Design and synthesis of novel tetrahydro-2H-Pyrano[3,2-c]Pyridazin-3(6H)- one derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2010.09.029

Polyfunctional tetrahydro-2H-pyrano[3,2-c]pyridazin-3(6H)-one derivatives were synthesized and biologically evaluated as novel anticancer agents. These motifs were produced by a five-step reaction sequence in which the Achmatowicz oxidative cyclization, i

Full text of DOI:10.1016/j.ejmech.2010.09.029

European Journal of Medicinal Chemistry 45 (2010) 5724e5731
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel tetrahydro-2H-Pyrano[3,2-c]Pyridazin-3(6H)-one
derivatives as potential anticancer agents
,b,
Taleb H. Al-Tel a
*
a College of Pharmacy, Department of Medicinal Chemistry, University of Sharjah, P. O. Box 27272, Sharjah, United Arab Emirates
b Sharjah Institute for Medical Research-Cancer Division, University of Sharjah, Sharjah, 27272, United Arab Emirates
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2010
Received in revised form
1 September 2010
Accepted 1 September 2010
Available online 17 September 2010
Polyfunctional tetrahydro-2H-pyrano[3,2-c]pyridazin-3(6H)-one derivatives were synthesized and bio-
logically evaluated as novel anticancer agents. These motifs were produced by a five-step reaction
sequence in which the Achmatowicz oxidative cyclization, is the basic core for such synthesis.
Compounds 15f, 16c, and 16d showed antiproliferative activity against the SK-BR-3 breast cancer cell line.
Importantly, 16c and 16d showed the highest efficacy, being approximately 30-fold more potent against
SK-BR-3 (IC50 0.21 and 0.15 mM, respectively) compared to other cancer cell lines tested. In addition, 16c
and 16d displayed about 295 fold less toxicity against normal breast cell line MCF10A compared to SK-
BR-3 breast cancer cells. These compounds form the foundation for further investigation in our
continuing efforts to develop potent anticancer agents.
Keywords:
Pyrenones
Tetrahydro-2H-pyrano[3,2-c]pyridazin-3
(6H)-one
Synthesis
Ó 2010 Elsevier Masson SAS. All rights reserved.
b-Ketoesters
Achmatowicz reaction
Anticancer activity
1. Introduction
starting point will be driven by novel synthetic strategies that can
lead to a chemical space of skeletally diverse molecules [8e10]. The
Proliferative disorders are expected to be a major cause of death
in the 21st century [1,2]. Tumor cells are a rapidly changing target
leading to the selection and overgrowth of drug-resistant tumor
cells, and their therapeutic uses are often hindered by the deve-
lopment of drug-resistance, myelosuppression and cytotoxicity
towards normal cells. On the other hand, anticancer drugs tend to
cause general toxicity to normally proliferating cells, which can
severely limit the therapeutic value of these regimens [3].
Thus, much effort is being made to increase tissue, cell, and target
selectivity for chemotherapy [4,5], and extensive synthesis and
design of new structural motifs in order to overcome the afore-
mentioned problems and improve antitumor activity are highly
desirable.
greatest challenge for synthetic chemists is therefore the improve-
ment of overall efficiency by using atom-, step-, and economic
procedures that proceed with high yield and selectivity [11,12].
In this respect, natural products can be viewed as evolved
privileged structures and biologically prevalidated leads that have
probably evolved evolutionarily to exert highly specialized func-
tions [13e15]. However, due to their structural complexity, this may
lead to limited commercial supplies and hinder mechanism of
action studies and clinical development [16e18]. For this reason,
diversity oriented synthesis to generate a plethora of novel scaf-
folds, is a powerful and highly productive tool for lead development
and analog design [19,20].
In a recent unambiguous stereochemical synthesis of cyclopenta
[b]pyranes fused to pyrazolones [21], has allowed us to gain access
to an increased diversity of closely related scaffolds. This versatile
synthesis has given us an opportunity to expand structure activity
studies significantly and thus to test the importance of subtle
skeleton alterations on anticancer activity.
To identify newchemical entities for moreeffectivetreatments of
cancer, medicinal chemists can adopt many strategies, but the
crucial decision is the selection of a suitable starting point from the
astronomically large chemical and biological space [6,7]. The ideal
In this report, the anticancer activities of a related group of
simplified pyrano[b]pyridazinones were designed and their poten-
tial as anticancer agents was investigated against different panels of
tumor cell lines. These efforts have been driven by our previous
* College of Pharmacy, Department of Medicinal Chemistry, University of Sharjah,
P. O. Box 27272, Sharjah, United Arab Emirates. Tel.: þ971 501732950; fax: þ971
65585812.
E-mail address: taltal@sharjah.ac.ae.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.029

T.H. Al-Tel / European Journal of Medicinal Chemistry 45 (2010) 5724e5731
5725
Ph
Ph
O
O
R1
O
MeO
O
O
MeO
O
OMe
O
O
R-NHNH2
Li +
R1
-78 oC
OMe
OMe
OMe
5: R1 = Me
6: R1 = Phenyl
: R1 = 4-Pyridyl
N
EtOH, pTSA
reflux
O
2: R1 = Me
3
N
R
HO
O
H
: R1 = Phenyl
4: R1 = 4-Pyridyl
12
15a: R = 3,5-Di-MeO-Ph
15c: R = 4-Me-Ph
15e: R = 5-Pyrimidyl
15b: R = 3-Cl-5-F-Phenyl
15d: R = 4-OMe-Ph
15f: R = 2-indolyl
1
7
R1
MeI, Ag O
mCPBA HO
DCM
O
2
Scheme 3. Synthesis of polyfunctional tetrahydro-2H-pyrano[3,2-c]pyridazin-3(6H)-
one motifs.
O
O
8
: R1 = Me
2. Results and discussion
9: R1 = Phenyl
10:R1 = 4-Pyridyl
2.1. Chemistry
R1
R1
MeO
OMe
O
OMe
MeO
O
In a recent article from our group [21], we reported on the
application of a protocol for combining the Achmatowicz oxidative
cyclization and the Knoevenagel regiocontrolled condensation on
the resulting cyclic enone scaffolds to deliver cyclopenta[b]pyr-
enones. In such a strategy, furfural derivatives were used as
+
O
O
O
O
8b
9b
8a
: R1 = Me
9a: R1 = Phenyl
10a:R1 = 4-Pyridyl
: R1 = Me
: R1 = Phenyl
10b:R1 = 4-Pyridyl
a nucleofuge and
b-ketoester as a nucleophile. In this report
however, -ketoesters 2-4 were used as the nucleofuge and furyl
lithium 1 as the nucleophile to generate systems of types 5-7
(Scheme 1)
b
Scheme1. . Diastereoselective Synthesis of 3-oxo-3,6-dihydro-2H-pyran-2-yl)acetate
derivatives.
Oxidative cyclization using m-chloroperbenzoic acid (mCPBA) or
N-bromo succinamide (NBS) of the latter, delivered compounds
8-10; these in turn form the building blocks for generating a library
of tetrahydro-2H-pyrano[3,2-c]pyridazin-3(6H)-one derivatives.
Therefore, all target compounds 14-16 were synthesized through
a five-step sequence (Schemes 1-4). Various esters 2-4 were reac-
ted at ꢀ78 ꢁC with furyl lithium intermediate 1 to generate furanol
esters 5-7 in quantitative yields (Scheme 1).Pyrenones 8-9 were
smoothly produced after the application of Achmatowicz oxidative
cyclization protocol using mCPBA in DCM. However, an attempt was
made to subject compound 7 to mCPBA oxidative cyclization
procedure; this attempt failed and tarry reaction products were
formed according to TLC analysis. Alternatively, the synthesis of
pyrenone 10 was achieved through the use of NBS.
The arrival at synthones 11-13 were made available through
standard two step reaction sequence. For this purpose, anomeric
protection of compounds 8-10 using Ag2O/MeI, delivered com-
pounds 8a-10a and 8b-10b as mixtures of diastereoisomers in fairly
good combined yield. Initial efforts to purify these mixtures on silica
gel using EtOAc/hexane (5% v/v) as two separate fractions, was
successful. However, the fractions containing compounds 8b-10b
were always contaminated to about 30% level with difficultly
separable unknown impurities. Nonetheless, since the goal is to
produce pyrano[b]pyridazinone derivatives, efforts were concen-
trated on handling these major products for such purpose and the
fractions that contain the minor products 8b-10b were neglected.
The stereochemistry around structures 8a-10a was confirmed
through NMR nOe experiments and based on the experience gained
experience in the reactivity of diketones [22] and profiting from the
wide applications resulting from the Achmatowicz oxidative cycli-
zation [23,24].
As shown in Scheme 1, this complexity-generating reaction [25]
transforms a relatively simple substrate such as 5, into a more
complex product 8 with potential for diversification via functionali-
zation of the resultant pyrenone scaffold. In connection with
a program directed toward the synthesis of natural product analogs
and finding leads to certain disease states [26,27], this report
describes a new chemical entity, substituted tetrahydro-2H-pyrano
[3,2-c]pyridazin-3(6H)-one derivatives, and their cytotoxic activity
against human tumor cell line panels. Many 1,2-diaza-3-one hetero-
cycles have been found to have potent anticonvulsant, antitubercu-
losis, antitumor, and herbicidal activities [28e33]. It’s quite
interesting that the electronic substituent effects on the bioactivity
changes at certainpositions of such heterocycles have been observed.
For instance, the bioactivities of pyridazinone and its 4,5-dihydro
derivatives were found to be quite sensitive to the electronic effects of
substituents at 2- or 6-positions [34]. To the best of our knowledge,
however, the systems described here, represent novel scaffolds that
have never been described before.
R1
R1
MeO
O
OMe
Pd/C, H2
MeO
O
OMe
O
O
O
O
11: R1 = Me
12: R1 = Phenyl
8a: R1 = Me
9a
10a: R1 = 4-Pyridyl
:
R1 = Phenyl
13
: R1 = 4-Pyridyl
N
N
CH3
R-NHNH2 MeO
O
O
11
MeO
O
OMe
R-NHNH2
MeO
O
O
EtOH, pTSA
reflux
N
R
N
EtOH, pTSA
reflux
O
N
H
O
N
R
14a: R = 4-Cl-Ph
14b: R = 4-Me-Ph
14d: R = 3,5-Di-Cl-Ph
14f: R = 5-Pyrimidyl
H
14c: R = 3,5-Di-MeO-Ph
14e: R = 3-Pyridyl
13
16a: R = 3,5-Di-Cl-Ph 16b: R = 4-Me-Ph
16c 16d
: R = 2-indolyl
14g: R = 2-indolyl
: R = 3,5-Di-MeO-Ph
Scheme 2. Synthesis of polyfunctional tetrahydro-2H-pyrano[3,2-c]pyridazin-3(6H)-
Scheme 4. Synthesis of polyfunctional tetrahydro-2H-pyrano[3,2-c]pyridazin-3(6H)-
one motifs.
one motifs.

5726
T.H. Al-Tel / European Journal of Medicinal Chemistry 45 (2010) 5724e5731
of other pyrano[b]pyridazinones 15a-16d, under the similar
conditions described above (Schemes 3 and 4).
R
H
MeO
O
Results from substituent effects on the ring-closure reaction
described in Schemes 2, 3 and 4 indicated that the electron rich
hydrazine, such as 4-methoxyphenyl hydrazine, provided higher
yields of dihydropyridazinone product (compounds 14b, 14c and
15a Schemes 2 and 3, respectively). On the other hand, reaction
process with electron deficient hydrazine, for instance chloro
substituted hydrazine 15b and 16a (Schemes 2 and 3), proceeded
slowly with low yields.
NH
NR1
17
O
Fig. 1. Structure of octahydro-3H-pyrano[30,20:3,4]cyclopenta[1,2-c]pyrazol-3-one
derivatives [21].
2.2. Biological assay and SAR
during the synthesis of such systems. H-2 of compound 8a reso-
nated at 4.95 ppm as doublets of doublets (J ¼ 0.6 and 3.6 Hz).
Irradiation of the anomeric proton produced a strong reciprocal
nOe enhancement for the methyl group resident at C-6, indicating
a cis relationship. Hydrogenation of derivatives 8a-10a in the
presence of Pd/C produced 11-13. Having secured pure samples
from compounds 11-13, their stereochemistry was further deduced
from NMR nOe experiments. In that, a strong reciprocal nOe
interactions between the 6-Me group in compound 11 and 2-H
were produced. This further confirmed the previously concluded
stereochemistry.
Although our previous studies provided some important infor-
mation about the cytotoxicity of related compounds such as 17
(Fig. 1), several questions remained unanswered: How do the
skeletal complexity and substitution patterns affect the activity and
selectivity? How does the tricylic framework in 17 contribute to
activity, and how will the activity and selectivity change by
simplification of previously described tricyclic motifs [21]. To
answer these questions, chemical and biological strategies were
followed. Prior article from our group, revealed that octahydro-3H-
pyrano[30,20:3,4]cyclopenta[1,2-c]pyrazol-3-one derivatives (e.g.,
17, Fig. 1), in which ring-B (cyclopentanone ring) of 17 is missing in
14, and the pyrazolone ring in 17 has been replaced by pyridazinone
ring in 14, are a new class of potent anticancer agents. These results
encouraged us to further simplify the scaffold of 17 and investigate
the structure activity relationships and selectivity of the newly
synthesized motifs 14a-16d.
In this regard, the newly synthesized tetrahydro-2H-pyrano
[3,2-c]pyridazin-3(6H)-one analogs 14ae14f were evaluated for in
vitro anticancer activity against a panel of tumor cell lines. Results
from 14ae14g (Table 1) showed that different substituent’s around
the pyrazolone ring were critical to the efficacy of these motifs.
Interestingly, compounds 14c,14f and 14g, indicated approximately
five-fold greater potency toward the breast cancer cell line SK-BR-3
and the human lung cancer cell line (A549) compared to other cell
lines tested. Enlightened by these findings, structural modifications
in the pyranopyridazinone ring system were explored with deriv-
atives 15ae15f (Scheme 3 and Table 1). Surprisingly, the intro-
duction of an aryl ring on the ring junction and indolyl group on the
pyrazolone ring; increased the potency to about twenty fold
A
tandem condensation and ring-closure reaction of
g-ketoesters with hydrazines is one of the practical processes
toward the construction of substituted 1,2-diaza-3-one heterocyclic
ring systems [35e37]. After optimization of reaction conditions, we
applied this protocol to a range of hydrazine substrates, including
aryl and heteroaryl hydrazines (Schemes 2, 3 and 4). This sequential
process, however, was reported to be sensitive and chemo-
selectively controlled by the percentage of the acidic catalyst that is
added to this reaction [38,39]. High conversions of substrates were
successfully realized when the condensations were carried out in
refluxed EtOH under the catalysis of Bronsted acids. In particular,
a good yield of the desired 4,5-dihydropyridazinone was obtained
when the amount of catalyst of p-TSA was 10 mol % and the reaction
time was 12 h. Thus, compounds 14a-g were easily prepared
following standard reaction protocols between compounds 11-13
and hydrazine derivatives in refluxing EtOH under acid catalysis
(Scheme 2).
After optimization of reaction conditions, the scope of this
sequential process was then successfully extended to the synthesis
Table 1
IC50 Values for Cellular Proliferation Inhibition results of Compounds 14a-16d in Different Cancer Cell Line Panel.a
IC50 ꢂ SD (
mM)
Compdb
HCT116
SKeNeSH
SK-BR-3
DU-145
A549
MCF10A
14a
14b
14c
14d
14e
14f
14g
15a
15b
15c
15d
15e
15f
35.70 ꢂ 3.30
28.70 ꢂ 1.20
24.30 ꢂ 5.30
19.40 ꢂ 1.10
17.10 ꢂ 0.90
15.10 ꢂ 1.40
18.30 ꢂ 1.70
18.10 ꢂ 1.10
16.20 ꢂ 1.00
10.50 ꢂ 0.80
11.30 ꢂ 0.20
9.13 ꢂ 0.14
8.20 ꢂ 0.30
9.10 ꢂ 0.10
7.40 ꢂ 0.10
6.50 ꢂ 0.10
4.35 ꢂ 0.02
31.70 ꢂ 2.00
25.30 ꢂ 1.80
23.70 ꢂ 4.00
20.20 ꢂ 1.10
19.00 ꢂ 0.80
16.60 ꢂ 1.90
19.50 ꢂ 1.20
19.50 ꢂ 1.00
17.40 ꢂ 0.90
11.10 ꢂ 0.30
10.40 ꢂ 0.10
8.20 ꢂ 0.09
9.10 ꢂ 0.70
8.30 ꢂ 0.230
8.10 ꢂ 0.13
6.40 ꢂ 0.09
3.90 ꢂ 0.04
28.70 ꢂ 2.30
29.70 ꢂ 1.20
5.70 ꢂ 1.30
18.80 ꢂ 1.00
15.20 ꢂ 1.10
3.40 ꢂ 1.20
4.10 ꢂ 0.30
1.20 ꢂ 0.11
17.60 ꢂ 0.13
9.20 ꢂ 0.18
8.40 ꢂ 0.10
5.30 ꢂ 0.12
0.53 ꢂ 0.04
5.50 ꢂ 0.08
6.20 ꢂ 0.10
0.21 ꢂ 0.005
0.15 ꢂ 0.009
24.20 ꢂ 2.50
23.20 ꢂ 1.70
22.20 ꢂ 1.90
21.29 ꢂ 0.80
16.43 ꢂ 0.30
17.10 ꢂ 1.40
14.40 ꢂ 2.30
18.30 ꢂ 1.10
16.10 ꢂ 1.20
11.20 ꢂ 0.20
10.10 ꢂ 0.09
7.33 ꢂ 0.13
7.90 ꢂ 0.03
7.60 ꢂ 0.12
7.10 ꢂ 0.13
5.90 ꢂ 0.02
4.10 ꢂ 0.07
25.47 ꢂ 2.10
21.49 ꢂ 1.40
6.15 ꢂ 1.20
17.48 ꢂ 1.10
14.35 ꢂ 0.30
4.10 ꢂ 1.80
3.92 ꢂ 0.30
17.25 ꢂ 1.40
14.14 ꢂ 2.40
8.11 ꢂ 0.50
7.10 ꢂ 0.13
5.20 ꢂ 0.11
8.67 ꢂ 0.15
7.50 ꢂ 0.20
5.90 ꢂ 0.08
6.22 ꢂ 0.10
4.05 ꢂ 0.002
>38
16a
16b
16c
16d
>51
>55
a
Data represent mean values (SD) for two independent determinations.
Evaluated as HCl salts.
b

T.H. Al-Tel / European Journal of Medicinal Chemistry 45 (2010) 5724e5731
5727
3. Conclusions
In conclusion, this study discovered a novel class of promising
anti-breast cancer agents; substituted tetrahydro-2H-pyrano[3,2-c]
pyridazin-3(6H)-one analogs. The IC50 values of the two most
potent analogs (16c and 16d) against SK-BR-3 were 0.21 and
0.15 mM, respectively. More importantly, 16c and 16d showed high
anti-breast cancer potency, being approximately 30-fold more
potent against SK-BR-3 compared to four other cancer cell lines
from other tissue types. Furthermore, compounds 16c and 16d
displayed a 290-fold increase in potency against the SK-BR-3 breast
cancer cell line when compared to the non-tumorigenic breast cell
line (MCF10A). These findings form the foundation for redirected
efforts aimed at developing novel analogs as preclinical trials
candidates for anti-breast cancer treatment.
Fig. 2. In vitro cytotoxicity of compounds 15f, 16c and 16d against MCF10A (normal
breast cell line).
(compound 15f) of these scaffolds against SK-BR-3 compared to
their cytotoxicity toward A549 and other cell lines. Accordingly,
compounds 16a-16d (Scheme 4) were synthesized and tested. From
these molecules, compounds 16c and 16d were found to have about
thirty-fold potency toward SK-BR-3 relative to the other cell lines
4. Experimental
4.1. General methods
with IC50 values of 0.21 and 0.15
At this junction, selected active compounds with IC50 values less
than 1 M were tested against non-tumorigenic cell lines derived
from breast tissue (MCF10A) vs SK-BR-3 as a positive cancer cell line
control. The tested compounds showed IC50 > 38 M against
MCF10A. These results indicating that, the tested compounds are
more cytotoxic against cancer cells compared to normal healthy
cells.
mM, respectively.
All reagents were used as purchased from commercial suppliers
without further purification. The reactions were carried out in oven
dried or flamed graduated vessels. Solvents were dried and purified
by conventional methods prior use. All reactions were monitored
by thin layer chromatography (TLC) using Merck aluminum plates
pre-coated with silica gel PF254; 20 ꢃ 20, 0.25 mm, and detected by
visualization of the plate under UV lamp (l¼ 254 or 365 nm). Spots
were detected by spraying with anisaldehydeesulpharic acid in
ethanol, followed by heating to 140 ꢁC. Compounds were purified
through recrystallization or using column chromatography which
was performed on silica gel, packed by the slurry method. Flash
column chromatography was performed with Silica gel 60,
0.040e0.063 mm (230e400 mesh). Aluminium backed plates pre-
coated with silica gel 60 (UV254) were used for thin layer chro-
matography. 1H and 13C NMR spectra were recorded on a 300 MHz/
75 MHz spectrometer. Splitting patterns are designated as s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
m
m
Figs. 2 and 3 show the results of cell viability studies carried out
on compound 15f, 16c and 16d using the MTT assay protocol. These
results further confirm not only the selectivity of these novel motifs
but also indicated their lower toxicity against normal cells.
At this stage, some structure activity relationships could be
construed from these results. For the compounds of type 14, in
which the ring junction carries a methyl group, there was no
noticeable effect on the potency with different substituents resi-
dent on the N-2 of the pyridazinone ring. However, a phenyl ring
on the ring junction as indicated in systems of type 15, electron
rich phenyl (e.g. 15a, 15c and 15d) and/or heteroaryl groups (e.g.
15e and 15f) increased the potency of these scaffolds as anticancer
agents compared to an electron poor phenyl substituents on N-2
(e.g. 15b).
Interestingly, when the ring junction of the bicyclic framework
carries a pyridyl moiety such as systems of type 16, the anticancer
activity of these motifs enhanced for both electron rich aryl and
heteroaryl rings compared to electron poor aryl systems. In general,
introduction of an aryl and/or heteroaryl ring at the ring junction
increases the potency of these systems toward breast cancer cell
line compared to other cell lines investigated.
Chemical shifts (d) are given in ppm relative to the resonance of
their respective residual solvent peak, CHCl3 (7.27 ppm, 1H;
77.16 ppm, the middle peak, 13C). Spectra were acquired in CDCl3
containing 1% TMS. High resolution Mass spectra (HRMS) were
recorded in positive ion mode by Electrospray Ionization (ESI) on
a Bruker instrument. The samples were dissolved in acetonitrile,
diluted in spray solution (methanol/water 1:1 v/v 0.1 % formic acid)
and infused using a syringe pump with a flow rate of 2 mL/min.
External calibrationwas conducted using Arginine cluster in a mass
range m/z 175e871. For all HRMS data, mass error: 0.00e0.50 ppm.
Melting points (m.p.) were determined on an Electrothermal
Melting point Apparatus and were reported uncorrected in oC.
4.2. Biology
4.2.1. Cell growth inhibition assay
All stock cultures are grown in T-25 flasks. Freshly trypsinized
cell suspensions were seeded in 96-well microtiter plates with
compounds added from DMSO diluted stock. The plates were
incubated for an additional 72 h after attachment and addition of
compounds, and the assay was terminated by 10% TCA. Then, 0.4%
SRB dye in 1% HOAc was added to stain the cells for 10 min.
Unbound dye was removed by repeated washing with 1% HOAc
and the plates were air dried. Bound stain was subsequently solved
with 10 mM trizma base, and the absorbance read at 515 nm. Stock
solutions of agents in DMSO at 1 mg/mL were prepared fresh for
each experiment and were diluted in cell culture medium to
achieve desired concentrations upon addition to the target cells.
Fig. 3. In vitro cytotoxicity of compounds 15f, 16c and 16d against SK-BR-3 (breast
cancer cell line).

5728
T.H. Al-Tel / European Journal of Medicinal Chemistry 45 (2010) 5724e5731
All six types of cells were incubated for 48 h at 37 ꢁC in 96-well
microtiter plates to assess inhibition of cell proliferation by
measuring changes in protein content in triplicate wells. Cytotox-
icity was assessed using sulforhodamine-B (SRB) by measuring the
extent of inhibition of protein synthesis in treated cells compared
with that in untreated cells. Following 48 h treatment, cells were
fixed by the addition of cold (4 ꢁC) 50% trichloroacetic acid (50 mL/
well). After a 1-h incubation at 4 ꢁC, plates were gently washed 5
times with deionized water and allowed to dry overnight. A 0.4%
solution of SRB in 1% acetic acid (50 mL) was added to each well,
and plates were incubated 10 min at room temperature. Wells
were then rinsed 5 times with 1% acetic acid and allowed to dry. To
solubilize the SRB dye, 150 mL/well of 10 mM Tris base (pH 10.5)
was incubated for 20 min at room temperature and plates were
gently vortex-mixed before absorbance at 570 nm was measured
using a spectrophotometer (Spectramax-Pro; Molecular Devices).
Wells containing untreated cells served as positive controls. The
mean absorbance of the corresponding set of blanks was sub-
tracted from the mean absorbance of wells incubated with each
test agent. This value was then divided by the difference between
the mean absorbance of the untreated cells and that of the blanks
in order to calculate a percent inhibition for each concentration of
agent. Growth inhibition of 50% (IC50) is calculated as the drug
concentration, which caused a 50% reduction in the net protein
increase during the drug incubation. The mean IC50 is the
concentration of agent that reduces cell growth by 50% under the
experimental conditions and is the average with SEM from at least
three independent determinations. The following human tumor
cell lines were used in the assay: A549 (non small cell lung cancer,
HCT116 (human colorectal cancer cells) and SKeNeSH (human
Caucasian bone marrow neuroblastoma) cells, SK-BR-3 (HER2
over-express in breast cancer), DU-145 (prostate cancer cell line).
Cells propagated in RPMI-1640 supplemented with 10% FBS,
temperature for 45 min, the mixture was stirred warmed to 0 ꢁC
after which the mixture was quenched with a saturated solution of
NH4Cl (15 mL), extracted with EtOAc (3 ꢃ 30 mL), dried over
Na2SO4 and concentrated under vacuum to give a yellowish crude
oil. The crude was used in the next step without further purifica-
tion. Compounds 5-7 were produced in quantitative yields as
yellowish oils.
4.2.3.2. General procedure for the synthesis of compounds 8-9. A
solution of furyl alcohols 5 (920 mg, 5 mmol) or 6 (1.23 g, 5 mmol)
in dicholomethane (40 mL) at 0 ꢁC was treated with m-chlor-
operbenzoic acid (1.376 g, 8.0 mmol of 80% aqueous slurry) which
was dissolved in 30 mL DCM, dried over Na2SO4, filtered, diluted
with 50 mL toluene and dried under reduced pressure) and stirred
at 0 ꢁC for 6 h. Sodium sulfite solution (30 mL of 10%) was intro-
duced, and the layers were separated after 1 h of rapid mixing. The
aqueous phase was washed with saturated sodium bicarbonate
solution (30 mL), brine and water (30 mL) prior to drying and
solvent evaporation. The crude viscous yellowish oils (8 and 9)
were used in the next step without further purification.
4.2.3.3. General procedure for the synthesis of compounds 8a, 9a and
10a. A solution of the anomeric mixture of 8a (600 mg, 3 mmol) or
9a (786 mg, 3 mmol) or 10a (789 mg, 3 mmol) in MeI (15 mL) at rt
was treated with 3.0 mmol of Ag2O. The progress of reaction was
monitored by TLC (12 h). When complete, the reaction mixture was
filtered over celite. The filtrate was washed with saturated solutions
of sodium sulfite, sodium bicarbonate and brine, dried and evap-
orated. The residue was purified on silica gel column using hexane/
EtOAc (9:1) as an eluent.
4.2.3.4. General procedure for the synthesis of compounds 11-13. To
a stirred solution of 8a-10a (2.0 mmol) in dry EtOAc/MeOH (1:1,
20 mL) was added Pd/C (56 mg, 15% Pd/C w/w). The mixture was
placed under 1.0 atm of H2 pressure, and the progress of reaction
was monitored by TLC. After 2 h, the solid was removed by filtration
through a celite pad, which was washed repeatedly with EtOAc.
After concentration of the filtrate, the residue was purified on silica
gel column (elution with 5% ethyl acetate in hexane) to give 11-13
as amorphous solids.
penicillin (100 IU/mL), streptomycin (1
mg/mL), and amphotericin B
(0.25
m
g/mL), and were cultured at 37 ꢁC in a humidified atmo-
sphere of 95% air/5% CO2.
4.2.2. Methodology of MTT assay
The cytotoxic activity of the cyclopenta[b]pyrane derivatives
15f, 16c and 16d was determined using a standard (MTT)-based
colorimetric assay. This assay quantifies viable cells by observing
the reduction of tetrazolium salt, MTT, to formazan crystals by the
cells. Based on the absorbance of the cell samples after the test is
carried out, cell viability can be measured. Cells were plated with
nutritional medium in 96 well plates (2000 and 5000 cells/well for
HCT116 and SKeNeSH). After 24 h, cells were treated with
4.2.3.5. General procedure for the synthesis of compound 10. Furyl
alcohol 7 (494 mg, 2 mmol), 5 mL of THF, and 2 mL of H2O were
cooled to 0 ꢁC. Solid NaHCO3 (460 mg, 4.15 mmol), NaOAc 3H2O
ꢄ
(420 mg, 3.11 mmol), and NBS (360 mg, 2.7 mmol) were added to
the solution and the mixture was stirred for 0.5 h at 0 ꢁC. The
reaction was quenched with saturated NaHCO3 (5 mL), extracted
(3 ꢃ 20 mL) with EtOAc, dried over Na2SO4, concentrated under
reduced pressure and the crude product was used in the next step
without further purification.
a different concentration (0.1, 0.3, 1.0, 3.0 and 10 mM) of the new
compounds, each concentration in 3 repetitions. The plates were
incubated with the pyrazolone derivatives for 72 h. At the end of
treatment, cells were washed with PBS solution. Then, 100
fresh medium and 50 L from a stock solution of MTT (3 mg/mL
PBS) were added to each well. After 4 h of incubation at 37 ꢁC, the
medium was discarded and 100 L of DMSO solution were added
mL of
m
4.2.3.6. General procedure for the synthesis of compounds 14a-
16d. The pyranone ketoesters 11-13 derived from the reduction
product of 8a-10-a (142 mg, 0.5 mmol) and the desired hydrazine
derivative (0.52 mmol) in 20 mL absolute ethanol was refluxed in
the presence of 10 mol% pTSA. The progress of reaction was
monitored by TLC. After 12 h, the reaction mixture was concen-
trated on a rotary evaporator; the residue was purified through
triturating with cold EtOH containing traces of water to give 14a-
16d as light yellowish solids.
m
to each well, in order to dissolve the crystals that were formed.
After a 30 min period, the absorbance of the samples was
measured by an Elisa reader. The absorbance data was converted to
% cell viability.
4.2.3. Synthesis
4.2.3.1. General procedure of the synthesis of compounds 5-7. A
suspension of furyl lithium was prepared under argon by slow
addition of n-BuLi (3.25 mL, 5.2 mmol of 1.6 M solution in hexane)
furan (340 mg, 5 mmol) in THF (15 mL) at e 78 ꢁC over 45 min
period. To this mixture, methyl acetoacetate (580 mg, 5 mmol in
5 mL THF) was added over 5 min period. After stirring at this
4.2.3.7. Methyl
3-(furan-2-yl)-3-hydroxybutanoate
(5). Yield:
7.10
quantitative, yellowish oil. 1H NMR (300 MHz, CDCl3, in ppm)
d
(s, 1H), 6.34 (d, J ¼ 3.0 Hz, 1H), 4.45 (s, 1H), 3.69 (s, 3H), 3.10 (d,
J ¼ 15.0 Hz, 1H), 3.0 (d, J ¼ 15.0 Hz, 1H), 1.44 (s, 3H). 13C NMR

T.H. Al-Tel / European Journal of Medicinal Chemistry 45 (2010) 5724e5731
5729
(75 MHz, CDCl3, in ppm)
d
168.6, 154.1, 140.5, 109.4, 105.3, 71.1, 54.3,
4.2.3.14. Methyl 2-((2S,6S)-6-methoxy-2-methyl-3-oxotetrahydro-
44.0, 25.9. ESIMS: m/z calculated for [M þ Naþ]: 207.063330, found
2H-pyran-2-yl)acetate (11). Yield: 91%, amorphous solid. 1H NMR
207.063210.
(300 MHz, CDCl3, in ppm)
d
4.52 (dd, J ¼ 5.3, 9.7 Hz,1H), 3.62 (s, 3H),
3.32 (s, 3H), 2.79-2-61 (m, 4H), 2.08e1.92 (m, 2H), 1.47 (s, 3H). 13C
4.2.3.8. Methyl
(6). Yield: quantitative, yellowish oil. 1H NMR (300 MHz, CDCl3, in
ppm)
3-(furan-2-yl)-3-hydroxy-3-phenylpropanoate
NMR (75 MHz, CDCl3, in ppm) d 207.1, 171.2, 93.1, 82.7, 55.4, 50.8,
41.9, 32.6, 28.8, 21.7. ESIMS: m/z calculated for [M þ Naþ]:
d
7.53e7.22 (m, 2H), 7.10e7.15 (m, 3H), 6.81 (t, J ¼ 0.2Hz, 1H),
239.089545, found 239.089531.
6.37 (d, J ¼ 3.0 Hz,1H), 6.17 (dd, J ¼ 0.2, 3.0 Hz,1H), 3.69 (s, 3H), 3.44
(d, J ¼ 16.0 Hz, 1H), 3.39 (d, J ¼ 16.1 Hz, 1H). 13C NMR (75 MHz,
4.2.3.15. Methyl 2-((2R,6S)-6-methoxy-3-oxo-2-phenyltetrahydro-
CDCl3, in ppm)
d
170.5, 154.1, 143.4, 139.9, 129.3, 128.7, 126.2, 110.3,
2H-pyran-2-yl)acetate (12). Yield: 88%, amorphous solid. 1H NMR
104.8, 81.7, 54.1, 44.1. ESIMS: m/z calculated for [M þ Naþ]:
(300 MHz, CDCl3, in ppm)
8.7 Hz, 1H), 3.61 (s, 3H), 3.33 (s, 3H), 3.03e2.80 (m, 4H), 2.11e2.04
(m, 2H). 13C NMR (75 MHz, CDCl3, in ppm)
205.1, 169.3, 140.1,
d
7.38e7.2 (m, 5H), 4.92 (dd, J ¼ 3.1,
269.078980, found 269.078851.
d
4.2.3.9. Methyl 3-(furan-2-yl)-3-hydroxy-3-(pyridin-4-yl)propanoate
128.3, 126.4, 124.9, 95.1, 83.1, 55.1, 50.8, 41.7, 34.8, 28.1. ESIMS: m/z
(7). Yield: quantitative, yellowish oil. 1H NMR (300 MHz, CDCl3, in
calculated for [M þ Naþ]: 301.105195, found 301.105168.
ppm)
d
8.72 (dd, J ¼ 3.0, 6.0 Hz, 2H), 7.51 (dd, J ¼ 3.0, 6.0 Hz, 2H), 6.92
(t, J ¼ 0.1 Hz, 1H), 6.41 (d, J ¼ 3.0 Hz, 1H), 6.11 (dd, J ¼ 0.2, 3.0 Hz, 1H),
4.2.3.16. Methyl 2-((2R,6S)-6-methoxy-3-oxo-2-(pyridin-4-yl)tetra-
3.63 (s, 3H), 3.41 (d, J ¼ 15.7 Hz, 1H), 3.28 (d, J ¼ 15.7 Hz,1H).13C NMR
hydro-2H-pyran-2-yl)acetate (13). Yield: 78%, amorphous solid. 1H
(75 MHz, CDCl3, in ppm)
d
171.1, 153.9, 147.5, 139.4, 123.9, 122.4, 114.3,
NMR (300 MHz, CDCl3, in ppm)
d
8.97 (d, J ¼ 5.4 Hz, 2H), 7.75 (d,
105.0, 81.6, 53.2, 44.1. ESIMS: m/z calculated for [M þ Naþ]:
J ¼ 6.6 Hz, 2H), 4.96 (dd, J ¼ 3.1, 8.9 Hz, 1H), 3.59 (s, 3H), 3.11 (s, 3H),
270.074229, found 270.074213.
3.09e2.80 (m, 4H), 2.13e2.09 (m, 2H). 13C NMR (75 MHz, CDCl3, in
ppm)
d 206.1, 170.3, 147.2, 121.8, 118.4, 94.1, 83.7, 55.5, 50.5, 41.2,
4.2.3.10. Methyl 2-(6-hydroxy-2-methyl-3-oxo-3,6-dihydro-2H-pyran-
36.0, 27.8. ESIMS: m/z calculated for [M þ Naþ]: 302.100444, found
2-yl)acetate (8). Amorphous solid. 1H NMR (300 MHz, CDCl3, in ppm)
302.100437.
d
6.85 (dd, J ¼ 2.3, 9.2 Hz, 1H, H-5), 6.79 (dd, J ¼ 3.1, 10.0 Hz, 1H, H-5),
6.10 (d, J ¼ 9.6 Hz, 1H, H-4), 6.07 (dd, J ¼ 1.1, 10.4 Hz, 1H, H-4), 5.65 (t,
J ¼ 4.0 Hz, 1H, H-6), 5.59 (t, J ¼ 6.9 Hz, 1H, H-6), 3.55 (s, 3H, OMe),
3.45 (s, 3H, OMe), 3.02 (d, J ¼ 15.7 Hz, 1H, H-7), 2.98 (d, J ¼ 15.4 Hz,
1H, H-7), 2.53 (d, J ¼ 15.7 Hz, 1H, H-7), 2.42 (d, J ¼ 15.5 Hz, 1H, H-7),
4.2.3.17. (4 aS,6S)-2-(4-Chlorophenyl)-6-methoxy-4a-methyl-4,4a,6,7-
tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (14a). Yield: 63%,
m.p. 176 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
7.73 (d, J ¼ 9.0 Hz,
2H), 7.24 (d, J ¼ 9.0 Hz, 2H), 4.86 (dd, J ¼ 2.6, 6.4 Hz, 1H), 4.49 (dd,
1.46, 1.33 (s, 6H, CH3). 13C NMR (75 MHz, CDCl3, in ppm)
d 197.5,197.4
J ¼ 2.9 Hz, 9.1 Hz,1H), 3.35 (s, 3H), 2.73 (d, J ¼ 12.7 Hz,1H), 2.48e2.27
(C-3), 170.5, 169.1 (CO2Me), 146.5, 143.9 (C-5), 126.3 (C-4), 87.7, 87.6
(C-6), 79.6, 79.3 (C-2), 55.5, 55.3 (OMe), 45.4, 45.2 (C-7), 27.0, 23.4
(CH3). ESIMS: m/z calculated for [M þ Naþ]: 223.058245, found
223.058197.
(m, 3 H), 1.44 (s, 3H). 13C NMR (75 MHz, CDCl3, in ppm)
d 169.1, 145.4,
141.3, 133.7, 130.6, 125.9, 105.8, 105.3, 77.9, 55.7, 43.2, 29.9, 26.1.
ESIMS: m/z calculated for [MþNaþ331.082541 :[, found 331.082532.
4.2.3.18. (4aS,6S)-6-methoxy-4a-methyl-2-p-tolyl-4,4a,6,7-tetrahy-
4.2.3.11. Methyl 2-((2S,6S)-6-methoxy-2-methyl-3-oxo-3,6-dihydro-
dro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (14b). Yield: 58%, m.p.
2H-pyran-2-yl)acetate (8a). Yield: 82%, amorphous solid. 1H NMR
162 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
7.71 (d, J ¼ 8.7 Hz, 2H),
(300 MHz, CDCl3, in ppm)
d
6.87 (dd, J ¼ 3.5, 10.3 Hz, 1H, H-3), 6.25
7.11 (d, J ¼ 8.7 Hz, 2H), 4.86 (dd, J ¼ 2.8, 6.3 Hz, 1H), 4.45 (dd, J ¼ 6.1,
9.1 Hz, 1H), 3.36 (s, 3H), 2.69 (d, J ¼ 13.1 Hz, 1H), 2.49e2.27 (m, 3 H),
(d, J ¼ 10.3 Hz, 1H, H-4), 4.95 (dd, J ¼ 0.6, 3.6 Hz, 1H, H-2), 3.62 (s,
3H), 3.41 (s, 3H, OMe), 3.07 (d, J ¼ 16.3 Hz, 1H, H-7), 2.54 (d,
J ¼ 16.3 Hz, 1H, H-7), 1.46 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3, in
2.22 (s, 3H), 1.44 (s, 3H). 13C NMR (75 MHz, CDCl3, in ppm)
d 167.1,
146.4, 141.5, 140.7, 130.8, 124.2, 106.2, 105.1, 77.9, 54.8, 41.6, 29.8,
26.4, 20.9. ESIMS: m/z calculated for [M þ Naþ]: 311.137163, found
311.137172.
ppm)
d 197.0 (C-3), 167.0 (CO2Me), 140.0 (C-5), 126.3 (C-4), 87.6 (C-
6), 80.2 (C-2), 45.6 (C-7), 26.7 (CH3). ESIMS: m/z calculated for
[M þ Naþ]: 237.073895, found 237.073791.
4.2.3.19. (4aS,6S)-2-(3,5-Dimethoxyphenyl)-6-methoxy-4a-methyl-
4.2.3.12. Methyl 2-((2R,6S)-6-methoxy-3-oxo-2-phenyl-3,6-dihydro-
4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (14c). Yield:
2H-pyran-2-yl)acetate (9a). Yield: 77%, amorphous solid. 1H NMR
55%, m.p. 189 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d 6.94 (d,
(300 MHz, CDCl3, in ppm)
d
7.44 (d, J ¼ 6.6 Hz, 1H), 7.29e7.21 (m,
J ¼ 5.8 Hz, 2H), 6.55 (d, J ¼ 2.7 Hz, 1H), 4.76 (dd, J ¼ 3.0, 5.7 Hz, 1H),
4.51 (dd, J ¼ 2.8, 5.4 Hz, 1H), 3.80 (s, 6H), 3.31 (s, 3H), 2.69 (d,
J ¼ 15.3 Hz, 1H), 2.46e2.25 (m, 34H), 1.42 (s, 3H). 13C NMR (75 MHz,
4 H), 6.78 (dd, J ¼ 3.3, 10.1 Hz, 1H, H-3), 6.56 (dd, J ¼ 0.6, 10.8 Hz,
1H, H-4), 5.29 (d, J ¼ 3.3 Hz, H-2), 3.65 (s, 3H, OMe), 3.31 (s, 3H),
3.18 (d, J ¼ 17.1 Hz, H-7), 2.92 (d, J ¼ 17.1 Hz. H-7). 13C NMR
CDCl3, in ppm) d 167.4, 165.7, 146.1, 140.9,110.6, 106.4, 104.9, 95.6, 77.8,
(75 MHz, CDCl3, in ppm)
d
192.7 (C-3), 169.9 (CO2Me), 145.3 (C-5),
55.7, 55.6, 42.4, 29.7, 26.6. ESIMS: m/z calculated for [M þ Naþ]:
140.3, 129.7, 127.5, 126.1, 126.0, 101.9, 83.8 (C-6), 54.5, 51.7, 42.1
(C-7). ESIMS: m/z calculated for [MþNaþ299.089545 :[, found
299.089511.
357.142643, found 357.142633.
4.2.3.20. (4aS,6S)-2-(3,5-Dichlorophenyl)-6-methoxy-4a-methyl-
4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (14d). Yield:
4.2.3.13. Methyl 2-((2R,6S)-6-methoxy-3-oxo-2-(pyridin-4-yl)-3,6-
62%, m.p. 194 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d 7.78 (d,
dihydro-2H-pyran-2-yl)acetate (10a). Yield: 69%, amorphous solid.
J ¼ 1.8 Hz, 2H), 7.42 (s, 1H), 4.84 (dd, J ¼ 5.7, 8.4 Hz, 1H), 4.53 (dd,
1H NMR (300 MHz, CDCl3, in ppm)
d
8.91 (d, J ¼ 5.4 Hz, 2H), 7.71 (d,
J ¼ 3.1, 6.2 Hz, 1H), 3.34 (s, 3H), 2.71e2.29 (m, 4H), 1.41 (s, 3H). 13C
J ¼ 6.6 Hz, 2H), 6.82 (d, J ¼ 12.9 Hz, 1H), 6.33 (d, J ¼ 11.8 Hz, 1H),
5.45 (d, J ¼ 3.1 Hz, 1H), 3.68 (s, 3H), 3.37 (s, 3H), 3.31 (d, J ¼ 16.8 Hz,
1H), 3.12 (d, J ¼ 16.8 Hz, 1H). 13C NMR (75 MHz, CDCl3, in ppm)
NMR (75 MHz, CDCl3, in ppm) d 167.2, 145.5, 139.1, 136.4, 134.2, 131.9,
124.6, 122.8, 106.7, 105.3, 79.1, 55.6, 42.3, 29.4, 26.5. ESIMS: m/z
calculated for [M þ Naþ]: 365.043569, found 365.043544.
d
192.6, 170.1, 150.1, 144.2, 128.0, 122.3, 120.1, 101.7, 83.6, 55.2, 50.1,
41.5. ESIMS: m/z calculated for [M þ Naþ]: 300.084794, found
4.2.3.21. (4aS,6S)-6-methoxy-4a-methyl-2-(pyridin-3-yl)-4,4a,6,7-
tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (14e). Yield: 68%,
300.084699.

5730
T.H. Al-Tel / European Journal of Medicinal Chemistry 45 (2010) 5724e5731
m.p. 181 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
9.25 (d, J ¼ 2.9 Hz,
J ¼ 2.2, 5.6 Hz, 1H), 4.71 (bd, J ¼ 8.6 Hz, 1H), 3.73 (s, 3H), 3.30 (s, 3H),
3.09 (d, J ¼ 13.7 Hz, 1H), 2.76 (d, J ¼ 13.7 Hz, 1H), 2.53 (dt, J ¼ 5.4,
14.3 Hz, 1H), 2.38 (dt, J ¼ 5.1, 14.3 Hz, 1H). 13C NMR (75 MHz, CDCl3, in
1H), 8.62 (dd, J ¼ 2.8, 5.8 Hz, 1H), 8.12 (dd, J ¼ 2.7, 5.9 Hz, 1H), 7.42
(dd, J ¼ 2.8, 5.7 Hz,1H), 4.78 (dd, J ¼ 2.6, 6.2 Hz,1H), 4.39 (dd, J ¼ 1.2,
3.2 Hz, 1H), 3.34 (s, 3H), 2.69 (d, J ¼ 15.7 Hz, 1H), 2.46e2.28 (m, 3H),
ppm) d 166.1, 161.6, 145.1, 142.3, 136.7, 126.3, 126.0, 124.9, 123.0, 115.1,
1.42 (s, 3H). 13C NMR (75 MHz, CDCl3, in ppm)
d
167.2, 146.4, 146.1,
107.9, 107.2, 82.7, 55.8, 55.1, 43.0, 29.3. ESIMS: m/z calculated for
143.4, 136.7, 127.9, 126.8, 106.5, 105.2, 78.3, 55.9, 42.1, 29.7, 26.7.
[M þ Naþ]: 389.147728, found 389.147719.
ESIMS: m/z calculated for [M
298.116759.
þ
Naþ]: 298.116762, found
4.2.3.28. (4aR,6S)-6-methoxy-4a-phenyl-2-(pyrimidin-5-yl)-4,4a,6,7-
tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (15e). Yield: 50%,
4.2.3.22. (4aS,6S)-6-methoxy-4a-methyl-2-(pyrimidin-5-yl)-4,4a,6,7-
m.p. 183 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d 9.33 (s, 2H), 8.92 (s,
tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (14f). Yield: 59%,
1H), 7.27e7.09 (m, 5H), 5.03 (dd, J ¼ 2.2, 5.1 Hz, 1H), 4.62 (dd, J ¼ 2.2,
5.3 Hz, 1H), 3.35 (s, 3H), 3.08 (d, J ¼ 14.2 Hz, 1H), 2.75 (d, J ¼ 14.2 Hz,
1H), 2.52 (dt, J ¼ 5.3, 14.1 Hz, 1H), 2.41 (dt, J ¼ 2.3, 14.2 Hz, 1H). 13C
m.p. 159 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d 9.33 (s, 2H), 8.91 (s,
1H), 4.85 (dd, J ¼ 2.6, 5.2 Hz, 1H), 4.39 (dd, J ¼ 1.1, 8.7 Hz, 1H), 3.36 (s,
3H), 2.76 (d, J ¼ 14.3 Hz, 1H), 2.47e2.29 (m, 3H),1.41 (s, 3H). 13C NMR
NMR (75 MHz, CDCl3, in ppm) d 165.7, 151.9, 148.2, 145.3, 142.2, 132.6,
(75 MHz, CDCl3, in ppm)
d
167.7,151.2,147.9,146.3,131.5,106.8,105.4,
126.8, 124.5, 122.8, 107.7, 107.5, 82.4, 55.3, 43.1, 29.4. ESIMS: m/z
79.2, 55.6, 43.0, 29.3, 25.9. ESIMS: m/z calculated for [M þ Naþ]:
calculated for [M þ Naþ]: 361.127661, found 361.127672.
299.112011, found 299.112023.
4.2.3.29. (4aR,6S)-2-(1H-indol-2-yl)-6-methoxy-4a-phenyl-4,4a,6,7-
4.2.3.23. (4aS,6S)-2-(1H-indol-2-yl)-6-methoxy-4a-methyl-4,4a,6,7-
tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (15f). Yield: 49%,
tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (14g). Yield: 48%,
m.p. 156 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d 10.6 (s, 1H), 8.4 (s,
m.p. 191 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
7.78 (d, J ¼ 8.9 Hz,
1H), 7.8 (s, 1H), 7.41e7.09 (m, 6H), 6.57 (t, J ¼ 8.6 Hz, 2H), 5.05 (t,
J ¼ 4.2 Hz, 1H), 4.61 (t, J ¼ 6.7 Hz, 1H), 3.33 (s, 3H), 3.11 (d,
J ¼ 12.7 Hz, 1H), 2.79 (d, J ¼ 12.7 Hz, 1H), 2.51 (bd, J ¼ 5.7 Hz, 1H),
1H), 7.43 (d, J ¼ 8.7 Hz, 1H), 7.21 (s, 1H), 7.12 (dd, J ¼ 5.4, 8.6 Hz, 1H),
6.67 (t, J ¼ 8.8 Hz, 1H), 4.85 (dd, J ¼ 2.7, 6.1 Hz, 1H), 4.37 (dd, J ¼ 1.0,
8.5 Hz, 1H), 3.30 (s, 3H), 2.71 (d, J ¼ 13.1 Hz, 1H), 2.48e2.28 (m, 3H),
2.41 (bd, J ¼ 4.5 Hz, 1H). 13C NMR (75 MHz, CDCl3, in ppm)
d 164.9,
1.39 (s, 3H). 13C NMR (75 MHz, CDCl3, in ppm)
d
165.7, 148.0, 141.5,
147.8, 144.1, 140.6, 133.7, 128.3, 126.5, 125.1, 124.8, 123.2, 121.9, 121.4,
110.3, 108.1, 107.8, 98.2, 83.1, 55.7, 43.2, 30.1. ESIMS: m/z calculated
for [M þ Naþ]: 398.148062, found 398.148058.
133.1, 129.3, 123.9, 121.7, 121.0, 109.9, 106.6, 105.1, 97.1, 78.7, 55.8,
42.3, 29.1, 25.9. ESIMS: m/z calculated for [M þ Naþ]: 336.132412,
found 336.132443.
4.2.3.30. (4aR,6S)-2-(3,5-Dichlorophenyl)-6-methoxy-4a-(pyridin-4-
yl)-4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one
(16a). Yield: 60%, m.p. 171 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
4.2.3.24. (4aR,6S)-2-(3,5-Dimethoxyphenyl)-6-methoxy-4a-phenyl-
4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (15a). Yield:
57%, m.p. 186 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
7.31e7.09 (m,
d
8.65 (d, J ¼ 5.3 Hz, 2H), 7.87 (d, J ¼ 2.1 Hz, 2H), 7.41 (t, J ¼ 2.1 Hz,
5H), 6.79 (d, J ¼ 2.8 Hz, 2H), 6.30 (s, 1H), 5.11 (dd, J ¼ 2. 4, 5.4 Hz, 1H),
4.66 (bs, 1H), 3.83 (s, 6H), 3.35 (s, 3H), 3.12 (d, J ¼ 15.1 Hz, 1H), 2.80 (d,
J ¼ 15.1 Hz, 1H), 2.50 (dt, J ¼ 5.7,10.4, 11.6 Hz, 1H), 2.34 (dt, J ¼ 5.3, 10.1,
1H), 7.32 (d, J ¼ 5.4 Hz, 2H), 5.12 (dd, J ¼ 2.0, 5.6 Hz, 1H), 4.60 (dd,
J ¼ 2.1, 5.4 Hz, 1H), 3.36 (s, 3H), 3.11 (d, J ¼ 14.6 Hz, 1H), 2.72 (d,
J ¼ 14.6 Hz,1H), 2.54 (dt, J ¼ 5.3, 8.9 Hz,1H), 2.41 (dt, J ¼ 2.3,12.1 Hz,
11.9 Hz, 1H). 13C NMR (75 MHz, CDCl3, in ppm)
d
166.2, 165.0, 145.1,
1H). 13C NMR (75 MHz, CDCl3, in ppm)
d 165.9, 146.1, 145.8, 144.2,
142.3, 139.7, 126.5, 123.9, 122.1, 110.0, 107.9, 107.3, 95.8, 82.1, 55.7, 55.2,
42.5, 29.7. ESIMS: m/z calculated for [M þ Naþ]: 419.158293, found
419.158281.
137.9, 136.1, 124.3, 123.1, 120.7, 108.2, 107.8, 82.9, 55.6, 43.2, 29.7.
ESIMS: m/z calculated for [M
þ
Naþ]: 428.054468, found
428.054454.
4.2.3.25. (4aR,6S)-2-(3,5-Dichlorophenyl)-6-methoxy-4a-phenyl-
4.2.3.31. (4aR,6S)-6-methoxy-4a-(pyridin-4-yl)-2-p-tolyl-4,4a,6,7-
4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (15b). Yield:
tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (16b). Yield: 46%,
51%, m.p. 166 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
7.92 (s, 2H),
m.p. 145 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
8.67 (d, J ¼ 5.7 Hz,
7.36e7.09 (m, 6H), 5.18 (t, J ¼ 2.7 Hz, 1H), 4.31 (bs, 1H), 3.31 (s, 3H),
3.15 (d, J ¼ 14.5 Hz, 1H), 2.78 (d, J ¼ 14.4 Hz, 1H), 2.54 (dd, J ¼ 4.2,
12.6 Hz, 1H), 2.42 (dd, J ¼ 4.5, 8.5 Hz, 1H). 13C NMR (75 MHz, CDCl3, in
2H), 7.61 (d, J ¼ 8.9 Hz, 2H), 7.33 (d, J ¼ 5.6 Hz, 2H), 7.22 (d,
J ¼ 10.5 Hz, 2H), 5.09 (dd, J ¼ 2.2, 5.6 Hz, 1H), 4.61 (t, J ¼ 5.7 Hz, 1H),
3.35 (s, 3H), 3.12 (d, J ¼ 14.3 Hz, 1H), 2.82 (d, J ¼ 14.3 Hz, 1H), 2.53
(dt, J ¼ 5.2, 14.5 Hz, 1H), 2.35 (dt, J ¼ 2.6, 13.7 Hz, 1H), 2.23 (s, 3H).
ppm) d 165.8, 145.0, 142.3, 138.1, 136.2, 126.4, 124.8, 123.9, 123.3, 122.8,
107.9, 107.5, 82.1, 55.7, 43.1, 29.8. ESIMS: m/z calculated for [M þ Naþ]:
13C NMR (75 MHz, CDCl3, in ppm)
d 165.8, 146.1, 145.3, 144.9, 142.6,
427.059219, found 427.059207.
141.0, 130.7, 123.6, 121.1, 107.9, 107.2, 83.2, 56.1, 42.3, 31.1, 21.2.
ESIMS: m/z calculated for [M
374.148071.
þ
Naþ]: 374.148062, found
4.2.3.26. (4aR,6S)-6-methoxy-4a-phenyl-2-p-tolyl-4,4a,6,7-tetrahy-
dro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (15c). Yield: 44%, m.p.
198 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d
7.62 (d, J ¼ 12.2 Hz, 2H),
4.2.3.32. (4aR,6S)-2-(3,5-Dimethoxyphenyl)-6-methoxy-4a-(pyr-
idin-4-yl)-4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one
(16c). Yield: 47%, m.p. 162 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
7.31e7.09 (m, 7H), 5.22 (t, J ¼ 5.4 Hz, 1H), 4.59 (dd, J ¼ 2.6, 5.3 Hz,
1H), 3.34 (s, 3H), 3.13 (d, J ¼ 14.6 Hz, 1H), 2.81 (d, J ¼ 14.6 Hz, 1H),
2.53 (dt, J ¼ 5.6, 17.2 Hz, 1H), 2.39 (dt, J ¼ 2.8, 17.2 Hz, 1H), 2.31 (s,
d
8.65 (d, J ¼ 5.7 Hz, 2H), 7.29 (d, J ¼ 5.5 Hz, 2H), 6.78 (d, J ¼ 2.4 Hz,
3H). 13C NMR (75 MHz, CDCl3, in ppm)
d
165.9, 145.1, 143.0, 140.6,
2H), 6.35 (bs, 1H), 5.04 (dd, J ¼ 2.7, 5.6 Hz, 1H), 4.65 (bs, 1H), 3.87 (s,
6H), 3.34 (s, 3H), 3.12 (d, J ¼ 14.3 Hz, 1H), 2.88 (d, J ¼ 14.3 Hz, 1H),
2.55 (dt, J ¼ 5.4, 12.2 Hz, 1H), 2.34 (dt, J ¼ 5.5, 12.3 Hz, 1H). 13C NMR
131.2, 126.9, 124.4, 123.5, 122.9, 108.0, 107.5, 82.3, 55.7, 42.4, 29.8,
20.9. ESIMS: m/z calculated for [MþNaþ373.152813 :[, found
373.152827.
(75 MHz, CDCl3, in ppm)
d 165.9, 165.3, 146.1, 145.5, 144.8, 141.1,
120.8, 109.9, 107.8, 107.1, 95.7, 83.2, 55.8, 55.3, 43.1, 29.0. ESIMS: m/z
4.2.3.27. (4aR,6S)-6-methoxy-2-(4-methoxyphenyl)-4a-phenyl-
calculated for [M þ Naþ]: 420.153542, found 420.153550.
4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (15d). Yield:
53%, m.p. 177 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
5.1 Hz, 2H), 7.32e7.07 (m, 5H), 6.92 (dd, J ¼ 2.1, 5.3 Hz, 2H), 5.08 (dd,
d
7.51 (dd, J ¼ 2.2,
4.2.3.33. (4aR,6S)-2-(1H-indol-2-yl)-6-methoxy-4a-(pyridin-4-yl)-
4,4a,6,7-tetrahydro-1H-pyrano[3,2-c]pyridazin-3(2H)-one (16d). Yield:

T.H. Al-Tel / European Journal of Medicinal Chemistry 45 (2010) 5724e5731
5731
41%, m.p. 178 ꢁC. 1H NMR (300 MHz, CDCl3, in ppm)
d 8.61 (d,
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Slambrouck, W.F.A. Steelant, N.M. Evdokimov, P.Y. Uglinskii, E.M. Elias,
E.J. Knee, P. Tongwa, M.Y. Antipin, J. Med. Chem. 51 (2008) 2561e2570.
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Chem. 51 (2008) 1530e1533.
[18] H. Ichikawa, Y. Nakamura, Y. Kashiwada, B.B. Aggarwal, Curr. Pharm. Des. 13
(2007) 3400e3416.
[19] K.H. Altmann, J. Gertsch, Nat. Prod. Rep. 24 (2007) 327e357.
[20] D.J. Newman, J. Med. Chem. 51 (2008) 2589e2599.
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1039/c0ob00289e.
J ¼ 5.6 Hz, 2H), 7.75 (d, J ¼ 5.4 Hz, 1H), 7.41e7.21 (m, 4H), 6.66 (d,
J ¼ 5.7 Hz, 1H), 5.07 (t, J ¼ 4.2 Hz, 1H), 4.61 (t, J ¼ 6.9 Hz, 1H), 3.11 (s,
3H), 3.09 (d, J ¼ 12.1 Hz, 1H), 2.81 (d, J ¼ 12.1 Hz, 1H), 2.55 (bd,
J ¼ 4.2 Hz, 1H), 2.42 (bd, J ¼ 4.2 Hz, 1H). 13C NMR (75 MHz, CDCl3, in
ppm) d 164.9, 148.1, 146.7, 146.0, 140.3, 133.4, 129.2, 125.0, 121.8, 121.6,
120.4, 110.1, 108.0, 107.2, 97.2, 81.4, 55.7, 43.1, 28.7. ESIMS: m/z calcu-
lated for [M þ Naþ]: 399.143311, found 399.143322.
[22] T.H. Al-Tel, R.A. Al-Qawasmeh, S.S. Sabri, W. Voelter, J. Org. Chem. 74 (2009)
4690e4696 (references cited therein).
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Tetrahedron 27 (1971) 1973e1996.
Acknowledgements
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14095e14104.
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The author is grateful to the National Research Fund (UAE) and
the College of graduate studies and research (University of Sharjah)
for funding this research project. Dr. Kenneth Banner (Transtech
Pharma, High Point, NC, USA) and Dr. Jareer Kassis (Transtech
Pharma, High Point, NC, USA) are greatly acknowledged for their
assistance in the in vitro data analysis, fruitful discussions and proof
reading.
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