Synthesis and cytotoxicity of novel hexahydrothieno- cycloheptapyridazinone derivatives

Article abstract of DOI:10.3390/molecules14093494

Designed as a new group of tricyclic molecules containing the thienocycloheptapyridazinone ring system, a number of 2N-substituted- hexahydrothieno- cycloheptapyridazinone derivatives were synthesized and their biological activity evaluated. Among the synthesized compounds, derivatives 7d and 7h were found to possess cytotoxic activity against non-small cell lung cancer and central nervous system cancer cell lines, respectively.

Full text of DOI:10.3390/molecules14093494

Molecules 2009, 14, 3494-3508; doi:10.3390/molecules14093494
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Cytotoxicity of Novel Hexahydrothieno-
cycloheptapyridazinone Derivatives
Amedeo Pau 1,*, Gabriele Murineddu 1, Battistina Asproni 1, Caterina Murruzzu 1, Giuseppe E.
Grella 1, Gérard A. Pinna 1, Maria M. Curzu 1, Irene Marchesi 2 and Luigi Bagella 2
1
Dipartimento Farmaco Chimico Tossicologico,Università di Sassari, Via Muroni 23/A, 07100
Sassari, Italy
2
Department of Biomedical Sciences, Division of Biochemistry and Biophysics, and National
Institute of Biostructures and Biosystems, University of Sassari, Viale San Pietro 43/b 07100
Sassari, Italy
* Author to whom correspondence should be addressed; E-mail: pau@uniss.it; Tel.: +39 079 228736;
Fax: +39 079 228720.
Received: 17 July 2009; in revised form: 26 August 2009 / Accepted: 9 September 2009 /
Published: 9 September 2009
Abstract: Designed as a new group of tricyclic molecules containing the
thienocycloheptapyridazinone ring system, a number of 2N-substituted-hexahydrothieno-
cycloheptapyridazinone derivatives were synthesized and their biological activity
evaluated. Among the synthesized compounds, derivatives 7d and 7h were found to
possess cytotoxic activity against non-small cell lung cancer and central nervous system
cancer cell lines, respectively.
Keywords: pyridazinones; synthesis; cytotoxicity
1. Introduction
Among diseases, cancer is not a single pathological state but a broad group of diseases
characterized by a high proliferative index and the spread of aberrant cells from their site of origin [1].
Clinically, the therapeutic treatment of cancer is a combination of surgery and/or radiotherapy with
chemotherapy [2,3].

Molecules 2009, 14
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Current chemotherapy consists of cytotoxic (cell-killing) agents and anti-hormonal drugs, which
reduce the proliferation of the tumors [2,3]. The therapeutic use of anticancer drugs is complicated by
systemic toxicity, usually observed in the bone narrow, the gastrointestinal (GI) tract and hair, and by
development of resistance. Therefore, the search for novel chemical structures with broader therapeutic
windows and acceptable resistance profiles is being actively pursued.
In discovering anticancer compounds, a notable role is played by polycondensed heterocycles
containing the pyridazinone moiety [4]. A wide spectrum of pharmacological activities has been
reported for these compounds. These include anticancer [5], antihypertensive, anti-thrombotic and
antiulcerative properties [6-9]. Pyridazinone derivatives also possess affinity for benzodiazepine
receptors [10] and the ability to inhibit the human matrix metalloproteinase [11] and aldose reductase
[12,13] enzymes.
A major interest in our group is the design, synthesis and evaluation of new antiproliferative
compounds as candidate cytotoxic and anticancer agents. In recent years, we have reported the
synthesis of novel derivatives of 1,4-dihydroindeno[1,2-b]pyrroles (1) [5], 1H-benzo[g]indoles (2) [5],
thieno[3,2-g]indoles (3) [14], 1,4,5,6-teterahydrobenzo[6,7]cyclohepta[1,2-b]pyrroles (4) [5],
naphto[2,3-d]imidazoles (5) [15], pyrrole[2,3-d]pyridazinones (6) [16] and their cytotoxic activities in
the NCI preclinical antitumor screen (Figure 1).
Figure 1. Chemical structures of some known anticancer agents synthesized by our group.
Q
Q
X
Y
Q
NH
N
H
N
Y
1
2
3
X
N N
O
N N
O
a
, X = CH2, Y = S
Q =
Q =
R
R
b, X = S, Y = CH2
H
a, R = H,
, R = Ph
R = H, Ph
O
N
Q = 2,3
b
a
, X-Y = single bond,
b
, X-Y = double bond
O
O
Q
N
Q
N
X
HN
N
N
H
N
R2
6
5
4
R
a
b
, X = H,
, X = CH3
N N
O
Q =
Q =
c, X = -CH2-
d, X = -(CH2)2-
R1
R
e
f
, X = -CH=CH-
, X = -(CH2)3-
a, R = H,
, R = Ph
R = H, Cl, CF3,
R1 = H, CH3, Cl, CF3, NO2, SPh,
b
R2 = H, CH3, CH2Ph

Molecules 2009, 14
3496
In continuation of our research in this field, we describe herein the synthesis of novel 2,4,4a,5,6,7-
hexahydro-3H-thieno[2’,3’:6,7]cyclohepta[1,2-c]pyridazinones bearing substituted piperazine,
piperidine and morpholine moieties, using a random screening approach [17], and the antitumor
activities of the resulting compounds 7a-l reported in Table 1.
Table 1. Novel thienocyclohepta[1,2-c]pyridazinones 7a-l.
Y
X
N
N
O
N
R
S
7 a
R H
X N
b
H
N
c
H
N
d
H
N
e
CH3
N
f
g
h
i
H
j
k
l
CH3
N
CH3 CH3
N
CH3 H CH3
N
CH2 CH2
O
-
O
-
Y Ph o-OCH3-Ph o-F-Ph CH3 Ph o-OCH3-Ph o-F-Ph CH3 CH3 CH3
2. Chemistry
The retrosynthetic analysis shown in Figure 2 shows how novel pyridazinone derivatives could be
prepared by condensation of a tricyclic ring system with formaldehyde and the appropriate substituted
piperazine synthon or its isosteres such as methylpiperidine and morpholine.
Figure 2. The retrosynthetic analysis of the target compounds 7.
Q
Y
N
H
O
3
O
N
1
N
N
Q
N
Y
9
+ HCHO
+
R
HN
R
S
S
7
Accordingly, the new 2,4,4a,5,6,7-exahydro-3H-thieno[2’,3’:6,7]cyclohepta[1,2-c]pyridazin-3-one
derivatives 7a-l were synthesized in according to Scheme 1. The reaction of thiophenes 8a,b with
glutaric anhydride to give ketoacids 9a,b was followed by Wolff- Kishner reduction to 10a,b, whose
cyclization with P2O5 over Celite® gave the ketones 11a,b. A Mannich reaction furnished 12a,b,
which were converted with NaCN in CH3OH into the nitriles 13a,b. Hydrolysis of these nitriles in
refluxing HCl/AcOH led to the γ-ketoacids 14a,b; condensation of the latter with hydrazine hydrate
afforded the pyridazinones 15a,b.

Molecules 2009, 14
3497
Preparation of the target compounds 7a-l was accomplished by treatment of the pyridazinones
15a,b with formaldehyde and appropriate amines.
Scheme 1. Synthesis of novel pyridazinone derivatives 7a-l.
O
O
HO
HO
c
b
R
a
R
R
S
S
S
8a R = H,
O
10a,b
9a,b
8b
R = CH3
O
O
CN
O
N
e
f
d
R
R
R
S
S
S
.
13a,b
11a,b
12a,b HCl
Y
X
N
H
N
HO
O
N
O
O
O
N
N
h
R
g
R
R
S
S
S
7a-l
X and Y as reported in Table 1
14a,b
15a,b
Reagents and conditions: a) AlCl3, CH2Cl2, glutaric anhydride, RT, 0.5h; b) DEG, KOH,
H2NNH2·H2O, reflux, 3h; c) Toluene, Celite®, P2O5, reflux, 2h; d) HN(CH3)2·HCl, CH2O, Ac2O,
75 °C, 3h; e) MeOH, NaCN, 55 °C, 4h; f) AcOH, HCl 37%, reflux, 3h; g) EtOH an., H2NNH2·H2O,
reflux, 3h; h) EtOH an., CH2O, amine, N2, reflux, 8h.
3. Results and Discussion
A new series of twelve substituted pyridazinones 7a-l were synthesized and eight of them (7c-e,h-l)
were evaluated at a single concentration of 10-5 M (10 μM) for their antitumor activities. The
evaluation established a primary screening where compounds were tested to determine their growth
inhibitory properties against sixty different human tumor cell lines in vitro [18-20]. The compounds
were added at a single concentration and the cell culture was incubated for 48 h. End point
determinations were made using a protein binding dye, sulforhodamine B (SRB), which was used to
estimate cell viability or growth [21]. The results for each compound are reported as percent growth of
treated cells when compared to untreated control cells (Tables 2-3). Range of growth % shows the
lowest and the highest growth % found among different cancer cell lines, where all tested compounds
have demonstrated being scarcely active or completely inactive in the antitumor screening in vitro
(Table 2). 5-Fluorouracil (5-FU) was used as reference compound with the mean growth inhibitory
effect (GI50) of 2.45 × 10-5 M which corresponds in logarithmic scale to 4.61 [22].

Molecules 2009, 14
Table 2. Anticancer screening data of selected pyridazinone derivatives 7c,d,e,h,i,j,k,l.a
3498
Panel/Cell Lines
Non-small cell
lung cancer
Mean growth %
Range of growth
%
Compounds
7c 7d
97.85
7e
7h
7i 7j
7k
7l
98.09
103.17
101.55
101.33
80.20
101.88
96.66
102.97
93.09 to
112.16
81.03 to
113.17
72.06 to 78.91 to 92.87 to
81.70 to 75.17 to
120.26
108.81
109.80
to110.82
119.13
113.32
Colon cancer
Mean growth %
Range of growth
%
107.29
105.03
109.01
104.88
103.17
104.91
101.79
123.28
99.22 to
110.16
100.56 to 96.19 to 97.01 to 93.70 to
93.76 to 95.50 to 93.53 to
117.52
111.60
119.39
123.28
117.4
112.76
114.38
Breast Cancer
Mean growth %
Range of growth
%
103.25
92.60 to
113.98
100.59
107.91
105.20
104.68
88.76
107.49
101.71
108.39
87.28 to
129.10
91.34 to 96.71 to 91.23 to
91.22 to 94.33 to
109.30
121.53
120.16
to122.29
124.23
110.47
Ovarian Cancer
Mean growth %
Range of growth
%
104.72
99.67 to
111.88
101.64
108.09
102.84
109.28
102.64
103.69
104.24
96.32 to
122.92
98.15 to 95.14 to 96.73 to
92.31to 94.48 to 92.28 to
108.92
119.38
120.49
170.96
117.11
117.79
Leukemia
Mean growth %
Range of growth
%
100.46
91.68 to
107.61
92.31
98.55
102.95
94.13
100.01
86.67
98.54
75.05 to
112.55
81.94 to 91.36 to 85.24 to
80.99 to 90.34 to 76.07 to
113.17
101.13
111.53
120.85
112.16
94.26
Renal Cancer
Mean growth %
Range of growth
%
101.77
93.09 to
110.89
103.67
95.02
103.58
94.14
100.19
104.36
101.80
85.76 to
112.31
95.38 to 94.16 to 77.38 to
86.51 to 93.91 to
108.40
102.46
114.16
114.45
to113.75
121.05
114.99
101.57
Melanoma
Mean growth %
Range of growth
%
106.50
100.28
106.62
101.06
92.73 to
112.29
102.95
87.54
107.10
104.80
93.82 to
112.20
88.88 to 101.92
92.35 to 91.52 to
to110.39
111.60
to116.2
4
to119.80
124.26
114.53
Prostate Cancer
Mean growth %
Range of growth
%
102.25
98.48 to
106.02
100.46
114.38
109.08
101.76
99.92
103.85
102.00
109.31
93.16 to 108.08 104.25 to 99.78 to 96.26 to
102.40 to
116.22
107.76
to
113.91
103.76
103.58
to105.70
120.68
CNS Cancer
Mean growth
Range of growth
%
99.49
91.08 to
108.91
100.85
100.78
82.03
111.34
98.38
107.23
97.20
82.07 to
106.60
77.31 to 89.96 to 72.14 to
131.16 110.76 115.77
a Assay at 1-dose 10-5 M (10 µM) concentration.
80.17 to 85.30 to 81.54 to
194.64 105.97 164.17
Nevertheless, compounds 7d and 7h displayed a higher anti-proliferative activity in the non-small
cell lung cancer cell line EKVX and in the CNS cancer cell line SNB-75, which showed growth
inhibitions of 27.94% and 27.86%, respectively (Table 3). Compounds 7k and 7l also showed cell

Molecules 2009, 14
3499
growth inhibitory activity, even if weaker than the one expressed by 7d and 7h. In particular,
compound 7l was found to be active as growth % inhibitor of the leukemia cell line RPMI-8226 with a
value of 24.95%; the derivative 7k was active on leukemia cell line SR with a value of 24.83% and
non-small cell lung cancer cell line EKVX with a value of 23.93%. Moreover, 7e was found to be
active as growth % inhibitor of the non-small cell lung cancer cell line EKVX with a value of 21.09%.
Finally, 7c and 7i showed a growth % inhibitor of non-small cell lung cancer cell line HOP-92 with
values of 18.97, and 19.80%, respectively.
Table 3. In vitro cancer lines growth % inhibition of pyridazinones 7d,e,h,k,l.
Panel/Cell
Non-small cell lung
cancer
Lines
Leukemia Renal
cancer
CNS
cancer
SNB-75*
22.69
Compd
EKVX* HOP-92
SR
RPMI-8226 CAKI-1 UO-31
7d
7e
7h
7k
7l
27.94
21.09
20.09
22.62
22.23
27.86
23.93
24.83
24.95
* The most sensitive cell lines.
4. Conclusions
As part of our continuous search for potential biologically active compounds, a series of
pyridazinone derivatives were synthesized and assessed for their anticancer activity. It was found that
all new eight compounds tested showed weak or incomplete activity without significant differences
between 9-substituted and unsubstituted derivatives. Specifically, two of them showed scant activity,
while others showed no activity in the cell growth inhibition assay against sixty different human
cancer cell lines panel in vitro. From these data, we may conclude compounds 7d and 7h were the
most effective molecules for anti-proliferative activity, specifically in non-small cell lung cancer and
CNS cancer respectively, so they might be useful as leads for designing new compounds with potential
antitumoral activity. This structure was derived from pyridazinone with hydrogen or methyl group in
the 9-position linked to the 4-methylpiperazine moiety by a methylene spacer. The obtained results
prove the necessity for further investigations to clarify the molecular mechanisms involved in
antitumor activities to acquire more information about the structural requirements for enhancing
anticancer activities and minimizing neurotoxicities, the synthesis of more new derivatives with
different substituents at other positions is needed.
5. Experimental
5.1. General
Melting points were determined using a Reichert-Köfler hot-stage apparatus and are uncorrected.
Infrared spectra were recorded with a Perkin-Elmer Paragon 500 FT IR spectrophotometer (KBr
1
pellets, in Nujol mulls, as well as in film). H-NMR spectra were recorded on a Varian XL 200 FT

Molecules 2009, 14
3500
NMR spectrometer using CDCl3 as solvent, unless otherwise specified. Chemical shifts are reported in
δ or ppm and coupling constants (J) in Hertz (Hz), downfield from tetramethylsilane (TMS).
Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), m
(multiplet). Reactions were monitored by analytical thin-layer chromatography (TLC) using SiO2
Polygram SIL and ALOX N/UV254 precoated plastic sheets and with visualization by irradiation with a
UV lamp and/or iodine vapor for detection. Flash chromatography was performed using Merck silica
gel type 60 (230-400 mesh ASTM). Electron ionization mass spectra (70 eV) were recorded on a
Hewlett-Packard 5790-5970 MSD gas chromatograph/mass spectrometer. Atmospheric Pressure
Ionization Electrospray (APIES) mass spectra, when reported, were obtained on a Agilent 1100 series
LC/MSD spectrometer. All moisture sensitive reactions were performed under nitrogen atmosphere,
using oven-dried glassware. Anhydrous DCM, THF and DMF was obtained from Aldrich, Lancaster
or Merck. All starting materials and reagents were commercially available from Aldrich, Lancaster and
Avocado. Evaporation was performed in vacuo (rotary evaporator). Anhydrous sodium or magnesium
sulfate was always used as the drying agent. Elemental analyses were performed in a Perkin-Elmer
240C elemental analyzer, and the results were within ± 0.4% of the theoretical values, unless otherwise
noted.
5.2. General procedure for the synthesis of 5-oxopentanoic acids 9a,b
To a suspension of anhydrous AlCl3 (17.54 mmol) in dry CH2Cl2 (20 mL) cooled with an ice bath, a
solution of glutaric anhydride (19 mmol) in dry CH2Cl2 (20 mL) was added dropwise under a N2
atmosphere, and the whole mixture was stirred at RT for 0.5 h. Then a solution of thiophene 8a,b
(17 mmol) in dry CH2Cl2 (15 mL) was added dropwise and the reaction mixture stirred at the same
temperature for an additional 0.5 h. The mixture was poured into crushed ice and conc. HCl was
slowly added followed by warming until the suspended materials dissolved. The aqueous phase was
separated and extracted with CH2Cl2. The combined organic phase was washed with H2O and then
extracted with 2N NaOH aqueous solution (5 × 7 mL): the solid separated upon acidification of the
alkali layer, was filtered off and air dried to yield the desired product.
5-(Thiophen-2-yl)-5-oxopentanoic acid (9a): Yield 2.12 g (60%) as a cream solid: mp 93-94 °C; Rf:
1
0.48 (CHCl3-MeOH 9:1); IR (Nujol, ν, cm-1): 1696 (COOH), 1654 (CO); H-NMR (CDCl3), δ ppm:
7.74 (d, 1H, J = 4 Hz, CH), 7.64 (d, 1H, J = 5.2 Hz, CH), 7.15 (t, 1H, J = 3.6 Hz, CH), 3.04 (t, 2H,
J = 7 Hz, CH2), 2.53 (t, 2H, J = 7.4 Hz, CH2), 2.09 (quint., 2H, J = 7 Hz, CH2); GC-MS m/z: 198
(M+); Calcd for C9H10O3S: C, 54.53; H, 5.08; S, 16.17. Found: C, 54.42; H, 4.89; S, 16.08.
5-(5-Methyl-2-thienyl)-5-oxopentanoic acid (9b): Yield 2.07 g (60%) as a cream solid:
mp 105-107 °C; Rf: 0.65 (CHCl3-MeOH 9:1); IR (Nujol, ν, cm-1): 1693 (COOH), 1650 (CO); 1H-NMR
(CDCl3), δ ppm: 7.54 (d, 1H, J = 3.6 Hz, CH), 6.80 (d, 1H, J = 3 Hz, CH), 2.95 (t, 2H, J = 7.4 Hz,
CH2), 2.54 (s, 3H, CH3), 2.5 (t, 2H, CH2), 2.09 (qu, 2H, CH2); GC-MS m/z: 212 (M+); Calcd for
C10H12O3S: C, 56.58; H, 5.70; S, 15.11. Found: C, 56.49; H, 5.63; S, 15.21.

Molecules 2009, 14
3501
5.3. General procedure for the synthesis of pentanoic acids 10a,b
A mixture of 5-oxopentanoic acid 9a,b (2.00 g, 9.3 mmol), diethylene glycol (DEG, 24 mL),
potassium hydroxide (0.035 mol) and hydrazine hydrate (0.045 mol) was refluxed with a Dean-Stark
apparatus for 3 h. The solution, after cooling to RT, was poured into cold water (50 mL), washed with
ether, acidified with 6 N HCl and then extracted with ether (4 × 5 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and evaporated in vacuo to give the title compounds.
5-(2-thienyl)pentanoic acid (10a): Yield 1.50 g (82%) as a yellow amber solid: mp 41 °C; Rf: 0.72
1
(CHCl3-MeOH 9:1); IR (Nujol, ν, cm-1): 1702 (COOH); H-NMR (CDCl3), δ ppm: 7.11 (d, 1H,
J = 4 Hz, CH), 6.93 (d, 1H, J = 4 Hz, CH), 6.79 (t, 1H, J = 3.8 Hz, CH), 2.77 (t, 2H, J = 6.6 Hz, CH2),
2.41 (s, 2H, J = 6.8 Hz, CH2), 1.74 (m, 4H, J = 3.6 Hz, 2CH2); GC-MS m/z: 184 (M+); Calcd for
C10H12O3S: C, 58.67; H, 6.56; S, 17.14. Found: C, 58.59; H, 6.49; S, 17.23.
5-(5-Methyl-2-thienyl)pentanoic acid (10b): Yield 1.50 g (82%) as yellow amber solid: mp 47-49 °C;
1
Rf: 0.72 (CHCl3-MeOH 9:1); IR (Nujol, ν, cm-1): 1693 (COOH); H-NMR (CDCl3), δ ppm: 6.54 (s,
2H, 2CH), 2.77 (t, 2H, CH2), 2.43 (s, 3H, CH3), 2.38 (m, 2H, CH2), 1.70 (m, 4H, 2CH2); GC-MS m/z:
198 (M+); Calcd for C10H12O3S: C, 60.57; H, 7.12; S, 16.17. Found: C, 60.49; H, 7.06; S, 16.12.
5.4. General procedure for the synthesis of ketones 11a,b
To a solution of pentanoic acid 10a,b (13 mmol) in toluene (35 mL), were added Celite® (4.52 g)
and phosphorus pentoxide (23 mmol). The mixture was refluxed for 2 h, then cooled and filtered. The
filtrate was washed with 5% aqueous NaHCO3 solution (2 × 5 mL), dried (Na2SO4), filtered and
concentrated in vacuo, to give the ketones as oils.
5,6,7,8-Tetrahydro-4H-cyclohepta[b]thiophen-4-one (11a): Yield 0.78 g (59%) as a yellow brown oil;
1
Rf: 0.87 (CHCl3-MeOH 9:1); IR (film, ν, cm-1): 1665 (CO). H-NMR (CDCl3), δ ppm: 7.33 (d, 1H,
J = 5.4 Hz, CH-2), 6.91 (d, 1H, J = 5.4 Hz, CH-3), 3.03 (t, 2H, J = 5.2 Hz, CH2-8), 2.65 (t, 2H,
J = 6.8 Hz, CH2-5), 2.39 (s, 3H, CH3), 1.92-1.16 (m, 4H, 2CH2); GC-MS m/z: 166 (M+); Calcd for
C9H10OS: C, 65.02; H, 6.06; S, 19.29. Found: C, 65.08; H, 6.12; S, 19.21.
5,6,7,8-Tetrahydro-2-methyl-4H-cyclohepta[b]thiophen-4-one (11b): Yield 0.79 g (59%) of the
compound 7 as yellow brown oil which was used for the next step without further purification. Rf: 0.88
(CHCl3-MeOH 9:1); IR (film, ν, cm-1): 1663 (CO); 1H-NMR (CDCl3), δ ppm: 7.05 (d, 1H, CH), 3.02
(t, 2H, CH2), 2.69 (t, 2H, CH2), 2.39 (s, 3H, CH3), 1.92 (m, 4H, 2CH2); GC-MS m/z: 180 (M+); Calcd
for C10H12OS: C, 66.63; H, 6.71; S, 17.79. Found: C, 66.57; H, 6.63; S, 17.71.
5.5. General procedure for the synthesis of Mannich bases 12a,b
Acetic anhydride (39 mmol) was added dropwise to a solution of dimethylamine hydrochloride
(10 mmol) and 37% formaldehyde (29 mmol) at 85-90 °C and the mixture was stirred for 0.5 h. Then
tetrahydrocyclohepta[b]thiophen-4-one 11a,b (7 mmol) was added to the mixture and the whole stirred

Molecules 2009, 14
3502
at 75 °C for 3h. After cooling, the mixture was evaporated under reduced pressure and the resulting
crude residue was crystallized from acetone (12a) or triturated with diisopropyl ether (12b) to afford
the desired product.
5,6,7,8-Tetrahydro-5-dimethylaminomethyl-4H-cyclohepta[b]thiophen-4-one⋅HCl (12a): Yield 0.76 g
(42%) as a crystalline solid; mp 155 °C; Rf: 0.23 (CHCl3-MeOH 9:1); IR (film, ν, cm-1): 1650 (CO);
1H-NMR (CDCl3), δ ppm: 7.37 (d, 1H, J = 5.4 Hz, CH), 7.04 (d, 1H, J = 5.4 Hz, CH), 4.02-3.59 (m,
2H, CH2-N+H (CH3)2), 3.30-3.00 (m, 3H, CH-5, CH2-8), 2.77 (m, 6H, 2CH3). 2.40-1.52 (m, 4H,
2CH2); GC-MS m/z: 259 (M+); Calcd for C12H18ClN OS; C, 55.48; H, 6.98; Cl, 13.65; S, 12.34. Found:
C, 55.57; H, 6.92; Cl, 13.69; S, 12.39.
2-Methyl-5,6,7,8-tetrahydro-5-dimethylaminomethyl-4H-cyclohepta[b]thiophen-4one⋅HCl (12b): Yield
0.14 g (51%) as a crystalline solid: mp 156 °C; Rf: 0.72 (CHCl3-MeOH 9:1); IR (Nujol), ν, cm-1): 1654
1
(CO); H-NMR (CDCl3), δ ppm: 6.96 (s, 1H, CH), 3.05 (t, 2H, CH2), 2.64 (t, 2H, CH2), 2.32 (s, 3H,
CH3), 2.02 (m, 4H, 2CH2); GC-MS m/z: ND (M+); Calcd for C13H20ClNOS: C, 57.02; H, 7.36; Cl,
12.95; N, 5.12; S, 11.71. Found: C, 57.10; H, 7.39; S, 11.65.
5.6. General procedure for the synthesis of nitriles 13a,b
To a solution of the Mannich base 12a,b (4 mmol) in methanol (8 ml), an aqueous solution of
NaCN (22 mmol, 10 ml) was dropwise added, at RT, and the mixture was stirred at 55 °C for 4 h, then
poured onto cold H2O and afterwards extracted with CH2Cl2 (3 × 5 mL). The resulting organic layer
was washed with H2O, brine, dried (Na2SO4), filtered and evaporated in vacuo.
5,6,7,8-Tetrahydro-5-cianomethyl-4H-cyclohepta[b]thiophen-4-one (13a): Yield 0.62 g (75%) as a
dark oil; Rf: 0.86 (CHCl3-MeOH 9:1); IR (film, ν, cm-1): 1660 (CO), 2246 (CN); 1H-NMR (CDCl3), δ
ppm: 7.43 (d, 1H, J = 5.4 Hz, CH), 7.03 (d, 1H, J = 4.8 Hz, CH), 3.38-2.80 (m, 4H, 2CH2), 2.7-2.5
(m, H, CH-5), 2.24-1.68 (m, 4H, 2CH3); GC-MS m/z: 205 (M+); Calcd for C11H11NOS: C, 64.36; H,
5.44; N, 6.82; S, 15.62. Found: C, 64.32; H, 5.49; N, 6.91; S, 11.69.
2-Methyl-5,6,7,8-Tetrahydro-5-cianomethyl-4H-cyclohepta[b]thiophen-4-one (13b): Yield 0.49 g
(47%) as an amorphous dark solid: mp 77-78 °C; Rf: 0.90 (CHCl3-MeOH 9:1); IR (Nujol, ν, cm-1):
1
1645 (CO), 2237 (CN); H-NMR (CDCl3), δ ppm: 7.01 (s, 1H, CH), 3.07 (t, 2H, CH2), 2.7 (m, 2H,
CH2), 2.40 (s, 3H, CH3), 2.26 (m, 4H, 2CH2); GC-MS m/z: 219 (M+); Calcd for C12H13NOS: C, 65.72;
H, 5.97; N, 6.39; S, 14.62. Found: C, 65.72; H, 5.93; S, 14.67.
5.7. General procedure for the synthesis of acids 14a,b
To a solution of nitrile 13a,b (3.5 mmol) in AcOH (3.6 ml), HCl conc. (2.5 ml) was dropwise added
at RT, then the reaction mixture was refluxed for 3 h (TLC). After cooling to RT, the mixture was
diluted with cold H2O and afterwards extracted with CH2Cl2 (4 × 5 mL). The resulting organic layer
was washed with H2O, brine, dried (Na2SO4), filtered and evaporated in vacuo.

Molecules 2009, 14
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4-Oxo-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-5-acetic acid (14a): Yield 0.67 g (85%) as a
brown dark solid: mp145-147 °C; Rf: 0.53 (CHCl3-MeOH 9:1); IR (Nujol, ν, cm-1): 1660 (CO), 1707
1
(COOH); H-NMR (CDCl3), δ ppm: 8.27 (bs, 1H, COOH, exchanged with D2O), 7.33 (d, 1H,
J = 5.2 Hz, CH), 6.92 (d, 1H, J = 5.4 Hz, CH), 3.36 (m, 5H, 2CH2, CH-5), 2.63-1.57 (m, 4H, 2CH2);
GC-MS m/z: 224 (M+); Calcd for C11H12O3S: C, 58.91; H, 5.38; S, 14.30. Found: C, 58.97; H, 5.43; S,
14.38.
2-Methyl-4-oxo-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-5-acetic acid (14b): Yield 0.88 g (82%)
as a coffee-black solid: mp 145-147 °C. Rf: 0.68 (CHCl3-MeOH 9:1); IR (Nujol, ν, cm-1): 1660 (CO),
1
1708 (COOH); H-NMR (CDCl3), δ ppm: 8.27(bs, 1H, COOH, exchanged with D2O), 7.01 (s, 1H,
CH), 3.07 (t, 2H, CH2), 2.7 (m, 2H, CH2), 2.40 (s, 3H, CH3), 2.26 (m, 4H, 2CH2); GC-MS m/z: 239
(M+); Calcd for C12H14O3S: C, 60.48; H, 5.92; S, 13.46. Found: C, 60.56; H, 5.98; S, 13.52.
5.8. General procedure for the synthesis of pyridazinones 15a,b
To the solution of acid 14a,b (3 mmol) in anhydrous EtOH (10 mL), H2NNH2·H2O 80% (3 mmol)
was added dropwise and the resulting mixture was refluxed for 3 h. After cooling at room temperature,
the solvent was evaporated in vacuo.
2,4,4a,5,6,7-Hexahydro-3H-thieno[2',3':6,7]cyclohepta[1,2-c]pyridazin-3-one (15a): Yield 0.59 g
(87%) as a brown dark solid: mp 147-148 °C; Rf: 0.66 (CHCl3-MeOH 9:1); IR (KBr, ν, cm-1): 1674
1
(CO), 3173 (NH); H-NMR (CDCl3), δ ppm: 8.89 (s, 1H, NH, exchanged with D2O), 7.26 (d, 1H,
J = 4.2 Hz, CH), 7.04 (d, 1H, J = 5.4 Hz, CH), 3.10-2.6 (m, 4H, 2CH2), 2.70-2.20 (m, 3H, CH2, CH),
2.43-2.213(m, 2H, CH2-6), 2.17-1.63 (m, 3H, CH, CH2); GC-MS m/z: 220 (M+); Calcd for
C11H12N2OS: C, 59.97; H, 5.49; N, 12.72; S, 14.56. Found: C, 59.92; H, 5.43; N, 12.67; S, 14.48.
9-Methyl-2,4,4a,5,6,7-hexahydro-3H-thieno[2',3':6,7]cyclohepta[1,2-c]pyridazin-3-one (15b): Yield
0.78 g (85%) as a colorless solid scales: mp 179-182 °C; Rf: 0.68 (CHCl3-MeOH 9:1); IR (KBr,
1
ν, cm-1): 1680 (CO), 3175 (NH); H-NMR (CDCl3), δ ppm: 8.39 (s, 1H, NH, exchanged with D2O),
3.10-2.82 (m, 4H, 2CH2), 2.70-2.20 (m, 3H, CH2, CH), 2.40 (s, 3H, CH3), 1.80-2.05 (m, 2H, CH2);
GC-MS m/z: 234 (M+); Calcd for C12H14N2OS: C, 61.51; H, 6.02; N, 11.96; S, 13.69. Found: C, 61.45;
H, 6.07; N, 11.91; S, 13.63.
5.9. General procedure for the synthesis of 2-N-substituted-2,4,4a,5,6,7-hexahydro-3H-
thieno[2’,3’:6,7]cyclohepta[1,2-c]pyridazin-3-one derivatives 7a-l
To a solution of pyridazinone (0.91 mmol) in anhydrous ethanol (5 mL), 37% formaldehyde (11
mmol) and appropriate amines (2 mmol) were added and the mixture was refluxed under nitrogen
atmosphere for 8 h. The reaction mixture was cooled to RT, then the solvent was evaporated under
reduced pressure and the residue was taken-up in water and extracted with chloroform (4 × 5 mL). The
combined organic phase was dried (Na2SO4), filtered and concentrated in vacuo, to give a crude oil
which was purified by flash chromatography (FC).

Molecules 2009, 14
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2-N-[(4-N-Phenylpiperazin-1-yl)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclohepta[1,2-
c]pyridazin-3-one (7a): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.19 g (52%) as a beige solid with
1
mp 121-123 °C; Rf: 0.35 (petroleum ether/EtOAc 6.5:3.5); IR: (KBr, ν, cm-1): 1661 (CO); H-NMR
(CDCl3), δ ppm: 7.28 (d, 1H, J = 5.8 Hz, CH), 7.05 (d, 1H, J = 5.6 Hz, CH), 7.00-6.80 (m, 5H, Ar-
H),4.84 (dd, 2H, J = 13.2 Hz, 2H, CH2), 3.38-3.12 (m, 4H, ArN(CH2)2,), 3.07-2.63 (m, 6H, , 2H, CH2-
7, 4H, N(CH2)2), 2.78-2.60 (m, 2H, CH2-4), 2.50-2.30 (m, 2H, CH2-6), 2.06-1.70 (m, 3H, CH-4a, CH2-
5); GC-MS m/z: 395 (M+); Calcd for C22H26N4OS: C, 66.97; H, 6.64; N, 14.20; S, 8.13. Found: C,
66.85; H, 6.53; N, 13.99; S, 8.02.
2-N-[(4-(o-Methoxy-phenylpiperazin-1-yl)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclo-
hepta[1,2-c]pyridazin-3-one (7b): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.186 g (48%) as a glassy
solid with mp 50-51 °C; Rf: 0.23 (petroleum ether/EtOAc 6.5:3.5); IR (Nujol, ν, cm-1): 1669 (CO);
1H-NMR (CDCl3), δ ppm: 7.20 (d, 1H, J = 5.4 Hz, CH-9), 6.94 (d, 1H, J = 5.4 Hz, CH-10), 6.90-6.70
(m, 4H, Ar-H), 4.75 (dd, 2H, CH2), 3.74 (s, 3H, OCH3), 3.15-2.75 (m, 10H, 5CH2, Ar-N(CH2)2, CH2,
N(CH2)2; 2,70-2.20 (m, 4H, 2CH2), 2.00-1.60 (m, 3H, CH-4a, CH2-5); GC-MS m/z: 425 (M+); Anal.
Calcd for C23H28N4O2S: C, 65.07; H, 6.65; N, 13.20; S, 7.55. Found: C, 64.86; H, 6.53; N, 13.09; S,
7.43.
2-N-[(4-(o-Fluoro-phenylpiperazin-1-yl)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclo-
hepta[1,2-c]pyridazin-3-one (7c): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.217 g (58%) as a white
1
solid with mp 170 °C; Rf: 0.32 (petroleum ether/EtOAc 6.5:3.5); IR (Nujol, ν, cm-1): 1671 (CO); H-
NMR (CDCl3), δ ppm: 7.28 (d, 1H, J = 5.6 Hz, CH), 7.04 (d, 1H, J=5.6 Hz, CH), 7.13-6.86 (m, 4H,
Ar-H), 4.82 (dd, 2H, J = 13 Hz, CH2), 3.20-2.85 (m, 10H, ArN(CH2)2, CH2, N(CH2)2), 2.76-2.30 (m,
4H, 2CH2), 2.05-1.70 (m, 7H, 3H, CH2, CH); GC-MS m/z: 413 (M+); Calcd for C22H25F N4OS: C,
64.05; H, 6.11; F, 4.61; N, 13.58; S, 7.77. Found: C, 64.17; H, 6.03; N, 13.49; S, 7.65.
2-N-[(4-(o-Methylpiperazin-1-yl)methy]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclohepta[1,2-
c]pyridazin-3-one (7d): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.020 g (5.3%) as a brown solid
1
with mp 118 °C; Rf: 0.32 (petroleum ether/EtOAc 6.5:3.5); IR (Nujol, ν, cm-1): 1673 (CO); H-NMR
(CDCl3), δ ppm: 7.29 (d, 1H, J = 5.4 Hz, CH-9), 7.05 (d, 1H, J = 5.2 Hz, CH-10), 5.26 (m, 2H, CH2-
a), 3.10-2.86 (m, 6H, CH2, N(CH2)4), 2.68-1.72 (m. 14H, N(CH2)2, 3CH2, CH3,CH); GC-MS m/z: 332
(M+); Calcd for C17H24N4OS: C, 61.41; H, 7.28; N, 16.85; S, 9.64. Found: C, 61.39; H, 7.13; N, 16.73;
S, 9.48.
9-Methyl-2-N-[(4-N-phenylpiperazin)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclo-
hepta[1,2-c]pyridazin-3-one (7e): FC: petroleum ether/acetone 6.5:3.5; Yield 0.070 g (20%) as a beige
solid with mp 51-52 °C; Rf: 0.42 (petroleum ether/EtOAc 6.5:3.5); IR (Nujol, ν, cm-1): 1671 (CO); 1H-
NMR (CDCl3), δ ppm: 7.35-6.80 (m, 6H, 5H, Ar, 1H, CH-10), 4.81 (dd, 2H, J = 13,2 Hz, 2H, CH2-a),
3.35-3.08 (t, 4H, ArN(CH2)2,), 3.00-2.80 (m, 6H, 4H, N(CH2)2, 2H, CH2), 2.75-1.67 (m, 10H, 6H,
CH2, 1H, CH3, 1H, CH); GC-MS m/z: 408 (M+); Calcd for C23H28N4OS: C, 67.61; H, 6.91; N, 13.71;
S, 7.85. Found: C, 67.52; H, 6.56; N, 13.58; S, 7.79.

Molecules 2009, 14
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9-Methyl-2-N-[(4-(o-methoxy-phenylpiperazin-1-yl)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno
[2’,3’:6,7]cyclohepta[1,2-c]pyridazin-3-one (7f): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.090 g
(24%) as a glassy solid with mp 51-53 °C; Rf: 0.23 (petroleum ether/EtOAc 6.5:3.5); IR (KBr, ν, cm-1):
1
1671 (CO); H-NMR (CDCl3), δ ppm: 6.97-6.65 (m, 5H, 4H, Ar, 1H, CH-10), 4.73 (dd, 2H, CH2),
3.74 (s, 3H, OCH3), 3.12-2.72 (m, 10H, 4H, ArN(CH2)2, 2H, CH2, 4H, N(CH2)2; 2,38 (s, 3H, CH3),
2.62-1.62 (m, 7H, 6H, CH2, 1H, CH); GC-MS m/z: 439 (M+); Calcd for C24H30N4O2S: C, 65.72; H,
6.89; N, 12.77; S, 7.31. Found: C, 65.79; H, 6.78; N, 12.65; S, 7.43.
9-Methyl-2-N-[(4-(o-fluoro-phenylpiperazin-1-yl)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno
[2’,3’:6,7]cyclohepta[1,2-c]pyridazin-3-one (7g): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.217 g
(47%) as a white solid with mp 138-140 °C; Rf: 0.29 (petroleum ether/EtOAc 6.5:3.5); IR (KBr, ν, cm-
1
1): 1665 (CO); H-NMR (CDCl3), δ ppm: 7.23-6.86 (m, 6H, 4H, Ar, 1H,CH-10), 4.81 (dd, 2H, CH2),
3.18-3.02 (t, 4H, ArN(CH2)2, 2.97-2.02 m, 6H, 4H, N(CH2)2, 2H, CH2), 2.41 (s, 3H, CH3),2.75-2.72
(m, 7H, 6H, CH2, 1H, CH); GC-MS m/z: 426 (M+); Calcd for C23H27F N4OS: C, 64.76; H, 6.38; F,
4.45; N, 13.13; S, 7.52. Found: C, 64.67; H, 6.25; F, 4.38; N, 13.19; S, 7.63.
9-Methyl-2-N-[(4-methylpiperazin-1-yl)methy]-2,4,4a,5,6,7-hexahydro-3H-tieno[2’,3’:6,7] cyclohepta
[1,2-c]pyridazin-3-one (7h): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.012 g (5%) as a brown solid
with mp 136-137 °C; Rf: 0.46 (ether); IR (Nujol, ν, cm-1): 1667 (CO); 1H-NMR (CDCl3), δ ppm: 6.97
(s, 1H, CH), 5.24 (dd, 2H, CH2), 3.15-2.84 (m, 6H, 3CH2), 2.41 (s, 3H, CH3), 2.28 (s, 3H, CH3), 2.73-
2.22 (m, 6H, 3CH2), 2.10-1.72 (m, 5H, 2CH2, CH ); GC-MS m/z: 347 (M+); Anal. Calcd for
C18H26N4OS: C, 62.40; H, 7.56; N, 16.17; S, 9.25. Found: C, 62.35; H, 7.48; N, 16.24; S, 9.36.
2-N-[(4-Methylpiperidin-1-yl)methy]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclohepta[1,2-c]-
pyridazin-3-one (7i): FC: petroleum ether/EtOAc 6.5:3.5; Yield 0.17 g (45%) as a white solid with mp
87 °C; Rf: 0.33 (petroleum ether/EtOAc 6.5:3.5); IR (Nujol, ν, cm-1): 1659 (CO); 1H-NMR (CDCl3), δ
ppm: 7.2 (d, 1H, J = 5.0 Hz, CH-9), 7.3 (d, 1H, J = 5.2 Hz, CH-10), 4.74 (dd, 2H, J = 12.8 Hz, CH2),
3.13-2.25 (m, 8H, CH2-7, N(CH2)2, CH2-4), 2.05-1.18 (m, 10H, 4CH2, 2CH), 0.91 (d, 3H, CH3); GC-
MS m/z: 331 (M+); Calcd for C18H25N3OS: C, 65.22; H, 7.60; N, 12.68; S, 9.67. Found: C, 65.15; H,
7.48; N, 12.57; S, 9.49.
9-Methyl-2-N-[(4-methylpiperidin-1-yl)methy]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclohepta
[1,2-c]pyridazin-3-one (7j): FC: petroleum ether/ethyl acetate 6.5:3.5; Yield 0.190 g (51%) as a white
1
solid with mp 133-135 °C; Rf: 0.42 (petroleum ether/EtOAc 6:4); IR (Nujol, ν, cm-1): 1667 (CO); H-
NMR (CDCl3), δ ppm: 6.92 (s, 1H, CH),4.73 (dd, 2H, J = 15.8 Hz, CH2), 3.12-2.25 (m, 8H, 4CH2),
2.41 (s, 3H, CH3), 2.05-1.18 (m, 10H, 4CH2, 2CH), 0.91 (d, 3H, CH3); GC-MS m/z: 345 (M+); Calcd
for C19H27N3OS: C, 66.05; H, 7.88; N, 12.16; S, 9.28. Found: C, 66.15; H, 7.78; N, 12.08; S, 9.36.
2-N-[(4-Morpholine-1-yl)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7]cyclohepta[1,2-c]
pyridazin-3-one (7k): FC: petroleum ether/ acetone 6.5:3.5; Yield 0.23 g (78%) as a brown solid with
mp 80 °C; Rf: 0.44 (petroleum ether/acetone 7:3); IR (Nujol, ν, cm-1): 1666 (CO); 1H-NMR (CDCl3), δ
ppm: 7.25 (d, 1H, J = 4.8 Hz, CH-9), 7.04 (d, 1H, J = 5Hz, CH-10), 4.73 (dd, 2H, J = 13.2 Hz, CH2),

Molecules 2009, 14
3506
3.7 (t, 4H, J = 4.4 Hz, O(CH2)2), 3.10 (m, 11H, CH2-7, N(CH2)2, CH2-4, CH2-6, CH-4a), 2.00-1.75 (m,
2H, CH2-5); GC-MS m/z: 319 (M+); Calcd for C16H21N3O2S: C, 60.16; H, 6.63; N, 13.16; S, 10.04.
Found: C, 60.02; H, 6.56; N, 13.02; S, 9.89.
9-Methyl-2-[(4-morpholine-1-yl)methyl]-2,4,4a,5,6,7-hexahydro-3H-thieno[2’,3’:6,7] cyclohepta[1,2-
c]pyridazin-3-one (7l): FC: petroleum ether/acetone 6.5:3.5; Yield 0.180 g (46%) as a white solid with
1
mp 108-110 °C; Rf: 0.46 (petroleum ether/acetone 7:3); IR (KBr, ν, cm-1): 1671 (CO); H-NMR
(CDCl3), δ ppm: 6.90 (s, 1H, CH), 4.72 (dd, 2H, J = 14.6 Hz, CH2), 3.69 (t, 4H, J = 4.4 Hz, 2CH2),
2.91 (t, 2H, J = 6.6 Hz, CH2), 2.71 (t, 4H, J = 4.2 Hz, 2CH2), 2.41 (s, 3H, CH3), 2.64- 1.78 (m, 7H,
3CH2 CH); GC-MS m/z: 333 (M+); Calcd for C17H23N3O2S: C, 61.23; H, 6.95; N, 12.60; S, 9.62.
Found: C, 61.15; H, 6.84; N, 12.54; S, 9.51.
Acknowledgements
The authors thank Drs. Maria Giovanna Mura and Antonella Lai, for the support in the preparation
of the compounds described herein. The authors wish to acknowledge Dr. Daniel Zaharevitz and the
National Cancer Institute, Bethesda, MD, USA, for the in vitro evaluation of anticancer activity. This
work was partially supported by a grant from Ministero dell'Università e della Ricerca Scientifica e
Tecnologica (MURST).
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Sample Availability: Samples of the compounds 7g and 7l are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).