Design and synthesis of some thieno[2,3-c]pyridazine derivatives of expected anticancer activity

Article abstract of DOI:10.1007/s00044-012-0258-9

Design and synthesis of some new thienopyridazine derivatives as anticancer agents were the goal of this work. Accordingly, a series of novel compounds were synthesized via reacting thienopyridazine carboxylic acid hydrazide with different organic reagents. Twelve novel compounds were selected by National Cancer Institute for a full anticancer screening assay where seven of the investigated compounds showed non-selective broad spectrum and promising activity almost against all cancer cell lines. One of the most active compounds was chosen to be evaluated against 60-cell line panel at five concentration levels and revealed a remarkable growth inhibition activity.

Full text of DOI:10.1007/s00044-012-0258-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2589–2601
DOI 10.1007/s00044-012-0258-9
ORIGINAL RESEARCH
Design and synthesis of some thieno[2,3-c]pyridazine derivatives
of expected anticancer activity
•
Afaf K. El-Ansary Aliaa M. Kamal
•
Mokhtar AbdHafiz Al-Ghorafi
Received: 22 February 2012 / Accepted: 21 September 2012 / Published online: 4 October 2012
Ó Springer Science+Business Media New York 2012
Abstract Design and synthesis of some new thienopyrid-
azine derivatives as anticancer agents were the goal of this
work. Accordingly, a series of novel compounds were syn-
thesized via reacting thienopyridazine carboxylic acid
hydrazide with different organic reagents. Twelve novel
compounds were selected by National Cancer Institute for a
full anticancer screening assay where seven of the investi-
gated compounds showed non-selective broad spectrum and
promising activity almost against all cancer cell lines. One of
the most active compounds was chosen to be evaluated
against 60-cell line panel at five concentration levels and
revealed a remarkable growth inhibition activity.
al., 1997; Pieretti et al., 2006), antimicrobial (Hassall et al.,
1979; Radwan and Bakhite, 1999; Kandile et al., 2009;
Deeb et al., 2004; Nagawade et al., 2005; Akbas and
Beber, 2005; Wu et al., 2009), antidiabetic (Mylari et al.,
2005; Rathish et al., 2009), positive inotropic activ-
ity(Mertens et al., 1990; Combs et al., 1992), antianxiety
(Lewis et al., 2006) and antitumour activity (Malinka and
Redzicka, 2004; Byth et al., 2004; Brana et al., 2005;
Shenvei et al., 2005). Likewise fused pyridazines, e.g.
imidazopyridazines were designed as potent selective
CDK2 inhibitors and provided useful leads for discovery
and development of CDK inhibitors (Byth et al., 2004).
Pyrazolopyridazines and their analogues were identified in
a high-throughput screening as potent inhibitor of CDK1/
cyclin B and have selectivity for the CDK family (Brana et
al., 2005). Moreover, thieno[2,3-d]pyridazines were pre-
pared and found to have I.Kappa.B kinase (IKKs) inhibi-
tors that are useful as anticancer (Shenvei et al., 2005).
Thieno[2,3-c]pyridazines bearing several pharmaco-
phores that hoping to show anticancer activity were syn-
thesized. Sulphonamide derivatives that have been reported
to show substantial antitumour activity in vitro and/or in
vivo (Owa et al., 1999; Abbate et al., 2004), anhydride
moiety that showed significant effect as cytotoxic agent for
T lymphoblastic leukaemia cell line (Urban et al., 2004)
and colon tumour cancer (McClusky et al., 2000), pyraz-
olyl, oxadiazolyl and thiadiazolyl moieties that proved to
have cytotoxic effect (Khalil et al., 2003; Aboraia et al.,
2006; Loetchutinat et al., 2003; Abadi et al., 2003; Rostom
et al., 2003; Abdel-Hamid et al., 2007) were used as
pharmacophores. Likewise, thiourea and thiosemicarba-
zones show potent antiproliferative [Khalil et al., 2003;
Esteves-Souza et al., 2006; Dilovic et al., 2008) and
ribonucleotide reductase inhibitory actions (French et al.,
1970; Quiroga et al., 1998). Moreover, one of the
Keywords Antitumour agents ꢀ
Thieno[2,3-c]pyridazines ꢀ Cytotoxic activity ꢀ
Pharmacophores ꢀ Improving chemotherapy regimen
Introduction
During the last four decades, intensive research have been
conducted on pyridazine chemistry because many deriva-
tives were found to have a wide variety of biological
activities, e.g. psychomimetic (Wermuth et al., 1989), anti
inflammatory (Tewari and Mishra, 2001; Takaya et al.,
1979), analgesics (Rohet et al., 1997), anticonvulsant
(Moreau et al., 1994), antiasthmatic and bronchodilator
(Yamaguchi et al., 1995), antihypertensive (Pifferi et al.,
1975; Tomil et al., 1998; McEvoy and Allen, 1974; Curran
and Ross, 1974), phosphodiesterase IV inhibitors (Piaz et
A. K. El-Ansary ꢀ A. M. Kamal (&) ꢀ M. A. Al-Ghorafi
Pharmaceutical Organic Chemistry Department,
Faculty of Pharmacy, Cairo University, Cairo 11561, Egypt
e-mail: K_aliaa2@hotmail.com; K_aliaa2511@hotmail.com
123

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Med Chem Res (2013) 22:2589–2601
promising class of compounds was the Schiff‘s bases as
they are hydrolyzed readily in vitro under mildly acidic
conditions, such compounds probably could be hydrolyzed
selectively by the tumour cells to liberate the active alde-
hydes that serve as alkylating agents while the active amine
is liberated to act as an antimetabolite (Popp, 1961; Popp
and Kirsch, 1961; Modi et al., 1970; Billman and
Schmidgall, 1970). All of the aforementioned stimulated
the interest in synthesizing compounds containing these
moieties hoping that these compounds show acceptable
value as anticancer agents.
appearance of a strong absorption bands at 1784,
1670 cm-1 (2C=O) while the 1H NMR revealed the
absence of singlet signal at d 8.24 ppm corresponding to
the azomethine (CH=N) of its precursor as well as the
disappearance of the signals at d 12.83 and d 13.07 ppm
corresponding to NH and OH, also the presence of two
singlet signals at d 1.42 and d 1.91 ppm corresponding to
the 2COCH3 groups. Synthesis of compound 6 was
achieved via reacting compound 2 with phthalic anhydride
in glacial acetic acid. Reacting ethyl acetoacetate with acid
hydrazide 2 in absolute ethanol in the presence of anhy-
drous sodium acetate afforded compound 7 in a moderate
yield.
Results and discussion
During the course of this study, oxadiazolylthienopyr-
idazinethiol 8 was prepared via reacting compound 2 with
carbon disulphide in basic medium. Alkylation of com-
pound 8 with the appropriate alkyl halide in alcoholic
potassium hydroxide yielded exclusively both N– and
O–alkylated oxadiazolylthienopyridazine 10a, b and this
was confirmed using IR spectra that showed disappearance
of (OH) band at 3,365–3,200 cm-1 and 1H NMR spectrum
of 10a, b indicated the presence of two singlet signals at d
2.79 (S–CH3), d 3.19 (O–CH3) and disappearance of OH
signal. It is worth to mention that, upon using anhydrous
sodium acetate instead of potassium hydroxide the mono-
alkylated as well as the dialkylated compounds 9a, 9b and
10a, 10b were obtained together in fair yield and they are
separated by fractional crystallization.
Chemistry
The synthesis of the target compounds is outlined in
Schemes 1, 2 and 3. A controlled hydrazinolysis of ben-
zil(1,2-diphenylethane-1,2-dione) gave benzil monohyd-
razone which upon reacting with diethyl malonate in
strongly basic medium followed by chlorination of the
resulted product using excess phosphorus oxychloride
afforded ethyl 3-chloro-5,6-diphenyl-4-pyridazine-carbox-
ylate 1 (Schmidt and Druey, 1954). In a reported multistep
pathway, preparation of compound 2 was carried out by
interacting compound 1 with thiourea in ethanol (Kamal
El-Dean et al., 1996) then the resulted ethyl 2,3-dihydro-
5,6-diphenyl-3-thioxo-2,3-dihydro-4-pyridazine-carboxylate
was reacted with ethyl chloroacetate to afford the corre-
sponding thieno[2,3-c]pyridazine-carboxylate (Radwan
and Bakhite, 1999) which upon hydrazinolysis turned into
compound 2 (Bakhite et al., 2002). Condensation of certain
sulphonamide derivatives with compound 1 in 1-butanol
gave compounds 3a, 3b in good yield. Heating the thie-
no[2,3-c]pyridazine carboxylic acid hydrazide 2 with aro-
matic aldehydes in boiling absolute ethanol yielded the
arylidene derivatives 4a, b. According to the literature, the
hydrazones may exist as Z/E geometrical isomers about
C=N and cis/trans amide conformers; besides hydrazones
derived from aldehydes and substituted hydrazides are
present in solution in the E form. It has been reported that
when hydrazones are dissolved in dimethyl sulfoxide
(DMSO)-d6 solution, the E geometrical isomers of these
compounds undergo a rapid cis/trans amide equilibrium, in
which the cis conformer predominates (Gerard et al., 1986;
Elzbieta and Prukaia, 1998; Salgin-Goksen et al., 2007).
Boiling 4a, b with excess acetic anhydride for 1 h affor-
ded compounds 5a, 5b. Regarding this short reaction time,
the N-acetyl hydroxy oxadiazolinyl thienopyridazine was
expected to be the product, but examining the spectral
data showed exclusive formation of the N– and O– diacetyl
oxadiazolinyl thienopyridazine 5a, b; IR spectra showed
The reaction of acid hydrazide 2 with either methyl
isothiocyanate or a mixture of aromatic carboxylic acids
and phosphorus oxychloride took place to give compounds
11, 15a and 15b, respectively, in a good yield. Reacting
thiosemicarbazide 11 with monochloroacetic acid in glacial
acetic acid or heating it with either conc. H2SO4 or 2N
NaOH afforded the goal compounds 12, 13 and 14
sequentially. Structure elucidation of the newly synthesized
compounds was confirmed using elemental analyses and
spectral data.
Preliminary in vitro anticancer screening
Out of 17 novel compounds, 12 compounds (3a, 3b, 5a, 5b,
7, 8, 9a, 9b, 13, 14, 15a and 15b) were selected by National
Cancer Institute (NCI) to be evaluated. The screening is a
two-stage process, beginning with the evaluation of all
compounds against the 60-cell lines at a single dose of
10 lM. The operation of this screening utilizes 60 different
human tumour cell lines, representing leukaemia, mela-
noma and cancers of the lung, colon, brain, ovary, breast,
prostate and kidney. The one-dose data was reported as a
mean graph of the percent growth of treated cells. Com-
pounds which exhibit significant growth inhibition are
evaluated against the 60-cell panel at five concentration
123

Med Chem Res (2013) 22:2589–2601
2591
Ph
N
Ph
N
Ph
Ph
N
OH
OH
OCOCH
Ph
Ar
3
Ph
Ph
Ph
COOEt
1- NH2CSNH2
N
NH
NCOCH
Ar
Ac
O
ArCHO
3
2
CONHNH
N
2
N
N
N
2- ClCH COOC
2
H
5
N
S
O
2
S
Cl
S
O
N
(1)
3- NH2NH2
(4a&b)
(5a&b)
(2)
Ar= 4-FC
Ar= 2-NO -C
H
4a)
4b)
6
5
Ar= 4-F-C
H
5
5a)
6
H
5
RNH
2
6
2
Ar= 2-NO -C
2
H
5
5b)
6
R=C H SO NH
2
3a)
3b)
6
4
2
Ph
R=
NHSO C H
6 4
2
Ph
COOEt
N
S
N
NHR
N
(3a&b)
O
Ph
OH
O
O
O
Ph
OH
Ph
N
O
N
Ph
CH CH COOC H
2 5
NH
3
2
O
O
N
(2)
S
S
N
N
N
N
O
(7)
CH
(6)
3
Scheme 1 Synthesis of compounds 3a&b, 4a&b, 5a&b,6 and 7
Ph
Ph
Ph
N
OR
OH
OH
Ph
Ph
Ph
N
O
N
N
N
N
CS / KOH
2
N
RX
+
2
N
N
N
N
S
SR
O
S
S
SH
N
R = a) CH3
O
SR
(9a&b)
9a) R = CH
R = b) CH Ph
2
(10a&b)
(8)
R = CH
10a)
3
3
R = CH Ph
2
10b)
R = CH Ph
2
9b)
Scheme 2 Synthesis of compounds 8, 9a&b and 10a&b
Ph
OH
N
N
Ph
N
N
N
S
SH
(14)
H C
3
2N NaOH
OH
Ph
OH
Ph
N
Ph
N
S
Ph
N
conc.H SO
2
4
CH NCS
3
CONHNHCSNHCH 3
N
2
S
N
N
NHCH
3
S
(13)
(11)
ClCH COOH
2
ArCOOH / POCl
3
Ph
N
OH
OH
O
Ph
Ph
N
N
Ph
S
N
N
NH
S
O
Ar
N
N
S
N
O
H
C
(15a&b)
Ar = Ph
3
(12)
15a)
Ar = 4-Cl-C
H
4
15b)
6
Scheme 3 Synthesis of compounds 12, 13, 14, and 15a&b
levels (Monk et al., 1991). The test compounds revealed
growth inhibition values approximately [40 %, broad
spectrum antitumour activity as well as distinctive pattern
of selectivity (Table 1). Compound 5b showed very good
activity against leukaemia cell lines; compound 9a
revealed good potency against most cancer cell lines while
compound 9b acted only against leukaemia cell lines.
Moreover, compound 7 showed remarkable activity against
123

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Med Chem Res (2013) 22:2589–2601
Table 1 The mean growth percent, delta value and the growth percent of the synthesized compounds
Cpd. Mean growth Delta Panel
Sub-panel cell lines (growth %)*
no.
percent
3a
100.57
29.40 Leukaemia
Non-small cell
CCRF-CEM (81.76), HL-60 (TB) (71.17), MOLT-4 (78.23), RPMI-8226 (80.92), SR (76.14)
A549/ATCC (90.89), EKVX (104.79), HOP-62 (108.15), HOP-92 (86.46), NCI-H226 (102.41), NCI-H23
(115.31), NCI-H460 (108.22), NCI-H522 (111.33)
lung cancer
Colon cancer
COLO 205 (113.16), HCC-2998 (96.78), HCT-116 (93.28), HCT-15 (93.37), HT29 (117.80), KM12 (104.15),
SW-620 (104.53)
CNS cancer
Melanoma
SF-268 (104.65), SF-295 (88.53), SF-539 (107.83), SNB-19 (107.85), SNB-75 (84.61), U251 (95.03)
LOX IMVI (99.43), M14 (104.44), MDA-MB-435 (107.35), SK-MEL-2 (122.90), SK-MEL-28 (107.03),
SK-MEL-5 (94.59), UACC-257 (101.44), UACC-62 (90.20).
Ovarian Cancer
Renal cancer
OVCAR-3 (118.14), OVCAR-5 (116.04), OVCAR-8 (99.79), NCI/ADR-RES (96.78), SK-OV-3 (107.94)
ACHN (97.61), CAKI-1 (83.81), RXF 393 (108.65), SN12C (103.81, TK-10 (108.36)
DU-145 (119.58)
Prostate cancer
Breast cancer
MDA-MB-231/ATCC (107.99), HS 578T (116.13), BT-549 (94.96), T-47D (91.33), MDA-MB-468 (97.89)
CCRF-CEM (91.26), HL-60 (TB) (104.16), MOLT-4 (79.33), RPMI-8226 (96.51), SR (76.19)
3b
106.03
29.84 Leukaemia
Non-small cell
lung cancer
A549/ATCC (110.47), EKVX (101.97), HOP-62 (116.93), HOP-92 (84.26), NCI-H226 (102.49), NCI-H460
(110.74), NCI-H522 (102.52)
Colon cancer
CNS cancer
Melanoma
COLO 205 (114.19), HCT-116 (98.58), HCT-15 (106.45), HT29 (116.43), KM12 (113.98), SW-620 (131.32)
SF-268 (111.63), SF-295 (101.35), SF-539 (116.80), SNB-19 (108.01), SNB-75 (104.30), U251 (98.51)
M14 (104.03), MDA-MB-435 (119.20), SK-MEL-2 (118.01), SK-MEL-28(113.16), SK-MEL-5 (97.63),
UACC-257 (104.13), UACC-62 (92.90)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (119.99), OVCAR-5 (116.92), OVCAR-8 (102.01), SK-OV-3 (105.59)
786-0 (109.27), ACHN (102.02), CAKI-1 (106.73), RXF 393 (120.22), SN12C (94.78), TK-10 (109.21)
DU-145 (114.49)
MDA-MB-231/ATCC (114.64), HS 578T (117.67), BT-549 (98.88) T-47D (104.38), MDA-MB-468 (110.22)
CCRF-CEM (39.13), HL-60 (TB) (83.79), MOLT-4 (55.07), RPMI-8226 (71.26), SR (79.30)
5a
88.10
48.97 Leukaemia
Non-small cell
lung cancer
EKVX (112.62), HOP-62 (120.56), HOP-92 (46.17), NCI-H226 (92.39), NCI-H23 (102.71), NCI-H322M
(85.72), NCI-H460 (101.37), NCI-H522 (80.41)
Colon cancer
COLO 205 (116.80), HCC-2998 (82.78), HCT-116 (80.50), HCT-15 (61.15) HT29 (101.51), KM12 (81.73),
SW-620 (97.41)
CNS cancer
Melanoma
SF-268 (89.62), SF-539 (104.41), SNB-19 (82.64), SNB-75 (78.06), U251 (104.67)
LOX IMVI (98.96), M14 (101.54), MDA-MB-435(79.63), SK-MEL-2 (99.88), SK-MEL-28 (82.09), SK-
MEL-5 (88.23), UACC-257 (113.18), UACC-62 (57.55)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (96.33), OVCAR-5 (95.35), OVCAR-8 (100.86), NCI/ADR-RES (85.35), SK-OV-3 (97.61)
786-0 (94.72), ACHN (71.97), RXF 393 (87.97), SN12C (80.24), TK-10 (94.06), UO-31 (83.34)
DU-145 (114.07)
MCF7 (73.65), MDA-MB-231/ATCC (85.91), HS 578T (119.61) BT-549 (65.26), T-47D (89.97), MDA-MB-
468 (84.04)
5b
89.12
69.51 Leukaemia
CCRF-CEM (19.61), HL-60 (TB) (58.85), MOLT-4 (47.20), RPMI-8226 (57.43), SR (49.02)
Non-small cell
lung cancer
EKVX (103.17), HOP-62 (108.98), HOP-92 (72.35), NCI-H226 (92.99), NCI-H23 (100.52), NCI-H460
(100.34), NCI-H522 (92.03)
Colon cancer
COLO 205 (108.41), HCC-2998 (119.54), HCT-116 (76.77), HCT-15 (78.03), HT29 (87.54), KM12 (108.16),
SW-620 (82.83)
CNS cancer
Melanoma
SF-268 (85.56), SF-295(85.05), SF-539 (107.22), SNB-19 (88.60), SNB-75 (76.33), U251 (80.55)
LOX IMVI (105.36). MALME-3M (93.18), M14 (91.79), MDA-MB-435 (97.87), SK-MEL-2 (101.08),
SK-MEL-28 (97.12), SK-MEL-5 (98.48), UACC-257 (100.74), UACC-62 (79.98)
Ovarian cancer
Renal cancer
OVCAR-3 (93.38), OVCAR-5 (95.13), OVCAR-8 (90.12), NCI/ADR-RES (98.39), SK-OV-3 (105.80)
786-0 (101.77), ACHN (98.01), CAKI-1 (86.82), RXF 393 (93.45), SN12C (76.26), TK-10 (106.67), UO-31
(78.88)
Prostate cancer
Breast cancer
DU-145 (104.34)
MCF7 (64.05), MDA-MB-231/ATCC (89.64), HS 578T (114.02), BT-549 (89.78), T-47D (97.53), MDA-
MB-468 (86.49)
123

Med Chem Res (2013) 22:2589–2601
2593
Table 1 continued
Cpd. Mean growth Delta Panel
Sub-panel cell lines (growth %)*
no.
percent
7
81.02
64.98 Leukaemia
Non-small cell
CCRF-CEM (49.49), HL-60 (TB) (69.40), MOLT-4 (67.75), RPMI-8226 (59.34), SR (68.57)
A549/ATCC (84.52), EKVX (74.35), HOP-62 (82.76), HOP-92 (69.54), NCI-H226 (91.86), NCI-H23 (82.61),
NCI-H460 (52.21), NCI-H522 (87.44)
lung cancer
Colon cancer
COLO 205 (111.27), HCC-2998 (59.67), HCT-116 (89.78), HCT-15 (49.03), HT29 (118.61), KM12 (71.30),
SW-620 (79.43)
CNS cancer
Melanoma
SF-268 (88.28), SF-295 (48.77), SF-539 (109.29), SNB-19 (60.71), SNB-75 (90.01), U251 (71.69)
LOX IMVI (97.61), M14 (87.40), MDA-MB-435 (83.66), SK-MEL-2 (109.63), SK-MEL-28 (83.45), SK-
MEL-5 (80.19), UACC-257 (86.80), UACC-62 (77.39)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (84.74), OVCAR-5 (116.72), OVCAR-8 (86.95), NCI/ADR-RES (16.04), SK-OV-3 (90.63)
786-0 (65.92), ACHN (73.19), CAKI-1 (31.00), RXF 393 (95.62), SN12C (93.85), TK-10 (103.38)
DU-145 (97.81)
MCF7 (85.12), MDA-MB-231/ATCC (121.74), HS 578T (89.56), BT-549 (104.40), T-47D (82.16), MDA-
MB-468 (80.49)
8
101.86
29.58 Leukaemia
CCRF-CEM (96.08), HL-60 (TB) (101.19), MOLT-4 (91.02), RPMI-8226 (102.43), SR (83.38)
Non-small cell
lung cancer
A549/ATCC (106.79), EKVX (101.64), HOP-62 (99.71), HOP-92 (72.28), NCI-H226 (91.52), NCI-H460
(116.24), NCI-H522 (101.95)
Colon cancer
CNS cancer
Melanoma
COLO 205 (106.14), HCT-116 (103.46), HCT-15 (103.25), HT29 (113.71), KM12 (118.62), SW-620 (123.96)
SF-268 (102.12), SF-295 (111.85), SF-539 (106.47), SNB-19 (103.01), SNB-75 (72.62), U251 (106.06)
M14 (99.51), MDA-MB-435 (93.59), SK-MEL-2 (106.13), SK-MEL-28 (102.19), SK-MEL-5 (94.73), UACC-
257 (97.02), UACC-62 (94.34)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (114.84), OVCAR-5 (101.82), OVCAR-8 (106.44), SK-OV-3 (96.37)
786-0 (108.48), ACHN (92.38), CAKI-1 (93.26), RXF 393 (108.60), SN12C (94.04), TK-10 (103.36)
DU-145 (112.35)
MCF7 (91.97), MDA-MB-231/ATCC (109.03), HS 578T (121.05), BT-549 (99.45), T-47D (104.90), MDA-
MB-468 (107.85)
9a
79.34
38.49 Leukaemia
CCRF-CEM (47.40), HL-60 (TB) (106.92), MOLT-4 (61.85), RPMI-8226 (67.23), SR (62.54)
Non-small cell
lung cancer
A549/ATCC (80.50), EKVX (77.45), HOP-62 (71.09), NCI-H226 (87.66), NCI-H460 (56.66), NCI-H522
(96.63)
Colon cancer
CNS cancer
Melanoma
COLO 205 (110.09), HCT-116 (60.87), HCT-15 (54.62), HT29 (106.98), KM12 (72.87), SW-620 (92.54)
SF-268 (80.58), SF-295 (40.85), SF-539 (84.57), SNB-19 (66.39), SNB-75 (78.42), U251 (62.91)
M14 (73.00), MDA-MB-435 (88.39), SK-MEL-2 (102.24), SK-MEL-28 (85.53), SK-MEL-5 (83.46), UACC-
257 (91.64), UACC-62 (58.86)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (86.83), OVCAR-5 (100.68), OVCAR-8 (85.77), SK-OV-3 (85.51)
786-0 (88.65), ACHN (83.64), CAKI-1 (43.46), RXF 393 (82.59), SN12C (75.67), TK-10 (100.32)
DU-145 (84.70)
MCF7 (83.55), MDA-MB-231/ATCC (90.40), HS 578T (98.44), BT-549 (74.10), T-47D (74.97), MDA-MB-
468 (78.93)
9b
100.09
47.62 Leukaemia
CCRF-CEM (66.76), HL-60 (TB) (69.81), MOLT-4 (85.98), RPMI-8226 (92.86), SR (106.06)
Non-small cell
lung cancer
EKVX (122.08), HOP-62 (104.80), HOP-92 (52.47), NCI-H226 (101.27), NCI-H23 (106.21), NCI-H322M
(93.59), NCI-H460 (110.68), NCI-H522 (88.49)
Colon cancer
COLO 205 (121.93), HCC-2998 (96.54), HCT-116 (83.35), HCT-15 (93.63), HT29 (89.80), KM12 (106.95),
SW-620 (109.04)
CNS cancer
Melanoma
SF-268 (102.87), SF-539 (121.97), SNB-19 (103.18), SNB-75 (87.82), U251 (122.20)
LOX IMVI (83.96), M14 (108.50), MDA-MB-435 (99.43), SK-MEL-2 (93.08), SK-MEL-28 (98.86), SK-
MEL-5 (99.71), UACC-257 (134.15), UACC-62 (78.64)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (126.46), OVCAR-5 (98.80), OVCAR-8 (103.87), NCI/ADR-RES (93.49), SK-OV-3 (100.41)
786-0 (105.20), ACHN (103.91), RXF 393 (114.20), SN12C (90.04), TK-10 (99.47), UO-31 (92.94)
DU-145 (111.58)
MCF7 (82.19), MDA-MB-231/ATCC (97.90), HS 578T (135.35), BT-549 (106.94), T-47D (104.20), MDA-
MB-468 (101.16)
123

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Med Chem Res (2013) 22:2589–2601
Table 1 continued
Cpd. Mean growth Delta Panel
Sub-panel cell lines (growth %)*
no.
percent
13
104.82
32.80 Leukaemia
Non-small cell
CCRF-CEM (109.75), HL-60 (TB) (106.84), MOLT-4 (93.30), RPMI-8226 (112.86), SR (78.25)
EKVX (112.00), HOP-62 (114.59), HOP-92 (116.37), NCI-H226 (106.31), NCI-H23 (115.18), NCI-H322M
(106.45), NCI-H460 (105.54), NCI-H522 (110.52)
lung cancer
Colon cancer
COLO 205 (131.27), HCC-2998 (98.36), HCT-116 (102.46), HCT-15 (98.86), HT29 (101.73), KM12 (125.99),
SW-620 (105.10)
CNS cancer
Melanoma
SF-268 (105.83), SF-295 (91.70), SF-539 (108.67), SNB-19 (105.21), SNB-75 (87.90)
LOX IMVI (103.37), MALME-3M (110.67), M14 (107.34), MDA-MB-435 (103.42), SK-MEL-2 (126.39),
SK-MEL-28 (111.12), SK-MEL-5 (104.34), UACC-257 (94.51), UACC-62 (97.50)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (116.37), OVCAR-5 (96.45), OVCAR-8 (104.14), NCI/ADR-RES (105.01), SK-OV-3 (97.70)
786-0 (108.26), ACHN (107.37), CAKI-1 (72.02), RXF 393 (103.04), SN12C (94.21), TK-10 (108.25)
DU-145 (135.07)
MCF7 (88.70), MDA-MB-231/ATCC (104.21), HS 578T (108.53), BT-549 (101.34), T-47D (97.60), MDA-
MB-468 (92.62)
14
15a
15b
a
106.58
51.52
79.27
20.91 Leukaemia
CCRF-CEM (105.31), HL-60 (TB) (100.69), MOLT-4 (101.26), RPMI-8226 (103.03), SR (107.94)
Non-small cell
lung cancer
A549/ATCC (95.93), EKVX (110.54), HOP-62 (102.20), HOP-92 (101.20), NCI-H226 (98.84), NCI-H23
(106.77), NCI-H460 (105.74), NCI-H522 (102.37)
Colon cancer
COLO 205 (115.02), HCC-2998 (97.65), HCT-116 (107.29), HCT-15 (108.76), HT29 (108.00), KM12
(112.09), SW-620 (103.31)
CNS cancer
Melanoma
SF-268 (104.66), SF-295 (88.89), SF-539 (108.81), SNB-19 (123.26),SNB-75 (85.68), U251 (108.41)
LOX IMVI (104.22), M14 (114.09), MDA-MB-435 (118.87), SK-MEL-2 (106.33), SK-MEL-28 (112.60),
SK-MEL-5 (108.06), UACC-257 (95.77), UACC-62 (128.45)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (108.96), OVCAR-5 (106.61), OVCAR-8 (99.73), NCI/ADR-RES (104.19), SK-OV-3 (119.09)
786-0 (117.73), ACHN (115.12), CAKI-1 (85.67), RXF 393(114.55), TK-10 (111.81)
DU-145 (117.49)
MCF7 (88.50), MDA-MB-231/ATCC (122.22), HS 578T (111.17), BT-549 (98.66), T-47D (106.78), MDA-
MB-468 (105.36)
35.99 Leukaemia
CCRF-CEM (47.72), HL-60 (TB) (83.15), MOLT-4 (47.93), RPMI-8226 (64.77), SR (46.84)
Non-small cell
lung cancer
EKVX (94.12), HOP-62 (73.69), HOP-92 (81.71), NCI-H226 (85.04), NCI-H23 (90.42), NCI-H322M (42.51),
NCI-H460 (39.83), NCI-H522 (97.24)
Colon cancer
COLO 205 (102.17), HCC-2998 (70.39), HCT-116 (47.39), HCT-15 (36.64), HT29 (103.44), KM12 (72.29),
SW-620 (64.41)
CNS cancer
Melanoma
SF-268 (85.51), SF-295 (42.89), SF-539 (68.47), SNB-19 (57.21), SNB-75 (84.39)
LOX IMVI (65.66), MALME-3M (83.48), M14 (69.63), MDA-MB-435 (78.42), SK-MEL-2 (106.28), SK-
MEL-28 (85.15), SK-MEL-5 (71.16), UACC-257 (99.78), UACC-62 (51.06)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (72.38), OVCAR-5 (106.37), OVCAR-8 (66.81), NCI/ADR-RES (18.75), SK-OV-3 (83.00)
786-0 (79.92), ACHN (67.04), CAKI-1 (29.12), RXF 393 (85.62), SN12C (61.41), TK-10 (90.60)
DU-145 (106.01)
MCF7 (58.02), MDA-MB-231/ATCC (89.19), HS 578T (97.05), BT-549 (74.89), T-47D (81.07), MDA-
MB-468 (64.74)
43.85 Leukaemia
CCRF-CEM (27.56), HL-60 (TB) (52.52), MOLT-4 (35.81), RPMI-8226 (40.41), SR (35.11)
Non-small cell
lung cancer
A549/ATCC (51.18), EKVX (58.45), HOP-62 (51.25), HOP-92 (54.54). NCI-H226 (67.24), NCI-H23 (56.55),
NCI-H460 (27.44), NCI-H522 (75.12)
Colon cancer
COLO 205 (67.44), HCC-2998 (54.66), HCT-116 (19.31), HCT-15 (32.00), HT29 (91.24), KM12 (39.18),
SW-620 (35.61)
CNS cancer
Melanoma
SF-268 (55.21), SF-295 (23.53), SF-539 (55.04), SNB-19 (43.91), SNB-75 (62.75), U251 (32.64)
LOX IMVI (28.10), M14 (55.28), MDA-MB-435(58.58), SK-MEL-2 (87.07), SK-MEL-28 (71.76), SK-MEL-
5 (53.66), UACC-257 (85.02), UACC-62 (37.98)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
OVCAR-3 (25.98), OVCAR-5 (84.85), OVCAR-8 (46.09), NCI/ADR-RES (15.53), SK-OV-3 (61.89)
786-0 (61.10), ACHN (53.96), CAKI-1 (24.50), RXF 393 (72.35), TK-10 (59.00)
DU-145 (71.93)
MCF7 (33.46), MDA-MB-231/ATCC (77.91), HS 578T (62.42), BT-549 (56.05), T-47D (52.95), MDA-MB-
468 (44.61)
Growth inhibition (GI %) = 100 % - growth %
123

Med Chem Res (2013) 22:2589–2601
2595
Table 2 GI50, TGI, LC50 of compound 15b
Table 2 continued
Panel/cell line
GI50
TGI
LC50
Panel/cell line
GI50
TGI
LC50
Leukaemia
CCRF-CEM
HL-60 (TB)
K-562
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
0.00258
0.018316
0.018316
0.018316
0.018316
0.00288
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.005042
0.004608
0.004169
0.004472
0.003966
0.004517
0.005858
0.005917
0.004254
0.001836
0.003661
0.004654
0.005144
0.003625
0.018316
0.012525
0.017422
0.009952
0.018316
0.017597
0.016907
0.00622
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
MOLT
A498
RPMI
ACHN
SR
CAKI-1
Non-small cell lung cancer
A549/ATCC
EKVX
RXF 393
SN12C
0.004654
0.004046
0.004992
0.003215
0.003058
0.004942
0.004169
0.003151
0.004339
0.014846
0.011221
0.018316
0.006738
0.018316
0.018316
0.010567
0.008067
0.009755
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
TK-10
HOP-62
UO-31
HOP-92
Prostate cancer
PC-3
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
0.004087
0.00622
0.018316
0.018316
0.018316
0.018316
DU-145
Breast cancer
MCF7
0.004427
0.004562
0.004427
0.001008
0.004893
0.005042
0.018316
0.012277
0.018316
0.018316
0.018316
0.010462
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
MDA-MB-231/ATCC
HS 578T
BT-549
0.004701
0.004992
0.003966
0.002265
0.004562
0.004701
0.004562
0.018316
0.018316
0.007155
0.018316
0.009095
0.014996
0.006605
0.018316
0.018316
0.014552
0.018316
0.018316
0.018316
0.018316
T-47D
MDA-MB-468
HT29
colon cancer HCC-2998, HCT-15 cell lines, CNS cancer
SF-295 cell lines and renal cancer CAKI-1 cell lines but
selectively acted against ovarian cancer NCI/ADR-RES
cell line in an excellent potency. It has to be mentioned that
compounds 15a and 15b are the most potent ones of the
target compounds as they showed activity against almost
all cancer cell lines (Table 1). Compound 5-hydroxy-3,4-
KM12
SW-620
CNS cancer
SF-268
0.005517
0.00328
0.018316
0.008148
0.015764
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
SF-295
SF-539
0.004748
0.004701
0.005742
0.003553
SNB-19
diphenyl-6-(5-(4-chloro
phenyl)-[1,3,4]oxadiazol-2-yl)-
SNB-75
thieno[2,3-c]pyridazine (15b) satisfied a pre-determined
threshold inhibition criteria in a minimum number of cell
lines, so it was passed to the full five-dose assay on to
further evaluation at five concentration levels (0.01 and
100 lM) (Table 2). It showed broad spectrum anticancer
activity against all tested sub-panel tumour cell lines, with
GI50 full panel mean graph mid-point (MG-MID) =
-5.4 lM, total growth inhibition (TGI) full panel (MG-
MID) = -4.28 lM and LC50 full panel (MG-MID) =
-4.01 lM.
U251
Melanoma
LOX IMVI
MALME-3M
M14
0.003735
0.004608
0.004992
0.004654
0.004128
0.001136
0.005407
0.006158
0.003773
0.009466
0.011333
0.012155
0.018316
0.00823
0.018316
0.018316
0.018316
0.018316
0.017597
0.018316
0.013569
0.018316
0.018316
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
0.018316
0.009095
0.014996
0.006605
Experimental
0.003661
0.003773
0.004748
0.002177
0.004383
0.006346
0.006097
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
0.018316
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
General
Reactions were routinely monitored by thin-layer chro-
matography (TLC) on silica gel sheets that precoated with
123

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Med Chem Res (2013) 22:2589–2601
General procedure for the preparation of compounds
4a and 4b
UV fluorescent silica (MERCK 60 F 254) and spots were
developed using I2 vapour/UV light as visualizing agents.
Solvent system was chloroform:methanol (in different
1
A mixture of 2 (3.62 g, 0.01 mol) and the appropriate
aromatic aldehyde (0.01 mol) in absolute ethanol (10 mL)
was heated under reflux for 3 h then cooled, filtered and the
solid obtained was crystallized from acetic acid.
ratios). H NMR spectra were determined in CDCl3, or
DMSO-d6 solvent with Varian Gemini 300 MHZ Spec-
trometer. Peak positions were given in parts per million (d)
downfield the tetramethylsilane (TMS) as internal standard.
IR spectra were recorded on a Shimadzu 435 Spectrometer,
using KBr discs and values were represented in cm-1. Mass
spectra were run on Hewlett Packard 5988 spectrometer at
the Microanalytical Center, Cairo University. Melting points
were determined on a Griffin instrument and are uncorrected.
All reported products showed 1H NMR spectra in agreement
with the assigned structures. Elemental analyses were per-
formed at the Micro-analytical Center, Cairo University,
Egypt. Compounds 1 and 2 were prepared adopting a
reported procedure (Radwan and Bakhite, 1999; Kamal
El-Dean et al., 1996; Bakhite et al., 2002).
5-Hydroxy-3,4-diphenyl-thieno[2,3-c]pyridazine-6-carbox-
ylic acid (ZE) (4-fluorobenzylidene)-hydrazide 4a Yield
80 %; mp 290–292 °C. IR (KBr): 3150 (NH), 3047–3022
1
(CH, arom), 1631 (C=O) cm-1; H NMR (DMSO-d6, d,
ppm):7.15–7.97 (m, 14H, Ar–H), 8.24 (s, 1H, N=CH),
12.54 (s, 1H, NH, D2O exchangeable), 13.22 (s, 1H, OH,
D2O exchangeable) ppm; EIMS: m/z (%) = 468 (M?, 6.4),
467 (M-1?., 36.0); Anal. Calcd for C26H17FN4O2S: C
66.65, H 3.66, N 11.96; Found: C 66.50, H 3.72, N
12.20 %.
5-Hydroxy-3,4-diphenylthieno[2,3-c]pyridazine-6-carbox-
ylic acid (ZE) (2-nitrobenzylidene)-hydrazide 4b Yield
65 %. mp 275–277 °C. IR (KBr): 3100 (NH), 3051–3020
Chemistry
General procedure for the preparation of compounds
3a and 3b
(CH, arom), 1639 (C=O), 1528, 1340 (NO2) cm-1 1H
;
NMR (DMSO-d6, d, ppm): 7.29–8.32 (m, 14H, Ar–H),
8.67 (s, 1H, N=CH), 12.83 (s, 1H, NH, D2O exchangeable),
13.07 (s, 1H, OH, D2O exchangeable) ppm; EIMS: m/
z (%) = 496(M?1?, 6.1), 495 (M?, 22.2), 331(100 %);
Anal. Calcd for C26H17N5O4S: C 63.02, H 3.46, N 14.13;
Found: C 63.09, H 3.81, N 14.12 %.
A mixture of 1 (1.69 g, 0.005 mol) and the appropriate
4-aminobenzenesulphonamide (0.005 mol) in 1-butanol
(20 mL) was refluxed for 3 h, the reaction mixture was
cooled, filtered and the separated solid was crystallized
from ethanol.
General method for preparation of compounds
5a and 5b
Ethyl 5,6-diphenyl-3-(4-sulfamoylphenylamino)-4-pyridazine-
carboxylate 3a Yield 85 %. mp 230–232 °C. IR (KBr):
3400, 3373, 3300 (NH2, NH), 3090–3040, (CH, arom),
A mixture of 4a and 4b (0.01 mol) and acetic anhydride
(3 mL) was refluxed for 1 h, allowed to cool, and then
ammonium hydroxide (5 mL) was added. The obtained
solid was filtered, washed with water and crystallized from
aqueous ethanol (50 %).
2910–2850 (CH, aliph), 1747 (C=O) cm-1 1H NMR
;
(DMSO-d6, d, ppm): 0.82–0.88(t, J = 7.2 Hz, 3H, –CH2–
CH3); 4.01–4.12 (q, J = 7.2 Hz, 2H, CH2–CH3); 7.17–7.94
(m, 15H, Ar–H? (1H, NH, D2O exchangeable); 9.03 (s, 2H,
NH2, D2O exchangeable) ppm; EIMS: m/z (%) = 474(M?,
35), 473(M?-1, 100); Anal. Calcd for C25H22N4O4S: C
63.28, H 4.67, N 11.80; Found: C 63.33, H 4.45, N 11.99 %.
Acetic acid 6-[4-acetyl-5-(4-fluorophenyl)-4,5-dihydro-[1,3,4]
oxadiazol-2-yl]-3,4-diphenylthieno[2,3-c]pyridazin-5-yl ester
5a Yield 72 %. mp 155–157 °C. IR (KBr): 3057–3032
(CH, arom), 2922–2850 (CH, aliph), 1782, 1676 (2C=O)
Ethyl 5,6-diphenyl-3-[4-(thiazol-2-ylsulfamoyl)-phenylamino]-
4-pyridazine-carboxylate 3b Yield 79 %. mp 191–193 °C.
IR (KBr): 3400 (NH), 3066–3026 (CH, arom), 2980–2900
1
cm-1; H NMR (DMSO-d6, d, ppm): 1.42 (s, 3H, –CO
CH3), 1.91 (s, 3H, –CO CH3), 6.93 (s, 1H, C-2 oxadiazo-
line), 7.00–8.10 (m, 14H, ArH) ppm; Anal. Calcd for
C30H21FN4O4S: C 65.21, H 3.83, N 10.14; Found: C 64.90,
H 3.61, N 10.01 %.
1
(CH, aliph), 1732 (C=O) cm-1; H NMR (DMSO-d6, d,
ppm): 0.71–0.79 (t, J = 7.2 Hz, 3H, –CH2–CH3);
4.01–4.10 (q, J = 7.2 Hz, 2H, –CH2–CH3); 6.82 (s, 2H,
C-4, C-5 thiazole), 7.14–7.82 (m, 14H, Ar–H); 9.09 (s, 1H,
NH, D2O exchangeable); 12.72 (s, H, NH, D2O
exchangeable) ppm; EIMS: m/z (%) = 558 (M?1?, 2.40);
Anal. Calcd for C28H23N5O4S2: C 60.31, H 4.16, N 12.56;
Found: C 60.30, H 4.00, N 12.64 %.
Acetic acid 6-[4-acetyl-5-(2-nitrophenyl)-4,5-dihydro-[1,3,4]
oxadiazol-2-yl]-3,4-diphenylthieno[2,3-c]pyridazin-5-yl ester
5b Yield 71 %. mp 228–230 °C. IR (KBr): 3057–3028
(CH, arom), 2910–2820 (CH, aliph) 1784, 1678 (2C=O),
123

Med Chem Res (2013) 22:2589–2601
2597
1
1531, 1344 (NO2) cm-1; H NMR (DMSO-d6, d, ppm):
water (30 mL), and dilute HCl (10 mL) was added. The
separated solid was filtered, dried and crystallized from
acetone to give 8 in 2.7 g (66 %) yield. mp 308–310 °C. IR
(KBr): 3321 (OH), 3055–3022 (CH, arom), 2534 (SH)
1.32(s, 3H, –CO CH3), 1.89 (s, 3H, –COCH3), 6.83(s, 1H,
C-2 oxadiazoline) 6.87–8.30 (m, 14H, ArH) ppm; EIMS:
m/z(%) = 580 (M?1?, 2.92), 579 (M?, 6.41), 536
(M?–COCH3, 2.12); Anal. Calcd for C30H21N5O6S: C
62.17, H 3.65, N 12.08; Found: C 62.60, H 3.80, N 11.88 %.
1
cm-1; H NMR (DMSO-d6, d, ppm): 7.1–7.3 (m, 10H,
Ar–H), 8.4 (s, 2H, OH and SH, D2O exchangeable) ppm;
EIMS: m/z (%) = 406 (M?2?, 4.43), 405 (M?1?, 8.64),
404 (M?, 25.14); Anal. Calcd for C20H12N4O2S2: C 59.39,
H 2.99, N 13.85; Found: C 59.19, H 3.08, N 13.62 %.
N-[5-Hydroxy-3,4-diphenylthieno[2,3-c]pyridazine-6-
carboxylic acid amido]isoindole-1,3-dione 6
To a mixture of 2 (3.62 g, 0.01 mol) in glacial acetic acid
(10 mL), phthalic anhydride (2.22 g, 0.015 mol) was
added and the mixture was heated under reflux for 6 h,
after cooling the reaction mixture was poured onto crushed
ice while stirring. The formed precipitate was filtered and
crystallized from ethanol to give compound 6 in 3.33 g
(68 %) yield. mp 215–217 °C. IR (KBr): 3305, 3200 (OH,
NH), 3082–3050 (CH, arom), 1743 (C=O), 1685
(C=O) cm-1; 1H NMR (DMSO-d6, d, ppm): 7.21–8.22 (m,
14 Ar–H), 10.28 (s, 1H, NH, D2O exchangeable), 11.51(s,
1H, OH, D2O exchangeable) ppm; EIMS: m/z (%) =
493(M?1?, 1.33), 492 (M?, 4.24 %); Anal. Calcd for
C27H16N4O4S: C 65.84, H 3.27, N 11.38; Found: C 65.84,
H 3.25, N 11.30 %.
General method for preparation of compounds 9a, 9b
and 10a, 10b
A mixture of 8 (4.04 g, 0.01 mol), the corresponding alkyl
halide (0.01 mol) and anhydrous sodium acetate (0.82 g,
0.01 mol) in absolute ethanol (30 mL) was refluxed for
1 h; the reaction mixture was allowed to cool, and poured
into ice-cold water (50 mL). The solid product was col-
lected and fractionally crystallized from ethanol; the
insoluble compounds were 9a and 9b while the soluble
compounds were 10a and 10b.
6-(5-Methylsulfanyl-[1,3,4]oxadiazol-2-yl)-3,4-diphenylthieno
[2,3-c]pyridazin-5-ol 9a Yield 30 %. mp 240–242 °C. IR
(KBr): 3267 (OH), 3057–3028 (CH arom), 2950–2850 (CH
5-Methyl-2-(5-hydroxy-3,4-diphenylthieno
[2,3-c]pyridazine-6-carbonyl)-2,4-dihydro-pyrazol-3-one 7
1
aliph) cm-1; H NMR (DMSO-d6, d, ppm): 2.90 (s, 3H,
–S–CH3), 7.10–7.30 (m, 10H, Ar–H), 7.96 (s, 1H, OH, D2O
exchangeable) ppm; EIMS: m/z (%) = 419(M?1?, 16.9),
418(M?, 45.8); Anal. Calcd for C21H14N4O2S2: C 60.27, H
3.37, N 13.39; Found: C 60.08, H 3.57, N 13.50 %.
A mixture of 2 (3.62 g, 0.01 mol), anhydrous sodium
acetate (0.82 g, 0.01 mol) and ethyl acetoacetate (2.6 g,
0.02 mol) in absolute ethanol (20 mL) was heated under
reflux for 8 h, then allowed to cool and poured into ice-
cold water (50 mL). The solid product was collected by
filtration, dried and crystallized from ethanol to give
compound 7 in 0.9 g (76 %) yield. mp 180–182 °C. IR
(KBr): 3200 (OH), 3055–3035 (CH, arom), 2993–2930
6-(5-Benzylsulfanyl-[1,3,4]oxadiazol-2-yl)-3,4-diphenylthieno
[2,3-c]pyridazin-5-ol 9b Yield 35 %. mp 190–192 °C. IR
(KBr): 3300 (OH), 3059–3028 (CH, arom) cm-1; 1H NMR
(DMSO-d6, d, ppm): 4.11 (s, 2H, CH2–Ph), 6.75–7.30 (m,
15H, Ar–H), 8.90 (s, 1H, OH, D2O exchangeable) ppm;
Anal. Calcd for C27H18N4O2S2: C 65.57, H 3.67, N 11.33;
Found: C 65.40, H 3.50, N 11.44 %.
(CH aliph), 1690 and 1660 (2C=O) cm-1
(DMSO-d6, d, ppm): 1.32 (s, 3H, CH3),4.15–4.40 (dd,
J = 8.3 Hz, J = 8.3 Hz, 2H, C-4 pyrazolone),
;
1H NMR
7.11–7.31(m, 10H, Ar–H), 10.57 (s, 1H, OH, D2O
exchangeable) ppm; EIMS: m/z (%) = 429(M?1?, 2.3),
428(M?, 2.5 %); Anal. Calcd for C23H16N4O3S: C 64.47,
H 3.76, N 13.08; Found: C 64.70, H 3.94, N 12.80 %.
5-Methoxy-6-(5-methylsulfanyl-[1,3,4]oxadiazol-2-yl)-3,4-
diphenylthieno[2,3-c]pyridazine 10a Yield 60 %. mp
200–202 °C. IR (KBr): 3066–3055 (CH, arom), 2972–2872
1
(CH, aliph) cm-1; H NMR (DMSO-d6, d, ppm): 2.79(s,
6-(5-Thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-5-
hydroxy-3,4-diphenylthieno[2,3-c]pyridazine 8
3H, –S–CH3), 3.19 (s, 3H, –O–CH3), 7.2–7.3(m, 10 H,
Ar–H) ppm; Anal. Calcd for C22H16N4O2S2: C 61.06, H
3.73, N 12.95; Found: C 61.34, H 3.88, N 12.67 %.
To a mixture of 2 (3.62 g, 0.01 mol) and potassium
hydroxide (0.56 g, 0.01 mol) in aqueous ethanol (50 %,
30 mL), carbon disulphide (12.5 mL) was added and the
reaction mixture was refluxed for 8 h, then evaporated
under reduced pressure. The residue was dissolved in cold
5-Benzyloxy-6-(5-benzylsulfanyl-[1,3,4]oxadiazol-2-yl)-3,
4-diphenylthieno[2,3-c]pyridazine 10b Yield 55 %. mp
185–187 °C. IR (KBr): 3047–3035 (CH, arom), 2950–2840
123

2598
Med Chem Res (2013) 22:2589–2601
1
(CH, aliph) cm-1; H NMR (DMSO-d6, d, ppm): 4.31 (s,
2H–CH2–Ph), 4.49 (s, 2H, O–CH2–Ph), 6.90–7.30 (m,
20H, Ar–H) ppm; Anal. Calcd for C34H24N4O2S2: C 69.84,
H 4.14, N 9.58; Found: C 69.41, H 4.29, N 9.03 %.
water and crystallized from ethanol to give compound 13 in
2.89 g (69 %) yield. mp 298–300 °C. IR (KBr): 3203,
3113 (OH, NH), 3050–3029 (CH, arom), 2940–2860 (CH,
1
aliph) cm-1; H NMR (DMSO-d6, d, ppm): 2.19 (s, 3H,
CH3),7.15–7.35 (m, 10H, Ar–H), 11.55 (s, H, NH, D2O
exchangeable), 13.47 (s, 1H, OH, D2O exchangeable) ppm;
EIMS: m/z (%) = 417(M?, 100); Anal. Calcd for C21H15
N5OS2: C 60.41, H 3.62, N 16.77; Found: C 60.10, H 3.80,
N 16.71 %.
1-(5-Hydroxy-3,4-diphenyl-thieno[2,3-c]pyridazin-6-yl)-
4-methylthiosemicarbazide 11
A mixture of the acid hydrazide 2 (3.62 g, 0.01 mol) and
methyl isothiocyanate (0.73 g, 0.01 mol) in dioxin
(30 mL) was refluxed for 6 h, the solvent was removed
under vacuum. The residue was washed with water, and
crystallized from acetone to give compound 11 in 3.4 g
(81 %) yield. mp 192–194 °C. IR (KBr): 3329, 3251 (OH,
NH), 3053–3030 (CH, arom), 2972–2870 (CH, aliph), 1633
6-(4-Methyl-5-thio-4H-1,2,4-triazol-5-yl)5-hydroxy-3,4-
diphenylthieno[2,3-c]pyridazine 14
A mixture of the thiosemicarbazide 11 (4.35 g, 0.01 mol)
and NaOH (2N, 5 mL) was refluxed for 3 h, after cooling
the reaction mixture was acidified with dilute HCl. The
solid formed was filtered and crystallized from ethanol to
give compound 14 in 3.33 g (80 %) yield. mp 142–144 °C.
IR (KBr): 3300–3200 (OH, NH), 3056–3024 (CH, arom),
1
(C=O), 1202 (C=S) cm-1; H NMR (DMSO-d6, d, ppm):
2.1 (s, 3H, CH3), 7.1–7.3 (m, 10H, Ar–H), 11.48, 12.20,
13.05 (3s, 3H, NH, D2O exchangeable),13.41 (s, 1H, OH,
D2O exchangeable) ppm; Anal. Calcd for C21H17N5O2S2:
C 57.91, H 3.93, N 16.08; Found: C 57.99, H 4.20, N
16.35 %.
2920–2855 (CH, aliph) cm-1 1H NMR (DMSO-d6, d,
;
ppm): 2.96 (s, 3H, CH3),7.1–7.2 (m, 10H, Ar–H), 8.00 (s,
2H, OH or SH, D2O exchangeable) ppm; Anal. Calcd for
C21H15N5OS2: C 60.41, H 3.62, N 16.77; Found: C 60.27,
H 3.89, N 16.42 %.
5-Hydroxy-3,4-diphenylthieno[2,3-c]pyridazine-6-
carboxylic acid (ZE) (3-methyl)-4-oxothiazolidin-2-
ylidene) hydrazide 12
General method for preparation of compounds
15a and 15b
A mixture of the thiosemicarbazide 11 (4.35 g, 0.01 mol),
monochloroacetic acid (0.94 g, 0.01 mol) and anhydrous
sodium acetate (0.82 g, 0.01 mol) in acetic acid (20 mL)
was heated under reflux for 10 h, the reaction mixture was
cooled and poured into ice-cold water. The precipitate was
filtered, washed with water and crystallized from ethanol to
give compound 12 in 3.08 (65 %) yield. mp 212–214 °C.
IR (KBr): 3300–3200 (OH, NH), 3055–3026 (CH, arom),
A mixture of acid hydrazide 2 (3.62 g, 0.02 mol) and the
respective aromatic acid (0.02 mol) in phosphorus oxy-
chloride (10 mL) was heated under reflux for 4 h. After
cooling, the reaction mixture was poured onto crushed ice
and sodium carbonate solution (20 %) (10 mL) was added.
The formed precipitate was filtered, washed with water,
dried and crystallized from DMF to give 15a and 15b.
2922–2850 (CH, aliph), 1728 (C=O), 1620 (C=O) cm-1
;
1H NMR (DMSO-d6, d, ppm): 2.23 (s, 3H, –CH3), 4.19 (s,
2H, C-5 thiazolidinone),7.11–7.38 (m, 10H, Ar–H), 11.67
(s, 1H, NH, D2O exchangeable), 13.48(s, 1H, OH, D2O
exchangeable) ppm; EIMS: m/z (%) = 476 (M?1?,
11.09), 475 (M)?, 31.47; Anal. Calcd for C23H17N5O3S2: C
58.09, H 3.60, N 14.73; Found: C 58.28, H 3.80, N
14.90 %.
5-Hydroxy-3,4-diphenyl-6-(5-phenyl-1,3,4-oxadiazol-2-yl)-
thieno[2,3-c]pyridazine 15a Yield 80 %.mp 122–124 °C.
1
IR (KBr): 3200 (OH), 3057–3020 (CH, arom) cm-1; H
NMR (DMSO-d6, d, ppm): 6.90–7.33 (m, 15H, Ar–H),
13.35 (s, 1H, OH, D2O exchangeable) ppm; EIMS:
m/z (%) = 449(M??1, 28.35), 448 (M?, 87.86); Anal.
Calcd for C26H16N4O2S: C 69.63, H 3.60, N 12.49; Found:
C 69.60, H 3.88, N 12.16 %.
5-Hydroxy-6-(5-methylamino-1,3,4-thiadiazol-2-yl)-3,4-
diphenylthieno[2,3-c]pyridazine 13
5-Hydroxy-3,4-diphenyl-6-(5-(4-chlorophenyl)-1,3,4-oxadiazol-
2-yl)-thieno[2,3-c]pyridazine 15b Yield 69 %. mp 192–194
An ice-cold solution of the thiosemicarbazide 11 (4.35 g,
0.01 mol) in concentrated sulphuric acid (10 mL) was
stirred for 10 min, then left at room temperature for
another 10 min. The resulting solution was poured slowly
into ice-cold water and made alkaline with conc. ammonia
(30 %).The precipitated product was filtered, washed with
°C. IR (KBr): 3330 (OH), 3093–3053 (CH, arom) cm-1
;
1H NMR (DMSO-d6, d, ppm): 7.11–7.93 (m, 14H, Ar–H),
13.20 (s, 1H, OH, D2O exchangeable) ppm; Anal. Calcd
for C26H15ClN4O2S: C 64.66, H 3.13, N 11.60; Found: C
64.32, H 3.39, N 11.74 %.
123

Med Chem Res (2013) 22:2589–2601
Antitumour screening
2599
exceeded, the value for that parameter is expressed as
greater or lesser than the maximum or minimum con-
centration tested.
Under a sterile condition, cell lines were grown in RPMI
1640 media supplemented with 10 % foetal bovine
serum, up to 105 cells/mL was used to test the growth
inhibition activity of the synthesized compounds. The
concentrations of the compounds ranging from 0.01 to
100 lM were prepared in phosphate buffer saline. Each
compound was initially solubilized in DMSO; however,
each final dilution contained less than 1 % DMSO.
Solutions of different concentrations (0.2 mL) were
pipetted into separate well of a microtitre tray in
duplicate. Cell culture (1.8 mL) containing a cell popu-
lation up to 104 cells/mL was pipetted into each well.
Controls, containing only phosphate buffer saline and
DMSO at identical dilutions, were also prepared in the
same manner. These cultures were incubated in a
humidified incubator at 37 °C. The incubator was sup-
plied with 5 % CO2 atmosphere. After 48 h, cells in
each well were diluted 10 times with saline and counted
by using a coulter counter. The counts were corrected for
the dilution (Monk et al., 1991). The anticancer activity
of tested compounds is given by three parameters for
each cell line: log GI50 value (GI50 = molar concentra-
tion of the compound that inhibits 50 % net cell growth),
log TGI value (TGI = molar concentration of the com-
pound leading to total inhibition) and log LC50 value
(LC50 = molar concentration of the compound leading to
50 % net cell death). Using the seven absorbance mea-
surements [time zero (Tz), control growth (C) and test
growth in the presence of drug at the five concentration
levels (Ti)], the percentage growth is calculated at each
of the drug concentrations levels. Percentage growth
inhibition is calculated as:
Acknowledgments Thanks are due to the NCI, Bethesda, MD, for
performing the antitumour testing of the synthesized compounds.
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½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢂ
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