Ureido-Pyridazinone Derivatives: Insights into the Structural and Conformational Properties for STAT3 Inhibition

Article abstract of DOI:10.1002/ejoc.201500599

Three new ureido-pyridazinone derivatives, which are structurally related to the known STAT3 inhibitor AVS-0288, were designed by taking into account the structure-activity relationships determined for several ureido-oxadiazole derivatives previously studied by our group. Their synthesis was first attempted through suitable 5-aminopyridazinone intermediates (6a and 6b), which molecular structures were confirmed by means of X-ray diffraction data on 6a. Amine functionalization was unsuccessful, therefore, an alternative method was devised. Dual-luciferase and AlphaScreen-based assays were used to test their activity. The obtained data were rationalized on the basis of a modeling study, which focused our attention on the geometrical preferences of the ureido moiety. Computational results seem to indicate that both the 1,2,5-oxadiazole ring and the extended ZZ arrangement are essential and probably act in a synergistic way to confer significant activity against STAT3.

Full text of DOI:10.1002/ejoc.201500599

FULL PAPER
DOI: 10.1002/ejoc.201500599
Ureido-Pyridazinone Derivatives: Insights into the Structural and
Conformational Properties for STAT3 Inhibition
Fiorella Meneghetti,[a] Stefania Villa,*[a] Daniela Masciocchi,[a] Daniela Barlocco,[a]
Lucio Toma,[b] Dong-Cho Han,[c] Byoung-Mog Kwon,[c] Naohisa Ogo,[d] Akira Asai,[d]
Laura Legnani,*[b] and Arianna Gelain[a]
Keywords: Medicinal chemistry / Cancer / Inhibitors / Signal transduction / Conformation analysis / Proteins
Three new ureido-pyridazinone derivatives, which are struc-
turally related to the known STAT3 inhibitor AVS-0288, were
method was devised. Dual-luciferase and AlphaScreen-
based assays were used to test their activity. The obtained
designed by taking into account the structure–activity rela- data were rationalized on the basis of a modeling study,
tionships determined for several ureido-oxadiazole deriva-
tives previously studied by our group. Their synthesis was
first attempted through suitable 5-aminopyridazinone inter-
mediates (6a and 6b), which molecular structures were con-
firmed by means of X-ray diffraction data on 6a. Amine func-
tionalization was unsuccessful, therefore, an alternative
which focused our attention on the geometrical preferences
of the ureido moiety. Computational results seem to indicate
that both the 1,2,5-oxadiazole ring and the extended ZZ ar-
rangement are essential and probably act in a synergistic
way to confer significant activity against STAT3.
STAT3 signaling leads to apoptosis of tumors cells[4,5] with
minimal effect on normal cells.[6–8] Therefore, STAT3 has
been proven to be a valid molecular target for cancer ther-
apy.
In the recent years, ureido-oxadiazole derivative AVS-
0288 (I)[9] was disclosed as a potent inhibitor of the STAT3
pathway (Figure 1), in a STAT3-dependant luciferase re-
porter assay.
Introduction
Signal transducer and activator of transcription 3
(STAT3) is a member of a STAT family that is made up of
seven isoforms (STAT1-4, 5a, 5b, 6).[1] They directly relay
signals from the cytoplasmic membrane to the nucleus and
regulate transcription of target genes.[2] STATs have several
retained domains of which the Src Homology 2 (SH2) do-
main is fundamental for STATs activation, which is regu-
lated by phosphorylation of a specific tyrosine residue
(Tyr705) by kinases. Phosphorylated STAT3 proteins form
homodimers and/or heterodimers with STAT1 through re-
ciprocal phosphotyrosine-SH2 interaction. The active di-
mers translocate into the nucleus where they bind to con-
sensus-promoter sequences of target genes[2] and modulate
their transcription. STAT3 is constitutively activated in a
wide variety of human solid and blood tumors.[3] Numerous
published reports have shown that a block on activated
[a] Dipartimento di Scienze Farmaceutiche, Università degli Studi
di Milano,
Via L. Mangiagalli 25, 20133 Milano, Italy
E-mail: stefania.villa@unimi.it
http://www.disfarm.unimi.it
[b] Dipartimento di Chimica, Università degli Studi di Pavia,
Via Taramelli 12, 27100 Pavia, Italy
[c] Laboratory of Chemical Biology and Genomics, Korea
Research Institute of Bioscience & Biotechnology and
Department of Biomolecular Science, University of Science and
Technology,
Eoun-Dong, Yuseong-gu, Daejeon 305-333, South Korea
[d] Center for Drug Discovery, Graduate School of Pharmaceutical
Sciences, University of Shizuoka,
52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500599.
Figure 1. Chemical structures of the reference compounds.
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 4907
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S. Villa, L. Legnani, et al.
FULL PAPER
Recently, we performed a modeling study on I to under- condensation with mucochloric acid to give 3b, which was
stand its conformational preferences.[10] Computational subsequently cyclized with hydrazine to give 4b. Replace-
data revealed, in the gas phase, complete preference of I for ment of the aryl chlorine with sodium azide led to 5b, which
a bent EZ arrangement around the two amide bonds, fa- was hydrogenated to give key intermediate 6b in the pres-
vored by formation of an intramolecular H-bond between ence of Pd/BaSO4 to avoid loss of the chlorine atom. How-
an ureido NH hydrogen and the oxadiazole N2 atom. How- ever, 6a and 6b did not react with 4-(trifluoromethyl)phenyl
ever, the solvent deeply influences the conformational pref- isocyanate under various experimental conditions, although
erences of I. In water, the extended ZZ arrangement is single-crystal X-ray analysis on 6a unambiguously sup-
largely preferred. 1H NMR spectroscopic studies in solvents ported their structure (see Supporting Information). The re-
with different polarity confirmed the modeling results. In activity of both 6a and 6b was tested through treatment
addition, an X-ray diffraction study showed for I a molecu- with acetic anhydride at reflux temperatures, but the ex-
lar feature that corresponds to the ZZ arrangement calcu- pected[13] acetylated products were not obtained.
lated in water.
Therefore, an alternative synthetic pathway was designed
Amongst many other compounds structurally related to that successfully led to desired pyridazinones 1 and 2
I, synthesized by us as possible STAT3 inhibitors, styryl (Scheme 1). Following a procedure already described for the
derivative II (Figure 1), for which the bent EZ arrangement preparation of 13a,[14] analogue 13b was prepared from
of the urea group was shown both in the solid state[11] and commercially available 4-chlorobenzoyl propionic acid
by theoretical calculations in vacuo and in water (unpub- through a hydroxymethylation reaction with formaldehyde
lished results), showed a significant drop in activity with and sodium hydroxide in water, and subsequent cyclization
respect to I.[11] The computational results show, both in in acidic conditions to give intermediate 7b.[15] Treatment
vacuo (100%) and in water (Ͼ99%), complete preference of the intermediate with a large excess of hydrazine mono-
for the EZ arrangement at the ureido moiety and the for- hydrate gave compound 8b, which was protected on the al-
mation of an intramolecular H-bond between an ureido coholic function by acetic anhydride (9b) and then dehydro-
NH hydrogen and the oxadiazole N2 atom.
genated with activated manganese dioxide to give aromatic
These results prompted us to assume that the extended derivative 10b. Removal of the protecting group (see 11b),
ZZ arrangement of the ureido moiety was a key geometri- followed by oxidation with activated manganese dioxide led
cal feature for STAT3 inhibition. On this basis, we have to corresponding aldehydic intermediate 12b, which under-
now synthesized compounds 1 and 2 by replacing the 1,2,5- went further oxidation to carboxylic acid 13b by treatment
oxadiazole with the pyridazinone ring and linking the ure- with silver oxide.
ido group at position 5 of the heterocycle, to prevent forma-
Both carboxylic acids 13a and 13b by means of a Curtius
tion of the intramolecular hydrogen bond. We took advan- rearrangement with diphenyl phosphoryl azide in toluene
tage of our experience in the chemistry of pyridazinone sys- led to isocyanate derivatives, which were not isolated but
tems, which was exploited in previous studies, in which this were directly converted into final ureidic products 1a and
nucleus was inserted in several benzocinnolinone derivatives 1b by coupling with 4-(trifluoromethyl)aniline. Similarly,
such as compound III (Figure 1) that showed the most 13a was converted into ureidic product 2 by coupling with
promising activity.[12]
2,4-dimethylphenyl aniline.
This paper describes the synthesis, conformational, and
biological properties of compounds 1 and 2 (Figure 2).
Modeling Studies
To determine the effects of replacement of the oxadiazole
with pyridazinone on the conformational properties of the
synthesized compounds, an in-depth modeling study of 1a
was performed. All its degrees of conformational freedom
were considered, with particular attention to: (i) the relative
orientation of the ureido chain with respect to the pyridaz-
inone ring (τ1: C4–C5–N1Ј–C2Ј); (ii) the different arrange-
ments of the ureido moiety (τ2: C5–N1Ј–C2Ј–N3Ј; τ3: N1Ј–
C2Ј–N3Ј–C1ЈЈ); and (iii) the relative orientation of the ure-
ido chain with respect to the phenyl ring (τ4: C2Ј–N3Ј–
C1ЈЈ–C2ЈЈ). After optimization of the various starting geo-
metries at the B3LYP level with the 6-311+G(d,p) basis
set,[16] the located conformers were grouped, on the basis
of the values of τ1, into two families, A and B, characterized
Figure 2. General structure of the title compounds.
Results and Discussion
Chemistry
Two different synthetic procedures were attempted for by having, respectively, the N1Ј hydrogen atoms pointing
compounds 1. In the first approach (see Supporting Infor- towards the aryl group or in the opposite direction. Within
mation) by following a procedure previously described for each family, the members differ for the arrangements of the
6a[13], analogue 6b was obtained from chlorobenzene by ureido moiety. In fact, depending on the dihedral angle val-
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Structural and Conformational Properties for STAT3 Inhibition
Scheme 1. Synthesis of compounds 1 and 2. Reagents and conditions: (a) 1. HCHO (37%), NaOH; 2. HCl, room temp., 65% for 7b;
(b) NH2NH2, EtOH, reflux, 20% for 8b; (c) Ac2O, Py, room temp., 96% for 9b; (d) MnO2, CHCl3, reflux, 20% for 10b; (e) HCl (2 n),
reflux, quantitative yield for 11b; (f) MnO2, THF, room temp., 55% for 12b; (g) 1. AgNO3, EtOH/H2O 2. NaOH (10%), room temp.,
70% for 13b; (h) 1. DPPA, Et3N, toluene, 80 °C; 2. 4-(trifluoromethyl)aniline, THF, reflux, 20% for 1a and 10% for 1b; (i) 1. DPPA,
Et3N, toluene, 80 °C; 2. 2,4-dimethylaniline, THF, reflux, 5%.
ues (τ2 and τ3) at the two pseudo-amide bonds, 1a can show angles. Figure 3 shows the three-dimensional plots of the
four different arrangements designated as ZZ, ZE, EZ, or most significant conformers. Only the members of the A
EE.
family (τ1 ≈ 0) are populated. In the gas phase, the preferred
The energy of the optimized conformations was recalcul- conformer is ZZ_A, which presents the ZZ arrangement at
ated by using a polarizable continuum solvent model[17] to the two amide bonds (τ2 = 176°, τ3 = 178°) and accounts
highlight the effect of water on the population distribution for about 93% of the overall population. Also conformers
of the conformers. In Table 1 the gas-phase and water-sol- EZ_A (τ2 = 19°, τ3 = –179°) and ZE_A (τ2 = 179°, τ3 =
vated energies of the minimum-energy conformers are re-
ported, together with the corresponding percentage contri-
butions to the overall population and the τ1–τ4 torsional
Table 1. Relative energy [kcal/mol], equilibrium percentages at
298 K, and τ1–τ4 torsional angles [°] of the B3LYP/6-311+G(d,p)
located conformers of compound 1a.
Conf. Erel
%
Erel
%
τ1
τ2
τ3
τ4
vacuo vacuo water water
ZZ_A 0.00
EZ_A 1.62
ZE_A 2.54
EE_A 6.10
ZZ_B 4.72
EZ_B 4.64
ZE_B 7.49
EE_B 9.49
92.7 0.0
99.1
0.3
0.4
0.0
0.2
0.0
0.0
–4
18
15
–2
176
19
179
42
178
–179
18
–3
4
6.0
1.3
0.0
0.0
0.0
0.0
0.0
3.53
3.20
7.13
3.72
5.73
7.11
40
18
–1
–2
57
–22
34
122 178
120
121 –176
143 –26
175
176
–2
1
10.17 0.0
–34
Figure 3. 3D plots of the most significant conformations of 1a.
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S. Villa, L. Legnani, et al.
FULL PAPER
18°) present a not negligible population percentage, even if the 1,2,5-oxadiazole ring might play two opposite roles. On
they are less stable than ZZ_A by 1.62 (6.0%) and one hand, it could favor the bent EZ ureido arrangement,
2.54 kcal/mol (1.3%), respectively. The relative energy of which would be detrimental for activity, and on the other
conformer EE_A (τ2 = 42°, τ3 = 34°) is much higher hand, it could play a specific role in the binding pocket of
(6.10 kcal/mol) so that it gives no contribution to the over- the SH2 domain by anchoring the ligand to the Arg609
all population. It should be noted that the preference for residue, as shown by previous docking studies.[20]
conformer ZZ_A is almost complete in water and its pop-
ulation percentage exceeds 99%, contrary to reference com-
pound I that showed, at the same level of calculations,[10]
a
Experimental Section
small preference for a conformation of the ZZ_B type more
stable by only 0.84 kcal/mol than its ZZ_A conformer.
Materials and Methods: Reagents were purchased from commercial
suppliers and used as received. Commercial plates on aluminum-
backed Silica Gel 60 plates (0.2 mm, Merck) were used for analyti-
cal TLC to follow the course of the reaction. Silica gel 60 (Merck
40–63 μm) was used for flash chromatography to purify intermedi-
ates and final compounds. The purity of final compounds was de-
termined by HPLC analysis and was Ն 95%. Melting points were
determined in open capillary tubes with a Buchi Melting Point 510.
1H NMR spectra were acquired at ambient temperature with a
Biological Evaluation
The STAT3 inhibitory activity of 1 and 2 was evaluated
by means of a modified dual-luciferase assay[18] in human
colorectal carcinoma cells HCT-116, characterized by un-
controlled expression of STAT3. After 24 h, inhibition per- 300 MHz Oxford-Varian instrument. Chemical shifts are expressed
in ppm from tetramethylsilane resonance in the indicated solvent
(TMS: δ = 0.0 ppm). Derivatives 3a–13a were synthesized accord-
ing to literature methods.[13–15] The structures of all compounds are
consistent with their analytical and spectroscopic data.
centages at 10 μm concentration of compounds 1a, 1b, and
2 were 26, 24, and 17%, respectively, whereas at 50 μm these
values slightly increased to 33, 35, and 26%, respectively.
Although the activity is approximately halved with respect
to compound I[10] (70% at 10 μm and 75% at 50 μm), they
still display moderate capability to interfere with the STAT3
pathway. Therefore, to investigate if these compounds are
able to bind the STAT3 SH2 domain, an AlphaScreen-
based assay was undertaken.[19] However, no significant re-
sults were obtained and the inhibition percentage was deter-
mined to be lower than Ϯ20% at 30 μm.
Synthetic Procedures
β-(4-Chlorobenzoyl)-γ-butyrolactone (7b): Formaldehyde (37%,
3.08 mmol) was added to a stirred solution of 3-benzoyl-4-chloro-
propionic acid (2.81 mmol) and NaOH (0.5 n, 3.1 mmol; molar ra-
tio of 6b/CH2O/NaOH = 1.0:1.1:1.1). After 1 h at room tempera-
ture, the mixture was acidified with concentrated hydrochloric acid
(0.30 mL) and stirred for an additional 12 h. The mixture was ex-
tracted with ethyl acetate (3ϫ 5 mL) and the combined organic
layers were washed with NaOH (1 n, 5 mL), dried with anhydrous
Na2SO4, and concentrated in vacuo. The crude mixture was puri-
fied by flash chromatography (eluent: dichloromethane/ethyl acet-
Conclusions
1
ate, 97:3) to give 7b (65% yield). H NMR (CDCl3): δ = 2.80 (m,
In this article, we describe the design, synthesis and bio-
logical properties of new potential STAT3 inhibitors 1a, 1b,
and 2 together with the modeling of 1a. These compounds
are characterized by a pyridazinone ring that replaces the
1,2,5-oxadiazole of reference compound I, a known agent
for prevention and treatment of cancer.[9] The previous
computational study carried out by us on I showed, for the
urea, the ZZ conformation to be favored in water.[10] Styryl
analogue II, which shows a significant drop in activity, pre-
fers, both in vacuo and in water, the bent EZ geometry,
which is stabilized by the formation of an intramolecular
H-bond between an ureido NH hydrogen and the N2 oxadi-
azole. The modeling study on new pyridazinone 1a re-
vealed, both in vacuo and in water, an almost complete
preference for the desired extended ZZ geometry. However,
once tested with a dual-luciferase assay for their inhibition
of the STAT3 signaling pathway, all compounds showed
only modest activity. Furthermore, the AlphaScreen-based
assay did not reveal any interaction with the STAT3 SH2
domain. These outcomes seem to indicate that neither the
1,2,5-oxadiazole ring, present in compound II, nor the ex-
tended ZZ arrangement, present in the new derivatives,
alone is sufficient to confer significant activity against
STAT3 and that they probably act in a synergistic way in
1 H, CH), 2.98 (m, 1 H, CH), 4.35 (m, 1 H, CH), 4.46 (m, 1 H,
CH), 4.62 (m, 1 H, CH), 7.50 (m, 2 H, ArH), 7.88 (m, 2 H,
ArH) ppm.
6-(4-Chlorophenyl)-5-(hydroxymethyl)-4,5-dihydro-3(2H)-pyridaz-
inone (8b): Hydrazine monohydrate (0.78 mmol) was added to a
solution of β-(4-chlorobenzoyl)-γ-butyrolactone (7b, 0.26 mmol) in
ethanol (2 mL), and the mixture was heated to reflux for 1.5 h. The
solvent was then removed in vacuo and the residue was extracted
with ethyl acetate (5ϫ 3 mL). The organic layer was dried with
anhydrous Na2SO4, and concentrated in vacuo. Compound 8b was
obtained upon purification by flash column chromatography (elu-
ent: dichloromethane/methanol, 95:5; 20 % yield). 1H NMR
(CDCl3): δ = 2.70 (m, 2 H, CH2), 3.57 (m, 2 H, CH2), 3.72 (m, 1
H, CH), 7.55 (m, 2 H, ArH), 7.86 (m, 2 H, ArH), 10.07 (s, 1 H,
NH) ppm.
6-(4-Chlorophenyl)-5-(hydroxymethyl)-4,5-dihydro-3(2H)-pyridaz-
inone Acetate (9b): Acetic anhydride (0.48 mmol) was added to a
solution of 6-(4-chlorophenyl)-5-(hydroxymethyl)-4,5-dihydro-
3(2H)-pyridazinone (8b, 0.25 mmol) in pyridine (2 mL), and the
mixture was stirred at room temp. for 4 h. The pyridine was then
removed under reduced pressure and the residue was extracted with
ethyl acetate (5ϫ 3 mL). The organic layer was dried with anhy-
drous Na2SO4, evaporated, and the crude mixture was purified by
flash chromatography (eluent: cyclohexane/ethyl acetate, 3:7) to
give 9b (96% yield). 1H NMR (CDCl3): δ = 2.0 (m, 3 H, CH3),
compound I. It should also be noted that the N2 atom of 2.75 (m, 2 H, CH2), 3.60 (m, 1 H, CH), 4.04 (m, 1 H, CH), 4.31
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Structural and Conformational Properties for STAT3 Inhibition
(m, 1 H, CH), 7.38 (m, 2 H, ArH), 7.75 (m, 2 H, ArH), 9.10 (s, 1
H, NH) ppm.
product 1a, which was obtained as a white solid after purification
by flash column chromatography (eluent: dichloromethane/meth-
1
anol, 95:5; 20% yield), m.p. 154–156 °C. H NMR (CD3OD): δ =
6-(4-Chlorophenyl)-5-(hydroxymethyl)-3(2H)-pyridazinone Acetate
(10b): A mixture of 6-(4-chlorophenyl)-5-(hydroxymethyl)-4,5-dihy-
dro-3(2H)-pyridazinone acetate (9b, 0.20 mmol) and activated
manganese dioxide (2.1 mmol) in dry dichloromethane (2 mL) was
heated to reflux for 24 h. After cooling, the oxidant was removed
by filtration through Celite and the manganese dioxide was washed
with dichloromethane. The filtrate was concentrated and the resi-
due purified by flash chromatography (eluent: dichloromethane/
7.55 (m, 9 H, ArH), 7.85 (s, 1 H, CH) ppm. HRMS (ESI): calcd.
for C18H13N4O2F3Na [M + 1] 397.08828; found 397.08940.
1-[3(4-Chlorophenyl)-6-oxo-1,6-dihydropyridazin-4-yl]-3-(4-tri-
fluoromethyl)phenylurea (1b): General procedure A was followed
for the synthesis of compound 1b, which was obtained as a pale
yellow solid after purification by flash column chromatography
(eluent: dichloromethane/methanol, 95:5; 10 % yield), m.p. 161–
ethyl acetate, 8:2; 20% yield). 1H NMR (CDCl3): δ = 2.13 (m, 3 162 °C. H NMR (CD3OD): δ = 7.59 (m, 8 H, ArH), 7.83 (s, 1 H,
1
H, CH3), 4.88 (m, 2 H, CH2), 7.05 (s, 1 H, CH), 7.35 (m, 2 H, CH) ppm. HRMS (ESI): calcd. for C18H12N4O2F3ClNa [M + 1]
ArH), 7.45 (m, 2 H, ArH), 10.8 (s, 1 H, NH) ppm.
431.0431; found 431.05017.
6-(4-Chlorophenyl)-5-(hydroxymethyl)-3(2H)-pyridazinones (11b): A
solution of 6-(4-chlorophenyl)-5-(hydroxymethyl)-3(2H)-pyridaz-
inone acetate (10b, 0.20 mmol) in ethanol (3 mL) was treated with
HCl (2 n, 1.5 mL) and the mixture was heated to reflux for 4 h.
After cooling, the solvent was removed in vacuo and the residue
extracted with ethyl acetate (4ϫ 3 mL). The organic layer was dried
with anhydrous Na2SO4 and concentrated in vacuo to give 11b in
quantitative yield. 1H NMR (CD3COCD3): δ = 2.85–3.05 (br. s,
1 H, OH), 4.45 (s, 2 H, CH2), 7.20 (s, 1 H, CH), 7.50 (m, 4 H,
ArH) ppm.
1-(6-Oxo-3-phenyl-1,6-dihydropyridazin-4yl)-3-[2,4-(dimethyl)-
phenyl]urea (2): General procedure A was followed for the synthesis
of compound 2, which was obtained as a white oil after purification
by flash column chromatography (eluent: dichloromethane/meth-
1
anol, 95:5; 5% yield). H NMR (CD3OD): δ = 2.11 (s, 3 H, CH3),
2.26 (s, 3 H, CH3), 6.95 (m, 2 H, ArH), 7.22 (s, 1 H, ArH), 7.50
(m, 5 H, ArH) ppm. HRMS (ESI): calcd. for C19H18N4O2 [M + 1]
335.15025; found 335.14865.
Dual Luciferase Assay
Cell Culture: Human colon cancer cell line (HCT-116) was ob-
tained from American Type Culture Collection and it was main-
tained in McCoy’s 5A (Gibco/BRL). The culture medium was sup-
plemented with heat-inactivated fetal bovine serum (10%; Gibco/
BRL). Cell cultures were maintained at 37 °C under a humidified
atmosphere of CO2 (5%) in an incubator.
6-(4-Chlorophenyl)-5-formyl-3(2H)-pyridazinone (12b): A solution
of 6-(4-chlorophenyl)-5-(hydroxymethyl)-3(2H)-pyridazinone (11b,
0.25 mmol) in dry tetrahydrofuran (THF; 2 mL) was stirred with
activated manganese dioxide (2.5 mmol) for 48 h at room temp.
The suspension was filtered through Celite and the manganese di-
oxide was washed with THF. The filtrate was concentrated and the
residue purified by flash chromatography (eluent: dichloromethane/
methanol, 95:5) to give 12b (55% yield). 1H NMR (CDCl3): δ =
7.33 (s, 1 H, CH), 7.43 (m, 4 H, ArH), 9.91 (s, 1 H, CHO) ppm.
Transient Transfection and Dual-luciferase Assays:[18] HCT-116 cells
were seeded at a density of 10ϫ105 cells in a 100 mm2 culture plate.
The cells were co-transfected with pSTAT3-TA-Luc (27 μg/plate)
and internal control plasmid pRL-TK (9 μg/plate) that contained
Renilla luciferase gene. All plasmids used in this experiment were
purchased from Promega. Transfection was carried out by using
TransFectin (Bio-Rad) in accordance with the manufacturer’s pro-
tocol. Then, 5 h after transfection, the cells were trypsinized and
seeded onto sterilized black bottom 96-well plates at a density of
1ϫ104 cells per well. The following day, cells were treated with test
compounds and incubated for 24 h. Firefly and Renilla luciferase
activities were measured by using dual-light reporter gene assay kit
(Promega) with a Wallac Victor2 (Perkin–Elmer, Inc., Wellesley,
MA). Renilla luciferase activity was determined to calibrate trans-
fection efficiency and cytotoxicity of chemicals. Relative STAT3
activity was calculated by dividing the firefly luciferase activity with
Renilla luciferase activity in each transfection experiment. The val-
ues of STAT3 inhibitory activity were the mean results of three
experiments and the maximum deviation from the mean was less
than 10%.
6-(4-Chlorophenyl)-5-carboxy-3(2H)-pyridazinone (13b): A solution
of 6-(4-chlorophenyl)-5-formyl-3(2H)-pyridazinone (12b,
0.25 mmol) and silver nitrate (1.25 mmol) in ethanol (0.5 mL) and
water (0.6 mL) was stirred rapidly under a nitrogen atmosphere. A
NaOH (10 %) solution was added until the reaction mixture
reached pH 12. The resulting black suspension was stirred at room
temperature for 15 h and then filtered. The filtrate was concen-
trated under reduced pressure, then acidified with HCl (6 n), ex-
tracted with diethyl ether (3 ϫ 2 mL), dried with anhydrous
Na2SO4, and concentrated in vacuo. The obtained crude mixture
was purified by flash chromatography (eluent: dichloromethane/
methanol, 9:1) to 13b (70% yield). 1H NMR (CDCl3): δ = 6.20–
6.82 (br. s, 1 H, COOH), 6.70 (s, 1 H, CH), 7.40 (d, 2 H, ArH),
7.60 (d, 2 H, ArH) 11.0 (s, 1 H, NH) ppm.
General Procedure A for the Synthesis of Compounds 1a and 1b:
Diphenylphosphoryl azide (DPPA; 0.36 mmol) was added to a sus-
pension of carboxylic acid (13, 0.23 mmol) in dry toluene (8 mL)
and dry THF (8 mL) under a nitrogen atmosphere. Triethylamine
(3.6 mmol) was dropped into the mixture that was stirred at room
temperature for 30 min. The solution was heated at 80 °C for 2 h
and then the appropriate substituted aniline (0.29 mmol) in THF
(3 mL) was added together with triethylamine (3.6 mmol). After
stirring at 80 °C overnight, the mixture was diluted with water
(10 mL) and extracted with ethyl acetate (3ϫ 10 mL). The organic
phase was washed with HCl (1 m, 1ϫ 10 mL), dried with anhy-
drous Na2SO4, and concentrated in vacuo to give the final prod-
ucts.
AlphaScreen-Based Assay: AlphaScreen is a bead-based nonradio-
active assay system to detect biomolecular interactions in microtiter
plate format. Binding of biological partners brings donor and ac-
ceptor beads into close proximity and as a result, a fluorescent
signal between 520 and 620 nm is produced. The AlphaScreen-
based assays[19] were performed in a final reaction volume of 25 μL
of the assay buffer that contained 4-(2-hydroxyethyl)-1-piperazine-
ethanesulfonic acid–NaOH (10 mm, pH 7.4), NaCl (50 mM), ethyl-
enediaminetetraacetic acid, (1 mm, pH 8.0), NP-40 (0.1%), and bo-
vine serum albumin (10 ng/μL) in a 96-well microtiter plate at
25 °C. The phospho-Tyr (pTyr) peptide probes used in this study
were 5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3, FITC-
GpYDKPHVL for STAT1, and FITC-PSpYVNVQN for Grb2.
1-(6-Oxo-3-phenyl-1,6-dihydropyridazin-4yl)-3-(4-trifluoromethyl)-
phenylurea (1a): General procedure A was followed to prepare final
Eur. J. Org. Chem. 2015, 4907–4912
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4911

S. Villa, L. Legnani, et al.
FULL PAPER
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Acknowledgments
The authors acknowledge the financial support of the Universities
of Milan and Pavia and the Italian Ministero dell’Università e della
Ricerca (MIUR) (funds PRIN 2010-11). B. M. K. was supported
by the KRIBB Research Initiative Program and the Bio-Synergy
Research Project (2012M3A9C404877).
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Published Online: June 30, 2015
4912
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 4907–4912