (Convergence limit = 0.01;
5
(
0–53°C; H-NMR (CDCl
t, 3H, –SCH CH CH ), 1.59 (m, 2H, –SCH
CH ), 2.43 (s, 3H, CH
.23 (d of t, 2H, –SCH –), 5.48 (s, 1H, CH pyridazine), 7.21–7.63
3
): δ 0.99 (t, 3H, –SCH
2
CH
CH
3
2
CH
), 1.65 (m,
–),
3
), 1.01
ꢀ
1
Iteration limit = 50; RMS gradient= 0.1 kcalmol ; Polak-Ribiere
optimizer algorithm). Crystal structures of AKT1 inhibitor
VIII were retrieved from RCSB Protein Data Bank (PDB
entry: 3O96).
2
2
3
2
CH
2
2H, SCH
2
CH
2
3
3
CO), 2.85 (t, 2H, –SCH
2
3
2
1
3
(m, 10H, phenyl); C-NMR (CDCl ): δ 13.4, 13.5, 22.6, 23.0,
3
2
1
1
4.9, 31.8, 32.8, 35.9, 105.9, 125.2, 125.3, 126.8, 127.6, 128.1,
28.2, 128.3, 128.4, 128.7, 128.8, 140.1, 141.4, 145.0, 151.8,
68.9, 169.2, 196.0; MS (EI): m/z (%) = 476 (M+), 433, 397; IR
Acknowledgments. The authors gratefully acknowledge Ferdowsi
University of Mashhad for partial support of this project (3/19530).
ꢀ
1
(KBr): ν 2958, 2925, 1686, 1511, 1493, 1351, 1135, 693 cm
;
Anal. Calcd for C H N S O: C, 65.51; H, 5.92; N, 11.75; S,
26 28 4 2
1
3.45. Found: C, 65.46; H, 5.87; N, 11.65; S, 13.40.
-(5,7-Bis(butylthio)-1,4-diphenyl-1,4-dihydropyrimido[4,5-c]
pyridazin-3-yl)ethanone (9d). Yellow powder, yield = 50%; mp
1
REFERENCES AND NOTES
1
6
8–70°C; H-NMR (CDCl ): δ 0.86 (t, 3H, –SCH CH CH CH ),
[1] Kettle, J. G.; Brown, S.; Crafter, C.; Davies, B. R.; Dudley, P.;
Fairley, G.; Faulder, P.; Fillery, S.; Greenwood, H.; Hawkins, J.; James, M.;
Johnson, K.; Lane, C. D.; Pass, M.; Pink, J. H.; Plant, H.; Cosulich, S. C. J
Med Chem 2012, 55, 1261.
3
2
2
2
3
0
.92 (t, 3H, SCH CH CH CH ), 1.29 (m, 2H, –SCH CH
2
2
2
3
2
2
CH
2
CH
CH
s, 3H, CH CO), 2.84–2.92 (m, 2H, –SCH –), 3.20 (d, 2H,
3
), 1.44 (m, 2H, SCH
CH
2 2
2 3
CH CH ), 1.52 (m, 2H,
–SCH
2
2
CH CH
2
3
), 1.62 (m, 2H, SCH
2
CH
2
CH CH ), 2.44
2
3
[
2] Altomare, C.; Cellamare, S.; Summo, L.; Catto, M.; Carotti, A.
J Med Chem 1998, 41, 3812.
3] Giovannoni P. M.; Vergelli, C.; Biancalani, C.; Cesari, N.;
(
3
2
J = 6.5 Hz, –SCH –), 5.48 (s, 1H, CH pyridazine), 7.23–7.63
2
[
1
3
(
m, 10H, phenyl); C-NMR (CDCl
3
): δ 13.7, 13.8, 22.0, 22.1,
Graziano, A.; Biagini, P.; Gracia, J.; Gavalda, A.; Piaz, V. D. J Med Chem
2006, 49, 5363.
[4] Rimaz, M.; Khalafy, J.; Pesyan, N. N.; Prager, R. H. Aust J
Chem 2010, 63, 507.
2
1
1
4
1
6
1
4.9, 29.6, 30.6, 31.2, 31.7, 35.9, 105.9, 125.2, 125.3, 126.8,
27.6, 128.1, 128.2, 128.3, 128.4, 128.7, 128.8, 140.1, 141.4,
44.9, 151.8, 168.9, 169.3, 196.0; MS (EI): m/z (%) = 504 (M+),
47, 425, 328; IR (KBr): ν 2958, 2929, 2871, 1686, 1511, 1493,
[5] Giovannoni, M. P.; Vergelli, C.; Cilibrizzi, A.; Crocettia, L.;
ꢀ
1
Biancalani, C.; Graziano, A.; Piaz, V. D., Loza, I. M.; Cadavid, M. I.;
Diazc, J. L.; Gavalda, A. Bioorg Med Chem 2010, 18, 7890.
32 4 2
351, 1136, 761, 694 cm ; Anal. Calcd for C28H N S O: C,
6.63; H, 6.39; N, 11.10; S, 12.71. Found: C, 66.59; H, 6.31; N,
1.08; S, 12.64.
1
[6] Duffy, J. P.; Harrington, E. M.; Salituro, F. G.; Cochran, J. E.;
Green, J.; Gao, H.; Bemis, G. W.; Evindar, G.; Galullo, V. P.; Ford, P. J.;
Germann, U. A.; Wilson, K. P.; Bellon, S. F.; Chen, G.; Taslimi, P.; Jones, P.;
Huang, C.; Pazhanisamy, S.; Wang, Y. M.; Murcko, M. A.; Su, M. S. S.
ACS Med Chem Lett 2011, 2, 758.
-(5,7-Bis(benzylthio)-1,4-diphenyl-1,4-dihydropyrimido[4,5-c]
pyridazin-3-yl)ethanone(9e). Yellow powder, yield= 34%; mp
): δ 2.42 (s, 3H, CH -CO), 5.44
s, 1H, CH pyridazine), 4.12 (d, 2H, –SCH –), 4.40 (d, 2H,
–), 7.02–7.66 (m, 20H, phenyl); C-NMR (CDCl ): δ
1
70–72°C; H-NMR (CDCl
3
3
[
7] Pollak, A.; Stanovnik, B.; Tisler, M. J Org Chem 1971, 36, 2457.
[8] Mallory, W. R.; Morrison, R. W. J J Org Chem 1980, 45, 3919.
9] Mallory, W. R.; Morrison, R. W. J.; Styles, V. L. J Org Chem
982, 47, 667.
(
2
1
3
–
2
1
1
1
SCH
2
3
[
4.9, 34.1, 35.0, 35.9, 106.0, 125.4, 125.5, 127.0, 127.1,
27.3, 127.7, 128.2, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8,
28.9, 128.8, 128.9, 129.0, 129.1, 129.2, 129.3, 136.9, 137.7,
39.9, 141.4, 145.0, 151.9, 168.3, 168.7, 195.8; MS (EI): m/z
1
[10] Styles, V. L.; Morrison, Jr. R. W. J Org Chem 1982, 47, 585.
[11] Morrison, R. W. J.; Styles, V. L. J Org Chem 1982, 47, 674.
[12] Turbiak, A. J.; Kampf, J. W.; Showalter, H. D. H. Tetrahedron
(
%) = 572 (M+), 541, 482, 403; IR (KBr): ν 3023, 1683,
Lett 2010, 51, 1326.
ꢀ
1
[13] Khalafy, J.; Rimaz, M.; Panahi, L.; Rabiei, H. Bull Korean
1
511, 1492, 1351, 1131, 693 cm
;
Anal. Calcd for
Chem Soc 2011, 32, 2428.
34 28 4 2
C H N S
C, 71.27; H, 4.91; N, 9.77; S, 11.17.
O: C, 71.30; H, 4.93; N, 9.78; S, 11.20. Found:
[
14] Eftekhari-Sis, B.; Zirak, M.; Akbari, A. Chem Rev 2013,
13, 2958.
15] Wu, W.; Voegtli, W. C.; Sturgis, H. L.; Dizon, F. P.; Vigers,
1
Theoretical calculations. In this study, the calculations for
molecular equilibrium geometry and vibrational spectra of all
molecules were performed by means of the Gaussian 03 W
software package [24]. The mPW1PW91 method and 6-31G(d)
basis set were utilized for optimization and NMR calculations
[
G. P. A.; Brandhuber, B. J PLOS ONE 2010, 5, e12913.
[16] Al-Awadi, N. A.; Ibrahim, M. R.; Al-Etaibi A. M.; Elnagdi, M. H.
ARKIVOC 2011, ii, 310.
[
17] Al-Mousawi, S. M.; Mustafa, M. S.; Elnagdi, M. H. Tetrahe-
dron Lett 2009, 50, 6411.
18] Sheibani, H.; Amrollahi, M. A.; Esfandiarpoor, Z. Mol Divers
010, 14, 277.
19] Rashad, A. E.; Hegab, M. I.; Abdel-Megeid, R. E.; Fathalla, N.;
[
25]. This level of density functional theory has been mentioned
[
as a powerful one in producing NMR spectra [26,27]. Gauge-
independent atomic orbital (GIAO) method [28–31] was applied
to provide NMR spectra. The chemical shift of TMS as the
reference was calculated by the same level. The chemical shifts
were obtained by the GaussView 4.1 [32], the graphical
2
[
Abdel-Megeid, F. M. E. Eur J Med Chem 2009, 44, 3285.
[20] Al-Afaleq, E. I.; Abubshait, S. A. Molecules 2001, 6, 621.
[21] Hassan, N. A. Molecules 2000, 5, 826.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Synthesis, Characterization, and Docking Evaluations of New Derivatives of
Pyrimido[4,5-c]pyridazine as Potential Human AKT1 Inhibitors
[
22] Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. J Chem Rev
Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.;
2
1
012, 112, 1839.
Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.;
Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford,
CT, 2004.
[
23] Katritzky, A. R. Adv Heterocycl Chem Academic Press 2010,
01, 162.
24] Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
[
[25] Adamo, C.; Barone, V. J Chem Phys 1998, 108, 664.
Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr., Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.;
Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson,
G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;
Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y. Kitao, O.; Nakai, H.;
Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo,
C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin,
A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma,
K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.;
Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.;
[
26] Bakavoli, M.; Eshghi, H.; Shiri, A.; Afrough, T.; Tajabadi, J. J
Tetrahedron 2013, 69, 8470.
[
[
[
[
27] Sarotti, A. M.; Pellegrinet, S. C. J Org Chem 2009, 74, 7254.
28] Ditchfield, R. J Chem Phys 1972, 56, 5688.
29] Ditchfield, R. Mol Phys 1974, 27, 789.
30] Rohlfing, C. M.; Allen, L. C.; Ditchfield, R. J Chem Phys
1
1
984, 87, 9.
[
31] Wolinski, K.; Hinton, J. F.; Pulay, P. J Am Chem Soc 1990,
12, 8251.
[32] Dennington, R.; Keith, T.; Millam, J. GaussView, Version 4.1,
Semichem Inc., Shawnee Mission, KS, 2007, 1, 93.
[33] Tomasi, J.; Persico, M. Chem Rev 1994, 94, 2027.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet